JP2004512806A - Plant centromere - Google Patents

Abstract

The present invention provides for the identification and cloning of functional plant centromeres in Arabidopsis. The present invention allows for the construction of stably inherited mini-chromosomes. Minichromosomes serve as vectors for the construction of transgenic plant and animal cells. In addition, information about the structure and function of these regions provides utility in isolating additional centromeres and centromere-related genetic elements and polypeptides from other species.

Description

プロテアーゼインヒビターはまた、昆虫耐性を提供し得（Ｊｏｈｎｓｏｎら、１９８９）、従って、植物における形質転換における利用を有する。トマトまたはジャガイモ由来のプロテアーゼインヒビタ−ＩＩ遺伝子であるｐｉｎＩＩの使用は、特に有用であると考察される。Ｂｔ毒素遺伝子と組み合わせた、ｐｉｎＩＩ遺伝子の使用は、さらにより都合が良く、その組み合わされた効果は、相乗作用の殺虫剤活性を生じることが発見されている。昆虫の消化系の阻害剤をコードする他の遺伝子、または阻害剤の産生を容易にする酵素または補因子をコードする遺伝子もまた有用であり得る、この群は、オリザシスタチン（ｏｒｙｚａｃｙｓｔａｔｉｎ）インヒビタ−およびアミラーゼインヒビター（例えば、小麦および大麦由来のそれらのインヒビタ−）によって例示され得る。
【０２０５】
また、レクチンをコードする遺伝子は、さらなる殺虫剤特性、または代替の殺虫剤特性を与え得る。レクチン（元々、フィトヘマグルチニンと呼ばれる）は、種の範囲から、赤血球を凝集させる能力を有する、炭水化物結合タンパク質である。レクチンは、ゾウムシ、ＥＣＢおよび根切り虫（ｒｏｏｔｗｏｒｍ）に対する活性を有する殺虫剤として最近同定された（Ｍｕｒｄｏｃｋら、１９９０；ＣｚａｐｌａおよびＬａｎｇ、１９９０）。有用であるよう意図されたレクチン遺伝子としては、例えば、大麦病原体凝集素（ｇｅｒｍ　ａｇｇｌｕｔｉｎｉｎ）および小麦病原体凝集素（ＷＧＡ）ならびにライスレクチン（Ｇａｔｅｈｏｕｓｅら、１９８４）が挙げられ、ＷＧＡが好まれる。
【０２０６】
昆虫ペスト中に導入される場合、昆虫に対して活性な大きいポリペプチドまたは小さいポリペプチド（例えば、溶解性（ｌｙｔｉｃ）ペプチド、ペプチドホルモンならびに毒物および毒液のような）の産生を制御する遺伝子は、本発明の別の局面を形成する。例えば、特定の昆虫ペストに対して指向される若年性（ｊｕｖｅｎｉｌｅ）ホルモンエステラーゼの発現は、殺虫活性も生じ、またはおそらく変態の停止を生じることが意図される（Ｈａｍｍｏｃｋら、１９９０）。
【０２０７】
昆虫の表皮の完全性（ｉｎｔｅｇｒｉｔｙ）に影響する酵素をコードする遺伝子を発現するトランスジェニック植物は、本発明のさらに別の局面を形成する。このような遺伝子としては、例えば、キチナーゼ、プロテアーゼ、リパーゼをコードする遺伝子およびニコマイシン（ｎｉｋｋｏｍｙｃｉｎ）（キチン合成を阻害する化合物）の産生ための遺伝子もまた挙げられ、これらのいずれかの導入が、昆虫耐性植物を産生するために意図される。昆虫の脱皮に影響する活性をコードする遺伝子（例えば、エクジステロイドＵＤＰグルコシルトランスフェラーゼの産生に影響する遺伝子）もまた、本発明の有用な導入遺伝子の範囲内にあたる。
【０２０８】
昆虫ペストに対する、宿主植物の栄養の質を低下する化合物の産生を容易にする酵素をコードする遺伝子もまた、本発明により包含される。例えば、そのステロール組成物を変更することにより、植物の殺虫活性を与えることを可能にし得る。ステロールは、昆虫の食物由来で、昆虫から得られ、そしてホルモン合成および膜安定性のために使用される。従って、新規の遺伝子（例えば、所望されないステロールの産生を直接促進する遺伝子、または所望のステロールを所望しない形態に変換する遺伝子）の発現による、植物ステロール組成物の変更が、昆虫の成長および／または発生においていて負の効果を有し、従って、植物に殺虫活性を賦与する。リポキシゲナーゼは、昆虫において抗栄養の効果を示し、そして昆虫の食物の栄養の質を低下するために示された天然に存在する植物酵素である。従って、さらに、本発明の実施形態は、昆虫の摂食に耐性であり得る増強されたリポキシゲナーゼ活性を有するトランスジェニック植物に関する。
【０２０９】
Ｔｒｉｐｓａｃｕｍ　ｄａｃｔｙｌｏｉｄｅｓは、トウモロコシ（ｃｏｒｎ）根切り虫を含む、特定の昆虫に対して耐性であるイネ科の植物の一種である。昆虫に対して毒性であり、または昆虫に対して毒性のある化合物の生合成に関与するタンパク質をコードする遺伝子は、Ｔｒｉｐｓａｃｕｍから単離され、そしてこれらの新規の遺伝子は、昆虫に対して耐性を与えるのに有用であると予測される。Ｔｒｉｐｓａｃｕｍにおいて昆虫耐性の根拠は、遺伝的なものであることが公知である。なぜなら、上記の耐性は、性的交配（ｓｅｘｕａｌ　ｃｒｏｓｓ）を介するＺｅａ　ｍａｙｓに伝達されたからである（ＢｒａｎｓｏｎおよびＧｕｓｓ、１９７２）。他の穀類の植物種、単子葉植物種または双子葉植物種が、昆虫耐性植物を産生するのに有用である、昆虫に対して毒性のあるタンパク質をコードする遺伝子を有し得ることが、さらに予測される。
【０２１０】
潜在的な殺虫活性を有するとして特徴付けられるタンパク質をコードするさらなる遺伝子もまた、これと一致して導入遺伝子として使用され得る。このような遺伝子としては、例えば、根切り虫の阻害物質として使用され得るカウピートリプシンインヒビター（ＣｐＴＩ；Ｈｉｌｄｅｒら、１９８７）；トウモロコシ根切り虫の阻害物質として特に有用と証明し得るアベルメクチンをコードする遺伝子（Ａｖｅｒｍｅｃｔｉｎ　ａｎｄ　Ａｂａｍｅｃｔｉｎ．、Ｃａｍｐｂｅｌｌ，Ｗ．Ｃ．、編、１９８９；Ｉｋｅｄａら、１９８７）；リボソーム不活性化タンパク質遺伝子；および植物構造を調節する遺伝子さえ、挙げられる。抗昆虫抗体遺伝子、および植物の外側に適用される非毒性殺虫剤（前毒性殺虫剤（ｐｒｏ−ｉｎｓｅｃｔｉｃｉｄｅ））を、植物の内側の殺虫剤に変換し得る酵素をコードする遺伝子を含むトランスジェニック植物もまた、意図される。
【０２１１】
（（ｉｉｉ）環境またはストレス耐性）
種々の環境ストレス（例えば、限定されないが、乾燥ストレス、過度の水分ストレス、寒冷ストレス、凍結ストレス、高温ストレス、塩ストレス、および酸化（ｏｘｉｄａｔｉｖｅ）ストレス）に耐性である植物の能力の改善もまた、新規の遺伝子の発現を通して、行われ得る。有益性は、Ｗｉｎｔｅｒ　Ｆｌｏｕｎｄｅｒ（Ｃｕｔｌｅｒら、１９８９）のような「抗凍結」タンパク質の導入またはその合成遺伝子誘導体の導入を介して、凍結温度に対する耐性の増加によって、実現され得る。改善された寒冷耐性はまた、クロロプラストにおけるグリセロール−３−ホスフェートアセチルトランスフェラーゼの発現の増加を介して与えられ得る（Ｗｏｌｔｅｒら、１９９２）。酸化ストレス（高い光強度と組み合わせて、寒冷温度のような条件によってしばしば悪化する）に対する耐性は、スーパーオキシドジスムターゼの発現によって与えられ得（Ｇｕｐｔａら、１９９３）、そしてグルタチオンレダクターゼによって改善され得る（Ｂｏｗｌｅｒら、１９９２）。このようなストラテジーは、新しく出現した領域における凍結に対する耐性、ならびに成熟がより遅く、より高い収量の種類を、相対的により早い成熟帯（ｚｏｎｅ）に広げることを考慮し得る。
【０２１２】
植物の水含有量、全水ポテンシャル、浸透圧ポテンシャルおよび膨圧を有利にもたらす新規遺伝子の発現が、乾燥に耐えられる植物の能力を増強することが、予期される。本明細書中で使用される場合、用語「旱魃抵抗性（ｄｒｏｕｇｈｔ　ｒｅｓｉｓｔａｎｃｅ）」および「旱魃耐性（ｄｒｏｕｇｈｔ　ｔｏｌｅｒａｎｃｅ）」は、正常な環境と比較する場合、水のアベイラビリティにおける減少により誘導されるストレスに対する植物の増加した抵抗性または耐性、ならびにより水の少ない環境で機能および生存する植物の能力をいうために使用される。本発明のこの局面では、例えば、浸透圧的に活性な溶質（例えば、ポリオール化合物）の生合成をコードする遺伝子の発現が、旱魃に対する防御を与えることが提唱される。このクラスには、マンニトール−Ｌ−リン酸デヒドロゲナーゼをコードする遺伝子（ＬｅｅおよびＳａｉｅｒ、１９８２）およびトレハロース−６−リン酸シンターゼをコードする遺伝子（Ｋａａｓｅｎら、１９９２）が存在する。細胞における本来のホスファターゼの引き続く作用を介して、または特定のホスファターゼの導入および共発現により、これらの導入された遺伝子は、各々、マンニトールまたはトレハロースのいずれかの蓄積を生じ、これらは両方とも、ストレスの効果を緩和し得る保護化合物として十分に示されている。トランスジェニックタバコにおけるマンニトール蓄積が立証されており、そして予備的な結果は、高レベルのこの代謝物を発現する植物が、適用された浸透圧的ストレスに耐え得ることを示す（Ｔａｒｃｚｙｎｓｋｉら、１９９２、１９９３）。
【０２１３】
同様に、酵素機能を保護することにおける他の代謝物（例えば、アラノパイン（ａｌａｎｏｐｉｎｅ）またはプロピオン酸）または膜統合性を保護することにおける他の代謝物（例えば、アラノパイン）のいずれかの効力は、示されており（Ｌｏｏｍｉｓら、１９８９）、そして従って、これらの化合物の生合成をコードする遺伝子の発現は、マンニトールに類似するか、または相補的な様式で旱魃抵抗性を与え得る。浸透圧的に活性であり、そして／または旱魃（ｄｒｏｕｇｈｔ）および／または乾燥（ｄｅｓｉｃｃａｔｉｏｎ）の間にいくらかの直接的保護効果を供給する、天然に存在する代謝生成物の他の例としては、フルクトース、エリスリトール（Ｃｏｘｓｏｎら、１９９２）、ソルビトール、ズルシトール（Ｋａｒｓｔｅｎら、１９９２）、グルコシルグリセロール（Ｒｅｅｄら、１９８４；ＥｒｄＭａｎｎら、１９９２）、スクロース、スタキオース（ＫｏｓｔｅｒおよびＬｅｏｐｏｌｄ、１９８８；Ｂｌａｃｋｍａｎら、１９９２）、ラフィノース（Ｂｅｒｎａｌ−ＬｕｇｏおよびＬｅｏｐｏｌｄ、１９９２）、プロリン（Ｒｅｎｓｂｕｒｇら、１９９３）、グリシン、ベタイン、オノニトール（ｏｎｏｎｉｔｏｌ）およびピニトール（ＶｅｒｎｏｎおよびＢｏｈｎｅｒｔ、１９９２）が挙げられる。連続したキャノピーの生育およびストレスの時間の間の増加した生殖適応度は、遺伝子（例えば、上記の浸透圧的に活性な化合物および他のそのような化合物を制御する遺伝子）の導入および発現により増強される。現在、浸透圧的に活性なポリオール化合物の合成を促進する好ましい遺伝子は、酵素（マンニトール−１−リン酸デヒドロゲナーゼ、トレハロース−６−リン酸シンターゼおよびミオイノシトール　０−メチルトランスフェラーゼ）をコードする遺伝子である。
【０２１４】
特定のタンパク質の発現がまた旱魃耐性を増加することが、予期される。後期胚形成タンパク質の３つクラスは、構造類似性に基づいて設定されている（Ｄｕｒｅら、１９８９を参照のこと）。ＬＥＡの全ての３つのクラスは、成熟（すなわち、乾燥）種子に存在することが実証された。ＬＥＡタンパク質のこれらの３つのクラスにおいて、ＩＩ型（デヒドリン型）は、一般に、栄養植物部分における旱魃耐性および／または乾燥耐性に関連している（すなわち、ＭｕｎｄｙおよびＣｈｕａ、１９８８；Ｐｉａｔｋｏｗｓｋｉら、１９９０；Ｙａｍａｇｕｃｈｉ−Ｓｈｉｎｏｚａｋｉら、１９９２）。最近、タバコにおけるＩＩＩ型ＬＥＡ（ＨＶＡ−１）は、草高、成熟度および旱魃耐性に影響することが見出された（Ｆｉｔｚｐａｔｒｉｃｋ、１９９３）。イネにおいて、ＨＶＡ−１遺伝子の発現は、水の欠乏および塩分に対する耐性に影響した（Ｘｕら、１９９６）。このように、すべての３つのＬＥＡ群由来の構造遺伝子の発現は、旱魃耐性を与えた。水ストレスの間に誘導される他のタイプのタンパク質としては、チオールプロテアーゼ、アルドラーゼ、および膜通過輸送体が挙げられ（Ｇｕｅｒｒｅｒｏら、１９９０）、これらは、旱魃ストレスの間の種々の保護および／または修復型の機能を与え得る。脂質生合成、そしてそれにより膜組成を達成する遺伝子もまた、植物に旱魃耐性を与える際に有用であることはまた、予期される。
【０２１５】
旱魃抵抗性を改良するためのこれらの遺伝子の多くは、相補的な作用形態を有する。従って、これらの遺伝子の組み合わせが、植物における旱魃抵抗性の改良において相加的および／または相乗的な効果を有し得ることが認識される。これらの遺伝子の多くはまた、凍結耐性（または、抵抗性）を改良する；凍結および旱魃の間に受ける物理的ストレスは、事実上類似し、そして同様の様式で緩和され得る。利点は、これらの遺伝子の構成的発現を介して与えられ得るが、これらの新規遺伝子を発現する好ましい手段は、膨圧誘導プロモーター（例えば、Ｇｕｅｒｒｅｒｏら、１９９０およびＳｈａｇａｎら、１９９３（これらは、参考として本明細書中に援用される）において記載される膨圧誘導遺伝子についてのプロモーター）の使用を介し得る。これらの遺伝子の空間的発現パターンおよび時間的発現パターンは、植物が、ストレスに対してより良好に耐えることを可能にする。
【０２１６】
乾燥土壌からの増加した水の吸い上げを可能にする特定の形態学的形質に関連する遺伝子の発現が有益であることが、提唱される。例えば、根の特徴を変化させる遺伝子の導入および発現は、水の取り込みを増強し得る。ストレスの時間の間に生殖適応度を増強する遺伝子の発現が有意な価値があることが、予期される。例えば、雌花部分（すなわち、毛（ｓｉｌｋ））の花粉脱粒（ｓｈｅｄ）および受容性（ｒｅｃｅｐｔｉｖｅｎｅｓｓ）の同調性を改良する遺伝子の発現は、有益であり得る。さらに、ストレスの時間の間に穀粒の発育不全を最小化する遺伝子の発現が、収穫される粒の量を増加し得、従って価値があることが提唱される。
【０２１７】
収穫高の決定における水の全体的な役割を考えると、新規遺伝子の導入および発現を介して植物がより効率的に水を利用することを可能にすることが、土壌水分のアベイラビリティが制限されない場合でも、全体的な効率を改良することが予期される。水のアベイラビリティに関するストレスの全ての範囲にわたって水の使用を最大にする植物の能力を改良する遺伝子を導入することにより、収穫安定性または収穫高の一貫性は、現実化し得る。
【０２１８】
（ｖｉ）疾患抵抗性
疾患に対する増加した抵抗性が、植物（例えば、単子葉植物（例えば、トウモロコシ））への遺伝子の導入を介して、現実化され得ることが、提唱される。ウイルス、細菌、菌類および線形動物により引き起こされる疾患に対する抵抗性を生じさせることが、可能である。マイコトキシンを産生する生物体の制御が、導入された遺伝子の発現を介して実現され得ることもまた、予期される。
【０２１９】
ウイルスに対する抵抗性は、新規遺伝子の発現を介して生じ得る。例えば、トランスジェニック植物におけるウイルス性コートタンパク質の発現が、ウイルスおよびおそらく他の近種のウイルスによる植物の感染に対する抵抗性を与え得ることが実証されている（Ｃｕｏｚｚｏら、１９８８、Ｈｅｍｅｎｗａｙら、１９８８、Ａｂｅｌら、１９８６）。必須のウイルス機能に標的化されたアンチセンス遺伝子の発現がまた、ウイルスに対する抵抗性を与え得ることが、予期される。例えば、ウイルス核酸の複製に応答可能な遺伝子に標的化されたアンチセンス遺伝子は、複製を阻害し、ウイルスに対する抵抗性を誘導し得る。アンチセンス遺伝子の使用を介する他のウイルス機能の妨害がまた、ウイルスに対する抵抗性を増加し得ると考えられる。さらに、サテライトウイルスの使用が挙げられるがこれに限定されない他の試みを介して、ウイルスに対する抵抗性を達成することが可能であり得ることが、提唱される。
【０２２０】
細菌および菌類により引き起こされる疾患に対する増加された抵抗性が、新規遺伝子の導入を介して実現され得ることが、提唱される。いわゆる「ペプチド抗生物質」、病因関連（ＰＲ）タンパク質、毒素抵抗性、および宿主病原相互作用（例えば、形態学的特徴）に影響するタンパク質をコードする遺伝子が有用であることが、予期される。ペプチド抗生物質は、細菌および他の微生物の増殖に対して阻害的であるポリペプチド配列である。例えば、セクロパイン（ｃｅｃｒｏｐｉｎ）およびマゲイニンといわれるペプチドのクラスは、細菌および菌類の多くの種の増殖を阻害する。単子葉植物（たとえば、トウモロコシ）におけるＰＲタンパク質の発現が、細菌性疾患に対する抵抗性を与えることにおいて有用であり得ることが、提唱される。これらの遺伝子は、宿主植物に対する病原体の攻撃の後に誘導され、そして少なくとも５つのタンパク質のクラスに分類される（Ｂｏｌ，ＬｉｎｔｈｏｒｓｔおよびＣｏｒｎｅｌｉｓｓｅｎ、１９９０）。β−１’３−グルカナーゼ、キチナーゼ、ならびにオスモチンおよび疾患生物体に対する抵抗性として植物において機能すると考えられるタンパク質が、ＰＲタンパク質に含まれる。抗菌特性を有する他の遺伝子は、同定されている（例えば、ＵＤＡ（有針イラクサ科のレクチン）およびヘベイン（ｈｅｖｅｉｎ）（Ｂｒｏａｋａｅｒｔら、１９８９；Ｂａｒｋａｉ−Ｇｏｌａｎら、１９７８））。特定の植物疾患がフィトトキシンの産生により引き起こされることは、公知である。これらの疾患に対する抵抗性が、フィトトキシンを分解するか、そうでなければ不活性化し得る酵素をコードする新規遺伝子の発現を介して達成されることが、提唱される。宿主植物と病原体との間の相互作用を変化させる新規遺伝子の発現が、宿主植物の組織を侵襲する疾患生物体の能力（例えば、葉クチクラのろう質（ｗａｘｉｎｅｓｓ）の増加または他の形態学的な特徴）を減少する際に有用であり得ることもまた、予期される。
【０２２１】
（ｖ）植物の農学的特徴
作物植物が生育され得る場所を決定する２つの要因は、生育期の間の平均の日中気温および霜の間の時間の長さである。特定の作物を生育することが可能である地域において、成熟まで生育しそして収穫が可能となる最大時間に関して、種々の制限が存在する。例えば、特定の地域で生育される変種は、最大の可能な収穫を伴って、必要とされる時間内で成熟し、そして収穫可能な水分含有量までドライダウンするその能力について選択される。従って、種々の成熟度の作物が、異なる生育場所に関して開発される。収穫を可能にするために十分にドライダウンされる必要性を除いて、収穫後のさらなる乾燥に必要とされる量のエネルギーを最小にするために、野外で最大に乾燥することが望ましい。また、より容易に産物（例えば、子実）がドライダウンし得るほど、生育および穀粒充填に入手可能である時間がより長くなる。成熟および／またはドライダウンに影響する遺伝子が同定され得、そして形質転換技術を使用して植物系統に導入され得、異なる生育場所または同じ生育場所に適用されるが、収穫の際の水分に対する収穫高の比が改良されている新たな変種を作出することが考えられる。植物開発の調節に関与する遺伝子の発現は、特に有用であり得る。
【０２２２】
立位性（ｓｔａｎｄａｂｉｌｉｔｙ）および他の植物生長特徴を改良し得る遺伝子が植物へ導入され得ることが、予期される。より強い柄、改良された根系を与えるか、または雌穂落下量を防ぐかもしくは減少させる新規遺伝子の植物における発現は、農家にとって大いに価値がある。例えば、光の分配および／または中断を増加することにより得られ得る光合成生成物の総量を増加する遺伝子の導入および発現が有利であることが、提唱される。さらに、光合成効率および／または林冠を増加する遺伝子の発現が、生産性をさらに増加する。作物植物中のフィトクロム遺伝子の発現が有利であり得ることは、予期される。このような遺伝子の発現は、頂芽優勢を減少し得、植物に半矮性を与え、そして耐陰性を増加する（米国特許第５，２６８，５２６号）。このような試みは、野外における増加した植物集団を可能にする。
【０２２３】
（ｖｉ）栄養の利用
利用可能な栄養を利用する能力は、作物植物の生育における制限因子であり得る。新規遺伝子の導入によって、栄養の取り込み、耐性ｐＨ極値、植物にわたる流動、貯蔵プール、および代謝活性に対するアベイラビリティを変化させることが可能であることが、提唱される。これらの改変は、植物（例えば、トウモロコシ）が、利用可能な栄養をより効率的に使用することを可能にする。例えば、植物中に正常に存在し、そして栄養の利用に関与する酵素の活性増加が栄養のアベイラビリティを増加することが、予期される。このような酵素の例は、フィターゼ（ｐｈｙｔａｓｅ）である。植物による増強された窒素の利用が望ましいことが、さらに予期される。植物におけるグルタミン酸デヒドロゲナーゼ遺伝子（例えば、Ｅ．ｃｏｌｉ　ｇｄｈＡ遺伝子）の発現は、有機化合物中の窒素の増加した固定化を誘導し得る。さらに、植物におけるｇｄｈＡの発現は、グルタミン酸の過剰なアンモニアの取り込みにより、除草剤グルフォシネート（ｇｌｕｆｏｓｉｎａｔｅ）に対する増強された抵抗性を誘導し得、従って、アンモニアを解毒する。新規遺伝子の発現が、以前にはアクセス可能ではなかった栄養供給源（例えば、より複雑な分子（恐らくは高分子）から栄養価値のある成分を放出する酵素）を利用可能にし得ることもまた、予期される。
【０２２４】
（ｖｉｉ）雄性不稔
雄性不稔は、ハイブリッド種子の産生において有用である。雄性不稔が新規遺伝子の発現を介して産生され得ることが、提唱される。例えば、雄性花序および／または配偶体の発達に干渉するタンパク質をコードする遺伝子の発現が、雄性不稔を生じることが、示された。トランスジェニックタバコおよびナタネ油（ｏｉｌｓｅｅｄ）のセイヨウアブラナ（ｒａｐｅ）の葯におけるキメラのリボヌクレアーゼ遺伝子は、雄性不稔を誘導することが実証されている（Ｍａｒｉａｎｉら、１９９０）。
【０２２５】
細胞質雄性不稔を与える多くの変異が、トウモロコシにおいて発見された。特にＴ細胞質といわれる１つの変異はまた、サザン（Ｓｏｕｔｈｅｒｎ）コーン夏疫病に対する感受性と相関する。Ｔ細胞質と相関するＴＵＲＦ−１３と称されるＤＮＡ配列（Ｌｅｖｉｎｇｓ、１９９０）が、同定された。形質転換を介してＴＵＲＦ−１３の導入を通じて、疾患感受性から雄性不稔を区別することが可能であることが、提唱される。育種目的および子実産生のために雄性不稔を回復し得ることが必要である場合、雄性稔性の回復をコードする遺伝子もまた導入され得ることが提唱される。
【０２２６】
（ｖｉｉｉ）改良された栄養含有量
遺伝子は、特定の作物の栄養の質または含有量を改良するために、植物に導入され得る。作物の栄養組成を変化する遺伝子の導入は、飼料価または食品価を非常に増強し得る。例えば、多くの子実のタンパク質は、特にブタ、家禽、およびヒトに与えられる場合、飼料および食品としての目的としては最適以下であり得る。このタンパク質は、これらの種の食餌において必須であるいくつかのアミノ酸を欠乏し、この子実への補充物の添加を必要とする。制限必須アミノ酸としては、リジン、メチオニン、トリプトファン、スレオニン、バリン、アルギニンおよびヒスチジンが挙げられ得る。いくつかのアミノ酸は、コーンが飼料処方のために他の投入物で補充される後のみに制限される。種子および子実の中のこれらの必須アミノ酸のレベルは、アミノ酸の生合成を増加するための遺伝子の導入、アミノ酸の分解を減少するための遺伝子の導入、タンパク質中のアミノ酸の貯蔵を増加するための遺伝子の導入、または種子もしくは子実へのアミノ酸の輸送を増加するための遺伝子の導入が挙げられるが、これらに限定されない機構により上昇し得る。
【０２２７】
作物のタンパク質組成は、種々の方法でアミノ酸の収支を改良するために変化され得、この方法としては、ネイティブなタンパク質の発現を上昇すること、乏しい組成を有する作物の発現を減少すること、ネイティブなタンパク質の組成物を変化すること、優れた組成を所有する全体的に新規なタンパク質をコードする遺伝子を導入することが挙げられる。
【０２２８】
作物植物の油含有量を変化する遺伝子の導入はまた、価値を有し得る。油含有量の増加は、食餌および食品の使用のための種子の代謝エネルギー含有量および密度の増加を生じ得る。導入された遺伝子は、脂肪酸または脂質の生合成における律速または制御された工程を取り除くかまたは減少する酵素をコードし得る。このような遺伝子としては、アセチル−ＣｏＡカルボキシラーゼ、ＡＣＰ−アセチルトランスフェラーゼ、β−ケトアシル−ＡＣＰシンターゼ、および他の周知の脂肪酸生合成活性をコードする遺伝子が挙げられるがこれらに限定されない。他の可能性は、アシル保有タンパク質のような酵素活性を有しないタンパク質をコードする遺伝子である。油中に存在する脂肪酸の収支を変化する遺伝子は、導入され得、これはより健康的であるかまたは栄養的な飼料を供給する。導入されたＤＮＡはまた、脂肪酸生合成に関与する酵素の発現をブロックする配列をコードし得、これは作物中に存在する脂肪酸の割合を変化する。
【０２２９】
例えば、枝分れの程度を増加することにより、作物のデンプン成分の栄養価を増強する遺伝子が導入され得、これはその代謝を遅延することによる家畜におけるデンプンの利用の改良を生じる。さらに、作物の他の主な構成要素が変化され得、これには、種々の他の栄養、プロセシング、または他の品質の局面に影響する遺伝子が挙げられる。例えば、色素沈着は、増加または減少され得る。
【０２３０】
飼料または食用作物はまた、不十分な量のビタミンしか有し得ず、十分な栄養価を提供するために補充を必要とする。ビタミン生合成を増強する遺伝子の導入は、例えば、ビタミンＡ、Ｅ，Ｂ_１２、コリンなどを含むと考えられる。ミネラル含有量もまた、至適以下であり得る。従って、とりわけ、リン、イオウ、カルシウム、マンガン、亜鉛および鉄を含む化合物の蓄積またはアベイラビリティに影響する遺伝子は、価値がある。
【０２３１】
作物の改良の多くの他の例は、本発明とともに使用され得る。この改良は、子実に関する必要はないが、例えば、サイレージについて作物価を改良し得る。このことを達成するためのＤＮＡの導入は、リグニン産生を変化する配列（例えば、ウシにとって優位な飼料価と関連する「褐色中央脈」表現型を生じる配列）を含み得る。
【０２３２】
飼料価および食品価における直接的な改良に加えて、作物の加工を改良し、そして加工から生じる生成物の価値を改良する遺伝子もまた、導入され得る。湿式ミルを介する場合、作物を使用する。従って、例えば、浸漬時間を減少することにより効率を増加し、そしてこのような加工の費用を減少する新規遺伝子はまた、用途を見出され得る。湿式製粉生成物の価値を改良することは、デンプン、油、コーングルテン粉またはグルテン食餌の成分の量または質を変化することを含み得る。デンプンの上昇は、デンプン生合成における律速工程の同定および除去を介するか、またはデンプンにおける割合の増加を生じる作物のほかの成分のレベルを減少することにより、達成され得る。
【０２３３】
油は、湿式ミルの別の生成物であり、その価値は、遺伝子の導入および発現により改良され得る。油の特性は、調理油、ショートニング、潤滑剤または他の油に由来する生成物の生成および使用、ならびに食品に関連する適用において使用される場合のその健康に関する性状の改良におけるその有効性を改良するために、変化され得る。抽出の際に、化学合成のための出発物質として機能し得る新規脂肪酸もまた、合成され得る。油の特性における変化は、その油に存在する脂肪酸のタイプ、レベル、または脂質の調整を変化することにより達成され得る。次に、このことは、新規脂肪酸およびそれらを有する脂質の合成を触媒する酵素をコードする遺伝子の添加によるか、またはネイティブな脂肪酸のレベルを増加し、一方でおそらく前駆体のレベルを減少することにより、達成され得る。あるいは、脂肪酸生合成における工程を遅延させるかまたはブロックするＤＮＡ配列が、導入され得、これは、前駆体脂肪酸中間体における増加を生じるる。添加され得る遺伝子としては、デサチュラーゼ、エポキシダーゼ、ヒドラターゼ、デヒドラターゼおよび脂肪酸中間体を巻き込む反応を触媒する他の酵素が挙げられる。ブロックされ得る触媒工程の代表的な例としては、ステアリン酸からオレイン酸への、およびオレイン酸からリノレン酸への不飽和化が挙げられ、これは、ステアリン酸およびオレイン酸の各々の蓄積を生じる。他の例は、伸長工程の妨害であり、これは、Ｃ_８〜Ｃ_１２飽和脂肪酸の蓄積を生じる。
【０２３４】
（ｉｘ）化学薬品および生物製剤の生成または同化作用
本発明に従って調製されるトランスジェニック植物が、全く生成されないか、または事前のコーン植物とは同じレベルでは生成されないかのいずれかである有用な生物学的化合物の生成または製造に使用され得ることが、さらに考えられる。あるいは、本発明に従って生成される植物は、特定の化合物（例えば、有害廃棄物）を代謝し得、これによりこれらの化合物のバイオレメディエーションを可能にするために作製され得る。
【０２３５】
これらの化合物を生成する新規植物は、本発明により提供される構築物を用いる、１つまたは潜在的に多くの遺伝子の導入および発現により可能にされる。可能な多くのアレイとしては、任意の生物体により現在生成される任意の生物学的化合物（例えば、タンパク質、核酸、初期代謝物および中間代謝物、炭水化物ポリマー、バイオレメディエーションにおける使用のための酵素、二次植物代謝物（例えば、フラボノイドまたはビタミン）を生成する経路を改変するための酵素、薬剤を生成し得る酵素、および導入について、製造業にとって目的の化合物（例えば、専門的な化学薬品およびプラスチック）を生成し得る酵素）が挙げられるが、これらに限定されない。これらの化合物は、植物により生成され得、収穫および／または加工に際して抽出され得、そして現在認識される任意の有用な目的（例えば、薬剤、芳香およびいくつかの名前がつけられる産業的酵素）のために使用され得る。
【０２３６】
（ｘ）非タンパク質発現配列
ＤＮＡは、植物の表現型に影響するための機能が未だタンパク質へ翻訳されてないＲＮＡ転写物を発現する目的のために植物に導入され得る。２つの例は、アンチセンスＲＮＡおよびリボザイム活性を有するＲＮＡである。両方とも、ネイティブな植物遺伝子または導入された植物遺伝子の発現の減少または除去において潜在的な機能を提供し得る。しかし、以下に詳細に述べるように、ＤＮＡは、植物の表現型をもたらすために必ずしも発現されない。
【０２３７】
（１．アンチセンスＲＮＡ）
遺伝子は、構築されるかまたは単離され得、これは、転写される場合、標的メッセンジャーＲＮＡの全てまたは部分に相補的であるアンチセンスＲＮＡを生成する。このアンチセンスＲＮＡは、メッセンジャーＲＮＡのポリペプチド生成物の生成を減少する。このポリペプチド生成物は、植物ゲノムによりコードされる任意のタンパク質であり得る。上述の遺伝子は、アンチセンス遺伝子といわれる。従って、アンチセンス遺伝子は、目的の選択されたタンパク質の発現を減少した新規トランスジェニック植物を生成するために、形質転換方法により植物へ導入され得る。例えば、このタンパク質は、植物において反応を触媒する酵素であり得る。酵素活性の減少は、植物中の任意の酵素的に合成される化合物を含む反応の生成物（例えば、脂肪酸、アミノ酸、炭水化物、核酸など）を減少するかまたは除去し得る。あるいは、このタンパク質は、貯蔵タンパク質（例えば、ゼイン）または構造タンパク質であり得、これらのタンパク質の減少した発現は、各々、種子のアミノ酸組成における変化または植物の形態学的変化を誘導し得る。上記の可能性は、例示によりのみ提供され、適用の完全な範囲を表さない。
【０２３８】
（２．リボソーム）
遺伝子はまた、構築または単離され得、転写される場合、エンドリボヌクレアーゼとして作用し得るＲＮＡ酵素（リボザイム）を産生し、そして選択された配列でＲＮＡ分子の切断を触媒し得る。選択されたメッセンジャーＲＮＡの切断は、それらのコードされたポリペプチド産物の産生を減少し得る。これらの遺伝子は、それらを保有する新規のトランスジェニック植物を調製するために使用され得る。このトランスジェニック植物は、上記で列挙されたポリペプチドを含むがこれらに限定されないポリペプチドの減少されたレベルを保有し得る。
【０２３９】
リボザイムは、部位特異的様式で核酸を切断するＲＮＡタンパク質複合体である。リボザイムは、エンドヌクレアーゼ活性を保有する特異的触媒ドメインを有する（ＫｉｍおよびＣｅｃｈ，１９８７；Ｇｅｒｌａｃｈら、１９８７；ＦｏｒｓｔｅｒおよびＳｙｍｏｎｓ，１９８７）。例えば、多数のリボザイムは、高度の特異性でリン酸エステル転移反応を促進し、しばしば、オリゴヌクレオチド基質における幾らかのリン酸エステルの１つのみを切断する（Ｃｅｃｈら、１９８１；ＭｉｃｈｅｌおよびＷｅｓｔｈｏｆ，１９９０；Ｒｅｉｎｈｏｌｄ−ＨｕｒｅｋおよびＳｈｕｂ，１９９２）。この特異性は、この基質が特定の塩基対相互作用を介して化学反応の前にリボザイムの内部ガイド（ｇｕｉｄｅ）配列（「ＩＧＳ」）に結合するという必要性に起因していた。
【０２４０】
リボザイム触媒は、最初、核酸を含む配列特異的切断／連結反応の一部として観測されてきた（Ｊｏｙｃｅ，１９８９；Ｃｅｃｈら、１９８１）。例えば、米国特許第５，３５４，８５５号は、特定のリボザイムが、公知のリボヌクレアーゼの配列特異性よりも優れた配列特異性を有するエンドヌクレアーゼとして作用し得、ＤＮＡ制限酵素の配列特異性に接近し得ると報告している。
【０２４１】
幾らかの異なるリボザイムモチーフは、ＲＮＡ切断活性を有すると記載されてきた（Ｓｙｍｏｎｓ，１９９２）。例は、Ｔｏｂａｃｃｏ　Ｒｉｎｇｓｐｏｔ　Ｖｉｒｕｓ（Ｐｒｏｄｙら、１９８６）、Ａｖｏｃａｄｏ　Ｓｕｎｂｌｏｔｃｈ　Ｖｉｒｏｉｄ（Ｐａｌｕｋａｔｉｓら、１９７９；Ｓｙｍｏｎｓ、１９８１）およびＬｕｃｅｒｎｅ　Ｔｒａｎｓｉｅｎｔ　Ｓｔｒｅａｋ　Ｖｉｒｕｓ（ＦｏｒｓｔｅｒおよびＳｙｍｏｎｓ，１９８７）を含むグループＩの自己スプライシングイントロン由来の配列を含む。これらウイルスおよび関連ウイルス由来の配列は、予期された折り畳み二次構造に基づくハンマーヘッド（ｈａｍｍｅｒｈｅａｄ）リボザイムとしていわれる。
【０２４２】
他の適切なリボザイムは、ＲＮＡ切断活性を有するＲＮａｓｅ　Ｐ由来の配列（Ｙｕａｎら、１９９２、ＹｕａｎおよびＡｌｔｍａｎ，１９９４，米国特許第５，１６８，０５３号および同第５，６２４，８２４号）、ヘアピンリボザイム構造（Ｂｅｒｚａｌ−Ｈｅｒｒａｎｚら、１９９２；Ｃｈｏｗｒｉｒａら、１９９３）およびＨｅｐａｔｉｔｉｓ　Ｄｅｉｔａウイルスベースのリボザイム（米国特許第５，６２５，０４７号）を含む。一般的な設計およびＲＮＡ切断活性に指向されたリボザイムの最適化は、詳細に議論されてきた（ＨａｓｅｌｏｆｆおよびＧｅｒｌａｃｈ，１９８８，Ｓｙｍｏｎｓ，１９９２，Ｃｈｏｗｒｉｒａら、１９９４；Ｔｈｏｍｐｓｏｎら、１９９５）。
【０２４３】
リボザイム設計に関する他の可変は、所与の標的ＲＮＡ上の切断部位の選択である。リボザイムは、贈呈（ｃｏｍｐｌｉｍｅｎｔａｒｙ）塩基対相互作用によって部位へのアニーリングという長所によって所与の配列に標的化される。相同性の２つの伸長は、この標的化にために必要である。相同性配列のこれらの伸長は、上記で規定された触媒的リボザイム構造に隣接する。相同性配列の各伸長は、７〜１５ヌクレオチド長に変更し得る。相同性配列を規定するための唯一の必要性は、標的ＴＮＡにおいて、それらが、切断部位である特異的配列によって分離されるということである。ハンマーヘッドリボザイムについて、切断部位は、標的ＲＮＡに関するジヌクレオチド配列であり、ウラシル（Ｕ）続いて、アデニンまたはシトシン、もしくはウラシル（Ａ，Ｃ，もしくはＵ）のいずれかである（Ｐｅｒｒｉｍａｎｎら、１９９２；Ｔｈｏｍｐｓｏｎら、１９９５）。任意の所与のＲＮＡにおけるこのジヌクレオチドが生じる頻度は、統計学的に３／１６である。従って、１，０００塩基の所与の標的メッセンジャーＲＮＡについて、１８７ジヌクレオチド切断部位が、統計学的に可能である。
【０２４４】
標的ＲＮＡの有効な切断のためのリボザイムの設計および試験は、当業者に周知のプロセスである。リボザイムを設計および試験するための科学的方法の例は、Ｃｈｏｗｒｉｒａら、（１９９４）およびＬｉｅｂｅｒおよびＳｔｒａｕｓｓ（１９９５）によって記載される（これらの各々は、参考として援用される）。所与の遺伝子を下方調製する際に使用するための操作的かつ好ましい配列の同定は、所与の配列を調製および試験するという単純な問題であり、当業者に公知の慣用的に実行される「スクリーニング」方法である。
【０２４５】
（３．遺伝子サイレンシングの誘導）
遺伝子は、同時抑制の機構によってネガティブな遺伝子産物の減少された発現を有する新規なトランスジェニック植物を産生するために導入され得るということもまた、可能である。タバコ、トマト、およびペチュニアにおいて、ネガティブな遺伝子のセンス転写の発現は、アンチセンス遺伝子で観測された様式と類似の様式でネガティブな遺伝子の発現を減少または排除することが、実証されてきた（Ｇｏｒｉｎｇら、１９９１；Ｓｍｉｔｈら、１９９０；Ｎａｐｏｌｉら、１９９０；ｖａｎ　ｄｅｒ　Ｋｒｏｌら、１９９０）。導入された遺伝子は、標的化ネガティブタンパク質のすべてまたは一部をコードし得るが、その翻訳は、そのネガティブタンパク質のレベルの減少について必要とされないかもしれない。
【０２４６】
（４．非ＲＮＡ発現性配列）
Ｄｓ、Ａｃ、またはＭｕのような転移性エレメントであるエレメントを含むＤＮＡエレメントは、遺伝子に挿入され、変異を生じ得る。これらのＤＮＡエレメントは、遺伝子を不活性化（または活性化）するために挿入され得、そしてそれによって特定の形質を「タグ化」し得る。この例において、転移性エレメントは、タグ化変異の不安定性を生じない。なぜなら、このエレメントの利用は、ゲノムにおいて移動するその可能性に依存しないからである。一旦、所望の形質が、タグ化されると、導入されたＤＮＡ配列は、例えば、ＰＣＲ遺伝子クローニング技術と一緒にＰＣＲプライマーとして導入されたＤＮＡ配列を用いて、対応する遺伝子をクローニングするために使用され得る（Ｓｈａｐｉｒｏ，１９８３；Ｄｅｌｌａｐｏｒｔａら、１９８８）。一旦同定されると、所望されるコントロールまたは調節領域を含む、特定の形質に対する全遺伝子は、所望の通りに、単離、クローニング、および操作され得る。遺伝子タグ化の目的のために、生物体に導入されたＤＮＡエレメントの利用性は、ＤＮＡ配列から独立し、そしてＤＮＡ配列の任意の生物学的活性、すなわちＲＮＡへの転写またはタンパク質への翻訳に依存しない。このＤＮＡエレメントの唯一の機能は、遺伝子のＤＮＡ配列を分裂させることである。
【０２４７】
発現されてないＤＮＡ配列（新規の合成的配列を含む）が、それらの細胞および植物およびその播種の独占的「標識」として細胞に導入され得ることは、予期される。標識ＤＮＡエレメントが、宿主生物体に対して遺伝子内因性の機能を崩壊することは必要ではない。なぜなら、このＤＮＡの唯一の機能が、生物体の起源を同定しなければならないからである。例えば、独自のＤＮＡ配列は、植物に導入され得、そしてこのＤＮＡエレメントは、その標識化供給源から惹起したような、すべての細胞、植物およびこれらの細胞の子孫を同定するからである。標識ＤＮＡの包含は、独占的生殖質またはこのような標識されてない生殖質由来の生殖質を区別することを可能にすることが、提案される。
【０２４８】
導入され得る別の可能なエレメントは、マトリックス付着領域エレメント（ＭＡＲ）（例えば、チキンリゾチームＡエレメント（Ｓｔｉｅｆ，１９８９））であり、これは、目的の発現可能な遺伝子のまわりで位置決めされ得、遺伝子の全体的な発現を増大させ、そして植物ゲノムへの組み込みの際に位置依存的効果を減少し得る（Ｓｔｉｅｆら、１９８９；Ｐｈｉ−Ｖａｎら、１９９０）。
【０２４９】
（５．その他）
本発明に関する使用を特異的に想定された非タンパク質発現配列の他の例としては、例えば、コドンの使用を変更するためのｔＲＮＡ配列、および例えば、種々の薬剤（例えば、抗体）に抵抗性を所与し得るｒＲＮＡ改変体が挙げられる。
【０２５０】
（ＩＸ．生物学的機能的同等物）
改善および変更は、本発明のセントロメア性ＤＮＡセグメントにおいて作製され得、そして所望の特徴づけを有する機能的分子をなお得ることができる。以下は、同等物、すなわち改善された、二次生成分子でさえ作成するための、セントロメアの核酸の変更に基づく議論である。
【０２５１】
本発明の特定の実施形態において、変異されたセントロメア配列は、セントロメアの利用性を増大させるために有用であると予期される。本発明のセントロメアの機能は、セントロメアおよび／またはこのセントロメアと相互作用するタンパク質のＤＮＡ配列の二次構造に基づき得ることが、特異的に予期される。セントロメアのＤＮＡ配列を変更することによって、セントロメアおよび／またはセントロメア配列の二次構造のための１以上のセントロメア関連タンパク質のアフィニティーを変更し得、それによって、セントロメアの活性を変更し得る。あるいは、変更は、このセントロメアの活性を実行しない、本発明のセントロメアにおいて作製され得る。セントロメア活性を所与するために必要なＤＮＡセグメントのサイズを減少するセントロメア配列における変更は、本発明において特に有用であると予期される。なぜなら、変更は、セントロメアが有糸分裂および減数分裂の間転移されたフィデェリティーを、増大するからである。
【０２５２】
（Ｘ．植物）
用語「植物」とは、本明細書中で使用される場合、植物の任意の型をいう。本発明者らは、本発明で使用され得る幾らかの植物の例示的説明を以下に提供した。しかし、この列挙は、任意の限定する様式ではない。なぜなら、植物の他の型は、当業者に公知であり、そして本発明において使用され得るからである。
【０２５３】
農学で活用される植物の共通の分類は、野菜園芸（朝鮮アザミ、コールラビ、キバナスズシロ、西洋にらねぎ、アスパラガス、レタス（例えば、ヘッド、リーフ、ロメイン）、白菜、タロイモ、ブロッコリ、メロン（例えば、マスクメロン、スイカ、クレンショウ、ハネデュー、カンタロープ）、ブラッセル芽、キャベツ、カードニ（ｃａｒｄｏｎｉ）、ニンジン、白菜、カリフラワー、オクラ、タマネギ、セロリ、パセリ、ヒヨコマメ、アメリカボウフウ、チコリー、中国産キャベツ、コショウ、コラード、ジャガイモ、キュウリ植物（インゲンマメ、キュウリ）、カボチャ、ウリ、ダイコン、乾燥球タマネギ、カブハボタン、ナス、サルシフィ、エスカロール、エシャロット、エンダイブ、ニンニク、ホウレンソウ、ネギ、カボチャ、緑色物（ｇｒｅｅｎ）、テンサイ、（砂糖ダイコンおよび飼料ビート）、サツマイモ、フダンソウ、西洋ワサビ、トマト、ケール、蕪、および香辛料を含む）である。
【０２５４】
市販の使用をしばしば見出す他の型の植物は、果実および蔓作物（例えば、リンゴ、アプリコット、チェリー、ネクタリン、モモ、西洋なし、西洋スモモ、プルーン、マルメロアーモンド、クリ、ハシバミ、ベカン、ピスタチオ、クルミ、シトリス、ブルーベリー、ボイセンベリー、クランベリー、干しブドウ、ローガンベリー、キイチゴ、イチゴ、黒イチゴ、ブドウ、アボカド、バナナ、キーウィ、カキ、ザクロ、パイナップル、熱帯果実、ナシ、メロン、マンゴ、パパイヤおよびレイチ）を含む。
【０２５５】
最も広範に増幅した多くの植物は、農作物植物（例えば、月見草、牧草フォーム（ｍｅａｄｏｗ　ｆｏａｍ）、穀草（フィールド、スイート、ポップコーン）、ホップ、ホホバ、ピーナッツ、米、ベニバナ、小穀物（オオムギ、カラスムギ、ライ麦、コムギなど）、サトウモロコシ、タバコ、カボック、マメ植物（マメ（ｂｅａｎ）、レンティル、エンドウマメ、ダイズ）、油料植物（セイヨウアブラナ、マスタード、アヘン、オリーブ、ヒマワリ、ココナツ、ヒマシ油植物、カカオの実、ラッカセイ）、繊維植物（ワタ、アマ、アサ、ジュート）、クスノキ科（シナモン、カンファー）、または植物（例えば、コーヒー、サトウキビ、茶、および天然ゴム植物））である。
【０２５６】
植物のなお別の例としては、花壇用植物（例えば、花、サボテン、多肉多重の植物、および装飾植物、ならびに木（例えば、森（広葉樹および針葉樹のような常緑樹）、果実、装飾樹、および木の実保有木、ならびに低木および他の苗木）が挙げられる。
【０２５７】
（ＸＩ．定義）
本明細書中で使用される場合、用語「自立性折り曲がり配列」または「ＡＲＳ」または「複製起源」とは、ＤＮＡ複製を開始するタンパク質によって認識されるＤＮＡ複製の起源をいう。
【０２５８】
本明細書中で使用される場合、用語「バイナリＢＡＣ」または「バイナリ細菌性人工染色体」とは、Ａｇｒｏｂａｃｔｅｒｉｕｍ媒介形質転換（例えば、Ｈａｍｉｌｔｏｎら、１９９６；Ｈａｍｉｌｔｏｎ，１９９７；およびＬｉｕら、１９９９）に必要なＴ−ＤＮＡボーダー配列を含む細菌性ベクターをいう。
【０２５９】
本明細書中で使用される場合、用語「候補セントロメア配列」とは、潜在的なセントロメア機能に対してアッセイすると意図する核酸配列をいう。
【０２６０】
本明細書中で使用される場合、「セントロメア」とは、細胞分裂を介して娘細胞に分離する能力を所与する任意のＤＮＡ配列をいう。１つの事情において、この配列は、約１％〜約１００％（娘細胞の約５％、１０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、または約９５を含む）の範囲の娘細胞への分離効率を産生し得る。このような分離効率における改変は、本発明の範囲内に重要な適用を見出す。例えば、１００％安定性を所与するミニ染色体輸送セントロメアは、選択内のすべての娘細胞に維持され得る一方で、１％安定性を所与するミニ染色体輸送セントロメアは、トランスジェニック生物体に一時的に導入され得るが、所望の場合、排除され得る。本発明の特定の実施形態において、セントロメアは、核酸配列の安定な分離を所与し、有糸分裂または減数分裂（有糸分裂および減数分裂の両方を含む）を介して、セントロメアを含む組み換え構築物を含む。植物セントロメアは、必ずしも植物由来ではないが、植物細胞におけるＤＮＡ分離を促進する能力を有する。
【０２６１】
本明細書中で使用される場合、用語「セントロメア関連タンパク質」とは、セントロメアの配列によってコードされるタンパク質または宿主ＤＮＡによってコードされかつこのセントロメアに対して比較的高いアフィニティーで結合するタンパク質をいう。
【０２６２】
本明細書中で使用される場合、「真核生物」とは、生存生物体の細胞が核を含む生存生物体をいう。真核生物は、核を欠損する生物体である「原核生物」と区別され得る。真核生物および原核生物は、それらの遺伝情報が組織される方法ならびにＲＮＡおよびタンパク質合成のそれらのパターンにおいて本質的に異なる。
【０２６３】
本明細書中で使用される場合、用語「発現」とは、構造的遺伝子が代表的にメッセンジャーＲＮＡ（ｍＲＮＡ）と称されるＲＮＡ分子を産生するプロセスをいう。このｍＲＮＡは、代表的ではあるがいつもではなく、ポリペプチドに転移される。
【０２６４】
本明細書中で使用される場合、「ゲノム」は、遺伝子のすべておよび生物体の所与の細胞内に遺伝的情報を含むＤＮＡ配列をいう。通常、これは、核内に含まれる情報を意味するが、細胞小器官をも含むように取られる。
【０２６５】
本明細書中で使用される場合、用語「高等真核生物」とは、多細胞真核生物をいい、代表的にそのより複雑な生理学的機構および比較的大きいサイズによって特徴付けられる。一般的に、植物および動物のような複雑な生物体は、このカテゴリーに含まれる。本発明によって形質転換される好ましい高等真核生物としては、例えば、単子葉被子植物種および双子葉被子植物種、裸子植物種、シダ植物種、これらの種の植物組織培養細胞、動物細胞および藻類細胞が挙げられる。真核生物および原核生物は、同様に本発明の方法によって形質転換され得ることが当然理解される。
【０２６６】
本明細書中で使用される場合、用語「宿主」とは、複製可能なプラスミドの複製である任意の生物体または植物染色体を含む発現ベクターをいう。理想的に、クローニング実験のために使用される宿主菌株は、使用される外来のＤＮＡを分解し得る任意の制限酵素活性がないべきである。本発明において有用な、クローニングのための宿主細胞に好ましい例としては、細菌（例えば、Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ、Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ、Ｐｓｅｕｄｏｍｏｎａｓ、Ｓｔｒｅｐｔｏｍｙｃｅｓ、Ｓａｌｍｏｎｅｌｌａ）および酵母細胞（例えば、Ｓ．ｃｅｒｅｖｉｓｉａｅ）が挙げられる。ミニ染色体の発現のために標的化され得る宿主細胞は、任意の供給源の植物細胞であり得、そして特異的にＡｒａｂｉｄｏｐｓｉｓ、トウモロコシ、米、サトウキビ、サトウモロコシ、オオムギ、ダイズ、タバコ、コムギ、トマト、ジャガイモ、シトリス、または任意の他の農学的または科学的重要な種を含む。
【０２６７】
本明細書中で使用される場合、「ハイブリダイゼーション」とは、ＲＮＡ−ＤＮＡハイブリッドを産生する相補的なＲＮＡおよびＤＮＡ鎖の対、あるいは二本鎖ＤＮＡ分子を産生するための一般的に異なるかまたは同一の供給源由来の２つのＤＮＡ一本鎖の対をいう。
【０２６８】
本明細書中で使用される場合、用語「リンカー」とは、一般的に５０または６０ヌクレオチド長までであり、そして化学的に合成されるかまたは他のベクターからクローニングされるＤＮＡ分子をいう。好ましい実施形態において、このフラグメントは、平滑切断酵素およびねじれ型切断酵素（例えば、Ｂａｍ　ＨＩ）のための１つ、好ましくは１つより多くの制限酵素部位を含む。このリンカーフラグメントの１つの末端は、直鎖状分子の１つの末端に結合可能であるように適応され、そして他の末端は、この直鎖状分子の他の末端に結合可能であるように適応される。
【０２６９】
本明細書中で使用される場合、「ライブラリー」とは、クローニングされるランダムＤＮＡフラグメントのプールである。原理的に、任意の遺伝子は、特異的ハイブリダイゼーションプローブでライブラリーをスクリーニングすることによって単離され得る（例えば、Ｙｏｕｎｇら、１９７７を参照のこと）。各ライブラリーは、分散した制限酵素で産生されたフラグメントとしてまたは何千のプラスミドベクターにランダムに向きを変えられたフラグメントとして挿入された所与の生物体のＤＮＡを含み得る。本発明の目的のために、Ｅ．ｃｏｌｉ、酵母およびＳａｌｍｏｎｅｌｌａプラスミドは、このゲノム挿入物が他の生物体からである場合に特に有用である。
【０２７０】
本明細書中で使用される場合、用語「下等真核生物」とは、比較的単純な生理機能および組成物と特徴付けられる真核生物、ならびに最も頻繁には単細胞をいう。
【０２７１】
本明細書中で使用される場合、「ミニ染色体」とは、セントロメアを含みかつ娘細胞に転移可能な組み換えＤＮＡ構築物である。細胞分裂を介するこの構築物の安定性は、約１％〜約１００％（５％、１０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、および９５％を含む）の間の範囲であり得る。このミニ染色体構築物は、環式または直鎖状の分子であり得る。１以上のテロメア、ＡＲＳ配列および遺伝子のようなエレメントを含み得る。含まれるこのような配列の数は、この構築物本体の物理的なサイズの限定によってのみ限定される。天然の染色体由来のＤＮＡを含み得るが、１〜１００％の範囲における分離効率を得ることを必要とされる最小量に対してＤＮＡの量を限定することが好ましくあり得る。このミニ染色体は、有糸分裂または減数分裂を通してか、または有糸分裂および減数分裂の両方を通して遺伝され得る。本明細書中で使用される場合、用語ミニ染色体は、用語「植物人工染色体」または「ＰＬＡＣ」およびこの用語ミニ染色体の意味の中にある構築物に特異的に適用するＰＬＡＣまたは植物人工染色体に関連するすべての技術を包含しかつ含む。
【０２７２】
本明細書中で使用される場合、「ミニ染色体でコードされたタンパク質」によって、本発明のミニ染色体の配列によってコードされるポリペプチドが意味される。これは、配列（例えば、選択マーカー、テロメアなど）、ならびにこのミニ染色体上の任意の他の選択された機能的遺伝子によってコードされるそれらのタンパク質を含む。
【０２７３】
「１８０塩基対の繰り返し」は、配列番号１８４〜２１２に開示される特定の繰り返しのいずれか１つまたは、それ由来の「連続した」配列として規定される。従って、所与の「１８０塩基対の繰り返し」は、１８０より多いかまたはこれ未満の塩基対を含み得、そしてそこに提供される特定の配列のいずれかによって示されない配列を反映し得る。
【０２７４】
本明細書中で示される場合、用語「植物」は、植物細胞、植物プロトプラスト、植物カルスなど、ならびにそれから再び産生された全植物を含む。
【０２７５】
本明細書中で使用される場合、用語「プラスミド」または「クローニングベクター」とは、共有結合で閉ざされた環式の染色体外ＤＮＡまたは直鎖状のＤＮＡ（宿主細胞において自立的に繰り返し得、そして細胞の生存に通常非必須である）をいう。広範な種々のプラスミドおよび他のベクターは、公知であり、そして当該分野で一般的に使用される（例えば、Ｃｏｈｅｎら、米国特許第４，４６８，４６４号（これは、ＤＮＡプラスミドの例を開示し、特異的に本明細書中で参考として援用される）を参照のこと）。
【０２７６】
本明細書中で使用される場合、「プローブ」は、任意の生物学的試薬（通常、同定の容易さのために同じ方法でタグ化される）であり、遺伝子、遺伝子産物、ＤＮＡセグメントまたはタンパク質を同定または単離するために使用される。
【０２７７】
本明細書中で使用される場合、用語「組み換え」とは、ＤＮＡ鎖の崩壊および再結合を含む任意の遺伝子の交換をいう。
【０２７８】
本明細書中で使用される場合、「調節配列」とは、任意の遺伝子の転写または翻訳の効率に影響を及ぼす任意のＤＮＡ配列をいう。この用語は、プロモーター、エンハンサーおよびターミネータを含むが、これらに限定されない。
【０２７９】
本明細書中で使用される場合、「選択マーカー」は、遺伝子の存在が、明確な表現型、およびこのマーカーを含む細胞に対する成長の利点を最もよく生じる遺伝子である。この成長の利点は、標準条件下、温度上昇のような変更された条件、または除草剤もしくは抗体のような特定の化学物質の存在下で存在し得る。選択マーカーの使用は、例えば、Ｂｒｏａｃｈら、（１９７９）に開示される。選択マーカーの例は、数ある中で、チミジンキナーゼ遺伝子、細胞性アデニンホスホリボシルトランスフェラーゼ遺伝子およびジヒドリルホレートレダクターゼ遺伝子、ハイグロマイシンホスホトランスフェラーゼ遺伝子、バー（ｂａｒ）遺伝子およびネオマイシンホスホトランスフェラーゼ遺伝子を含む。本発明における好ましい選択マーカーは、遺伝子の発現が宿主細胞に対して抗菌性の抵抗または除草剤抵抗を所与する遺伝子を含む。この遺伝子は、宿主細胞内のベクターの維持を可能にするために十分であり、そして新たな宿主細胞にプラスミドの操作を容易にする。本発明の特定の目的としては、例えば、アンピシリン、クロラムフェニコール、テトラサイクリン、Ｇ−４１８、ビアラフォス（ｂｉａｌａｐｈｏｓ）、およびグリフォセートに対して細胞性抵抗を所与するタンパク質である。
【０２８０】
本明細書中で使用される場合、「スクリーニングマーカー」は、遺伝子の存在が、同定可能な表現型を生じる遺伝子である。この表現型は、標準条件下、温度上昇のような変更された条件下、またはこの表現型を検出するために使用される特定の化学物質の存在下で観測可能であり得る。
【０２８１】
本明細書中で使用される場合、用語「部位特異的組み換え」とは、特定のＤＮＡ配列にてＤＮＡ鎖の崩壊および再結合を含む任意の遺伝子交換をいう。
【０２８２】
本明細書中で使用される場合、「構造的遺伝子」は、ポリペプチドまたはＲＮＡをコードする配列であり、５’末端および３’末端を含む。構造的遺伝子は、宿主に構造的遺伝子が形質転換される宿主または別の種由来である。構造的遺伝子は、好ましいが、必要ではなく、構造的遺伝子の発現を調節する１以上の調節配列（例えば、プロモーター、ターミネーターまたはエンハンサー）を含む。構造的遺伝子は、好ましいが、必要ではなく、構造的遺伝子を含む生物体に関する幾らかの有用な表現型（例えば、除草剤抵抗）を所与する。本発明の１つの実施形態において、構造的遺伝子は、タンパク質（例えば、ｔＲＮＡまたはｒＲＮＡ遺伝子）に翻訳されないＲＮＡ配列をコードし得る。
【０２８３】
本明細書中で使用される場合、用語「テロメア」とは、染色体の末端をキャッピングし、それによって染色体末端の分解を予防し得、複製を確実にし、そして他の染色体配列に対する融合を予防し得る配列をいう。
【０２８４】
本明細書中で使用される場合、用語「形質転換」または「トランスフェクション」とは、ＤＮＡの染色体性付加または染色体外性付加を介して新たなＤＮＡの細胞における獲得（ａｃｑｕｉｓｉｔｉｏｎ）をいう。これは、プロセスによって、裸のＤＮＡ、タンパク質でコートされたＤＮＡ、または全ミニ染色体が細胞に導入される、このプロセスであり、潜在的な遺伝的変化を生じる。
【０２８５】
（ＸＩＩ．実施例）
以下の実施例は、本発明の好ましい実施例を実証することを含む。本技術は、本発明の実行の際に十分な機能を本発明者によって開示される例示的技術に従う実施例において開示され、従ってその実行のために好ましい様式を構築することを考慮され得ることが、当業者に理解されるべきである。しかし、当業者は、本開示を考慮して、多くの変更が、開示される特定の実施形態において作製され得、そして本発明の概念、精神および範囲から逸脱することなく同様なまたは類似の結果をなお得ることができることを理解するべきである。より詳細には、化学的にかつ生理学的に関連する特定の薬剤は、本明細書中で記載される薬剤と置換され得る一方で、同一または類似の結果が達成されることは、明らかである。当業者に明らかなすべてのこのような類似の置換および改善は、添付の特許請求の範囲によって規定される通りの、本発明の精神、範囲および概念の中にあるとみなされる。
【０２８６】
（実施例１）
（Ａｒａｂｉｄｏｐｓｉｓ　ｔｈａｌｉａｎａ　マッピング（ｍａｐｐｉｎｇ）集団の産生）
花粉ドナー植物を生成するために、２つの親株輸送ｑｒｔｌを、互いに交差させる。ｑｒｔｌ−１対立遺伝子は、Ｌａｎｄｓｂｅｒｇ生態型バックグラウンドにあり、そしてｑｒｔｌ−２対立遺伝子は、Ｃｏｌｕｍｂｉａ生態型バックグラウンドにあった。Ｌａｎｄｓｂｅｒｇ生態型は、Ｃｏｌｕｍｂｉａ生態型から用意に識別可能である。なぜなら、それは、劣性の変異である、ｅｒｅｃｔａを輸送し、ニワトリに対する幹、より緻密である開花、およびより丸くそして野生型より小さい葉を生じる。ドナー植物のマーカーとしてこれを利用するために、ｑｒｔｌ−２対立遺伝子を、ａｒｔｌ−１メススチグマに交差した。Ｆ_１子孫は、すべての分子のマーカーでへテロ接合性であるが、この子孫は、融合された対立遺伝子グレインの四分子のｑｕａｒｔｅｔ表現型を維持する。さらに、Ｃｏｌｕｍｂｉａ植物のＥＲＥＣＴＡ表現型を表示する。この可視的マーカーは、交差が生態型特異的マーカーを分離する植物を生成することにおいて功を奏することの同定として役立つ。さらなる試験を、子孫が分子遺伝子座でへテロ接合性であるこを保証するためにＰＣＲ分析を行うことにより、ドナー植物に対して行った。
【０２８７】
花粉粒がマーカー分離について直接的にアッセイされ得ないという事実に起因して、そして複数のマーカーアッセイに利用可能な長期供給源を作製したいという願望のために、ドナー植物によって生成される個々の四分子を交雑することが必要であった。これは、大量の組織および種子の両方を生じる後代植物のセットを作製した。これらの交雑は、雄性不稔性（ｍｓ１）について同型接合のレシピエント植物を生成することによって効果的に達成された。劣性変異体ｍｓ１を、レシピエント植物が自家受精したり、後代が四分子植物と間違えられたりすることを防ぐために選択した。同型接合性植物は自己受精しないという事実に起因して、異型接合の雄性不稔性１（ｍａｌｅ　ｓｔｅｒｉｌｉｔｙ　１）植物によって生成された原種（これから不稔性レシピエント植物が選択され得る）は、維持される必要がある。
【０２８８】
（実施例２）
（四分子受粉）
四分子受粉を以下のように実施した。成熟した花をドナー植物から取り除き、そして成熟した四分子花粉粒を放出するようにガラスの顕微鏡用スライド上でトントンとたたいた。次いでこのスライドを２０〜４０倍のＺｅｉｓｓ解剖顕微鏡下に配置した。個々の四分子花粉粒を単離するために、小さな木製のだぼを使用し、このだぼにはゴム糊で眉毛を取り付けた。光学顕微鏡を使用して、四分子花粉ユニットを選択し、そして眉毛に接触させた。四分子は、優先的に眉毛に付着し、従って顕微鏡用スライドから釣り上げられ、そしてレシピエント植物の柱頭表面に運ばれる。この移動は、顕微鏡の使用を伴わずに実施し、次いで四分子が付着した眉毛をレシピエントの柱頭表面に対して配置し、そしてこの毛を、柱頭表面を横切るように手で引きずった。次いで、四分子は優先的にレシピエントの柱頭に付着し、そして交差受粉を完了した。
【０２８９】
最初に、各々３〜４個の種子からなる５７の四分子種子のセットを採集した。植物をこれらの四分子種子のセットから生育させ、そして組織を採集した。ＤＮＡを、ＰＣＲベースの分離分析のために、貯蔵した組織の少量から抽出した。さらに、可視的なｅｒｅｃｔａ表現型の分離を記録した。この植物が種子を生じる場合、この種子を、サザンブロッティングによってＲＦＬＰ分離を分析するために必要とされる、大量のＤＮＡの供給源として採集した。
【０２９０】
（実施例３）
（セントロメアに及ぶコンティグ（ｃｅｎｔｒｏｍｅｒｅ−ｓｐａｎｉｎｇ−ｃｏｎｔｉｇ）の調製および分析）
以前に、２つの細菌人工染色体（ＢＡＣ）ライブラリーのＤＮＡフィンガープリントおよびハイブリダイゼーション分析により、Ａｒａｂｉｄｏｐｓｉｓゲノムの単一コピー部分のほぼ全てをカバーする物理的地図の構築がなされた（Ｍａｒｒａら、１９９９）。しかし、Ａｒａｂｉｄｏｐｓｉｓセントロメア近傍の反復ＤＮＡ（１８０ｂｐの反復、レトロ要素（ｒｅｔｒｏｅｌｅｍｅｎｔ）、および中間反復配列を含む）の存在は、特定の染色体にセントロメアＢＡＣコンティグを固定するための取り組みを複雑にした（Ｍｕｒａｔａら、１９９７；Ｈｅｓｌｏｐ−Ｈａｒｒｉｓｏｎら、１９９９；Ｂｒａｎｄｅｓら、１９９７；Ｆｒａｎｚら、１９９８；Ｗｒｉｇｈｔら、１９９６；Ｋｏｎｉｅｃｚｎｙｔら、１９９１；Ｐｅｌｉｓｓｉｅｒら、１９９５；ＶｏｙｔａｓおよびＡｕｓｕｂｅｌ、１９８８；Ｃｈｙｅら、１９９７；Ｔｓａｙら、１９９３；Ｒｉｃｈａｒｄｓら、１９９１；Ｓｉｍｏｅｎｓら、１９８８；Ｔｈｏｍｐｓｏｎら、１９９６；Ｐｅｌｉｓｓｉｅｒら、１９９６）。本発明者らは、これらの固定されていないコンティグを特定のセントロメアにはっきりと割り当てるために遺伝子マッピングを使用し、ゲノム全体についての情報を与える交差（Ｃｏｐｅｎｈａｖｅｒら、１９９８）を用いて、４８の植物において多形性マーカーを記録した。このようにして、いくつかのセントロメアコンティグを染色体腕の物理的地図に関連付け（実施例６および表４を参照のこと）、そしてセントロメア境界を規定するＤＮＡマーカーの大きなセットを生成した。ＤＮＡ配列分析は、第ＩＩ染色体および第ＩＶ染色体についてのコンティグの構造（Ｌｉｎら、１９９９）を確証した。
【０２９１】
ＣＥＮ２およびＣＥＮ４を、特に分析のために選択した。両方とも、構造的に類似した染色体上に存在し、３．５Ｍｂ　ｒＤＮＡアレイをそれらの遠位端上に有し、それぞれ３Ｍｂおよび２Ｍｂを測定する領域をｒＤＮＡとセントロメアとの間に有し、そして１６Ｍｂ領域および１３Ｍｂ領域をそれらの長腕上に有する（ＣｏｐｅｎｈａｖｅｒおよびＰｉｋａａｒｄ、１９９６）。
【０２９２】
第ＩＩ染色体および第ＩＶ染色体の実質的に完全な、かつ注釈付きの配列を用いて、ヌクレオチドレベルでセントロメアの分析を実施した（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ｅｎｔｒｅｚ／ｎｕｃｌｅｏｔｉｄｅ．ｈｔｍｌ）。遺伝的に規定されたセントロメア境界内で配列組成を分析し、そして近接したセントロメア周囲領域と比較した（図１２Ａ〜Ｔ）。２つのセントロメアの分析は、配列パターンの比較および保存された配列エレメントの同定を容易にした。
【０２９３】
セントロメア配列は、１８０ｂｐ反復配列を有することが見出された。これらの配列は、各セントロメアコンティグのギャップ中に存在することが見出され（図３、図１２Ｂ、１２Ｌ）、ゲノムの他の部分には、繰り返しはほとんどなく、そして長アレイも存在しなかった。これらのギャップ近傍のＢＡＣクローンは、コンティグ間のＤＮＡの大部分（１８０ｂｐ反復、５Ｓ　ｒＤＮＡ、または１６０ｂｐ反復を含む）を構成すると思われる反復エレメントに対応する末端配列を有する（図３）。蛍光インサイチュハイブリダイゼーションは、これらの反復配列がＡｒａｂｉｄｏｐｓｉｓセントロメアの豊富な成分であることを示した（Ｍｕｒａｔａら、１９９７；Ｈｅｓｌｏｐ−Ｈａｒｒｉｓｏｎら、１９９９；Ｂｒａｎｄｅｓら、１９９７）。遺伝子マッピングおよびパルスフィールドゲル電気泳動は、多くの１８０ｂｐ反復が、セントロメア領域中の０．４Ｍｂと１．４Ｍｂとの間を測定する長アレイに存在する（Ｒｏｕｎｄら、１９９７）ことを示し；配列分析は、ギャップ近傍のさらに散在したコピーを明らかにした。本発明者らは特に、ミニ染色体の構築のためのこのような１８０ｂｐ反復の使用を意図する。第ＩＩ染色体および第ＩＶ染色体の注釈付き配列は、中間反復ＤＮＡに対する相同性を有する領域を同定し、共に、機能的セントロメア内、および隣接領域中に存在する（図１２Ｂ〜１２Ｅおよび１２Ｌ〜１２Ｏ）。
【０２９４】
ＣＥＮ２および２．８Ｍｂ配列決定領域（ＣＥＮ４を含む）を含む４．３ｋｂ配列決定領域において、レトロトランスポゾン相同性が、ＤＮＡ配列の１０％より多くを占めることが見出された。それぞれ最大で６２％および７０％である（図１２Ｃ、１２Ｍ）。トランスポゾンまたは中間反復エレメントに対する類似性を有する配列が、同様の帯域を占めることが見出されたが、しかし、あまり共通ではなかった（それぞれ、第ＩＩ染色体、第ＩＶ染色体について最大で２９％および１１％の密度（図１２Ｄ〜１２Ｅおよび図１２Ｂ〜１２Ｏ））。最終的に、ＤｒｏｓｏｐｈｉｌａおよびＮｅｕｒｏｓｐｏｒａセントロメアの場合（Ｓｕｎら、１９９７；Ｃａｍｂａｒｅｒｉら、１９９８）とは異なり、Ａｒａｂｉｄｏｐｓｉｓのセントロメア中では、低コンプレキシティーＤＮＡ（マイクロサテライト、ホモポリマー域、およびＡＴリッチイソコア（ｉｓｏｃｈｏｒｅｓ）を含む）が富化されていることは見出されなかった。ＣＥＮ２近傍では、単純反復配列密度は、遠位の染色体腕上の密度に匹敵し、セントロメア内の配列の１．５％を占め、フランキング領域では３．２％であり、そして長さが２０〜３１９ｂｐの範囲であった（平均７１ｂｐ）。ＣＥＮ２でのミトコンドリアＤＮＡの挿入を除いて、セントロメア中および周囲のＤＮＡは、約６４％Ａ＋Ｔのゲノム平均から有意に逸脱する大きな領域を含まなかった（図１２Ｆ、１２Ｐ）（Ｂｅｖａｎら、１９９９）。
【０２９５】
１８０ｂｐ反復とは異なり、ＣＥＮ２およびＣＥＮ４の近傍では、他の反復エレメントは全て、遺伝的に規定されたセントロメア内で、フランキング領域よりも豊富ではなかった。機能的セントロメアドメインの外側の高濃度の反復エレメントは、セントロメア活性について不充分であり得ることを示唆する。従って、これらの反復配列中で富化されたＡｒａｂｉｄｏｐｓｉｓゲノムのセグメントを同定することは、セントロメア機能を提供する領域を正確に突き止めなかった；同様の状況は、他の高等真核生物のゲノムにおいても起こり得る。
【０２９６】
セントロメアに隣接する反復ＤＮＡは、重要な役割を演じ得、これは、セントロメア構造を凝集させるか、または安定化するように働く、変化したクロマチン構造を形成する。あるいは、他の機構が、セントロメア近傍での反復エレメントの蓄積を生じ得る。進化モデルは、反復ＤＮＡが低い組換えの領域中に蓄積することを推定するが（Ｃｈａｒｌｅｓｗｏｒｔｈら、１９８６；Ｃｈａｒｌｅｓｗｏｒｔｈら、１９９４）、多くのＡｒａｂｉｄｏｐｓｉｓ反復エレメントは、セントロメア自体よりも、組換えが活性なセントロメア周囲領域において豊富である。代わりに、レトロエレメントおよび他のトランスポゾンは、セントロメアに隣接する領域に優先的に挿入し得るか、またはより高い速度でゲノムの残りの部分から除去され得る。
【０２９７】
（実施例４）
（セントロメアの遺伝子マッピング）
セントロメアをマッピングするために、数百の多形ＤＮＡマーカーについて異型接合性であるＦ_１植物を、Ｌａｎｄｓｂｅｒｇ生態型およびＣｏｌｕｍｂｉａ生態型由来のｑｕａｒｔｅｔ変異体を交雑することにより生成した（Ｃｈａｎｇら、１９８８；Ｅｃｋｅｒ、１９９４；ＫｏｎｉｅｃｚｙおよびＡｕｓｕｂｅｌ、１９９３）。これらの植物由来の四分子において、遺伝的マーカーは、２：２比で分離する（図６；Ｐｒｅｕｓｓら、１９９４）。次いで、マーカーの分離を、Ｌａｎｄｓｂｅｒｇ同型接合体においてＦ_１植物由来の花粉四分子を交雑させることにより生成した植物において決定した。四分子内の花粉粒の遺伝子型を後代の遺伝子型から推測した。まず、種子を１００より多くの成功した四分子受粉から生成し、そして組織および種子をこれらのうち５７から採集した。これは、ＰＣＲのための十分な材料、ならびにサザンハイブリダイゼーションおよびＲＦＬＰマッピングに必要な大量の組織を生成するために必要な種子を提供した。セントロメアのより正確な位置付けを得るために、元の四分子受粉を、５７の四分子から１０００より多くの四分子まで増大させた。
【０２９８】
マーカー分離を決定するために、ＰＣＲ分析を行った。雌親からのＬａｎｄｓｂｅｒｇバックグラウンドの寄与を説明するために、４つの四分子植物の各々か１つのＬａｎｄｓｂｅｒｇ相補物を差し引いた。図５に示すように、ゲノム全体にわたる部位からのマーカーを、全ての他のマーカーのペアでの比較のために使用した。テトラ型は、一方または両方のマーカーとそれらのセントロメアとの間の交差を示し、一方、二型は、交差がなかったこと（または二重交差の存在）を示す。
【０２９９】
従って、あらゆる遺伝子座において、得られた二倍体後代は、Ｌ／ＣまたはＣ／Ｃのいずれかであった。これらの植物内で生成されたマップは、既存のマップ（雄と雌との両方における組換えの平均を表す）とは異なり、雄性減数分裂にのみ基づく。従って、いくつかの十分に確立された遺伝的距離を再計算した。これによって、組換え頻度が有意に変化したか否かを決定する。
【０３００】
分析により得られた大量の遺伝的データは、四分子分析を行うために、対で比較されなければならない。これらのデータ全部をＭｉｃｒｏｓｏｆｔ　Ｅｘｃｅｌ表計算フォーマットで管理し、Ｌａｎｄｓｂｅｒｇ対立遺伝子を「１」の値に、そしてＣｏｌｕｍｂｉａ対立遺伝子を「０」の値に割り当てた。四分子において、１つの染色体上のマーカーの分離を、異なる染色体上のセントロメア連鎖参照遺伝子座と比較した（以下の表２を参照のこと）。各遺伝子座の値を適当な参照で掛け合わせ、そして各四分子についての結果を足すことによって、ＰＤ、ＮＰＤ、およびＴＴ四分子を、それぞれ２、０、および１の値で容易に区別した。
【０３０１】
この集団中の交差の位置をモニタリングすることは、セントロメアから組換えにより分離し得る染色体領域（テトラ型）、およびセントロメアと常に共分離する領域（二型）を同定した（Ｃｏｐｅｎｈａｖｅｒら、１９８８；Ｃｏｐｅｎｈａｖｅｒら、１９９９）。セントロメアでは、テトラ型頻度は０にまで減少した；結果として、セントロメア境界は、小さいが検出可能な数のテトラ型パターンを示す位置として規定された。約４００の四分子におけるセントロメア連鎖マーカーの分離を記録することにより、セントロメア１〜５を、それぞれ５５０ｋｂ、１４４５ｋｂ、１６００ｋｂ、１７９０ｋｂ、および１７７０ｋｂのコンティグに対応する物理的地図上の領域に位置決めした（図３）。さらに、各セントロメア間隔について、多数の有用な組換え体を同定した。分析の結果は、セントロメアが、組換え機構活性を制限する大きなドメイン内に存在すること、およびこれらのドメインと周囲の組換えに熟練したＤＮＡ（ｒｅｃｏｍｂｉｎａｔｉｏｎ−ｐｒｏｆｉｃｉｅｎｔ　ＤＮＡ）との間の転移が著しく急激であることを示した。
【０３０２】
【表２】

１８０ｂｐ反復に対応する多形の分析（ＲＣＥＮマーカー、Ｒｏｕｎｄら、１９９７）は、これらの反復が、遺伝的に規定されたセントロメア内にマッピングされることを確証した。１８０ｂｐ反復と関連した多形を、以前に記載（Ｒｏｕｎｄら、１９９７）のようにパルスフィールドゲル電気泳動によって分析した。情報を与える交差を伴う四分子におけるこれらの多形の分離は、各セントロメアでの１８０ｂｐ反復アレイの完全な連鎖を確証した。遺伝単位において、セントロメア間隔は、平均して０．４４ｃＭであった（組換え％＝１／２テトラ型頻度）。このことは、組換え率が、２２１ｋｂ／ｃＭのゲノム平均（ＳｏｍｅｒｖｉｌｌｅおよびＳｏｍｅｒｖｉｌｌｅ、１９９９；ｈｔｔｐ：／／ｎａｓｃ．ｎｏｔｔ．ａｃ．ｕｋ／ｎｅｗ＿ｒｉ＿ｍａｐ．ｈｔｍｌ）を少なくとも１０〜３０倍下回ったことを反映する。
【０３０３】
高等真核生物セントロメア近傍で代表的に観察される低い組換え頻度は、ＤＮＡ改変またはクロマチン状態の異常に起因し得る（Ｃｈｏｏ，１９９８；Ｐｕｅｃｈｂｅｒｒｙ、１９９９；ＭａｈｔａｎｉおよびＷｉｌｌａｒｄ、１９９８；Ｃｈａｒｌｅｓｗｏｒｔｈら、１９８６；Ｃｈａｒｌｅｓｗｏｒｔｈら、１９９４）。これらの状態を改変するために、そして従って組換え頻度を上昇させることによりセントロメアマッピング解像能を改善するために、Ｆ１　Ｌａｎｄｓｂｅｒｇ／Ｃｏｌｕｍｂｉａ植物を、ＤＮＡ損傷を引き起こすこと、クロマチン構造を改変すること、またはＤＮＡ改変を変化させることが知られる一連の化合物の１つで処理した。Ｆ１　Ｌａｎｄｓｂｅｒｇ　ｑｒｔ１／Ｃｏｌｕｍｂｉａ　ｑｒｔ１植物を、１’’平方ポットで２４時間光下で生育し、そしてメタンスルホン酸エチルエステル（０．０５％）、５−アザ−２’−デオキシシチジン（２５または１００ｍｇ／ｌ）、ゼオシン（Ｚｅｏｃｉｎ）（１μｇ／ｍｌ）、メタンスルホン酸メチルエステル（７５ｐｐｍ）、ｃｉｓ−ジアミンジクロロ白金（２０μｇ／ｍｌ）、マイトマイシンＣ（１０ｍｇ／ｌ）、ｎ−ニトロソ−ｎ−エチル尿素（１００μＭ）、ｎ−酪酸（２０μＭ）、トリコスタチンＡ（１０μＭ）、または３−メトキシベンズアミド（２ｍＭ）で処理した。植物に潅水し、そして花を有する茎をこれらの溶液中に浸漬した。あるいは、植物を３５０ｎｍのＵＶ（７秒または１０秒）、または熱ショック（３８℃または４２℃で２時間）に曝露した。これらの植物由来の花粉四分子を使用して、各処理の３〜５日後にＬａｎｄｓｂｅｒｇ柱頭に受粉させた；続いて、Ｆ１植物をさらなる処理に供した（１植物当たり５回まで、３〜５日毎）。
【０３０４】
処理植物由来の四分子を、Ｌａｎｄｓｂｅｒｇ柱頭に交雑し、そして各処理に供した８〜１０７四分子からの後代を回収し、そして分析し、６００を超えるさらなる四分子を得た。サンプルサイズは、個々の処理が顕著な影響を有したかどうかを決定するには不充分であったが、これらの四分子は、セントロメアに直に隣接する領域においてより高い組換えを示した（それぞれ、未処理植物および処理植物において１．６対３．４％組換え）。セントロメアのマップ位置を、第２染色体から第５染色体上で精製した（図１）。これは、それぞれ８８０ｋｂ、１１５０ｋｂ、１２６０ｋｂ、および１０７０ｋｂのコンティグが及ぶ間隔を生じ、全ての四分子が一貫して、同じ領域に対してセントロメア機能を位置付けした（Ｃｏｐｅｎｈａｖｅｒら，１９９９）。
【０３０５】
組換えを増大させる試みは、セントロメア近傍で交差を伴う多数の四分子を生じた；これらの交差は、セントロメア境界の狭い領域内に群がっていた。５つの交差がＣＥＮ２近傍の７０ｋｂ領域にわたって起こり、そして７つが、ＣＥＮ１近傍の２００ｋｂ領域にわたって起こり、しかもそれぞれ８８０ｋｂおよび５５０ｋｂの近接したセントロメア間隔においては交差は検出されなかった（図３）。従って、セントロメアは、組換え機構活性を制限する大きなドメイン内で見出された；これらのドメインと周囲の組換えに熟練したＤＮＡとの間の転移は、著しく急激である（図１２ＡおよびＫ）。より多くの四分子の分析がさらなる組換え事象を生じたが、観察された交差分布は、セントロメア位置が、有意に精製されなかったことを示す。
【０３０６】
（実施例５）
（Ａｒａｂｉｄｏｐｓｉｓセントロメアの配列分析）
（Ａ．セントロメア領域における遺伝子の量）
Ｓ．ｃｅｒｅｖｉｓｉａｅにおいては、発現遺伝子は、１ｋｂの必須セントロメア配列内に位置し、そしてＳ．ｐｏｍｂｅにおいては、多コピーのｔＲＮＡ遺伝子は、セントロメア機能に必要である８０ｋｂフラグメント内に存在する（Ｋｕｈｎら、１９９１）。対照的に、高等真核生物のセントロメアにおいては、遺伝子は、著しい例外はあるが、比較的まれであると考えられる。Ｄｒｏｓｏｐｈｉｌａ　ｌｉｇｈｔ、ｃｏｎｃｅｒｔｉｎａ、ｒｅｓｐｏｎｄｅｒ、およびｒｏｌｌｅｄ遺伝子座の全てが、第２染色体のセントロメア領域にマッピングされ、そしてその本来のヘテロクロマチン背景からｌｉｇｈｔを取り出す転座が、遺伝子発現を阻害する。対照的に、ユークロマチンに通常存在する多くのＤｒｏｓｏｐｈｉｌａ遺伝子およびヒト遺伝子は、それらがセントロメア近傍に挿入されたとき不活性となる。従って、セントロメア近傍に存在する遺伝子は、発現を可能にする特別な制御エレメントを有すると考えられる（Ｋａｒｐｅｎ、１９９４；ＬｏｈｅおよびＨｉｌｌｉｋｅｒ、１９９５）。本明細書で提供されるＡｒａｂｉｄｏｐｓｉｓ　ＣＥＮ２およびＣＥＮ４の配列は、遺伝子密度および発現が、セントロメア位置および関連したクロマチンとどのように相関するかを理解するための強力な供給源を提供する。
【０３０７】
第ＩＩ染色体および第ＩＶ染色体の注釈（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ｅｎｔｒｅｚ／ｎｕｃｌｅｏｔｉｄｅ．ｈｔｍｌ）は、ＣＥＮ２およびＣＥＮ４内の、およびそれに近接した多くの遺伝子を同定した（図８、図１２Ａ〜１２Ｔ）。Ａｒａｂｉｄｏｐｓｉｓ染色体腕上における推定遺伝子の密度は、１００ｋｂ当たり平均して２５であり、そしてＣＥＮ２およびＣＥＮ４に隣接する反復リッチ領域では、それぞれ、これは、１００ｋｂ当たり９遺伝子および７遺伝子にまで減少する（Ｂｅｖａｎら、１９９９）。多くの推定遺伝子はまた、組換えの不充分な（ｒｅｃｏｍｂｉｎａｔｉｏｎ−ｄｅｆｉｃｉｅｎｔ）遺伝的に規定されたセントロメア内に存在する。ＣＥＮ２内では、１００ｋｂ当たり５つの推定遺伝子が存在し；一方、ＣＥＮ４は顕著に異なっており、１００ｋｂ当たり１２の遺伝子であった。
【０３０８】
推定セントロメア遺伝子のいくつかが転写されることの強力な証拠が存在した。ホスホエノールピルビン酸遺伝子（ＣＵＥ１）は、１つのＣＥＮ５ボーダーを規定し；そしてこの遺伝子における変異は、光調節遺伝子発現において欠陥を引き起こす（Ｌｉら、１９９５）。ＣＥＮ２およびＣＥＮ４の配列決定された部分内では、推定遺伝子の１７％（２７／１６０）が、クローン化されたｃＤＮＡ（ＥＳＴ）と９５％より大きい同一性を有し、ＣＥＮ４において、ＣＥＮ２においてよりも３倍大きくマッチングした（ｈｔｔｐ：／／ｗｗｗ．ｔｉｇｒ．ｏｒｇ／ｔｄｂ／ａｔ／ａｇａｄ／）。これらの遺伝子のうち２４個が、複数のエキソンを有しており、そして４つが、既知の機能を有する単コピー遺伝子に対応する。同定された推定遺伝子のリストを以下の表３に示す。ＣＥＮ４の境界内でコードされたさらなる遺伝子のリストを、表４に列挙する。これらの遺伝子の同定は、その遺伝子が、それ自体、独特の調節エレメントを含み得るか、またはセントロメア機能または遺伝子発現に関与する独特の制御または調節エレメントに隣接するゲノム位置に存在し得るという点で、有意である。特に、本発明者らは、ミニ染色体における選択した遺伝子への挿入のための、これらの遺伝子、またはこれらの配列の０〜５ｋｂ上流または下流のＤＮＡ配列の使用を意図する。このようなエレメントは、セントロメアの独特の環境中にある場合でさえ、これらの遺伝子の発現の有益な調節制御をおそらく生じ得ることが期待される。
【０３０９】
残りの２３の遺伝子がセントロメアで独特にコードされたか否かを調べるために、注釈付きのゲノムＡｒａｂｉｄｏｐｓｉｓ配列のデータベースにおいて検索を行った。２つの遺伝子を除いて、９５％より大きい同一性を有するホモログは、配列決定されたゲノムの８０％中のどの場所においても見出されなかった。単コピー遺伝子に対応する独立したｃＤＮＡクローンの数は、遺伝子発現のレベルの概算値を提供する。第ＩＩ染色体上で、ｃＤＮＡデータベースに対して高い質のマッチング（９５％より大きい同一性）を有する推定遺伝子が、平均して４つの独立したｃＤＮＡクローンとマッチングした（レンジ１〜７８）。ＣＥＮ２およびＣＥＮ４内では、２７のうち１１の遺伝子がこの平均を超える（表３）。最終的に、ＣＥＮ２およびＣＥＮ４でコードされた遺伝子は、単一遺伝子ファミリーのメンバーではなく、しかもそれらは、セントロメア機能においてある役割を演じると推定される遺伝子にも対応せず、代わりに、多様な役割を有する。
【０３１０】
Ａｒａｂｉｄｏｐｓｉｓセントロメア領域における多くの遺伝子は、早期の停止コドンまたは破壊されたオープンリーディングフレームに起因して機能的でないが、染色体腕上に偽遺伝子はほとんど見られなかった。これらの偽遺伝子の大部分は、可動エレメントに対して相同性を有するが、多くは、典型的には可動性ではない遺伝子に対応する（図１２Ｉ〜Ｊおよび図１２Ｓ〜Ｔ）。遺伝的に規定されたセントロメア内では、１００ｋｂ当たり、１．０（ＣＥＮ２）および０．７（ＣＥＮ４）のこれらの非可動性偽遺伝子が存在した；セントロメアに隣接する反復リッチ領域はそれぞれ、１００ｋｂ当たり１．５および０．９を有する。偽遺伝子および転移性エレメントの分布は重複しており、これらの領域におけるＤＮＡ挿入が遺伝子破壊に寄与したことを示す。
【０３１１】
【表３】

【０３１２】
【表４】

（Ｂ．セントロメアＤＮＡの保存）
ＣＥＮ２およびＣＥＮ４配列の保存を研究するために、ＰＣＲプライマー対を設計した。これらのプライマー対は、Ｃｏｌｕｍｂｉａ配列内の独特の領域に対応し、そして約２０ｋｂの間隔でＬａｎｄｓｂｅｒｇおよびＣｏｌｉｍｂｉａのセントロメア領域の調査に使用される（図１４Ａ、Ｂ）。分析に使用されたプライマーは、図１５Ａ、Ｂに列挙される。適切な長さの増幅産物を、大部分のプライマー対（８５％）に関して両方の生態型において得た。このことは、以下のことを示す。増幅された領域が高度に類似であることを示す。残りの場合、プライマー対は、Ｃｏｌｕｍｂｉａ　ＤＮＡを増幅したが、Ｌａｎｄｓｂｅｒｇ　ＤＮＡを増幅しなかった（非常に低いストリンジェンシーでさえも）。これらの領域において、さらなるプライマーを設計し、非相同性の程度を決定した。ＣＥＮ２におけるミトコンドリアＤＮＡの大きな挿入の加えて、２つの他の非保存領域を同定した（図１４Ａ、Ｂ）。このＤＮＡは、Ｌａｎｄｓｂｅｒｇセントロメアを含まないので、セントロメア機能のために必要であるようである；従って、セントロメア配列の関連する部分が、５７７ｋｂ（ＣＥＮ２）および１２５０ｋｂ（ＣＥＮ４）に減少した。ＬａｎｄｓｂｅｒｇとＣｏｌｕｍｂｉａセントロメアとの間の高い程度の配列の保存は、以下のことを示した。組換え頻度の抑制は、非相同性の大きな領域に起因しないが、代わりにセントロメア自体の特性に起因することを示した。
【０３１３】
（Ｃ．ＣＥＮ２とＣＥＮ４との間の配列類似性）
セントロメア機能を識別するために、ＣＥＮ２とＣＥＮ４との間の新規の配列モチーフついて検索を行った。レトロエレメント、トランスポゾン、特徴付けられたセントロメア反復、および可動性遺伝子に似ているコード配列の比較を除いて検索を行った。単純な反復配列（ホモポリマートラクト（ｔｒａｃｔ）およびミクロサテライトを含む）を覆った後、ＣＥＮ２およびＣＥＮ４について４１７ｋｂおよび８５１ｋｂをそれぞれ測定する独特の配列のコンティグをＢＬＡＳＴを用いて比較した（ｈｔｔｐ：／／ｂｌａｓｔ．ｗｕｓｔｌ．ｅｄｕ）。
【０３１４】
この比較は、以下のことを示した。セントロメア領域内の複合ＤＮＡは、全体配列の長さに渡って相同ではなかったことを示した。しかし、ＣＥＮ２における１６のＤＮＡセグメントは、６０％以上の同一性でＣＥＮ４の１１の領域に一致したことを示した（図１６）。この配列は、関連した配列のファミリーにグループ化され、そしてＡｔＣＣＳ１〜７（Ａｒａｂｉｄｉｐｓｉｓ　ｔｈａｌｉａｎａセントロメア保存配列１〜７）が設計された。これらの配列は、Ａｒａｂｉｄｏｐｓｉｓゲノムにおいて反復されると以前に公知ではなかった。この配列は、合計１７ｋｂ（４％）のＣＥＮ２　ＤＮＡを含み、１０１７ｂｐの平均の長さを有した。そして６５％のＡ＋Ｔの含量であった。類似性に基づいて、一致する配列を、各々が８の配列（ＡｔＣＣＳ１およびＡｔＣＣＳ２；配列番号１〜１４）、３の配列（推定オープンリーディングフレームをコードする小さなファミリー由来）（ＡｔＣＣＳ３；配列番号２１〜２２）、ならびに４の配列（セントロメア内に一度見出された）（ＡｔＣＣＳ−ＡｔＣＣＳ７；配列番号１５〜２０）、予測されたＣＥＮ２およびＣＥＮ４タンパク質に対応する配列の１つ（エキソンおよびイントロン全体に渡った類似性を有する）（ＡｔＣＣＳ６；配列番号１７）を含む２つのファミリーを含む群に分類された（図１６）。
【０３１５】
Ａｒａｂｉｄｏｐｓｉｓゲノム配列データベースの検索は、以下のことを実証した。セントロメアおよびセントロメア周囲の領域において出現するＡｔＣＣＳ１〜ＡｔＣＣＳ５が、中程度に反復した配列であったことを実証した。この残りの配列は、遺伝的に定義されるセントロメアのみに存在する。全ての１６のＳ．ｃｅｒｅｖｉｓｉａｅセントロメアの同様の比較は、以下を定義した。保存された８ｂｐのＣＤＥＩモチーフ、ＡＴリッチな８５ｂｐのＣＤＥＩＩエレメント、および７の高度に保存されたヌクレオチドを有する２６ｂｐのＣＤＥＩＩ領域からなるコンセンサスを定義した（Ｆｌｅｉｇら、１９９５）。対照的に、３つのＳ．ｐｏｍｂｅセントロメアの調査は、全体のセントロメア構造の保存を明らかにしたが、広く保存されたモチーフを明らかにしなかった（Ｃｌａｒｋ、１９９８）。
【０３１６】
（実施例６　マッピングの結果：Ａｒａｂｉｄｏｐｓｉｓ　第１〜５染色体）　第１染色体上のセントロメアをｍｉ３４３（５６．７ｃＭ）とＴ２７Ｋ１２（５９．１ｃＭ）との間にマッピングした。より洗練された位置は、マーカーＴ２２Ｃ２３−ｔ７（約５８．３ｃＭ）とＴ３Ｐ８−ｓｐ６（約５９．１ｃＭ）との間に、セントロメアを配置する。この間隔に含まれるのは、以前に記載されたマーカーＥＫＲＩＶおよびＲＣＥＮ１であった。
【０３１７】
第２染色体上のセントロメアをｍｉ３１０（１８．６ｃＭ）とｇ４１３３（２３．８ｃＭ）との間にマッピングした。より洗練された位置は、マーカーＦ５Ｊ１５−ｓｐ６（約１９．１ｃＭ）とＴ１５Ｄ９（約１９．３ｃＭ）との間に、セントロメアを配置する。以下の配列決定された（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ｅｎｔｒｅｚ／ｎｕｃｌｅｏｔｉｄｅ．ｈｔｍｌ）ＢＡＣ（細菌人工染色体）クローンは、以下のために公知である。マーカーＦ５Ｊ１５−ｓｐ６とＴ１５Ｄ９との間の領域（Ｔ１３Ｅ１１、Ｆ２７Ｃ２１、Ｆ９Ａ１６、Ｔ５Ｍ２、Ｔ１７Ｈ１、Ｔ１８Ｃ６、Ｔ５Ｅ７、Ｔ１２Ｊ２、Ｆ２７Ｂ２２、Ｔ６Ｃ２０、Ｔ１４Ｃ８、Ｆ７Ｂ１９およびＴ１５Ｄ１９）を測定するために公知である。
【０３１８】
Ｔ１２ＪとＦ２７Ｂ２２との間に、ＢＡＣ適用範囲におけるギャップが存在する。ＲＡＲＥ切断、パルスフィールドゲル（ｐｕｌｓｅ　ｆｉｅｄｌ　ｇｅｌ）またはＤＮＡ配列タイリング（ｔｉｌｉｎｇ）が、使用され、配列決定のためのギャップにおけるＤＮＡを単離する。
【０３１９】
第３染色体上のセントロメアを、ａｔｐｏｘ（４８．６ｃＭ）とＡＴＡ（５３．８ｃＭ）との間にマッピングした。より洗練された位置は、マーカーＴ９Ｇ９−ｓｐ６（約５３．１ｃＭ）とＴ５Ｍ１４−ｓｐ６（約５３．３ｃＭ）との間にセントロメアを配置された。この間隔内に含まれるのは、以前に記載されたマーカーＲＣＥＮ３である。
【０３２０】
第４染色体上のセントロメアをｍｉ２３３（１８．８ｃＭ）とｍｉ１６７（２１．５ｃＭ）との間にマッピングした。より洗練された位置は、マーカーＴ２４Ｈ２４．３０ｋ３（約２０．３ｃＭ）とＦ１３Ｈ１４−ｔ７（約２１．０ｃＭ）との間にセントロメアを配置する。以下の配列決定された（ｈｐｐｔ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ｅｎｔｒｅｚ／ｎｕｃｌｅｏｔｉｄｅ．ｈｔｍｌ）ＢＡＣ（細菌人工染色体）クローンは、マーカーＦ５Ｊ１５−ｓｐ６とＴ６Ａ１３−ｓｐ６（Ｔ２７Ｄ２０、Ｔ１９Ｂ１７、Ｔ２６Ｎ６、Ｆ４Ｈ６、Ｔ１９Ｊ１８、Ｔ４Ｂ２１、Ｔ１Ｊ１、Ｔ３２Ｎ４、Ｃ１７Ｌ７、Ｃ６Ｌ９、Ｆ６Ｈ８、Ｆ２Ｉ１２、Ｆ１４Ｇ１６、およびＦ２８Ｄ６）との間の領域を測ることが公知である。
【０３２１】
Ｆ２Ｉ１２とＦ１４Ｇ１６との間のＢＡＣ切断内のギャップが存在する。ＲＡＲＥ切断、パルスフィールドゲルまたはＤＮＡ配列タイリングが使用され、配列決定のためのギャップ内のＤＮＡが単離される。
【０３２２】
第５染色体上のセントロメアをｎｇａ７６（７１．６ｃＭ）とＰｈｙＣ（７４．３ｃＭ）との間にマッピングした。より洗練された位置はマーカーＦ１３Ｋ２０−ｔ７（約６９．４ｃＭ）とＣＵＥ１（約６９．５ｃＭ）との間にセントロメアを配置する。この間隔に含まれるのは、公に利用可能なマーカー、ｕｍ５７９Ｄ、ｍｉ２９１ｂ、ＣＭｓ１である。
【０３２３】
所定の第１〜５染色体上のセントロメア内に位置することが公知のＢＡＣクローンを示す表、ならびにこれらのクローンの配列についてのＧｅｎｂａｎｋ目録番号を以下の表５および表６に示す。
【０３２４】
Ｌｉｓｔｅｒ　ａｎｄ　Ｄｅａｎ　Ｒｅｃｏｍｂｉｎａｎｔ　Ｉｎｂｒｅｄ　Ｇｅｎｅｔｉｃ　ｍａｐに対応する遺伝的な位置（すなわち、ｃＭ値）は、ｈｔｔｐ：／／ｎａｓｃ．ｎｏｔｔ．ａｃ．ｕｋ／ｎｅｗ＿ｒｉ＿ｍａｐ．ｈｔｍｌでオンラインで利用可能である。マーカーは、ｈｔｔｐ：／／ｇｅｎｏｍｅ−ｗｗｗ．ｓｔａｎｆｏｒｄ．ｅｄｕ／Ａｒａｂｉｄｏｐｓｉｓ／ａｂｕｏｕｔｃａｐｓ．ｈｔｍｌで利用可能である。
【０３２５】
【表５】

【０３２６】
【表６】

（実施例７　セントロメアの機能を試験するためのＢＡＣベクターの構築）
ＢＡＣクローンは、１つ以上の植物テロメアおよび選択マーカーを用いて、Ａｇｒｏｂａｃｔｅｒｉｕｍ形質転換に必要なＤＮＡエレメントと一緒になって逆根管充填（ｒｅｔｒｏｆｉｔｔｉｎｇ）され得る（図９）。この方法は、任意のＢＡＣクローンを植物細胞に送達する手段を提供し、セントロメアの機能について試験する。
【０３２７】
この方法は、以下の方法で働く。転換ベクターは、逆根管充填カセットを含む。この逆根管充填カセットは、Ｔｎ１０、Ｔｎ５、Ｔｎ７、Ｍｕまたは他の転移性エレメントに隣接しており、そして複製起点およびＡｇｒｏｂａｃｔｅｒｉｕｍのための選択マーカー含み、植物テロメアアレイ、続いて、Ｔ−ＤＮＡ右および左ボーダー、続いて第２の植物テロメアアレイ、および植物選択マーカー（図９）を含む。この転換ベクターは、標的ＢＡＣを保有するＥ．ｃｏｌｉ株に形質転換され得る。次いで、逆根管充填カセットに隣接する転移性エレメントは、カセットのＢＡＣクローンへの転移をランダムに媒介する。この逆根管充填されたＢＡＣクローンは、今や適切なＡｇｒｏｂａｃｔｅｒｉｕｍの株に形質転換され、次いで植物再に形質転換され得る。ここで高度のフィデリティー（ｆｉｄｅｌｉｔｙ）の減数分裂および有糸分裂の伝達ついて試験され得、この伝達は、このクローンが完全な機能性植物セントロメアを含んだことを示す。
【０３２８】
（実施例８　植物ミニ染色体の構築）
ミニ染色体を以下のように構築する。以前に単離された必須の染色体エレメントを組み合わせることによって構築する。例示的なミニ染色体ベクターとしては、「シャトルベクター」であるように設計されたベクターが挙げられる。シャトルベクターは、すなわち簡便な宿主（例えば、Ｅ．ｃｏｌｉ、Ａｇｒｏｂａｃｔｅｒｉｕｍ、または酵母）ならびに植物細胞において維持され得る。
【０３２９】
（Ａ．ミニ染色体の構築のための一般的な技術）
ミニ染色体は、Ｅ．ｃｏｌｉまたは他の細菌細胞において、環状分子として維持され得る。テロメア配列ブロックの間の移動性ｓｔｕｆｆｅｒフラグメントと配置することによって維持され得る。このｓｔｕｆｆｅｒフラグメントは、不必要なＤＮＡ配列であり、独特の制限部位に隣接する。このフラグメントは、環状ＤＮＡの制限消化によって除去され、テロメア末端を有する直鎖状分子を作製する。次いで、この直鎖状ミニ染色体は、例えばゲル電気泳動によって単離され得る。ｓｔｕｆｆｅｒフラグメントおよび植物テロメアに加えて、ミニ染色体は、複製起点および選択マーカーを含む。選択マーカーは植物において機能し得、環状分子が細菌細胞において維持されることを可能にする。ミニ染色体はまた、植物選択マーカー、植物セントロメア、および植物ＡＲＳを含み、植物細胞においてＤＮＡ分子の複製および維持が可能である。最後に、ミニ染色体はいくつかの独特の制限部位を含み、ここでさらなるＤＮＡ配列挿入物がクローン化され得る。例えば、ミニ染色体の物理的な構築の最も迅速な方法、すなわち種々の必須のエレメントの連結は、総合すると例えば、当業者に明瞭である。
【０３３０】
多数のミニ染色体ベクターが、現在の発明者によって設計され、そして本明細書中に例示の目的で開示されている（図７Ａ〜図７Ｈ）。しかし、これらのベクターは限定ではなく、当業者に明らかなように、多くの変化および変更がなされ得、そしてさらに機能的ベクターが得られ得る。
【０３３１】
（Ｂ．ミニ染色体の構築のための変更された技術）
２段階の工程の方法が、ミニ染色体の構築のために開発された。この方法は、セントロメアＤＮＡを含むＢＡＣクローンに必須のエレメントを加えることを可能にする。これらの手順は、インビボで起こり得、染色体の切断の問題を排除する。この染色体の切断の問題は、しばしば試験管内で生じる。この技術の詳細および利点は、以下のようである：
１）１つのプラスミドは、作製され得る。このプラスミドはマーカー、Ａｇｒｏｂａｃｔｅｒｉｕｍの転移のための起点および植物におけるボーダー配列、選択およびスクリーニングのためのマーカー、植物テロメア、およびｌｏｘＰ部位またはインビボまたはインビトロでの部位特異的組換えに有用な他の部位を含む。第２のプラスミドは、ＢＡＣクローンであり得、入手可能なゲノムライブラリーより単離される（図１１Ａ）。
【０３３２】
２）単一のＥ．ｃｏｌｉ内でか、または試験管内でのいずれかで２つのプラスミドが混合され、そして部位特異的組換えｃｒｅが導入される。これは２つのプラスミドがｌｏｘＰ部位で融合することを引き起こす（図１１Ｂ）。
【０３３３】
３）必要であるとみなされる場合、有用な制限部位（ＡｓｅＩ／ＰａｃＩまたはＮｏｔ　Ｉ）は、過剰の物質（例えば、他の選択マーカーまたは複製起点）を除去するために含まれる。
【０３３４】
４）変異は、Ｋａｎ^Ｒ遺伝子（有するか、または有さない）（図１１Ｂ、１１Ｃ）、ＬＡＴ５２　ＧＵＳ遺伝子（有するか、または有さない）、ＬＡＴ５２ＧＦＰ遺伝子を含み、そしてＧＵＳ遺伝子を含む（図１１Ｃ、１１Ｄおよび１１Ｅ）。他の植物プロモーターの制御下で。
【０３３５】
（Ｃ．安定な組み込まれないミニ染色体の調製方法）
ミニ染色体が宿主ゲノム内に組み込まれないことを確認するための技術が開発された（図１１Ｆ）。特に、ミニ染色体は、宿主の染色体とは別の異なるエレメントとして維持されなければならない。導入されたミニ染色体が組み込まれないことを確認するために、本発明者らは、以下の多様性を想像する。この多様性は、致死植物遺伝子をコードする（例えば、ジフテリア（ｄｉｐｔｈｅｒｉａ）毒素または任意の他の遺伝子産物（発現された際に、植物に致死を引き起こす））。この遺伝子は、右Ａｇｒｏｂａｃｔｅｒｉｕｍボーダーとテロメアとの間に位置し得る。植物核に入り、そして宿主染色体に組み込まれるミニ染色体は、致死を生じる。しかし、ミニ染色体が別のままである場合、そしてさらにこの構築物の端がテロメアまで分解される場合、致死遺伝子が除去され、そして細胞は生存する。
【０３３６】
（実施例９　二セントロメア染色体の分析によるセントロメア活性のインビボスクリーニング）
方法を、セントロメア活性のスクリーニングのために設計した（図１０）。この方法において、植物は、２成分のＢＡＣクローン（遺伝的に定義されたセントロメア領域のＤＮＡを含む）で初め形質転換される。ＤＮＡが宿主染色体中に組み込まれることを可能にすることによって、この組み込みが２つのセントロメアを有する染色体を生じると期待される。これは不安定な状況である。この状況は、しばしば染色体の破損につながる。有糸分裂および減数分裂減少の間に、逆の極に引かれる場合、２以上の機能的セントロメアを保有する単一の染色体が、２つのセントロメアの間での接合部で頻繁に破損するように、染色体の破損につながる。これは、重い増殖欠失および生存不能な子孫に引き起こし得る。細胞プロセスおよび発達プロセスに重要な、または必須な遺伝子が、破損現象によって破壊される場合に。従って、セントロメア機能を有する領域は、クローンの探索によって同定され得る。ここでこのクローンは、宿主植物に導入される際に、任意の以下の予測された特性を示す：形質転換の減少された効率；形質転換された植物が、異常なセクターおよび増加した致死を示すように天然の染色体に組み込まれた際の遺伝的不安定性の因果関係；特に形質転換された植物が、導入遺伝子の維持について選択しない条件下で増殖する際の維持の困難；染色体の遠位の先端でか、またはセントロメア領域でゲノムに組み込まれる傾向。対照的に、非セントロメアＤＮＡを含むクローンは、よりランダムなパターンを組み込むと予測される。生じる組み込みの分布およびパターンの確認は、挿入されたＤＮＡの末端の配列決定によって決定され得る。
【０３３７】
スクリーニングを、ＢｉＢＡＣライブラリー（２成分の細菌人工染色体）においてセントロメアＤＮＡをコードする１００ｋｂより大きいクローンの同定によって行った（Ｈａｍｉｌｔｏｎ、１９９７）。これは、セントロメア由来のＤＮＡをコードするクローンについてのＢｉＢＡＣゲノムライブラリーを含むスクリーニングフィルターによってなされる（図１０、工程１）。ＢｉＢＡＣベクターが使用されるのは、Ａｒａｂｉｄｏｐｓｉｓゲノム物質の大きな挿入物を含み得るからであり、またＡｇｒｏｂａｃｔｅｒｉｕｍ媒介形質転換に必要な２成分の配列をコードするからである。次いで、ＢｉＢＡＣベクターを含むセントロメア配列を、Ａｇｒｏｂａｃｔｅｒｉｕｍ媒介形質転換によって直接染色体に組み込んだ（図１０、工程２）。コントロールとして、非セントロメアＤＮＡを含むＢｉＢＡＣ構築物をまた、形質転換に使用した。セントロメア機能を有する配列を保有するＢｉＢＡＣは、二セントロメア染色体の形成を生じる。形質転換された植物由来の子孫は、以下について分析される。二セントロメア染色体の形成に起因する染色体の破壊に寄与する生育不能および全体の形態学的な差について分析される（図１０、工程３）。非セントロメア配列は、野生型植物との表現型の差をほとんど示さないと予測される。
【０３３８】
（実施例１０　増加した組換えのための処理での洗練されたセントロメアマッピング）
Ａｒａｂｉｄｏｐｓｓ　ｔｈａｌｉａｎａにおけるセントロメアについてのより洗練されたマップ位置を達成するために、種々の化学および環境処理を使用し、組換えを刺激した。これらの処理を、四分子セットの植物を作製するために達成された交雑種（ｃｒｏｓｓ）の花粉ドナーに使用した（実施例２を参照のこと）。花粉ドナー植物を、１インチ四方のポットに個別に植え、そして開花まで２４時間の光で生育室で生長させた。次いで開花植物を以下の溶液の１つに漬け、そして５０ｍｌの同じ溶液で灌漑した。
【０３３９】
【表７】

処理の後、次いで植物を生育室に戻し、そして標準条件下で２〜５日育てた。次いで花粉を、新しく開いた花より収集し、そして使用し、実施例２に記載されるように柱頭を受粉させた。次いで花粉ドナー植物を再び上記のように処理し、そして別の回の受粉に使用した。花粉ドナー植物は、代表的に５〜１０回の処理および花粉の収集に供された。
【０３４０】
処理をまた、非化学試薬を使用して行った。上記のように、処理を使用し、Ａｒａｂｉｄｏｐｓｉｓにおけるセントロメアについてのより洗練されたマップの位置を、さらなる花粉ドナー植物において組換えを刺激することによって達成した。処理は以下のようであった。
【０３４１】
【表８】

水で満たされた浅い皿に花粉ドナー植物を含むポットを配置することによって（乾燥を防ぐために）、ヒートショック処理を行った。そして適切な温度の生育機に植物を含む皿を配置した。花粉ドナー植物を、ＢｉｏＲａｄ　ＵＶチャンバーに配置することによって、ＵＶ照射を行い、そして異なる量の時間、適切な波長で植物を照射した。ＵＶ植物およびヒートショック植物の両方を、何回かの処理および花粉の収集に供した。ガンマ放射源（コバルト−６０）に曝露した植物を１度のみ処理し、次いで有害な染色体の再編成の蓄積を防ぐために処分した。
【０３４２】
処理の後、次いで植物を生育室に戻し、そして標準条件下で２〜５日生育した。次いで花粉を新しく開いた花より収集し、そして実施例２に記載のように感受性の柱頭を受粉するために使用した。次いで花粉ドナー植物を再び、上記のように処理し、そして別の回の受精に使用した。花粉ドナー植物は、代表的に５〜１０回の処理および花粉の収集に供された。結果を以下の表９に示す。
【０３４３】
【表９】

（実施例１１　遺伝性遺伝子移入の促進）
本発明者よって、以下のこともまた企図される。遺伝子移入を促進するために組換えを刺激する技術または処理を使用し得ることも、企図される。遺伝子移入は、育種技術を記述し、これによって１つ以上の所望の形質が別の系統（Ｂ）から１つの系統（Ａ）に、移される。次いでこの形質は、同じ系統（Ａ）への一連の戻し交配によって所望の系統（Ａ）の遺伝的バックグラウンドに単離される。所望の遺伝的バックグラウンドにおける所望の形質を単離するために必要とされる多数の戻し交配は、各戻し交配における組換えの頻度に依存する。
【０３４４】
戻し交配は、特定の所望の形質を、１つの供給源から近交系またはその形質がない他の植物に転移する。これは、例えば、以下のように初めに交配することによって、達成され得る。優位な近交系（Ａ）（反復性の親）を、ドナー近交系（非反復性の親）と交配することによって。それらは、問題の形質のための適切な遺伝子（例えば、本発明に従って調製された構築物）を保有する。この交配の子孫は、反復性の子孫において、所望の性質（非反復性の親から移される）について最初に選択され、次いで選択された子孫は、優位な反復性の親（Ａ）に戻し交配される。所望の性質についての選択された５以上の戻し交配世代の後、その子孫は、移された特徴を調整する座についてヘミ接合であるが、大部分か、またはほとんど全ての他の遺伝子について優位な親と似ている。最後の戻し交配世代を自家受粉し、子孫を得る。その子孫は、移された遺伝子について純粋育種である（すなわち、１つ以上の形質転換現象）。
【０３４５】
従って、一連の育種操作を通じて選択された導入遺伝子は、１つの系統から、全く異なる系統に、さらなる組換え操作の必要もなく移動され得る。導入遺伝子は、以下において役立つ。代表的に、任意の他の遺伝子として遺伝的に振舞う際に役立つ。そして任意の他のコーン（ｃｏｒｎ）遺伝子に同一な様式における育種技術によって操作され得る。従って、１以上の導入遺伝子を本当に育種する近交系植物を産生し得る。異なる近交系植物との交配によって、異なる導入遺伝子の組み合わせを有する多数の異なるハイブリッドを産生し得る。この様式において、ハイブリッドと頻繁に関連する所望の農学特性（「ハイブリッド効力」）ならびに１以上の導入遺伝子によって与えられる所望の特徴を有する植物が、産生され得る。
【０３４６】
育種はまた、全体のミニ染色体を１つの植物から別の植物に移すために使用され得る。例えば、ミニ染色体を有する第１の植物を、ミニ染色体を有さない第２の植物交配させることによって、この交配の任意の世代の子孫が得られ得る。この子孫は、ミニ染色体を有するか、または任意のさらなる数の所望のミニ染色体を有する。一連の戻し交配を通じて、植物が得られ得る。この植物は、第２の植物の遺伝的バックグラウンドを有するが、しかし第１の植物由来のミニ染色体を有する。
【０３４７】
本明細書中で開示および請求された全ての組成物および方法が、本開示の観点における過度の実験法を有さないでなされ得、そして作成され得る。本発明の組成物および方法が、好ましい実施形態の用語において記載されるが、当業者には以下のことが明瞭である。変更が、本発明の、概念、精神および範囲から逸脱しないで以下に適用され得ることが明瞭ある。本明細書中に記載される組成物ならびに方法に適用され得、そして本明細書中に記載される工程または方法の工程の順序に適応され得ることが明瞭である。より具体的には、以下のことが明らかである。化学的および生理的の両方に関連した特定の薬剤が、本明細書中に記載される薬剤と置換され得るが、しかし、同じか、または類似の結果が達成され得ることが明らかである。当業者に明瞭なすべてのこのような類似の置換および変更は、添付の請求項によって定義されるように本発明の精神、範囲および概念内である。
【０３４８】
（参考文献）
以下の参考文献は、それらが本明細書中に示される例示的な手順または他の詳細な補遺を提供する程度まで、本明細書中に参考として援用される。
【０３４９】
【表１０】

【図面の簡単な説明】
【図１】
図１は、整列していない四分染色体についてのセントロメア染色体地図である：２つの親（ＡＡＢＢ×ａａｂｂ）の交配、ここで「Ａ」は、一つの染色体のセントロメア上に存在し、そして「Ｂ」は、第２の染色体のセントロメアと連結される。減数分裂において、ＡおよびＢ染色体は、独立して交際（ａｓｓｏｒｔ）し、同数の親のダイタイプ（ｄｉｔｙｐｅ）（ＰＤ）および非親ダイタイプ（ＮＰＤ）四分染色体（組換え子孫は、灰色で示される）を生じる。テトラタイプ（ｔｅｔｒａｔｙｐｅ）（ＴＴ）は、「Ｂ」とそのセントロメアとの間の乗換えからのみ生じる。
【図２】
図２は、Ａｒａｂｉｄｏｐｓｉｓセントロメアの位置の低解像度地図である。三染色体地図作製を使用して、５つのＡｒａｂｉｄｏｐｓｉｓ染色体のうちの４つのセントロメアの地図の位置を決定した（Ｋｏｏｒｎｎｅｅｆ、１９８３；Ｓｅａｒｓら、１９７０）。染色体４について、有用な三染色体種は得られなかった。ＫｏｏｒｎｎｅｅｆおよびＳｅａｒｓら、１９８３（これは、低解像度欠失地図作製を信頼する）の方法を用いて、染色体１上のセントロメアが、５ｃＭだけ離れている、目に見える２つのマーカー（ｔｔｌおよびｃｈｌ）の間に位置することが見出された。他の染色体上のセントロメアの位置は、低解像度に対して地図作製される。
【図３】
図３は、遺伝子的に定義されるＡｒａｂｉｄｏｐｓｉｓセントロメアの位置の物理的地図である。各セントロメア領域は、スケールに引き寄せられる；物理的サイズは、ＤＮＡ配列決定（染色体ＩＩおよびＩＶ）に由来するか、またはＢＡＣフィンガープリント法（Ｍａｒｒａら、１９９９；Ｍｏｚｏら、１９９９）に基づく評価に由来する（染色体Ｉ、ＩＩＩ、およびＶ）。各染色体について、フィンガープリント分析（Ｍａｒｒａら、１９９９；Ｍｏｚｏら、１９９９）に由来する、マーカー（上）の位置、それらのマーカーにおけるテトラタイプ／全四分染色体の数（下）、セントロメアの境界（太い黒棒）およびコンティグの名前が示される。各コンティグについて、２よりも多くの遺伝子マーカー（ＢＡＣ末端配列のデータベースから開発された）（ｈｔｔｐ：／／ｗｗｗ．ｔｉｇｒ．ｏｒｇ／ｔｄｂ／ａｔ／ａｂｅ／ｂａｃ　ｅｎｄ　ｓｅａｒｃｈ．ｈｔｍｌ）に得点をつけた。これらの配列に対応するＰＣＲプライマーを使用して、ＣｏｌｕｍｂｉａおよびＬａｎｄｂｅｒｇ生態型（ＢｅｌｌおよびＥｃｋｅｒ、１９９４；ＫｏｎｉｅｃｚｎｙおよびＡｕｓｕｂｅｌ、１９９３）におけるサイズまたは制限部位多型を同定した；プライマー配列は入手可能である（ｈｔｔｐ：／／ｇｅｎｏｍｅ−ｗｗｗ．ｓｔａｎｆｏｒｄ．ｅｄｕ／Ａｒａｂｉｄｏｐｓｉｓ／ａｂｏｕｔｃａｐｓ．ｈｔｍｌ）。乗換えを刺激する処理から生じるテトラタイプ四分染色体（ボックス）；組換え近交系（ＲＩ）地図（楕円形）に分配されたセンチモルガン（ｃＭ）のマーカーの位置（ｈｔｔｐ：／／ｎａｓｃ．ｎｏｔｔ．ａｃ．ｕｋ／ｎｅｗ　ｒｉ　ｍａｐ．ｈｔｍｌ；ＳｏｍｅｒｖｉｌｌｅおよびＳｏｍｅｒｖｉｌｌｅ、１９９９）；および１８０ｂｐ反復に対応する物理的地図における配列境界ギャップ（オープンサークル）（Ｒｏｕｎｄら、１９９７）、５ＳｒＤＮＡ（黒の円）または１６０ｂｐ反復（灰色円）が示される（Ｃｏｐｅｎｈａｖｅｒら、１９９９）。
【図４】
図４は、Ａｒａｂｉｄｏｐｓｉｓ　ｔｈａｌｉａｎａにおける四分染色体分析のために使用される種子ストックの例示的リストである。個々の株は、株番号により同定される（欄Ｂ）。四分染色体メンバー番号（欄Ａ）は、四分染色体供給源を示す（すなわち、Ｔ１は、四分染色体番号１に由来する種子を示す、そしてその番号−１、−２、−３または−４は、四分染色体の個々のメンバーを示す）。列挙された株は、Ｄａｐｈｎｅ　Ｐｒｅｕｓｓの名前でオハイオ州立大学においてＡｒａｂｉｄｏｐｓｉｓ　Ｂｉｏｌｏｇｉｃａｌ　Ｒｅｓｏｕｒｃｅｓ　Ｃｅｎｔｅｒ（ＡＢＲＣ）に寄託されている。
【図５】
図５は、セントロメア地図作製のためのマーカー情報である。セントロメアを位置付けするために使用されるＤＮＡ多型は、染色体によって示される（欄１）。各マーカーの名前は、欄２に示され、セントロメアを位置付けするためにＣｏｐｅｎｈａｖｅｒら、１９９９によって使用されるマーカーの名前は、欄３に与えられ、そしてマーカー型は、欄４にて示される。ＣＡＰＳ（同時優性増幅多型部位）は、ＰＣＲで増幅され得、そして適切な制限酵素で消化することによって検出され得るマーカーである（また、欄３において示される）。ＳＳＬＰ（単純配列長多型）は、異なる長さのＰＣＲ産物を増幅することによって多型を検出する。欄５は、そのマーカーが公共のウェッブサイト（例えば、ｈｔｔｐ：／／ｇｅｎｏｍｅ−ｗｗｗ．ｓｒａｎｆｏｒｄ．ｅｄｕ／Ａｒａｂｉｏｐｓｉｓ）で入手可能である場合を特に言及する。公共のウェッブサイト上で入手できないそれらのマーカーについて、そのマーカーを増幅するために使用される正方向プライマーおよび逆方向プライマーの配列が、それぞれ欄６および７に列挙される。
【図６】
図６は、四分染色体分析のための、ＰＣＲベースのマーカーを得点付けである。一つの花粉四分子染色体（Ｔ２）に由来する子孫の遺伝子型を、２つの遺伝子マーカー（ＳＯ３９２およびｎｇａ７６）について決定した。ＰＣＲを使用する４つの子孫植物の分析（Ｔ２−１〜Ｔ２−４）およびゲル電気泳動は、植物の遺伝子型が決定されること、および親花粉の遺伝子型が推測されることを可能にする。
【図７Ａ】
図７Ａは、例示的なミニ染色体ベクターである。図７Ａに示されるベクターは、高コピー数、低コピー数または１コピーであり得るＥ．ｃｏｌｉ複製起点を有する。図７Ａにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ａは、植物ＡＲＳを有するＥ．ｃｏｌｉ植物環状シャトル（ｃｉｒｃｕｌａｒ　ｓｈｕｔｔｌｅ）ベクターを示す。
【図７Ｂ】
図７Ｂは、例示的なミニ染色体ベクターである。図７Ｂに示されるベクターは、高コピー数、低コピー数または１コピーであり得るＥ．ｃｏｌｉ複製起点を有する。図７Ｂにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ｂは、植物ＡＲＳを伴わない植物円ベクターを示す。そのベクターは、他の植物ＤＮＡ配列（例えば、選択可能またはスクリーニング可能なマーカー）内に見出される複製機能の植物起点に依存する。
【図７Ｃ】
図７Ｃは、例示的なミニ染色体ベクターである。図７Ｃにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ｃは、植物ＡＲＳを有する酵母植物環状シャトルベクターを示す。この酵母ＡＲＳは、２倍含まれており、多重クローニング部位のいずれかの側で一旦、大きな挿入片が安定であることを確実にする。
【図７Ｄ】
図７Ｄは、例示的なミニ染色体ベクターである。図７Ｄにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ｄは、植物ＡＲＳを伴わない酵母−植物環状シャトルベクターを示す。このベクターは、他の植物ＤＮＡ配列（例えば、選択マーカー）内に見出される複製機能の植物起点に依存する。この酵母ＡＲＳは、２倍含まれており、多重クローニング部位のいずれかの側で一旦、大きな挿入片が安定であることを確実にする。
【図７Ｅ】
図７Ｅは、例示的なミニ染色体ベクターである。図７Ｅに示されるベクターは、高コピー数、低コピー数または１コピーであり得るＥ．ｃｏｌｉ複製起点を有する。図７Ｅにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ｅは、植物ＡＲＳを有するＥ．ｃｏｌｉ−Ａｇｒｏｂａｃｔｅｒｉｕｍ植物環状シャトルベクターを示す。Ｔ−ＤＮＡ転移のためのＶｉｒ機能は、適切なＡｇｒｏｂａｃｔｅｒｉｕｍ株を使用して、イントランスで提供される。
【図７Ｆ】
図７Ｆは、例示的なミニ染色体ベクターである。図７Ｆに示されるベクターは、高コピー数、低コピー数または１コピーであり得るＥ．ｃｏｌｉ複製起点を有する。図７Ｆにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ｆは、植物ＡＲＳを伴わないＥ．ｃｏｌｉ−Ａｇｒｏｂａｃｔｅｒｉｕｍ植物環状シャトルベクターを示す。このベクターは、他の植物ＤＮＡ配列（例えば、選択マーカー）内に見出される複製機能の植物起点に依存する。Ｔ−ＤＮＡ転移のためのＶｉｒ機能は、適切なＡｇｒｏｂａｃｔｅｒｉｕｍ株を使用して、イントランスで提供される。
【図７Ｇ】
図７Ｇは、例示的なミニ染色体ベクターである。図７Ｇにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ｇは、植物ＡＲＳを有する線状植物ベクターを示す。この線状ベクターは、インビトロで構築され得、次いで例えば、機械的手段（例えば、微小発射体衝撃（ｍｉｃｒｏ　ｐｒｏｊｅｃｔｉｌｅ　ｂｏｍｂａｒｄｍｅｎｔ）、エレクトロポレーションまたはＰＥＧ媒介性形質転換）によって植物に転移され得る。
【図７Ｈ】
図７Ｈは、例示的なミニ染色体ベクターである。図７Ｈにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ｈは、植物ＡＲＳを伴わない線状植物ベクターを示す。この線状ベクターは、インビトロで構築され得、次いで例えば、機械的手段（例えば、微小発射体衝撃、エレクトロポレーションまたはＰＥＧ媒介性形質転換）によって植物に転移され得る。
【図７Ｉ】
図７ＡＩは、例示的なミニ染色体ベクターである。図７Ｉに示されるベクターは、高コピー数、低コピー数または１コピーであり得るＥ．ｃｏｌｉ複製起点を有する。図７Ｉにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ｉ。この図は、それらが植物テロメアを含まないということを除いて、それぞれ図７Ａ−７Ｆと同一である。これらのベクターは、一旦植物細胞に送達されると環状のままであり、それゆえそれらの末端を安定にするためのテロメアを必要としない。
【図７Ｊ】
図７Ｊは、例示的なミニ染色体ベクターである。図７Ｊに示されるベクターは、高コピー数、低コピー数または１コピーであり得るＥ．ｃｏｌｉ複製起点を有する。図７Ｊにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ｊ。この図は、それらが植物テロメアを含まないということを除いて、それぞれ図７Ａ−７Ｆと同一である。これらのベクターは、一旦植物細胞に送達されると環状のままであり、それゆえそれらの末端を安定にするためのテロメアを必要としない。
【図７Ｋ】
図７Ｋは、例示的なミニ染色体ベクターである。図７Ｋにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ｋ。この図は、それらが植物テロメアを含まないということを除いて、それぞれ図７Ａ−７Ｆと同一である。これらのベクターは、一旦植物細胞に送達されると環状のままであり、それゆえそれらの末端を安定にするためのテロメアを必要としない。
【図７Ｌ】
図７Ｌは、例示的なミニ染色体ベクターである。図７Ｌにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ｌ。これらの図は、それらが植物テロメアを含まないということを除いて、それぞれ図７Ａ−７Ｆと同一である。これらのベクターは、一旦植物細胞に送達されると環状のままであり、それゆえそれらの末端を安定にするためのテロメアを必要としない。
【図７Ｍ】
図７Ｍは、例示的なミニ染色体ベクターである。図７Ｍにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ｍ。これらの図は、それらが植物テロメアを含まないということを除いて、それぞれ図７Ａ−７Ｆと同一である。これらのベクターは、一旦植物細胞に送達されると環状のままであり、それゆえそれらの末端を安定にするためのテロメアを必要としない。
【図７Ｎ】
図７Ｎは、例示的なミニ染色体ベクターである。図７Ｎにおいて、そのベクターは、４−８ｂｐの特異性を有する従来の制限エンドヌクレアーゼの認識配列ならびに非常に稀な切断酵素（例えば、Ｉ−Ｐｐｏ　Ｉ、Ｉ−Ｃｕｅ　Ｉ、ＰＩ−Ｔｌｉ、ＰＩ−Ｐｓｐ　Ｉ、Ｎｏｔ　ＩおよびＰＩ　Ｓｃｅ　Ｉ）の認識配列を含み得る、多重クローニング部位を含み、セントロメアは、部位特異的リコンビナーゼＣｒｅに対する標的として作用し得るＬｏｘ部位に隣接される。図７Ｎ。これらの図は、それらが植物テロメアを含まないということを除いて、それぞれ図７Ａ−７Ｆと同一である。これらのベクターは、一旦植物細胞に送達されると環状のままであり、それゆえそれらの末端を安定にするためのテロメアを必要としない。
【図８】
図８は、ＣＥＮ２（Ａ）およびＣＥＮ４（Ｂ）における配列の特徴である。中心の棒は、示されたＢＡＣクローンの注釈されているゲノム配列を示す；黒は、遺伝子的に定義されたセントロメア；白は、セントロメアに隣接する領域。遺伝子および反復特徴に対応する配列（ボックスで占められる（それぞれ、上棒および下棒））は、図１２Ａ−Ｔとして定義される；推定非移動性遺伝子、赤；移動性要素によって運ばれる遺伝子、黒；非移動性偽遺伝子、ピンク；移動性要素によって運ばれる偽遺伝子、灰色；レトロ要素、黄色；トランスポゾン、緑；これまで定義されたセントロメア反復、紺青；１８０ｂｐ反復、うす青。染色体特異的セントロメア特徴としては、大きなミトコンドリアＤＮＡ挿入（オレンジ；ＣＥＮ２）およびタンデム反復の新規なアレイ（紫；ＣＥＮ４）が挙げられる。物理的地図のギャップ（／／）、注釈されていない領域（ハッチボックス）および発現された遺伝子（フィルドサークル）が示される。
【図９】
図９は、ＢＡＣクローン（または、任意の他の細菌クローン）をミニ染色体に変えるための方法である。一部の転換ベクターは、非相同組換え（交換可能要素媒介性）を介してか、または部位特異的リコンビナーゼ系（例えば、Ｃｒｅ−ＬｏｘまたはＦＬＰ−ＦＲＴ）の作用によって、ＢＡＣクローン（または、目的の他の細菌クローン）内に組み込まれる。
【図１０】
図１０は、Ａｒａｂｉｄｏｐｓｉｓにおける二セントロメア染色体の分析のための方法である。ＢｉＢＡＣベクター含有セントロメアフラグメント（〜１００ｋｂ）は、Ａｇｒｏｂａｃｔｅｒｉｕｍ媒介性形質転換手順を使用してＡｒａｂｉｄｏｐｓｉｓゲノム内に組み込まれ、そして二セントロメア染色体の形成に起因する不利な影響について研究される。１）ＢｉＢＡＣ含有セントロメアフラグメントは、標準的なプロトコルを使用して同定される。２）植物形質転換。３）植物含有二セントロメア染色体の生長および発達における欠陥の分析。
【図１１Ａ】
図１１Ａは、ＢＡＣクローン（または任意の他の細菌クローン）をミニ染色体に転換するための方法である。必要な選択マーカーおよびＥ．ｃｏｌｉ、ＡｇｒｏｂａｃｔｅｒｉｕｍおよびＡｒａｂｉｄｏｐｓｉｓにおける遺伝子物質の増殖のための複製起点ならびにＡｒａｂｉｄｏｐｓｉｓへのＡｇｒｏｂａｃｔｅｒｉｕｍ媒介性形質転換のために必要な遺伝子座は、転換ベクター内にクローン化される。Ｃｒｅ／ｌｏｘＰ組換えを使用して、転換ベクターを、ミニ染色体を形成するようにＢＡＣ含有セントロメアフラグメントに組換える。
【図１１Ｂ】
図１１Ｂは、ＢＡＣクローン（または任意の他の細菌クローン）をミニ染色体に転換するための方法である。必要な選択マーカーおよびＥ．ｃｏｌｉ、ＡｇｒｏｂａｃｔｅｒｉｕｍおよびＡｒａｂｉｄｏｐｓｉｓにおける遺伝子物質の増殖のための複製起点ならびにＡｒａｂｉｄｏｐｓｉｓへのＡｇｒｏｂａｃｔｅｒｉｕｍ媒介性形質転換のために必要な遺伝子座は、転換ベクター内にクローン化される。Ｃｒｅ／ｌｏｘＰ組換えを使用して、転換ベクターを、ミニ染色体を形成するようにＢＡＣ含有セントロメアフラグメントに組換える。
【図１１Ｃ】
図１１Ｃは、ＢＡＣクローン（または任意の他の細菌クローン）をミニ染色体に転換するための方法である。必要な選択マーカーおよびＥ．ｃｏｌｉ、ＡｇｒｏｂａｃｔｅｒｉｕｍおよびＡｒａｂｉｄｏｐｓｉｓにおける遺伝子物質の増殖のための複製起点ならびにＡｒａｂｉｄｏｐｓｉｓへのＡｇｒｏｂａｃｔｅｒｉｕｍ媒介性形質転換のために必要な遺伝子座は、転換ベクター内にクローン化される。Ｃｒｅ／ｌｏｘＰ組換えを使用して、転換ベクターを、ミニ染色体を形成するようにＢＡＣ含有セントロメアフラグメントに組換える。
【図１１Ｄ】
図１１Ｄは、ＢＡＣクローン（または任意の他の細菌クローン）をミニ染色体に転換するための方法である。必要な選択マーカーおよびＥ．ｃｏｌｉ、ＡｇｒｏｂａｃｔｅｒｉｕｍおよびＡｒａｂｉｄｏｐｓｉｓにおける遺伝子物質の増殖のための複製起点ならびにＡｒａｂｉｄｏｐｓｉｓへのＡｇｒｏｂａｃｔｅｒｉｕｍ媒介性形質転換のために必要な遺伝子座は、転換ベクター内にクローン化される。Ｃｒｅ／ｌｏｘＰ組換えを使用して、転換ベクターを、ミニ染色体を形成するようにＢＡＣ含有セントロメアフラグメントに組換える。
【図１１Ｅ】
図１１Ｅは、ＢＡＣクローン（または任意の他の細菌クローン）をミニ染色体に転換するための方法である。必要な選択マーカーおよびＥ．ｃｏｌｉ、ＡｇｒｏｂａｃｔｅｒｉｕｍおよびＡｒａｂｉｄｏｐｓｉｓにおける遺伝子物質の増殖のための複製起点ならびにＡｒａｂｉｄｏｐｓｉｓへのＡｇｒｏｂａｃｔｅｒｉｕｍ媒介性形質転換のために必要な遺伝子座は、転換ベクター内にクローン化される。Ｃｒｅ／ｌｏｘＰ組換えを使用して、転換ベクターを、ミニ染色体を形成するようにＢＡＣ含有セントロメアフラグメントに組換える。
【図１１Ｆ】
図１１Ｆは、ＢＡＣクローン（または任意の他の細菌クローン）をミニ染色体に転換するための方法である。必要な選択マーカーおよびＥ．ｃｏｌｉ、ＡｇｒｏｂａｃｔｅｒｉｕｍおよびＡｒａｂｉｄｏｐｓｉｓにおける遺伝子物質の増殖のための複製起点ならびにＡｒａｂｉｄｏｐｓｉｓへのＡｇｒｏｂａｃｔｅｒｉｕｍ媒介性形質転換のために必要な遺伝子座は、転換ベクター内にクローン化される。Ｃｒｅ／ｌｏｘＰ組換えを使用して、転換ベクターを、ミニ染色体を形成するようにＢＡＣ含有セントロメアフラグメントに組換える。
【図１１Ｇ】
図１１Ｇは、ＢＡＣクローン（または任意の他の細菌クローン）をミニ染色体に転換するための方法である。必要な選択マーカーおよびＥ．ｃｏｌｉ、ＡｇｒｏｂａｃｔｅｒｉｕｍおよびＡｒａｂｉｄｏｐｓｉｓにおける遺伝子物質の増殖のための複製起点ならびにＡｒａｂｉｄｏｐｓｉｓへのＡｇｒｏｂａｃｔｅｒｉｕｍ媒介性形質転換のために必要な遺伝子座は、転換ベクター内にクローン化される。Ｃｒｅ／ｌｏｘＰ組換えを使用して、転換ベクターを、ミニ染色体を形成するようにＢＡＣ含有セントロメアフラグメントに組換える。
【図１２】
図１２Ａ−Ｔは、染色体ＩＩおよびＩＶ上のセントロメア領域の特性である。（頂部）遺伝的に定義されたセントロメア（影のかかった灰色、ＣＥＮ２、左；ＣＥＮ４、右）の図面、近接する周囲セントロメアＤＮＡ、および各染色体の遠位セグメントは、ＤＮＡ配列決定により決定されるようにＭｂにて測定される（物理的地図内のギャップに対応する影のかかった灰色のギャップ）。ＲＩ地図上のｃＭにおける位置（ｈｔｔｐ：／／ｎａｓｃ．ｎｏｔｔ．ａｃ．ｕｋ／ｎｅｗ　ｒｉ　ｍａｐ．ｈｔｍｌ）およびＭｂにおける物理的距離（ノザンテロメアにおける始まり、およびセントロメアギャップにおいて）が示される。（底部）各特徴の密度は（図１２Ａ−１２Ｔ）は、Ｍｂにおける染色体上の位置に関してプロットされる。（図１２Ａ、１２Ｋ）ＲＩ地図上のマーカーについてのｃＭ位置（塗りつぶした四角）および１ｃＭ／２２１ｋｂのゲノム平均を表す曲線（ダッシュライン）。ＲＩ地図作製集団における、ＣＥＮ４内の一つの乗換え（ｈｔｔｐ：／／ｎａｓｃ．ｎｏｔｔ．ａｃ．ｕｋ／ｎｅｗ　ｒｉ　ｍａｐ．ｈｔｍｌ；ＳｏｍｅｒｖｉｌｌｅおよびＳｏｍｅｒｖｉｌｌｅ、１９９９）は、本明細書中でモニターされた雄性減数分裂組換えと雌性減数分裂における組換えとの間の違いを反映し得る。（図１２Ｂ−１２Ｅおよび図１２Ｌ−１２Ｏ）反復要素で占められているＤＮＡの％は、１０ｋｂのスライディングインターバル（ｓｌｉｄｉｎｇ　ｉｎｔｅｒｖａｌ）を用いて１００ｋｂのウインドウについて計算された。（図１２Ｂ、１２Ｌ）１８０ｂｐ反復；（図１２Ｃ、１２Ｍ）レトロ要素と類似性を有する配列（ｄｅｌ、Ｔａｌ、Ｔａｌｌ、ｃｏｐｉａ、Ａｔｈｉｌａ、ＬＩＮＥ、Ｔｙ３、ＴＳＣＬ、１０６Ｂ（Ａｔｈｉｌａ様）、Ｔａｔｌ、ＬＴＲおよびＣｉｎｆｕｌを含む）；（図１２Ｄ、１２Ｎ）トランスポゾンと類似性を有する配列（Ｔａｇｌ、Ｅｎ／Ｓｐｍ、Ａｃ／Ｄｃ、Ｔａｍｌ　ＭｕＤＲ、Ｌｉｍｐｅｔ、ＭＩＴＥＳおよびＭａｒｉｎｅｒを含む）；（図１２Ｅ、１２Ｏ）前述のセントロメア反復（１６３Ａ、１６４Ａ、１６４Ｂ、２７８Ａ、１１Ｂ７ＲＥ、ｍｉ１６７、ｐＡＴ２７、１６０ｂｐ、１８０ｂｐおよび５００ｂｐ反復ならびにテロメア配列を含む（Ｍｕｒａｔａら、１９９７；Ｈｅｓｌｏｐ−Ｈａｒｒｉｓｏｎら、１９９９；Ｂｒａｎｄｅｓら、１９９７；Ｆｒａｎｚら、１９９８；Ｗｒｉｇｈｔら、１９９６；Ｋｏｎｉｅｃｚｎｙら、１９９１；Ｐｅｌｉｓｓｉｅｒら、１９９５；ＶｏｙｔａｓおよびＡｕｓｕｂｅｌ、１９８８；Ｃｈｙｅら、１９９７；Ｔｓａｙら、１９９３；Ｒｉｃｈａｒｄら、１９９１；Ｓｉｍｏｅｎｓら、１９８８；Ｔｈｏｍｐｓｏｎら、１９９６；Ｐｅｌｉｓｓｉｅｒら、１９９６Ｆｒａｎｚら、１９９８；Ｐｅｌｉｓｓｉｅｒら、１９９５；ＶｏｙｔａｓおよびＡｕｓｕｂｅｌ、１９８８；Ｔｈｏｍｐｓｏｎら、１９９６）。（図１２Ｆ、１２Ｐ）％（アデノシンおよびチミジン）は、２５ｋｂのスライディングインターバルを用いて５０ｋｂのウインドウについて計算された（図１２Ｇ−１２Ｊ、１２Ｑ−１２Ｔ）。推定される遺伝子または偽遺伝子の数は、１０ｋｂのスライディングインターバルを用いて１００ｋｂのウインドウに渡ってプロットされた。（図１２Ｇ、１２Ｉ、１２Ｑ、１２Ｓ）推定された遺伝子（図１２Ｇ、１２Ｑ）および偽遺伝子（図１２Ｉ、１２Ｓ）は、代表的に移動性ＤＮＡ要素上で見出されない；（図１２Ｈ、１２Ｊ、１２Ｒ、１２Ｔ）推定された遺伝子（図１２Ｈ、１２Ｒ）および偽遺伝子（図１２Ｊ、１２Ｔ）は、しばしは移動性ＤＮＡ上で運ばれる（逆転写酵素、トランスポザーゼ、およびレトロウイルスポリタンパク質を含む）。ダッシュラインは、領域を示し、その領域において、配列決定または注釈（ａｎｎｏｔａｔｉｏｎ）が進行中であり、注釈は、ＧｅｎＢａｎｋ記録（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ｅｎｔｒｅｚ／ｎｕｃｌｅｏｔｉｄｅ．ｈｔｍｌ）、ＡＧＡＤデータベース（ｈｔｔｐ：／／ｗｗｗ．ｔｉｇｒ．ｏｒｇ／ｔｄｂ／ａｔ／ａｇａｄ／．）および反復Ａｒａｂｉｄｏｐｓｉｓ配列のデータベースとのＢＬＡＳＴ比較（ｈｔｔｐ：／／ｎｕｃｌｅｕｓ．ｃｓｈｌ．ｏｒｇ／ｐｒｏｔａｒａｂ／ＡｔＲｅｐＢａｓｅ．ｈｔｍ）から得られた；最新の注釈記録は、個々のエントリを変更し得るが、その領域の全体構造は、有意に変更されない。
【図１３】
図１３は、植物細胞への導入のために、ＢＡＣクローン含有セントロメアＤＮＡをミニ染色体に変えるための方法である。記載される特定の要素が、例示の目的のために提供され、これらに限定されない。Ａ）ＢＡＣクローンの図、セントロメアＤＮＡの位置（赤）、部位特異的組換え部位（例えば、ｌｏｘＰ）、およびＦ複製起点を言及する。Ｂ）転換ベクター含有選択マーカーおよびカラーマーカー（例えば、３５Ｓ−Ｂａｒ、ｎｐｔＩＩ、ＬＡＴ５２−ＧＵＳ、Ｓｃａｒｅｃｒｏｗ−ＧＦＰ）、テロメア、部位特異的組換え部位（例えば、ｌｏｘＰ）、抗生物質耐性マーカー（例えば、ａｍｐまたはｓｐｃ／ｓｔｒ）、Ａｇｒｏｂａｃｔｅｒｉｕｍ　Ｔ−ＤＮＡ境界（Ａｇｒｏ　ＬｅｆｔおよびＲｉｇｈｔ）および複製起点（ＲｉＡ４）。Ｃ）ｌｏｘＰ部位にてＣｒｅリコンビナーゼを用いる部位特異的組換えの産物が、セントロメアＤＮＡおよびテロメアが隣接するマーカーを有する環状産物を生じる。Ｄ）植物への形質転換直後のミニ染色体；次いで、左および右の境界が、おそらく植物細胞により除去されて、そして植物テロメア−ゼによってさらなるテロメア配列が付加される。
【図１４】
図１４Ａ−Ｂは、セントロメアＤＮＡの転換である。配列ＣＥＮ２（図１４Ａ）およびＣＥＮ４（図１４Ｂ）に対して使用されるＢＡＣクローン（棒）が示される；矢印は、遺伝子的に定義されるセントロメアの境界を意味する。Ｃｏｌｕｍｂｉａ（フィルドサークル）のみ、またはＬａｎｄｓｂｅｒｇおよびＣｏｌｕｍｂｉａ（オープンサークル）の両方から産物を生じるＰＣＲプライマー対；ミトコンドリアゲノムに対する相同性を有するＢＡＣコードＤＮＡ（灰色棒）；１８０ｂｐ反復（灰色ボックス）；未配列決定ＤＮＡ（ダッシュライン）；ならびに物理的地図内のギャップ（二重スラッシュ）が示される。
【図１５Ａ】
図１５Ａは、Ａ．ｔｈａｌｉａｎａ　ＣｏｌｕｍｂｉａおよびＬａｎｄｓｂｅｒｇ生態型におけるセントロメア配列の転換を分析するために使用されるプライマーである。図１５Ａ：染色体２配列の増幅のために使用されるプライマー。
【図１５Ｂ】
図１５Ｂは、Ａ．ｔｈａｌｉａｎａ　ＣｏｌｕｍｂｉａおよびＬａｎｄｓｂｅｒｇ生態型におけるセントロメア配列の転換を分析するために使用されるプライマーである。図１５Ｂ：染色体４配列の増幅のために使用されるプライマー。
【図１６】
図１６は、ＣＥＮ２およびＣＥＮ４に共通の配列である。遺伝子的に定義されるセントロメア（太線）、配列決定されたＢＡＣクローン（細線）、および未注釈のＢＡＣクローン（ダッシュライン）が、図１４Ａ、Ｂにおいて示される。反復ＡｔＣＣＳ１（Ａ．ｔｈａｌｉａｎａセントロメア保存配列）およびＡｔＣＣＳ２（それぞれ、クローズドサークルおよびオープンサークル）、ＡｔＣＣＳ３（三角形）、およびＡｔＣＣＳ４−７（それぞれ、４−７）が示され（ＧｅｎＢａｎｋ登録番号ＡＦ２０４８７４〜ＡＦ２０４８８０）、そしてＢＬＡＳＴ２．０（ｈｔｔｐ：／／ｂｌａｓｔ．ｗｕｓｔｌ．ｅｄｕ）を使用して同定された。
【図１７】
セントロメア２由来の配列決定されたＢＡＣクローン。配列決定されたＢＡＣクローンを、図の上部付近の水平線に示す（例えば、Ｔ１４Ａ４を参照のこと）。赤のボックスは、セントロメア２の境界、そしてそのＢＡＣクローンについてセントロメアを含むことを示し、ＧｅｎＢａｎｋ登録番号を右下のパネルに提供する。赤のボックス内の連続配列を、配列番号２０９および配列番号２１０に提供する。配列決定されたクローンの下部の水平線は、さらなるＢＡＣクローンを示し；これらのＢＡＣの配列決定された終点を閉円で示す。１つ以上の未確定の終点を有するクローンを赤のテキスト文書で示す。
【図１８】
セントロメア４由来の配列決定されたＢＡＣクローン。セントロメア４由来の配列決定されたＢＡＣクローンを、図の上部付近の水平線に示す（例えば、Ｔ２４Ｍ８を参照のこと）。赤のボックスは、セントロメア４の境界、そしてそのＢＡＣクローンについてセントロメアを含むことを示し、ＧｅｎＢａｎｋ登録番号を右下のパネルに提供する。赤のボックス内の連続配列を、配列番号２１１および配列番号２１２に提供する。配列決定されたクローンの下部の水平線は、さらなるＢＡＣクローンを示し；これらのＢＡＣの配列決定された終点を閉円で示す。１つ以上の未確定の終点を有するクローンを赤のテキスト文書で示す。
【図１９】
セントロメア１、３、および５の配列タイリング方針（ｔｉｌｉｎｇ　ｐａｔｈ）。これらのセントロメアの境界を、Ｃｏｐｅｎｈａｖｅｒら（１９９９）に記載のように決定した。コンティグ数は、Ｍａｒｒａら（１９９９）によってまとめられたフィンガープリントコンティグをいう。これらのクローンのいくつかを、配列決定し、そして登録番号を提供する（添付の表を参照のこと）。他の場合には、配列決定は、Ａｒａｂｉｄｏｐｓｉｓゲノムプロジェクトによって終えられた。
【図２０】
ＢｉＢＡＢベクター中に保有されるセントロメア２由来のＤＮＡの位置。クローンを、フィンガープリントおよびＰＣＲ分析によって物理的地図上に配置し、そして配列決定されたＢＡＣクローンと比較した。
【図２１】
選択可能マーカーまたはスクリーニングマーカーを、ＢｉＢＡＣクローンに付加するための例示的方法。所望されるマーカーをトランスポゾン境界に隣接させ、そしてトランスポザーゼの存在下でＢｉＢＡＣと共にインキュベートした。その後、このＢｉＢＡＣを植物に導入する。しばしば、これらのＢｉＢＡＣは天然の染色体に組み込まれ得、安定性を改変され得かつ染色体切断を引き起こし得る二セントロメア染色体を作製し、新規な染色体フラグメントを生じる。
【図２２】
染色体安定性のアッセイ。天然の染色体、構築されたミニ染色体、または二セントロメア染色体の安定性は、細胞分裂を通した呈色マーカーの組合せをモニタリングすることによって評価され得る。これらのマーカーを、改変ＢＡＣまたはＢｉＢＡＣベクター中のセントロメアに連鎖させ、そして植物に導入する。適切なプロモーターによるマーカー遺伝子の調整が、どの組織がアッセイされるかを決める。例えば、根特異的プロモーター（例えば、ＳＣＡＲＥＣＲＯＷ）は、根細胞におけるファイルにおいて組合せをモニターすることを可能にし；減数分裂後花粉特異的プロモーター（例えば、ＬＡＴ５２）は、減数分裂を通した組合せのモニタリングを可能にし、そして一般的なプロモーター（例えば、３５Ｓカリフラワーモザイクウイルスプロモーター）は、多くの他の植物組織における組合せをモニターすることを可能にする。定量的アッセイは、安定性の一般的パターンを評価し、そしてマーカーの損失に対応する切片のサイズを測定する。一方、定性的アッセイは、細胞系列の見識を必要とし、そして有糸分裂および減数分裂の間の染色体損失事象数を算出することを可能にする。
【図２３Ａ】
セントロメア１〜４由来の１８０ｂｐ反復についての配列アラインメント。左側の欄は、反復コピーのＢＡＣ供給源およびその配列に与えられた任意の割り当て番号を示す。例えば、名称ｆ１２ｇ６−１は、ＢＡＣ番号ｆ１２ｇ６からの反復コピーおよび任意の所定反復番号１を示す。ｆ１２ｇ６、ｆ５ａ１３、ｔ２５ｆ１５、ｔ１２ｊ２、ｔ１４ｃ８、ｔ６ｃ２０、ｆ２１ｉ２およびｆ６ｈ８と称される、反復コピーを含むＢＡＣの核酸配列を、それぞれ、配列番号１８４、配列番号１９１、配列番号１８９、配列番号２０５、配列番号２０６、配列番号１８６、配列番号２０８、および配列番号２０７に提供する。（図２３Ａ）セントロメア１由来の１８０ｂｐ反復のアラインメント。
【図２３Ｂ】
セントロメア１〜４由来の１８０ｂｐ反復についての配列アラインメント。左側の欄は、反復コピーのＢＡＣ供給源およびその配列に与えられた任意の割り当て番号を示す。例えば、名称ｆ１２ｇ６−１は、ＢＡＣ番号ｆ１２ｇ６からの反復コピーおよび任意の所定反復番号１を示す。ｆ１２ｇ６、ｆ５ａ１３、ｔ２５ｆ５、ｔ１２ｊ２、ｔ１４ｃ８、ｔ６ｃ２０、ｆ２１ｉ２およびｆ６ｈ８と称される、反復コピーを含むＢＡＣの核酸配列を、それぞれ、配列番号１８４、配列番号１９１、配列番号１８９、配列番号２０５、配列番号２０６、配列番号１８６、配列番号２０８、および配列番号２０７に提供する。（図２３Ｂ）セントロメア２由来の１８０ｂｐ反復のアラインメント。
【図２３Ｃ】
セントロメア１〜４由来の１８０ｂｐ反復についての配列アラインメント。左側の欄は、反復コピーのＢＡＣ供給源およびその配列に与えられた任意の割り当て番号を示す。例えば、名称ｆ１２ｇ６−１は、ＢＡＣ番号ｆ１２ｇ６からの反復コピーおよび任意の所定反復番号１を示す。ｆ１２ｇ６、ｆ５ａ１３、ｔ２５ｆ５、ｔ１２ｊ２、ｔ１４ｃ８、ｔ６ｃ２０、ｆ２１ｉ２およびｆ６ｈ８と称される、反復コピーを含むＢＡＣの核酸配列を、それぞれ、配列番号１８４、配列番号１９１、配列番号１８９、配列番号２０５、配列番号２０６、配列番号１８６、配列番号２０８、および配列番号２０７に提供する。（図２３Ｃ）セントロメア３由来の１８０ｂｐ反復のアラインメント。
【図２３Ｄ】
セントロメア１〜４由来の１８０ｂｐ反復についての配列アラインメント。左側の欄は、反復コピーのＢＡＣ供給源およびその配列に与えられた任意の割り当て番号を示す。例えば、名称ｆ１２ｇ６−１は、ＢＡＣ番号ｆ１２ｇ６からの反復コピーおよび任意の所定反復番号１を示す。ｆ１２ｇ６、ｆ５ａ１３、ｔ２５ｆ５、ｔ１２ｊ２、ｔ１４ｃ８、ｔ６ｃ２０、ｆ２１ｉ２およびｆ６ｈ８と称される、反復コピーを含むＢＡＣの核酸配列を、それぞれ、配列番号１８４、配列番号１９１、配列番号１８９、配列番号２０５、配列番号２０６、配列番号１８６、配列番号２０８、および配列番号２０７に提供する。（図２３Ｄ）セントロメア４由来の１８０ｂｐ反復のアラインメント。[0001]
(Background of the Invention)
This application is filed with U.S. Provisional Patent Application No. 60 / 125,219 (filed on March 18, 1999), U.S. Provisional Patent Application No. 60 / 127,409 (filed on April 1, 1999), U.S. Provisional Patent Application No. 60/125. 134,770 (filed on May 18, 1999), US Provisional Patent Application No. 60 / 153,584 (filed on September 13, 1999), US Provisional Patent Application No. 60 / 154,603 (filed on September 17, 1999) And U.S. Provisional Patent Application No. 60 / 172,493 (filed December 16, 1999). The disclosure of each of these is hereby incorporated by reference in its entirety, and in particular herein.
[0002]
The government has rights under this invention under U.S. Department of Agriculture Grant No. 96-35304-3491, National Science Foundation Grant No. 9872641, and Grant No. DOEDE-FG05-920R22072 from the Consortium for Plant Biotechnology. Having.
[0003]
(I. Field of the Invention)
The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to plant chromosome compositions and methods of using the plant chromosome compositions.
[0004]
(II. Description on Related Fields)
Two general approaches have been used to introduce new genetic information into cells ("transformation"). The first approach is to introduce new genetic information as part of another DNA molecule (called a "vector", which can be maintained as an independent unit (episosome) separate from the chromosomal DNA molecule). It is. Episomal vectors have all the essential DNA sequence elements required for DNA replication and maintenance of the vector in the cell. Many episomal vectors are available in bacteria (see, eg, Maniatis et al., 1982). However, very few episomal vectors have been developed that function in higher eukaryotic cells. The available higher eukaryotic episomal vectors are based on naturally occurring viruses and, in most cases, function only in mammalian cells (Willard, 1997). In higher plant systems, only geminiviruses are known as double-stranded DNA viruses that replicate through a double-stranded intermediate that can be based on episomal vectors, but the length of the insert that can be inserted into geminiviruses is approximately Up to 800 bp. Episomal vectors for plants based on cauliflower mosaic virus have been developed, but the vector's ability to carry new genetic information is also limited (Brisson et al., 1984).
[0005]
Another common method of gene transformation involves integrating the introduced DNA sequence into the recipient cell chromosome. This causes the new information to be replicated and distributed to progeny as part of the natural chromosome. The most common form of the transformation method utilizing DNA integration is called "transfection" and is frequently used in a mammalian cell culture system. Transfection uses the introduction of relatively large amounts of protein-depleted DNA into cells. In most cases, the introduced DNA is degraded and spliced in various combinations before being integrated into random locations on the cell's chromosome (see, eg, Wigler et al., 1977). Common problems with this method include rearrangement of the introduced DNA sequence and unexpected levels of expression due to the location of the transgene in the genome (so-called "position effect variation"). ) ") (Shingo et al., 1986). Furthermore, unlike episomal DNA, integrated DNA cannot usually be accurately removed. Transformations utilizing more sophisticated forms of integration can be accomplished by utilizing naturally occurring viruses, such as retroviruses, which integrate into the host chromosome as part of the virus life cycle (Cepko). Et al., 1984). In mice, homologous integration has recently become common in mice. However, this method is much more difficult to use in plants (Lam et al., 1996).
[0006]
The most commonly used gene transformation method in higher plants is based on the transfer of bacterial DNA to the plant chromosome upon infection with the phytopathogenic soil bacterium Agrobacterium (Nester et al., 1984). By replacing the gene of interest with a naturally-transferred bacterial sequence (called T-DNA), it has become possible to introduce new DNA into plant cells. However, even this more "sophisticated" integration-based transformation system is limited in three main respects. First of all, DNA sequences introduced into plant cells using the Agrobacterium T-DNA system are frequently rearranged (see Jones et al., 1987). Second, the expression of the introduced DNA sequence varies between individual transformants (see Jones et al., 1985). This change is probably due to transgene methylation and the effects of surrounding sequences on rearranged and plant chromosomes (ie, position effects). A third disadvantage of the Agrobacterium T-DNA system is its reliance on a "gene addition" mechanism. That is, the new genetic information is added to the genome (that is, all the genetic information of a certain cell), and does not replace information already existing in the genome.
[0007]
One attractive method for commonly used transformation methods is a method using artificial chromosomes. Artificial chromosomes are artificially created linear or circular DNA molecules constructed from cis-acting DNA sequence elements necessary for proper replication and distribution of the native chromosome (Murray et al., 1983). See). Desirable elements include: (1) self-replicating sequences (ARS), which have the properties of an origin of replication, a site for initiation of DNA replication, (2) centromeres (centromere assembly) And telomeres (special DNA structures located at the ends of linear chromosomes, which provide stabilization of the ends and DNA molecules). Which functions to promote complete replication of the very terminal portion of).
[0008]
At present, the chromosomal elements essential for constructing artificial chromosomes are precisely characterized only in lower eukaryotic species. ARS was isolated from single-cell molds, including Saccharomyces @ cerevisiae (brewer's yeast) and Schizosaccharomyces @ pombe (see Stinchcomb et al., 1979, and Hisao et al., 1979). ARS behaves like an origin of replication that allows DNA molecules carrying it to be replicated as episomes after being introduced into the nucleus of these molds. Plasmids with these sequences replicate. However, in the absence of a centromere, the plasmid will be randomly distributed to daughter cells.
[0009]
Artificial chromosomes were constructed in yeast using three essential chromosomal elements cloned. Murray et al. In 1983 disclosed a cloning system based on the in vivo construction of linear DNA molecules that could be transformed into yeast. In this yeast, the linear DNA is maintained as an artificial chromosome. These yeast artificial chromosomes (YACs) contain cloned genes, origins of replication, centromeres and telomeres, and YACs at least up to 100 kB in length are distributed with high fidelity to daughter cells. Small vectors containing CEN can also be stably isolated, but only if they are circular.
[0010]
However, none of the essential elements identified in unicellular organisms function in higher eukaryotic systems. For example, the yeast CEN sequence does not confer stable inheritance properties on vectors introduced into higher eukaryotes. Such a DNA fragment can be easily introduced, but is not stably present as an episome in the host cell. This seriously hinders artificial chromosome production in higher organisms.
[0011]
In one case, plant artificial chromosomes were discussed (Richards et al., US Pat. No. 5,270,201). However, this vector was based on plant telomeres and no functional plant centromeres were disclosed. Although telomeres are important for maintaining chromosome end stability, telomeres do not encode the information necessary to ensure stable inheritance of artificial chromosomes. It has been well established that centromere function is critical for stable chromosomal inheritance in almost all eukaryotes (Nicklas review, 1988). For example, fragments containing centromeres are faithfully isolated, but broken chromosomes lacking centromeres (centerless chromosomes) are immediately lost from cell lines. During mitosis and meiosis, centromeres complete chromosome segregation by attaching to the spine via centromere binding proteins. This ensures proper gene isolation during cell division.
[0012]
S. cerevisiae and S. cerevisiae. In contrast to the detailed studies in pombe, little is known about the molecular structure of functional centromeric DNA of higher eukaryotes. Ultrastructural studies have shown that higher eukaryotic centromeres, a special protein complex formed on chromosomes later in prophase, are large structures with numerous microtubule attachment sites (mammalian centromere plates). Has a diameter of approximately 0.3 μm) (Rieder review, 1982). Therefore, the centromeric DNA region of these organisms can be correspondingly large. However, the minimum amount of DNA required for centromere to function may be smaller.
[0013]
Although such studies are useful for elucidating the structure and function of centromeres, no centromeres cloned from higher eukaryotes have been provided. Cloning of functional centromeres from higher eukaryotes, with the vast body of literature indicating both the need for centromeres for stable chromosomal inheritance and the inability of yeast centromeres to function in higher organisms, It has been shown to be the first necessary step in the production of artificial chromosomes suitable for use in higher plants and higher animals. Producing a centromeric artificial chromosome that functions in higher eukaryotes will represent a major breakthrough in biotechnology research, while overcoming many of the problems associated with the art.
[0014]
(Summary of the Invention)
In one aspect of the invention, a method is provided for identifying a plant centromere. In one embodiment of the present invention, the method may include a tetrad analysis. Briefly, tetrad analysis measures the frequency of recombination between genetic markers and centromeres by analyzing all four products of individual meiosis. A specific average results from the quartet (qrt1) mutation in Arabidopsis, which leaves the four products of pollen mother meiosis in Arabidopsis attached. Quartet mutations may also find use in accordance with the invention in species other than Arabidopsis. For example, several naturally occurring plant species (water lily, cattail, heather (Ericaceae and Epacridceae), primrose (Onagraceae), moss (Droseraceae), orchid (Orchidaceae), and also include acacia (Mimosea) It is known to release clusters (Preuss @ 1944; Smyth @ 1994). However, none of these species have been developed in experimental systems, thus significantly limiting their use for genetic analysis. However, it is contemplated by the inventors that a quartet mutation can be introduced into a host plant, potentially allowing the use of tetrad analysis in any species. When used to pollinate flowers, one tetrad can result in the formation of four seeds, and the plants derived from these seeds can be genetically analyzed. However, with disordered tetrads (eg, tetrads produced by Arabidopsis), genetic mapping using tetrad analysis requires that two markers be scored simultaneously.
[0015]
In another aspect, the invention provides a recombinant DNA construct comprising a plant centromere. The recombinant DNA construct may further include any other desired sequence, including, for example, telomeres, including plant telomeres (eg, Arabidopsis thaliana telomeres, or yeast or any other type of telomere). . It may also be desirable to include an autonomously replicating sequence (ARS), such as a plant ARS (including the ARS of Arabidopsis thaliana). And furthermore, including a structural gene on the construct, or less than or equal to the maximum number of structural genes (approximately 5000) that can be physically located on the recombinant DNA construct (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, 1000). Examples of structural genes that may be desired to be used include selectable or screening marker genes, antibiotic resistance genes, herbicide resistance genes, nitrogen fixation genes, plant pathogen defense genes, plant stress inducible genes, toxins Genes, receptor genes, ligand genes, hormone genes, enzyme genes, interleukin genes, coagulation factor genes, cytokine genes, antibody genes, growth factor genes, and seed storage genes. In one embodiment of the invention, the construct may express a structural gene, for example, in a prokaryote or eukaryote, including a lower eukaryote or a higher eukaryote, such as a plant.
[0016]
In yet another aspect, the present invention provides a recombinant DNA construct comprising a plant centromere, wherein the recombinant DNA construct is a plasmid. The plasmid may include any desired sequence, including, for example, an origin of replication, including origins of replication function in bacteria such as E. coli and Agrobacterium or plants or yeasts such as S. cerevisiae. . Plasmids can also include selectable markers that can function in bacteria (including E. coli and Agrobacterium), as well as those that function in plants or yeast (eg, S. cerevisiae).
[0017]
In yet another aspect, the invention provides a recombinant DNA construct comprising a plant centromere, which can be maintained as a chromosome, wherein the chromosome is transmitted to dividing cells. Plant centromeres can be from any plant.
[0018]
In yet another aspect, the present invention provides a plant centromere further defined as Arabidopsis thaliana centromere. In yet another embodiment of the present invention, the plant centromere is a chromosome 1 centromere of Arabidopsis thaliana and may be further defined as flanked by the genetic markers T22C23-T7 and T3P8-SP6, or It can be defined as flanked by T22C23-T7 and T5D18, T22C23-T7 and T3L4, T5D18 and T3P8-SP6, T5D18 and T3L4, and T3L4 and T3P8-SP6. In yet another embodiment of the invention, the plant centromere comprises a chromosome 2 centromere of Arabidopsis thaliana. The centromere of chromosome 2 is, for example, about 100 to about 611,000, about 500 to about 611,000, about 1,000 to about 611,000, about 10,000 to about 611 of the nucleic acid sequence of SEQ ID NO: 209. 000, about 20,000 to about 611,000, about 40,000 to about 611,000, about 80,000 to about 611,000, about 150,000 to about 611,000, or about 300,000 to about 611. 2,000 consecutive nucleotides, including those comprising the nucleic acid sequence of SEQ ID NO: 209. The centromere can also include about 100 to about 50,959, about 500 to about 50,959, about 1,000 to about 50,959, about 5,000 to about 50,959, about 10% of the nucleic acid sequence of SEQ ID NO: 210. 50,000 to 50,000 to 50,000, about 50,000 to 50,000, about 30,000 to about 509, or about 40,000 to about 50,959 consecutive nucleotides, and the sequence No. 210. The centromere may include sequences from both SEQ ID NOs: 209 and 210 (including the aforementioned fragments) or the entirety of SEQ ID NOs: 209 and 210. In certain embodiments, we can fuse the 3 'fragment of SEQ ID NO: 209 to the 5' fragment of SEQ ID NO: 210 (optionally, one or more 180 bp repeats located between them). (Including sequences).
[0019]
In still yet another embodiment, the invention provides a chromosome 3 centromere of Arabidopsis thaliana. In one embodiment of the invention, the centromere may be further defined as flanked by genetic markers T9G9-SP6 and T5M14-SP6, and furthermore, T9G9-SP6 and T14H20, T9G9-SP6 and T7K14, T9G9-SP6. And T21P20, T14H20 and T7K14, T14H20 and T21P20, T14H20 and T5M14-SP6, T7K14 and T5M14-SP6, T7K14 and T21P20, and defined as flanked by a pair of genetic markers selected from the group consisting of T21P20 and T5M14-SP6. Can be done.
[0020]
In still yet another embodiment, the invention provides a chromosome 4 centromere of Arabidopsis thaliana. In certain embodiments of the invention, the centromere comprises from about 100 to about 1,082,000, from about 500 to about 1,082,000, from about 1,000 to about 1,082, of the nucleic acid sequence of SEQ ID NO: 211. 000, about 5,000 to about 1,082,000, about 10,000 to about 1,082,000, about 50,000 to about 1,082,000, about 100,000 to about 1,082,000, From about 200,000 to about 1,082,000, from about 400,000 to about 1,082,000, or from about 800,000 to about 1,082,000 contiguous nucleotides (including the nucleic acid sequence of SEQ ID NO: 211) ). The centromere may also include about 100 to about 163,317, about 500 to about 163,317, about 1,000 to about 163,317, about 5,000 to about 163,317, about 10% of the nucleic acid sequence of SEQ ID NO: 212. 20,000 to about 163,317, about 30,000 to about 163,317, about 50,000 to about 163,317, about 80,000 to about 163,317, or about 120,000 to about 163,317 continuous And may be defined as including the nucleic acid sequence of SEQ ID NO: 212. The centromere may include sequences from both SEQ ID NOs: 211 and 212 (including the aforementioned fragments) or the entirety of SEQ ID NOs: 211 and 212. In certain embodiments, we are able to fuse the 3 ′ fragment of SEQ ID NO: 211 to the 5 ′ fragment of SEQ ID NO: 212 (optionally, with one or more 180 bp repeats located between them). (Including sequences).
[0021]
In yet another embodiment, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194 , SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, Sequence SEQ ID NO: 207, SEQ ID NO: 208, or a fragment thereof, provided as a centromere on Chromosome 1, Chromosome 3, or Chromosome 5 of Arabidopsis thaliana. In one embodiment, the construct comprises at least 100 base pairs (within full length) of one of the foregoing sequences. Further, the construct can include one or more 180 base pair repeats.
[0022]
In yet another embodiment, the invention provides a chromosome 5 centromere of Arabidopsis thaliana. This centromere may be further defined as flanked by genetic markers F13K20-T7 and CUE1, and furthermore, F13K20-T7 and T18M4, F13K20-T7 and T18F2, F13K20-T7 and T24I20, T18M4 and T18F2, T18M4 and T24I20, It may be defined as flanked by a pair of genetic markers selected from the group consisting of T18M4 and CUE1, T18F2 and T24I20, T18F2 and CUE1, and T24I20 and CUE1.
[0023]
In yet another embodiment, the present invention relates to a recombinant DNA construct comprising a plant centromere and further defined as comprising n copies of a repetitive nucleotide sequence, wherein n is at least 2. provide. Potentially, any number of repetitive copies (about 5, 10, 15, 20, 30, 50, 75, 100, 150, 200, 300, 400, 500, 750) that can be physically placed on the recombinant construct , 1,000, 1,500, 2,000, 3,000, 5,000, 7,500, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70 , Including 000, 80,000, 90,000, and about 100,000 (including all such intermediate ranges of copy numbers) can be included on this construct. In one embodiment, the repeating nucleotide sequence is SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193 SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, Sequence SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211 or SEQ ID NO: 212. Examples of such arrays that can be used are provided in FIGS. The length of the repeat used can vary, but preferably is from about 20 bp to about 250 bp, about 50 bp to about 225 bp, about 75 bp to about 210 bp, about 100 bp to about 205 bp, about 125 bp to about 200 bp, about 150 bp to about 150 bp. 195 bp, about 160 bp to about 190 bp, and about 170 bp to about 185 bp (including about 180 bp).
[0024]
In combination with SEQ ID NOs: 209, 210, 211, and 212, repeats can be included as part of the centromere structure. The number of iterations can vary and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 300, 400, 500 or more.
[0025]
In yet another aspect, the invention provides a minichromosome vector comprising a plant centromere and telomere sequence. Any additional desired sequences, such as an autonomously replicating sequence, a second telomere sequence and a structural gene, can be added to the minichromosome. Such sequences can be added up to the maximum number that one or more of the foregoing sequences can be physically located on the minichromosome. A minichromosome can include any of the centromere compositions disclosed herein. In one embodiment of the invention, the mini chromosome is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 It may include a selected nucleic acid sequence. The minichromosome also confers susceptibility to antibiotics, herbicides, or other drugs, thereby allowing the selection of a plant, plant cell, or cell of any other organism of interest containing the minichromosome, It may include a "negative" selectable marker. The minichromosome can also contain genes that control the copy number of the minichromosome in the cell. One or more structural genes may also be included on the minichromosome. Particularly contemplated as useful are any number of structural genes that can be inserted into a minichromosome while still maintaining a functional vector. It can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or more structural genes.
[0026]
In still yet another embodiment, the invention provides a recombinant DNA construct comprising a plant centromere. The cell can be any type of cell, including a prokaryotic or eukaryotic cell. If the cell is a eukaryotic cell, the cell can be, for example, a yeast cell or a higher eukaryotic cell (eg, a plant cell). The plant cells can be derived from dicotyledonous plants (eg, tobacco, tomato, potato, soybean, canola, sunflower, alfalfa, cotton, and Arabidopsis), or can be monocotyledonous cells (eg, wheat, corn, rye, rice) , Turfgrass, oats, barley, sorghum, millet, and sugarcane). In one embodiment of the invention, the plant centromere is a Arabidopsis thaliana centromere, and the cell may be an Arabidopsis thaliana cell. The recombinant DNA construct can include additional sequences such as telomeres, autonomously replicating sequences (ARS), structural genes, or selectable or screening markers, and as many as can be physically located on the recombinant DNA construct. Including such sequences. In one embodiment of the invention, the cell is further defined as being capable of expressing the structural gene. In another embodiment of the present invention, there is provided a plant comprising the aforementioned cell.
[0027]
In still yet another embodiment, the invention provides a method for preparing a transgenic plant cell. The method includes contacting a starting plant cell (starting plant cell) with a recombinant DNA construct comprising a plant centromere, whereby the starting plant cell is transformed with the recombinant DNA construct. A recombinant DNA construct can include any desired gene, such as as many structural genes as can be physically located on the recombinant DNA construct. In certain embodiments, the centromere may be an Arabidopsis thaliana centromere, and the plant cell may be an Arabidopsis thaliana cell.
[0028]
In yet another aspect, the invention provides a transgenic plant comprising a minichromosome vector, wherein the vector comprises plant centromere and telomere sequences. The minichromosome vector further comprises an autonomously replicating sequence, a second telomere sequence, or a structural gene (eg, an antibiotic resistance gene, a herbicide resistance gene, a nitrogen fixation gene, a plant pathogen defense gene, a plant stress inducible gene, a toxin gene, a receptor). Gene, ligand gene, seed storage gene, hormone gene, enzyme gene, interleukin gene, coagulation factor gene, cytokine gene, antibody gene, and growth factor gene). As many such sequences as can be physically located on the minichromosome can be included. The mini-chromosome vector further comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, Including a nucleic acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 obtain. Can the transgenic plant be any type of plant, such as dicotyledonous plants (eg, tobacco, tomato, potato, pea, carrot, cauliflower, broccoli, soybean, canola, sunflower, alfalfa, cotton, and Arabidopsis) Or monocotyledonous plants (eg, wheat, corn, rye, rice, turf, oat, barley, sorghum, millet, and sugarcane).
[0029]
In yet another aspect, the present invention provides a method for producing a minichromosome vector. The method comprises the steps of (a) obtaining a first vector and a second vector, wherein the first or second vector comprises a selectable or screening marker, an origin of replication, a telomere and a plant centromere; Wherein the first and second vectors comprise sites for site-specific recombination at: and (b) contacting the first and second vectors to form sites on the first vector. Causing a site-specific recombination between the site for specific recombination and the site for site-specific recombination on the second vector, the selectable or screening marker, the origin of replication, Making a minichromosome vector comprising the telomere and the plant centromere. The contacting step may be in vitro or in vivo (here, performing the contacting step in a prokaryotic cell, such as an Agrobacterium or E. coli cell, or a lower eukaryotic cell, such as a yeast cell). May be implemented. This contacting step can be further performed in higher eukaryotic cells, such as plant cells, including Arabidopsis thaliana cells. This contacting step can be performed in the presence of potentially any recombinase, including Cre, Flp, Gin, Pin, Sre, pinD, Int-B13, and R. The first or second vector can include a border sequence for Agrobacterium-mediated transformation. In one embodiment of the invention, the plant centromere is Arabidopsis thaliana centromere. The telomeres can be plant telomeres. Any plant selectable or screening marker, including GFP, GUS, BAR, PAT, HPT, or NPTII, may be used.
[0030]
In yet another aspect, a method is provided for screening a candidate centromere sequence for plant centromere activity. The method comprises the steps of: (a) obtaining an isolated nucleic acid sequence comprising a candidate centromere sequence; (b) transforming the isolated nucleic acid to be incorporated into a plant cell. And (c) screening the candidate centromere sequences for centromere activity. In this method, the step of screening can include obtaining a phenotypic effect present in a plant cell transformed to be integrated or a plant containing the plant cell, wherein the phenotypic effect comprises: It is not present in control plant cells that have not been transformed to integrate with the isolated nucleic acid sequence, or in plants that contain the control plant cells. The types of phenotypic effects that can be screened include reduced viability, reduced efficiency of this transformation, genetic instability in nucleic acids that have been transformed to integrate, abnormal plant sections, increased ploidy, Increasing integrative transformation in aneuploid and distal or centromeric chromosomal regions. An isolated nucleic acid sequence can include a bacterial artificial chromosome, which can be further defined as a binary bacterial artificial chromosome. Transforming to be integrated may involve the use of any type of transformation, such as Agrobacterium-mediated transformation. In one embodiment of the invention, control plant cells are transformed to integrate with a nucleic acid sequence other than the candidate centromere sequence.
[0031]
In yet another aspect, the invention provides a recombinant DNA construct comprising an Arabidopsis polyubiquitin 11 promoter, wherein the promoter comprises about 25 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 180. including. In a further embodiment of the invention, the promoter is from about 75 to about 2,000, about 125 to about 2,000, about 200 to about 2,000, about 400 to about 2,000 of the nucleic acid sequence of SEQ ID NO: 180. About 800 to about 2,000, about 1,000 to about 2,000, or about 1,500 to about 2,000 contiguous nucleotides, or may comprise the nucleic acid sequence of SEQ ID NO: 180. The promoter containing this construct can be any additional desired sequence (eg, enhancer sequence, telomere sequence, plant centromere sequence, ARS, or structural gene (antibiotic resistance gene, herbicide resistance gene, nitrogen fixation gene, plant pathogen defense). Gene, plant stress inducible gene, toxin gene, receptor gene, ligand gene, seed storage gene, hormone gene, enzyme gene, interleukin gene, coagulation factor gene, cytokine gene, antibody gene, and growth factor gene)) May be included. In one embodiment of the invention, a promoter may be operably linked to the 5 'end of the structural gene.
[0032]
In yet another aspect, the invention provides a recombinant DNA construct comprising the Arabidopsis 40S ribosomal protein S16 promoter, wherein the promoter comprises about 25 to about 2,000 contiguous sequences of the nucleic acid sequence of SEQ ID NO: 182. Nucleotides. In certain embodiments of the invention, the promoter is from about 75 to about 2,000, about 125 to about 2,000, about 200 to about 2,000, about 400 to about 2,000 of the nucleic acid sequence of SEQ ID NO: 182. 000, about 800 to about 2,000, about 1,000 to about 2,000, or about 1,500 to about 2,000 contiguous nucleotides, or may comprise the nucleic acid sequence of SEQ ID NO: 182. The promoter containing this construct can be any additional desired sequence (eg, enhancer sequence, telomere sequence, plant centromere sequence, ARS, or structural gene (antibiotic resistance gene, herbicide resistance gene, nitrogen fixation gene, plant pathogen defense). Gene, plant stress inducible gene, toxin gene, receptor gene, ligand gene, seed storage gene, hormone gene, enzyme gene, interleukin gene, coagulation factor gene, cytokine gene, antibody gene, and growth factor gene)) May be included. In one embodiment of the invention, a promoter may be operably linked to the 5 'end of the structural gene.
[0033]
In yet another aspect of the invention, there is provided a recombinant DNA construct comprising a 3 'regulatory sequence of Arabidopsis polyubiquitin 11 comprising a terminator sequence, wherein the 3' regulatory sequence comprises about 3% of the nucleic acid sequence of SEQ ID NO: 181. From 25 to about 2001 contiguous nucleotides. In one embodiment of the invention, the 3 'regulatory sequence comprises a nucleic acid sequence of SEQ ID NO: 181 from about 75 to about 2001, about 125 to about 2001, about 200 to about 2001, about 400 to about 2001, about 800 to about 2001. It may be further defined as comprising about 2001, or about 1,000 to about 2001 contiguous nucleotides, and may comprise the nucleic acid sequence of SEQ ID NO: 181. The recombinant sequence may further comprise any other sequence (eg, enhancer, telomere sequence, plant centromere sequence, ARS, and structural gene (antibiotic resistance gene, herbicide resistance gene, nitrogen fixation gene, plant pathogen defense gene, plant Stress inducible genes, toxin genes, receptor genes, ligand genes, seed storage genes, hormone genes, enzyme genes, interleukin genes, coagulation factor genes, cytokine genes, antibody genes, and growth factor genes). In one embodiment of the invention, a terminator may be operably linked to the 3 'end of the structural gene.
[0034]
In yet another aspect, the invention provides a recombinant DNA construct comprising a 3 'regulatory sequence of Arabidopsis 40S ribosomal protein S16, comprising a terminator sequence, wherein the 3' regulatory sequence comprises a nucleic acid of SEQ ID NO: 183. It comprises about 25 to about 2,000 contiguous nucleotides of the sequence. In certain embodiments of the invention, the 3 'regulatory sequence comprises from about 75 to about 2,000, from about 125 to about 2,000, from about 200 to about 2,000, from about 400 to about 2,000 of the nucleic acid sequence of SEQ ID NO: 183. It may include about 2,000, about 800 to about 2,000, or about 1,000 to about 2,000 contiguous nucleotides, and may include the nucleic acid sequence of SEQ ID NO: 183. The recombinant sequence may further comprise any other sequence (eg, enhancer, telomere sequence, plant centromere sequence, ARS, and structural gene (antibiotic resistance gene, herbicide resistance gene, nitrogen fixation gene, plant pathogen defense gene, plant Stress inducible genes, toxin genes, receptor genes, ligand genes, seed storage genes, hormone genes, enzyme genes, interleukin genes, coagulation factor genes, cytokine genes, antibody genes, and growth factor genes). In one embodiment of the invention, a terminator may be operably linked to the 3 'end of the structural gene.
[0035]
In yet another aspect, the present invention provides a method for expressing a foreign gene in a plant, plant cell, or cell of any other organism of interest. Foreign genes can be from any organism, including plants, animals, and bacteria. It is further contemplated that mini-chromosomes can be used to transfer multiple foreign genes simultaneously into a plant that contains a complete biochemical or regulatory pathway. In yet another embodiment of the present invention, it is contemplated that mini-chromosomes may be used as a DNA cloning vector. Such vectors can be used in plant and animal sequencing projects. The present invention may be particularly useful in cloning sequences that are "unclonable" in yeast and bacteria, but can be easily cloned in plant-based systems.
[0036]
In still yet another aspect of the invention, the minichromosomes disclosed herein are used to generate functional segments of DNA (eg, DNA origins of replication, telomeres, telomere associated genes, nuclei). The substrate adhesion region (nuclear matrix attachment region) (MAR), backbone binding region (SAR), boundary element (boundary element), enhancer, silencer, promoter, recombination hotspot and centromere) can be cloned. This embodiment may be practiced by cloning the DNA into a defective minichromosome that is defective for one or more types of functional elements. Sequences that complement such missing elements give rise to stably inherited minichromosomes. A selectable or screening marker on the minichromosome is then used to select for viable minichromosome-containing cells containing the type of cloned functional element that was non-functional in the defective minichromosome. obtain.
[0037]
In yet a further aspect of the invention, the sequences disclosed herein can be used for the isolation of centromere sequences from plants different from Arabidopsis. Such techniques may use, for example, hybridization assays or sequence-based assays. In one embodiment of the present invention, the centromere is an agriculturally important species, such as vegetable crops (arthur thistle, kohlrabi, yellow croaker, white leeks, asparagus, lettuce (eg, heads, leaves, romaine), bok choy, , Taro, broccoli, melon (eg, muskmelon, watermelon, crenshaw, sugar solution, cantaloupe), brussels sprouts, cabbage, carbans, cardoni, carrots, Chinese cabbage, cauliflower, okra, onion, celery, parsley, chickpea , Parsnip, chrysanthemum, body vegetables, pepper, collard, potato, cucumber plant (marlow, cucumber), squash, gourd, radish, dried spherical onion, kabukanran, eggplant, baramonjin, escalo , Shallots, endive, garlic, spinach, green onions, squash, greens, beet (sugar beet and sample beat) sweet potatoes, chard, horseradish, tomatoes, kale, turnips, and may be isolated from including condiments). Alternatively, centromeres include fruits and vines (e.g., apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, quince almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries, Berry, dried grape, loganberry, raspberry, strawberry, blackberry, grape, avocado, banana, kiwi, oyster, pomegranate, pineapple, tropical fruit, pear fruit, melon, mango, papaya and litchi).
[0038]
In yet another aspect of the present invention, centromeres are used according to the present invention for field crop plants (eg, primrose, meadowfoam), corn (corn (field @ corn), sweet corn, popcorn), hops, jojoba, peanuts. , Rice, safflower, small grains (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legume plants (legumes, lentils, peas, soybeans), oil plants (oilseed rape, mustard, poppies, olives, sunflowers) Isolated from plants like coconut, coconut, castor, cocoa nuts, jujube, fiber plants (cotton, flax, masa, jute), camphor (cinnamon, camphor) or coffee, sugar cane, tea and natural Indian rubber plant Still other examples of plants from which centromeres can be isolated include plants for flower beds (eg, flowers, cacti, juicy and ornamental plants) and forests (broad and evergreen, eg, conifers), fruit trees, Includes ornamental trees, and trees such as fruit set, as well as shrubs and other seedling stocks.
[0039]
In yet another aspect of the invention, an efficient gene replacement study can be performed using the minichromosome vectors described herein. At present, gene replacement has been detected in only a few cases in plant systems and only infrequently in mammalian tissue culture systems (Thomas et al., 1986; see Smithies et al., 1985). ). The reason for this is a high frequency of non-orthodox non-homologous recombination events compared to the frequency of homologous recombination (the former is responsible for gene replacement). Artificial chromosomes may be preferentially involved in homologous recombination. Artificial chromosomes remain intact upon delivery, so that no recombination-produced broken ends are generated that serve as substrates for highly efficient illegitimate recombination machinery. Thus, the artificial chromosome vectors disclosed herein are maintained in the nucleus during meiosis and are available to participate in homology-dependent meiotic recombination. Furthermore, since in principle artificial chromosomes of any length can be constructed using the teachings of the present invention, the vectors can be used to make very long chromosomes derived from the same organism or any other organism. Length DNA can be introduced into cells. Particularly contemplated inserts are approximately several base pairs to 100 megabase pairs (approximately 1 kb, 25 kb, 50 kb, 100 kb, 125 kb, 150 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1 MB). , 1.25 Mb, 1.5 Mb, 2 Mb, 3 Mb, 5 Mb, 10 Mb, 25 Mb, 50 Mb and 100 Mb).
[0040]
In yet another aspect, the present invention provides a method for the construction of a minichromosome vector for genetic transformation of a plant cell, the use of the vector, and an organism transformed thereby. Standard references showing the general principles of recombinant DNA technology include Lewin1985. Other works describe methods and products of genetic manipulation. See, e.g., Maniatis et al., 1982; Watson et al., 1983; Setlow et al., 1979; and Dillon et al., 1985.
[0041]
In yet another aspect, the present invention provides a method for preparing a transgenic cell. In one embodiment of the invention, the method comprises the steps of a) obtaining a nucleic acid molecule comprising Arabidopsis thaliana centromere DNA having the following characteristics: 1) mi342 and T27K12, mi310 and g4133, atox and ATA, mi233 and mi167 and F13K20-t7 and mapping of positions on the Arabidopsis thaliana chromosome defined by a pair of genetic markers selected from the group consisting of CUE 1 and 2; and 2) to the meiotic 1 spindle pole in a pattern showing segregation of homologous chromosomes B) preparing a recombinant construct comprising the nucleic acid molecule; and c) transforming a recipient cell with the recombinant construct.
[0042]
For example, the cell may be a lower eukaryotic cell (including a yeast cell) or may be a higher eukaryotic cell. If the cell is a higher eukaryotic cell, the cell can be an animal or plant cell. In one embodiment of the invention, the cell is not an Arabidopsis thaliana cell. In another embodiment of the present invention, the Arabidopsis thaliana centromere is defined by the marker pair mi342 and T27K12, which is further defined by the genetic marker pairs T22C23-t7 and T3P8-sp6; and / or by the marker pairs mi310 and g4133. Defined by the genetic marker pairs F5J15 and T15D9; and / or defined by the marker pairs atpox and ATA, which may be further defined by the genetic marker pairs T9G9-sp6 and T5M14-sp6; and / or It is defined by mi233 and mi167, which are additionally genetic marker pairs T24H24.30k3 and F13H14- 7 and / or defined by the genetic marker pair F13K20-t7 and CUE1, which is furthermore F13K20-T7 and T18M4, F13K20 and T18F2, F13K20-T7 and T18F2, F13K20-T7 and T24I20, T18M4 and T18F2, T18M4. And T24I20, T18M4 and CUE1, T18F2 and T24I20, T18F2 and CUE1, and T24I20 and CUE1 and may be defined by a pair of genetic markers.
[0043]
In one embodiment of the invention, the transformation comprises the use of a method selected from the group consisting of Agrobacterium-mediated transformation, protoplast transformation, electroporation, or bombardment. The recombinant construct may include the desired elements, including telomeres (Arabidopsis thaliana telomeres or yeast telomeres). The recombinant construct may also include an autonomously replicating sequence (ARS) (eg, Arabidopsis thaliana ARS). The recombinant construct may also include a prokaryotic or eukaryotic selectable or screening marker gene. It may also be desirable to include one or more structural genes with the recombinant construct. Exemplary structural genes include antibiotic resistance genes, herbicide resistance genes, nitrogen fixation genes, plant pathogen defense genes, plant stress inducing genes, toxin genes, seed storage genes, hormone genes, enzyme genes, interleukin genes, coagulation Examples include a gene selected from the group consisting of a factor gene, a cytokine gene, an antibody gene, and a growth factor gene. The method may further include the step of regenerating the transgenic plant from the cells.
[0044]
In yet another aspect, the present invention provides a method for identifying a nucleic acid molecule capable of conferring centromere activity, comprising the steps of: a) obtaining a nucleic acid molecule comprising Arabidopsis thaliana centromere DNA, wherein Arabidopsis is included. thaliana centromere is defined by a pair of genetic markers selected from the group consisting of mi342 and T27K12, mi310 and g4133, atox and ATA, mi233 and mi167, and F13K20-t7 and T17M11-sp6; b) And c) determining the ability of the recombinant construct to demonstrate a stable genetic pattern. In this way, the ability to demonstrate a stable genetic trait pattern can be determined by preparing a recombinant cell containing the recombinant construct. In another embodiment of the present invention, the Arabidopsis thaliana centromere is defined by the marker pair mi342 and T27K12, which is further defined by the genetic marker pairs T22C23-t7 and T3P8-sp6; and / or by the marker pairs mi310 and g4133. Defined by the genetic marker pair F5J15-sp6 and T15D9; and / or defined by the marker pair atpox and ATA, which may be further defined by the genetic marker pair T9G9-sp6 and T5M14-sp6; and / or Defined by the marker pairs mi233 and mi167, which further comprise the genetic marker pairs T24H24.30k3 and F13H 4-t7; and / or defined by the genetic marker pair F13K20-t7 and CUE1, which are furthermore F13K20-T7 and T18M4, F13K20-T20 and T18F2, F13K20-T7 and T24I20, T18M4 and T18F2, T18M4 and T24I20. , T18M4 and CUE1, T18F2 and T24I20, T18F2 and CUE1, and a gene marker pair selected from the group consisting of T24I20 and CUE1.
[0045]
In one embodiment of the invention, the recombinant construct is integrated into a chromosome. The obtaining step includes a step of obtaining a BAC clone or a YAC clone containing Arabidopsis thaliana centromere DNA. This DNA can be obtained by methods involving the use of pulsed-field gel electrophoresis, and can be obtained by methods involving positional cloning. In another embodiment of the invention, the positional cloning may include identifying a continuous set of clones containing the Arabidopsis thaliana centromere DNA, wherein the set of clones is mi342 and T27K12, mi310 and g4133, atox and ATA, mi233 and mi167, and a pair of genetic markers selected from the group consisting of F13K20-t7 and T17M11-sp6.
[0046]
A continuous set of clones can span Arabidopsis thaliana centromeres. The recombinant construct may include a selectable or screenable marker, and the step of determining includes determining the phenotype conferred by the selectable or screenable marker. The step of determining can include, for example, determining the ability of the recombinant construct to demonstrate a stable hereditary pattern in mitosis and / or meiosis. In yet another embodiment, the invention provides a transgenic cell prepared by a method provided by the invention. The present invention also provides a transgenic plant, a transgenic plant part, and a tissue culture comprising the transgenic cell. In another embodiment of the present invention, the Arabidopsis thaliana centromere is defined by the marker pair mi342 and T27K12, which is further defined by the genetic marker pairs T22C23-t7 and T3P8-sp6; and / or by the marker pairs mi310 and g4133. Defined by the genetic marker pair F5J15-sp6 and T15D9; and / or defined by the marker pair atpox and ATA, which may be further defined by the genetic marker pair T9G9-sp6 and T5M14-sp6; and / or Defined by the marker pairs mi233 and mi167, which are further genetic marker pairs T24H24.30k3 and F13 14-t7; and / or defined by the genetic marker pair F13K20-t7 and CUE1, which is further defined as F13K20-T7 and T18M4, F13K20-T7 and T18F2, F13K20-T7 and T24I20, T18M4 and T18F2, T18M4 and T24I20. , T18M4 and CUE1, T18F2 and T24I20, T18F2 and CUE1, and a gene marker pair selected from the group consisting of T24I20 and CUE1.
[0047]
In yet another aspect of the present invention, the centromere used in accordance with the present invention is not derived from Arabidopsis (eg, Arabidopsis thaliana). Similarly, a plant or plant cell comprising a centromere composition according to the present invention may also be derived from a plant different from Arabidopsis.
[0048]
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings, in combination with the detailed description of specific embodiments set forth herein. The file of this patent contains drawings made in at least one color. Copies of this patent with color drawings will be provided by the Patent and Trademark Offices upon request and payment of the necessary fee.
[0049]
(Detailed description of the invention)
The inventors have overcome the deficiencies in the prior art for the first time by providing a nucleic acid sequence of the plant centromere. The importance of this achievement compared to the prior art is illustrated by the general lack of detailed information in the art on the centromere of multicellular organisms in general. To date, the most extensive and definitive characterization of centromere sequences is cerevisiae and S. cerevisiae. It has been obtained from studies of lower eukaryotes such as pombe, where the ability to analyze centromere function has provided a clear picture of the desired DNA. S. The cerevisiae centromere consists of three essential regions, CDEI, CDEII, and CDEIII, totaling only 125 bp (ie, about 0.006-0.06% of each yeast chromosome) (Carbon et al., 1990; Bloom, 1993). S. The pombe centromere is between 40 and 100 kB in length and is composed of repetitive elements that comprise 1-3% of each chromosome (Baum et al., 1994). Subsequent studies using tetrad analysis to track the segregation of artificial chromosomes have shown that naturally occurring S. aureus may It has been demonstrated that less than 1/5 of the centromere of pombe is sufficient for centromere function (Baum et al., 1994).
[0050]
In contrast, the centromeres of mammals and other higher eukaryotes are poorly defined. Although DNA fragments that hybridize to the centromeric region of higher eukaryotes have been identified, little is known about their functionality (see Tyler-Smith et al., 1993). In many cases, using probes for both cytological and genetic repeat mapping to the centromere region, centromere repeats correlate to centromere position. Many of these sequences are satellite elements that are randomly repeated and are interspersed with sequences ranging from 300 kB to 5000 kB in length (Willard, 1990). To date, only one of these repeats, a 171 bp element known as alphoid satellite, has been shown by in situ hybridization to be present in each human centromere (Tyler-Smith et al., 1993). Since many other genomic DNAs need to confer inheritance based on certain regions of the DNA, it is still controversial whether repeats themselves represent functional centromeres (Willard, 1997). ). Alternatively, the location of some higher eukaryotic centromeres has been estimated by analyzing segregation of chromosomal fragments. However, this approach is inaccurate. Because a limited set of fragments can be obtained, and normal centromere function is affected by the surrounding chromosomal sequences (see, eg, Koorneef, 1983; FIG. 2).
[0051]
A more accurate method for mapping centromeres that can be used on intact chromosomes is tetrad analysis (Mortimer et al., 1981). This provides a functional definition of the centromere in its native chromosomal context. Currently, the few centromeres mapped in this manner are from lower eukaryotes, including the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe and Kluyveromyces lactis (Carbon et al., 1990; Hegman, et al., 1990). In these systems, accurate mapping of centromeres has made it possible to clone centromere DNA using a chromosomal walking strategy (Clarke et al., 1980). Subsequently, centromere sequences were more accurately defined using artificial chromosome assays (Hegmann et al., 1993; Baum et al., 1994).
[0052]
Attempts to develop a reliable centromere assay in mammals have yielded ambiguous results. For example, Hadlaczky et al. (1991) identified a 14 kB human fragment that could cause infrequent and novel centromere formation in a mouse cell line. However, in situ hybridization studies have shown that this fragment is not present in the naturally occurring centromere, questioning the reliability of this approach for testing centromere function (Tyler-Smith et al.). , 1993). Similarly, transfection of alphoid satellites into cell lines results in the formation of new chromosomes, which also contain host sequences that can contribute to centromere activity (Haaf et al., 1992; Willard, 1997). ). Furthermore, the new chromosome may have a dispersion of alphoid DNA over its entire length, but still has only a single centromere waist, and blocks of alphoid DNA alone are insufficient for centromere function. (Tyler-Smith et al., 1993).
[0053]
Plant centromeres can be easily visualized on condensed chromosomes, but plant centromeres are not as widely characterized as yeast or mammalian-derived centromeres. Genetic characterization is by segregation analysis of chromosomal fragments, and in particular, by analysis of trisomal strains with terminally labeled terminal centromere fragments (see, eg, Koorneef, 1983; FIG. 2). In addition, repetitive elements have been identified that are either genetically (Richards et al., 1991) or physically (Alfenito et al., 1993; Maluszynska et al., 1991) linked to centromeres. However, no examples have tested the functional significance of these sequences.
[0054]
Arabidopsis thaliana cytology has been instrumental in correlating centromere structure with repetitive sequences. DAPI, a fluorescent dye, allows visualization of the centromere chromatin domain on metaphase chromosomes. Fluorescent in situ hybridization (FISH) probes based on the 180 bp pAL1 repeat sequence were co-localized with DAPI indicia near the centromere of all five Arabidopsis chromosomes (Maluzzynska et al., 1991; Martinez-Zapator et al., 1986). Although a functional role for pAL1 has been proposed, more recent studies have failed to detect this sequence near the centromere in species closely related to Arabidopsis thaliana (Maluszynska et al., 1991). These results are particularly troublesome. Because one of the tested species, A. pumila is described in Thaliana is considered to be a diploid from a cross between another related species (Maluszynska et al., 1991; Price et al., 1995). Another repeat pAtT12 has been genetically mapped within 5 cM of the centromere on chromosome 1 and to the central region of chromosome 5 (Richards et al., 1991), but its presence on other chromosomes has not been established. . Like pAL1, the role of pAtT12 in centromere function has not yet been demonstrated.
[0055]
Due to the fact that centromeres constitute the essential link between centromeric DNA and the spindle apparatus, proteins related to these structures have recently become the center of intense research. (Bloom, 1993; Earnshaw, 1991). Human autoantibodies that specifically bind in the vicinity of the centromere have been cloned from centromere-related proteins (CENP, Rattner, 1991) and at least one of these proteins belonging to the kinesin superfamily of microtubule-based motors (Yen, 1991). Made it easier. Yeast centromere binding proteins have also been identified through both genetic and biochemical studies (Bloom, 1993; Lechner et al., 1991).
[0056]
The Arabidopsis thaliana centromere was mapped using a trisomal strain. Here, segregation of chromosomal fragments (Koorneef, 1983) or whole chromosomes (Sears et al., 1970) was used to map the four centromeres within 5, 12, 17, and 38 cM, respectively (FIG. 2). These locations have not been refined by more recent studies. This is because this method is limited by the difficulty of obtaining viable trisomic strains (Koorneef, 1983). These factors introduce a significant error in the calculated centromere position, and in Arabidopsis, where 1 cM corresponds to approximately 200 kB (Koorneef, 1987; Hwang et al., 1991), this method provides a practical chromosome walking strategy. We did not map any centromeres with sufficient accuracy to do so. The mapping of the Arabidopsis genome was also discussed by (Hauge et al., 1991).
[0057]
(I. tetrad analysis)
Using tetrad analysis, the frequency of recombination between genetic markers and centromeres can be measured directly (FIG. 1). Since this method requires the analysis of all four products of individual meiosis, and the meiotic products of multicellular eukaryotes typically dissociate, this method has previously been used for multicellular eukaryotes. Had not been applied. The identification of the quartet mutation marker enables tetrad analysis for the first time in higher eukaryotic systems (Preuss et al., 1994). This quartet (qrt1) mutation leaves four products of meiosis of pollen mother cells attached in Arabidopsis. When used to pollinate flowers, one tetrad can result in the formation of four seeds, and the plants derived from these seeds can be genetically analyzed.
[0058]
With disordered tetrads (eg, tetrads produced by S. cerevisiae or Arabidopsis), genetic mapping using quadruple analysis requires that two markers be scored simultaneously (Whitehouse, 1950). ). The tetrads are classified into different classes depending on whether the marker is a parallel (non-recombinant) or non-parallel (recombinant) sequence (FIG. 1). A tetrad having only non-recombinant members is called parental type (PD); a tetrad having only recombinant members is called non-parental type (NPD); and two recombinant members and two Four molecules with non-recombinant members are called tetra-type (TT) (Perkins, 1953). If the two loci are located on different chromosomes and are therefore categorized independently, the frequency of tetra (cross-product) versus parental or non-parental combination (non-cross-product) is the two loci And its respective centromere.
[0059]
Tetratype tetrads only occur when a crossover occurs between the marker and its centromere. Therefore, to identify genes that are closely linked to the centromere, the markers are tested in a pairwise fashion until the TT frequency approaches zero. The genetic distance between a marker and its individual centromere (centimorgan, cM) is defined by a function of [(1/2) TT] / 100 (Mortimer et al., 1981). As the position information obtained by tetrad analysis represents the physical distance between two points, the opportunity for recombination events decreases as the centromere is approached.
[0060]
Four molecule analysis was used to genetically track centromeres in yeast and other fungi, where the products of a single meiosis could be collected. Saccharomyces cerevisiae, a budding yeast, lacks mitotic condensation, and therefore cytogenetics (Hegemann et al., 1993) was used as a vehicle for the discovery of centromere function, still due to tetrad analysis. Following meiosis, four spores are generated that are retained in the ascus and can be assayed directly for gene segregation.
[0061]
The recessive qrt1 mutation allows one to perform tetrad analysis in Arabidopsis by keeping the four products of meiosis attached (Preuss et al., 1994; and Smythe, 1994; Incorporated herein by reference). As previously shown, in each of the four molecules, the loci segregate in a 2: 2 ratio (FIG. 6). Individual tetrads can be manipulated in flowers using a fine brush (at a rate of 20 tetrads per hour), and 30% of such crosses yield 4 viable seeds (Preuss et al., 1994).
[0062]
Mapping centromeres with high precision requires high density genetic maps, and current Arabidopsis maps contain many viable markers, but crossing each to a qrt1 background is a daunting task It is. Alternatively, hundreds of DNA polymorphisms can be introduced simultaneously by crossing two different strains, both containing the qrt1 mutation. High-density RFLP maps (Chang et al., 1988) and PCR-based maps (Konieczny et al., 1993; Bell et al., 1994) have been generated in Arabidopsis from crosses of Landsberg and Colimbia strains (Arabidopsis maps and genetic markers). Data is available from the Internet at http://genome-www.stanford.edu/Arabidopsis and http://cbil.humgen.uppen.edu/atgc/sslp_info/sslp.html). These strains differ by as much as 1% at the DNA sequence level and have a colinear genetic map (Chang et al., 1988; Koorneef, 1987).
[0063]
Centromere mapping in tetrad analysis requires the simultaneous analysis of two markers, one of which must be linked to the centromere (FIG. 1). To identify these centromere-linked markers, markers distributed over all five chromosomes were scored and compared in a paired fashion.
[0064]
First, genetic markers that could be scored by PCR analysis were tested (Konieczny et al., 1993; Bell et al., 1994). Such markers are currently dense enough to map any locus. This is because further PCR identifies detectable polymorphisms and incorporates them into the analysis. In addition, as described in FIG. 5, new CAPS and SSLP markers useful for mapping centromeres can be easily identified.
[0065]
A collection of Arabidopsis tetrad sets was prepared by the inventors for use in tetrad analysis. To date, progeny plants from more than 1,000 isolated tetrad seeds have been germinated, and leaf tissue has been collected and preserved from each tetrad progeny plant. DNA from leaf tissues from individual plants was used for PCR-based marker analysis. The plants were also selfed and the seeds they produced were collected. From each of these individual seed sets, seedlings could be germinated and their tissues used for the production of genomic DNA. Tissues pooled from multiple seedlings are useful for generating Southern genomic DNA blots for analysis of restriction enzyme DNA fragment length polymorphisms (RFLP). An exemplary list of beneficial individual seed stocks used for tetrad analysis is provided in FIG.
[0066]
(II. Mapping Strategy)
Previous DNA fingerprinting and hybridization analyzes of two bacterial artificial chromosome (BAC) libraries have led to the construction of a physical map covering almost every single copy portion of the Arabidopsis genome (Marra et al., 1999). ). However, the presence of repetitive DNA (including 180 bp repeats, retroelements, and medium frequency repeats) near the Arabidopsis centromere complicated attempts to anchor the centromere BAC contig to specific chromosomes (Murata et al., 1997). Heslop-Harrison et al., 1999; Brandes et al., 1997; Franz et al., 1998; Wright et al., 1996; Konieczynyet et al., 1991; Pelissier et al., 1995; Voytas and Ausubel, 1988; Chye et al., 1997; Et al., 1991; Simoens et al., 1988; Thompson et al., 1996; Pelissier et al., 1996).
[0067]
We used genetic mapping to clearly assign these non-sticky contigs to specific centromeres and scored polymorphic markers in 48 plants with informative crossings for the entire genome. (Copenhaver et al., 1998). In this manner, several centromere contigs were associated with a physical map of the chromosome arm (see Example 6) and generated many sets of DNA markers defining centromere boundaries. DNA sequence analysis confirmed the structure of the chromosome II and IV contigs (Lin et al., 1999).
[0068]
CEN2 and CEN4 were specifically selected for analysis. Both have a 3.5 Mb rDNA array at their distal end, a structural 3 and 2 Mb region between the rDNA and centromere, respectively, and a 16 and 13 Mb region on its long arm. (Copenhaver and Pikaard, 1996).
[0069]
Centromere analysis was performed at the nucleotide level using the substantially complete and annotated sequences of chromosomes II and IV (http: //www.ncbi.nlm.nih. gov / Entrez / nucleotide.html). Sequence composition was analyzed at genetically defined centromere boundaries and compared to adjacent pericentric regions (FIGS. 12A-T). Analysis of the two centromeres facilitated comparison of sequence patterns and identification of conserved sequence elements.
[0070]
The centromere sequence was found to carry a 180 bp repeat. These sequences were found to be present in gaps in each centromere contig (FIGS. 3, 12B, 12L) with few repeats elsewhere in the genome and no long sequences at all. Was done. BAC clones near these gaps have terminal sequences corresponding to repetitive elements that probably contribute to the bulk of DNA between contigs, including 180 bp repeats, 5SΔrDNA or 160 bp repeats (FIG. 3). Fluorescence in situ hybridization showed that these repeats were abundant components of Arabidopsis centromeres (Murata et al., 1997; Heslop-Harrison et al., 1999; Brandes et al., 1997). Genetic mapping and pulsed-field gel electrophoresis have shown that there are many 180 bp repeats in long sequences between 0.4 and 1.4 Mb measured in the centromere region (Round et al., 1997); Analysis revealed additional copies scattered around the gap. We specifically contemplate the use of such 180 bp repeats for the construction of minichromosomes. The annotated sequences on chromosomes II and IV identified regions with homology to moderately repeated DNA in both the functional centromere and its flanking regions (Figures 12B-12E and 12L-12O).
[0071]
In the 4.3 Mb sequenced region containing CEN2 and the 2.8 Mb sequenced region containing CEN4, retrotransposon homology was greater than 10% (up to 62% and 70%, respectively) of the DNA sequence. It was found to explain (FIGS. 12C, 12M). Sequences with similarity to the transposon or medium repeat element were found to occupy a similar zone, but were less common (29% and 11% for chromosomes II and IV, respectively). % Maximum density) (FIGS. 12D-12E and 12N-12O). Finally, unlike the Drosophila and Neurospora centromeres (Sun et al., 1997; Cambareni et al., 1998), low complexity DNA (microsatellites, homopolymeric tracts and AT-enriched isochores). Was not found to be enriched in Arabidopsis centromeres. Around CEN2, the simple repeat sequence density is comparable to the density on the distal chromosome arm, accounting for 1.5% of the sequence in the centromere, 3.2% in the flanking region, and range from 20-319 bp in length. (Average: 71 bp). Except for the insertion of mitochondrial DNA at CEN2, the DNA around the centromere did not contain any large regions prominently derived from the A + T genome on average about 64% (FIGS. 12F, 12P) (Bevan et al., 1999).
[0072]
Unlike the 180 bp repeat, all other repetitive elements near CEN2 and CEN4 were less abundant within genetically defined centromeres than in adjacent regions. The high density of repetitive elements outside the functional centromere domain suggests that they may be insufficient for centromere activity. Thus, identifying segments of the Arabidopsis genome that are enriched in these repeats does not precisely map the region that provides centromere function; similar situations can occur in other higher eukaryotic genomes.
[0073]
The repetitive DNA adjacent to the centromere can play an important role, forming an altered chromatin conformation that acts to produce a nucleus or to stabilize the centromere structure. Alternatively, other mechanisms may cause accumulation of repetitive elements near the centromere. Although evolutionary models have predicted accumulation of repetitive DNA in regions of low recombination (Charlesworth et al., 1986; Carlsworth et al., 1994), many Arabidopsis repetitive elements are more recombinant than in the centromere itself. It is more abundant in the area around the active centromere. Alternatively, retroelements and other transposons can be inserted preferentially into regions adjacent to the centromere or, at a higher rate, eliminated from genomic pauses.
[0074]
(III. Centromere composition)
Certain aspects of the present invention relate to isolated nucleic acid segments and recombinant vectors, including plant centromeres. In one embodiment of the invention, the plant centromere is the Arabidopsis thaliana centromere. In a further embodiment of the present invention, A nucleic acid sequence comprising a thaliana chromosome 2 centromere is provided. The sequence of the chromosome 2 centromere of Arabidopsis thaliana is exemplified by the nucleic acid sequences of SEQ ID NO: 209 and SEQ ID NO: 210. As shown in FIG. 17, the nucleic acid sequences of SEQ ID NO: 209 and SEQ ID NO: 210 flanking a series of 180 bp repeats on chromosome 2 of T. thaliana. Thus, a chromosome 2 centromere may be further defined as comprising n number of repeats linked to a nucleic acid sequence contained in SEQ ID NO: 209 or SEQ ID NO: 210, or a sequence isolated from both of these sequences. . In certain embodiments of the invention, the number of iterations (n) is about 2, 4, 8, 15, 25, 40, 70, 100, 200, 400, 600, 800, 1,000, 1,500, 2, 000, 4,000, 6,000, 8,000, 10,000, 30,000, 50,000, or about 100,000. The actual repetitive sequence used may vary. Representative samples of repetitive sequences that can be used are provided in FIGS. 23A-23D, and are included in the nucleic acid sequences provided in SEQ ID NOs: 184-208. The length of the repeats used can also vary, and for example, repeats of about 10 bp, 20 bp, 40 bp, 60 bp, 80 bp, 100 bp, 120 bp, 140 bp, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, or about 200 bp, or it Longer repeats (eg, as listed in FIGS. 23A-23D) may be included and are included in the nucleic acid sequences provided in SEQ ID NOs: 184-208.
[0075]
Isolated segments of the nucleic acid sequences of SEQ ID NO: 209 and SEQ ID NO: 210 are also contemplated for use in the present invention, which may or may not be linked in a series of repeats. In particular, about 100, 200, 400, 800, 1,500, 3,000, 5,000, 7,500, 10,000, 15,000, 25,000, of the nucleic acid sequence of SEQ ID NO: 209 or SEQ ID NO: 210. 40,000, 75,000, 100,000, 125,000, 150,000, 250,000, 350,000, 450,000, 600,000, 700,00, and about 800,000 bp contiguous nucleic acid segments Especially form part of the invention. In certain embodiments of the invention, such a nucleic acid sequence may be linked to n number of repetitive sequences, where n is, for example, 2, 4, 8, 15, 25, 40, 70, 100, 200 , 400, 600, 800, 1,000, 1,500, 2,000, 4,000, 6,000, 8,000, 10,000, 50,000 or about 100,000. The repeats are, for example, about 10 bp, 20 bp, 40 bp, 60 bp, 80 bp of the contiguous nucleotides of the repeats listed in FIGS. 23A-23D and the repeats contained in the nucleic acid sequences provided by SEQ ID NOs: 184-208. , 100 bp, 120 bp, 140 bp, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, or about 200 bp, or longer segments.
[0076]
In another embodiment of the present invention, A nucleic acid sequence comprising a thaliana chromosome 4 centromere is provided. The sequence of Arabidopsis thaliana chromosome 4 centromere is exemplified by the nucleic acid sequences of SEQ ID NO: 211 and SEQ ID NO: 212. As shown in FIG. 18, the nucleic acid sequences of Arabidopsis SEQ ID NO: 211 and SEQ ID NO: 212 are flanked by a series of repetitive sequences. Thus, the chromosome 4 centromere further comprises n number of repeats linked to a nucleic acid sequence contained in SEQ ID NO: 211 or SEQ ID NO: 212, or a sequence derived from both SEQ ID NO: 211 and SEQ ID NO: 212. Can be defined as In certain embodiments of the invention, the number of iterations (n) is approximately 2, 4, 8, 15, 25, 40, 70, 100, 200, 400, 600, 800, 1,000, 1,500, 2,000, 4,000, 6,000, 8,000, 10,000, 50,000 or about 100,000. The actual repeat sequences used may vary. Representative samples of repetitive sequences that can be used are shown in FIGS. 23A-23D, where these sequences are included in the nucleic acid sequences shown in SEQ ID NOs: 184-208. The length of the repeats used can also vary and include, for example, repeats of about 10 bp, 20 bp, 40 bp, 60 bp, 80 bp, 100 bp, 120 bp, 140 bp, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, or about 200 bp or more. obtain.
[0077]
Isolated segments of the nucleic acid sequences of SEQ ID NO: 211 and SEQ ID NO: 212 are also contemplated for use in the present invention, either linked or unlinked to a series of repetitive sequences. You. Specifically, about 100, 200, 400, 800, 1,500, 3,000, 5,000, 7,500, 10,000, 15,000, 25, of the nucleic acid sequence of SEQ ID NO: 211 or SEQ ID NO: 212. 000, 40,000, 75,000, 100,000, 125,000, 150,000, 250,000, 350,000, 450,000, 600,000, 700,000 bp contiguous nucleic acid segments of the invention Specifically form a part of In certain embodiments of the invention, such a nucleic acid sequence comprises n number of repetitive sequences (eg, where n is 2, 4, 8, 15, 25, 40, 70, 100, 200, 400, 600, 800, 1,000, 1,500, 2,000, 4,000, 6,000, 8,000, 10,000, 50,000 or about 100,000). The repetitive sequence is, for example, about 10 bp, 20 bp, 40 bp, 60 bp, 80 bp, 100 bp, 120 bp, 140 bp, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, or about 200 bp or more of the contiguous nucleotides of the sequence of SEQ ID NOs: 184 to 208. Of segments.
[0078]
In addition, a regulatory region (including its promoter and terminator sequence) derived from the Arabidopsis polyubiquitin 11 gene is provided by the present invention. The nucleic acid sequences of these regulatory regions are exemplified by the nucleic acid sequences of SEQ ID NO: 180 and SEQ ID NO: 181. In addition, about 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 300, 500, 750, 1,000, 1, Consecutive stretches from 500 and about 2,000 nucleotides are included in such sequences. In certain embodiments of the invention, it may be desirable to operably link the Arabidopsis polyubiquitin 11 promoter sequence to the 5 'end of the coding sequence. It may also be desirable to operably link the Arabidopsis polyubiquitin 11 terminator sequence to the 3 'end of the coding sequence.
[0079]
Still further, a regulatory region (including its promoter and terminator) from the Arabidopsis 40S ribosomal protein S16 gene is provided by the present invention. The nucleic acid sequences of these regulatory regions are exemplified by the nucleic acid sequences of SEQ ID NO: 182 and SEQ ID NO: 183. About 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 300, 500, 750, 1,000, 1,500 of the nucleic acid sequence of SEQ ID NO: 182 and SEQ ID NO: 183 Contiguous stretches from about 2,000 nucleotides are also included in such sequences. In certain embodiments of the invention, it may be desirable to operably link the Arabidopsis 40S ribosomal protein S16 gene sequence to the 5 'end of the coding sequence. It may also be desirable to operably link the Arabidopsis 40S ribosomal protein S16 gene sequence to the 3 'end of the coding sequence.
[0080]
Still further, gene sequences and associated regulatory sequences, and other functional sequences from the centromere region are provided by the invention. Specifically, the present invention relates to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, and about 5 of these sequences , 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 250, 300, 350, 400, 500, 550, 590, 1 It includes a centromere sequence represented by 2,000 and about 1,500 contiguous nucleotides (up to and including the full length of the sequence).
[0081]
Centromere-containing nucleic acid sequences can be provided along with other sequences for the production and use of recombinant minichromosomes. Such nucleic acid sequences specifically within the scope of the present invention include those listed in the Sequence Listing provided herein.
[0082]
The present invention provides a method for producing a recombinant molecule that is enriched for total genomic DNA or other nucleic acids and that may confer centromere activity on recombinant molecules when taken up into host cells. thaliana cells. As used herein, "nucleic acid segment" refers to a nucleic acid molecule that has been purified from total genomic nucleic acid of a particular species. Thus, a nucleic acid segment that confers a centromere function includes a centromere sequence and still contains A. thaliana refers to a nucleic acid segment that has been isolated from the total genomic nucleic acid or has been purified to be free of it. Nucleic acid segments and smaller fragments of such segments, as well as recombinant vectors (including, for example, BACs, YACs, plasmids, cosmids, phages, viruses, etc.) are included within the term "nucleic acid segment."
[0083]
Similarly, a nucleic acid segment comprising an isolated or purified centromeric sequence is a nucleic acid segment comprising a centromeric sequence, and in certain aspects, a regulatory sequence substantially isolated from other naturally occurring sequences, That is, it refers to another nucleic acid sequence. In this aspect, the term "gene" is used for simplicity to refer to a unit that encodes a functional nucleic acid segment, protein, polypeptide or peptide. As will be appreciated by those skilled in the art, this functional term refers to both genomic sequences, cDNA sequences, and smaller, engineered, capable of expressing or being adapted to express a protein, polypeptide or peptide. Gene segments.
[0084]
"Substantially isolated from other sources" means that the sequence of interest (in this case, the sequence of the centromere) is included in the genomic nucleic acid clones provided herein. Of course, this refers to a nucleic acid segment as originally isolated, and does not exclude genes or coding regions which were later added to the segment by human hand.
[0085]
In certain embodiments, the present invention relates to isolated nucleic acid segments and recombinant vectors that incorporate a nucleic acid sequence encoding a centromere functional sequence, including contiguous sequences from the centromere of the present invention. In certain other embodiments, the present invention provides a method comprising: The present invention relates to isolated nucleic acid segments and recombinant vectors that contain contiguous nucleic acid sequences from C. thaliana within those sequences. Also, nucleic acid segments that exhibit centromere functional activity are most preferred.
[0086]
The nucleic acid segments of the present invention can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., regardless of the length of the sequences themselves. As a result, their overall length can vary considerably. Thus, it is contemplated that nucleic acid fragments of almost any length may be used, where the total length is preferably limited by ease of preparation and use in the intended recombinant DNA protocol.
[0087]
((I) primer and probe)
In addition to their use in the construction of recombinant constructs (including minichromosomes), the nucleic acid sequences disclosed herein may find various other uses. For example, the centromere sequences disclosed herein may find use as probes or primers in nucleic acid hybridization embodiments. Thus, a contiguous sequence of at least 14 nucleotides in length having the same sequence as the centromere sequences of the invention (e.g., the sequences set forth in SEQ ID NOs: 1-212, and especially SEQ ID NOs: 1-21 and 180-212). Or nucleic acid segments that include a sequence region that is complementary to a 14 nucleotide long DNA segment of their centromere sequence find particular utility. Longer contiguous identical or complementary sequences, including all intermediate lengths of the sequences set forth in SEQ ID NOs: 1-212, and up to and including the full length sequence of those sequences (eg, about 20, 30 , 40, 50, 100, 200, 500, 1,000, 2,000, 5,000 bp, etc.) are also useful in certain embodiments.
[0088]
As described in detail herein, the ability of such nucleic acid probes to specifically hybridize to centromere sequences is a function of the presence of similar partially complementary sequences from other plants or animals. This allows the use of this probe in detection. However, other uses are contemplated, including the use of variant primers, or centromeres for the preparation of primers for use in preparing other genetic constructs.
[0089]
8, 9, 10, 11, 12, 13, 14, 15, which are identical to or complementary to the centromere sequences of the present invention (including the sequences set forth in SEQ ID NOs: 1-212). 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or even 101 to 200 nucleotides contiguous Nucleic acid fragments having sequence regions consisting of stretches of nucleotides are particularly useful as hybridization probes, for example, for use in Southern and Northern blotting. Is Fig. Smaller fragments generally find use in hybridization embodiments, where the length of the contiguous complementary region can vary (eg, between about 10-14 and about 100 or 200 nucleotides). , Larger continuous complementary stretches can also be used, depending on the length of the complementary sequence desired to be detected.
[0090]
Of course, fragments can also be obtained by other techniques, such as by mechanical shearing or by digestion with restriction enzymes. Small nucleic acid segments or fragments can be readily prepared, for example, by directly synthesizing the fragment by chemical means, as commonly performed using an automated oligonucleotide synthesizer. In addition, fragments can be obtained by applying a nucleic acid regeneration technique (for example, PCR described in U.S. Pat. Nos. 4,683,195 and 4,683,202).^TMTechniques, each of which is incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, as well as others commonly known to those skilled in molecular biology. Can be obtained by recombinant DNA technology.
[0091]
Thus, the centromere sequences of the present invention can be used for complementary stretches of DNA fragments and their ability to selectively form duplex molecules. Depending on the intended application, it may be desirable to use different conditions of hybridization to achieve different degrees of selectivity of the probe for the target sequence. For applications requiring high selectivity, it is typically desirable to use relatively stringent conditions to form hybrids. For example, select relatively low salt and / or high temperature conditions (eg, to provide 0.02M to about 0.15M @NaCl at a temperature of about 50 ° C to about 70 ° C). Such selection conditions allow little, if any, mismatch between the probe and the template or target strand, and are particularly suitable for isolating centromere DNA segments. Nucleic acid sequences that hybridize to the nucleic acid sequences provided by the invention under these conditions and under the following conditions, including the sequences represented by SEQ ID NOs: 1-212, form part of the invention. Detection of nucleic acid segments by hybridization is well known to those of skill in the art, and is described in US Pat. Nos. 4,965,188 and 5,176,995, which are hereby incorporated by reference in their entirety. The teachings of the present invention are exemplary of the method of hybridization analysis. The teachings are particularly relevant, for example, those found in the texts of Maloy et al., 1991; Segal, 1976; Prokop, 1991; and Kuby, 1994.
[0092]
Of course, for some applications, for example, if it is desired to prepare a mutant using a mutant primer strand that hybridizes to the underlying template, or to a centromere function-conferring sequence from a related species, a functional equivalent, etc. When trying to isolate, lower stringent hybridization conditions are typically required to allow heteroduplex formation. In these situations, it may be desirable to use conditions (eg, about 0.15M to about 0.9M salt) at a temperature in the range of about 20C to about 55C. Thus, cross-hybridizing species are easily identified as hybridizing signals that are positive for control hybridization. It is generally understood that in any case, the conditions can be made more stringent by the addition of increasing amounts of formamide, which acts to destabilize the hybrid duplex in the same manner as increasing the temperature or decreasing the salt. You. Thus, hybridization conditions can be readily manipulated, thereby generally selecting a method depending on the desired result.
[0093]
In certain embodiments, it is advantageous to use the nucleic acid sequences of the invention in combination with appropriate means (eg, labels) to determine hybridization. A wide variety of suitable indicator means, including fluorescent, radioactive, enzymatic or other ligands (eg, avidin / biotin), capable of producing a detectable signal are known in the art. In a preferred embodiment, it appears to be desirable to use a fluorescent label or an enzyme tag (eg, urease, alkaline phosphatase or peroxidase) instead of radioactive or other environmentally undesirable reagents. In the case of an enzyme tag, a colorimetric indicator substrate that can be seen by the human eye or used to provide a spectrophotometric means to identify specific hybridization with a sample containing complementary nucleic acids Is known.
[0094]
In general, the hybridization probes described herein are intended to be useful both as reagents in hybridizations in solution, as well as in embodiments using solid phases. In embodiments involving a solid phase, test DNA (or RNA) is adsorbed or otherwise added to a selected matrix or surface. This immobilized single-stranded nucleic acid is then subjected to specific hybridization with the selected probe under desired conditions. The conditions selected will depend on the particular environment required based on the particular criteria required (eg, depending on G + C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). After washing the hybridized surface to remove non-specifically bound probe molecules, specific hybridization is detected or even quantified by the label.
[0095]
((Ii) Large nucleic acid segment)
Using markers adjacent to each centromere (see FIG. 3), it may be possible to purify contiguous DNA fragments containing both adjacent markers and the centromeres encoded between these markers. . To do this, very large DNA fragments up to the size of the entire chromosome are prepared by embedding Arabidopsis tissue in agarose using, for example, the method described in Copenhaver et al. (1995). . These large pieces of DNA can be digested in agarose with any restriction enzymes. Restriction enzymes that are particularly useful for isolating intact centromeres include those that produce very large DNA fragments. Such restriction enzymes include enzymes having specificity greater than 6 base pairs (eg, AscI, BaeI, BbvCI, FseI, NotI, PacI, PmeI, PpuMI, RsrII, SanDI, SapI, SexAI, SfiI, SgfI, SgrAI, Sbfl, SrfI, Sse8387I, Sse8647I, Swa, UbaDI, and UbaEI), or any other enzyme that cleaves less frequently in the Arabidopsis genome and is specific within the centromere region. Alternatively, partial digestion with restriction enzymes that cut at a higher frequency may be used.
[0096]
Alternatively, large DNA fragments spanning some or all of the centromere can be produced using RecA-assisted restriction endonuclease (RARE) cleavage (Ferrin, 1991). To do this, very large DNA fragments up to the size of the entire chromosome are prepared, for example, by embedding Arabidopsis tissue in an agarose gel using the method described by Copenhaver et al. (1995). Is done. A single-stranded DNA oligomer having a sequence homologous to the region adjacent to the region of the DNA to be purified is prepared using the recombinase enzyme RecA to form a triple-stranded complex with the DNA embedded in agarose. Is done. The DNA is then treated with a site-specific methylase such as, for example, AluI methylase, BamHI methylase, dam methylase, EcoRI methylase, HaeIII methylase, HhaI methylase, HpaII methylase, or Msp methylase. The methylase modifies all sites specified by its recognition sequence except those in the triplex region protected by the RecA / DNA oligomer complex. The RecA / DNA oligomer complex is then removed from the DNA embedded in agarose, and the DNA is then removed from the restriction enzyme corresponding to the methylase used (eg, EcoRI restriction enzyme if an EcoRI methylase is used). Endonuclease is used to effect the cleavage). Only sites protected from modification are subject to cleavage by restriction endonucleases. Thus, by selecting a target adjacent to the centromere region that contains the recognition sequence for the site-specific methylase / restriction endonuclease pair, RARE can be used to cut the entire region from the rest of the chromosome. It is important to note that this method is used to isolate DNA fragments of unknown composition by using sequence information adjacent to it. Thus, this method is used to isolate DNA contained within any gap in the physical map of the chromosome. The DNA isolated by this method can then be sequenced.
[0097]
The large DNA fragments generated by digestion with restriction enzymes or by RARE cleavage are then separated by size using pulsed-field gel electrophoresis (PFGE) (Schwartz et al., 1982). Specifically, Contour-clamped Homogeneous Electric Field (CHEF) electrophoresis (various PFGE) can be used to separate DNA molecules as large as 10 Mb (Chu et al., 1985). The large DNA fragments that are separated on a CHEF gel can then be analyzed using standard Southern hybridization techniques, identifying and measuring the size of both centromere flanking markers and thus the fragment containing the centromere. After determining the size of the centromere-containing fragment by comparison with a standard of known size, the region from the gel containing the centromere fragment can be cut from the duplicate gel. The centromere DNA is then analyzed, sequenced, and used in various applications (including mini-chromosome construction) as described below. As shown in detail below, mini-chromosomes attach telomerase and selectable markers to centromere fragments excised from agarose gels using standard techniques that allow DNA ligation in gel slices. Can be constructed by Plant cells can then be transformed with the hybrid DNA molecule using the techniques described herein below.
[0098]
(IV. Recombinant Constructs Containing Centromere Sequence)
In view of the disclosure of the present invention, it is possible for one skilled in the art to construct the recombinant DNA constructs described herein. Useful construction methods are well known to those skilled in the art (see, eg, Maniatis et al., 1982). As constructed, the minichromosome preferably comprises an autonomously replicating sequence (ARS) functional in a plant, a centromere functional in a plant, and a telomere functional in a plant.
[0099]
The basic elements in addition to plant centromeres that can be used in the construction of minichromosome vectors are known to those skilled in the art. For example, one type of telomere sequence that can be used is the Arabidopsis telomere, which consists of an array of head-to-tail tails of monomeric repeat CCCTAAA totaling several kb (eg, 3-4 kb) in length. is there. Like telomeres in other organisms, Arabidopsis telomeres vary in length and do not appear to have strict length requirements. An example of a cloned telomere can be found in GenBank Accession No. M20158 (Richards and Ausubel, 1998). Yeast telomere sequences have also been described (eg, Louis, 1994; Genbank accession number S70807). In addition, methods for isolating higher eukaryotic telomeres from Arabidopsis thaliana have been described by Richards and Ausubel (1988).
[0100]
Higher eukaryotes do not possess certain sequences used as origins of replication, but are instead generally thought to replicate their DNA from random sites distributed along the chromosome. In Arabidopsis, cells are thought to form an origin of replication approximately once every 70 kb (Van't @ Hof, 1978). Thus, higher eukaryotes have replication origins at potentially random locations on each chromosome, making it impossible to describe a specific origin sequence, but having a sufficient size of plant DNA. Is generally recognized by the cell, and the origin is generally assumed to be created on the construct. For example, any piece of Arabidopsis genomic DNA larger than 70 kb is predicted to contain ARS. By including such a segment of DNA on a recombinant vector, ARS function can be provided in the vector. In addition, many S.A. cerevisiae autonomously replicating sequences have been sequenced and can be used to fulfill ARS function. One example is the Saccharomyces @ cerevisiae autonomously replicating sequence ARS131A (GenBank number L25319). Many origins of replication have also been sequenced and E. coli. It has been cloned from E. coli and can be used in the present invention (eg, the Col @ E1 origin of replication (Ohmori and Tomizawa, 1979; GenBank number V00270)). One Agrobacterium origin that can be used is RiA4. The localization of the replication origin in the plasmid of Agrobacterium rhizogenes strain A4 was described by Jouanin et al. (1985).
[0101]
((I) Considerations in the preparation of recombinant constructs)
In addition to the basic elements, positive or negative selection plant markers (eg, antibiotic or herbicide resistance genes), and cloning sites for insertion of foreign DNA can be included. Further, a visible marker, such as a green fluorescent protein, may also be desired. E. FIG. In order to propagate the vector in E. coli, it is necessary to convert the linear molecule into a circle by adding a stuffer fragment between the telomeres. E. FIG. It is also preferred to include an E. coli plasmid origin of replication and a selectable marker. It may also be desirable to include Agrobacterium sequences to improve replication and transfer into plant cells. Although we have described a number of exemplary minichromosome constructs in FIGS. 7A-7H, many changes can be made in the order and type of elements present in these constructs, and within the scope of the invention It will be apparent to those skilled in the art that still obtain a functional minichromosome.
[0102]
Artificial plant chromosomes replicating in yeast can also be constructed taking advantage of the large insertion capacity and stability of repetitive DNA inserts provided by this system (see Burke et al., 1987). In this case, the yeast ARS and CEN sequences can be added to the vector. Artificial chromosomes are maintained in yeast as circular molecules using stuffer fragments that separate telomeres.
[0103]
DNA fragments from any any source can be purified and inserted into the minichromosome at any suitable restriction endonuclease cleavage site. The DNA segment usually contains various regulatory signals for expression of the protein encoded by the fragment. Alternatively, regulatory signals present in the minichromosome can be utilized.
[0104]
The techniques and procedures required to effect insertion are well known in the art (see Maniatis et al., 1982). Typically, this is achieved by incubating the circular plasmid or linear DNA fragment in the presence of a restriction endonuclease, such that the restriction endonuclease cleaves the DNA molecule. Endonucleases preferentially break internal phosphodiester bonds in polynucleotide chains. They can cleave polynucleotide bonds relatively nonspecifically, regardless of the surrounding nucleotide sequence. However, an endonuclease that cleaves only a specific nucleotide sequence is called a restriction enzyme. Restriction endonucleases generally internally cleave DNA molecules at specific recognition sites, thereby often (but not all) exhibiting a two-fold symmetry around a given point. Breakage occurs in the "recognition" sequence.
[0105]
Many of these enzymes produce a staggered cut, resulting in a DNA fragment with a protruding single-stranded 5 'or 3' end. Such edges are referred to as "sticky" or "cohesive." Because they hydrogen bond to the complementary 3 'or 5' end. As a result, the end of any DNA fragment generated by an enzyme (eg, EcoRI) may anneal to any other fragment generated by the enzyme. This suitably allows, for example, splicing of foreign genes into plasmids. Some restriction endonucleases that may be particularly useful in the present invention include HindIII, PstI, EcoRI, and BamHI.
[0106]
Some endonucleases create fragments with blunt ends, ie, lacking any protruding single strands. Another way to create blunt ends is to use a restriction enzyme that leaves an overhang but fills in this overhang with a polymerase (eg, Klenow), thereby producing a blunt end. If the DNA was cut with restriction enzymes that cut across both strands at the same location, blunt-end ligation can be used to ligate the fragments directly together. The advantage of this technique is that any pair of ends can be linked together regardless of the sequence.
[0107]
Nucleases that preferentially destroy terminal nucleotides are called exonucleases. For example, a small deletion starts at each 3 'end of the DNA and removes the single strand in the 3' to 5 'direction, leaving a population of DNA molecules with single-stranded fragments at each end (some , Including terminal nucleotides), can be produced in any DNA molecule by treatment with an exonuclease. Similarly, exonucleases that digest DNA from the 5 'end, or enzymes that remove nucleotides from both strands, may often be used. Some exonucleases that may be particularly useful for the present invention include Bal31, SI, and ExoIII. These nucleolytic reactions can be controlled by varying the incubation time, temperature, and enzyme concentration required to create the deletion. Phosphatases and kinases can also be used to control which fragments have ends that can be ligated. Examples of useful phosphatases include shrimp alkaline phosphatase, and calf intestinal alkaline phosphatase. An example of a useful kinase is T4 polynucleotide kinase.
Once the source DNA and vector sequences have been cleaved and modified to yield the appropriate termini, they are incubated with an enzyme that can mediate the ligation of the two DNA molecules. Particularly useful enzymes for this purpose include T4 ligase, E. coli. coli ligase, or other similar enzymes. The action of these enzymes results in the sealing of linear DNA, producing larger DNA molecules containing the desired fragment (eg, US Pat. Nos. 4,237,224; 4,264,731; Nos. 4,273,875; 4,322,499 and 4,336,336, which are specifically incorporated herein by reference).
[0108]
It is understood that the ends of the linear plasmid and the end of the inserted DNA fragment must be complementary or blunt ended for a successful ligation reaction. Appropriate complementation can be achieved by selecting the appropriate restriction endonuclease (ie, the same overhang as the fragment is produced by the same restriction endonuclease or used to linearize a plasmid ( When produced by a restriction endonuclease that produces an overhang, the ends of both molecules are complementary). As previously discussed, in one embodiment of the present invention, at least two classes of vectors used in the present invention are adapted to accept foreign oligonucleotide fragments in only one orientation. The resulting hybrid DNA can then be selected from a large population of clones or libraries, after joining the DNA segments to the vector.
[0109]
Useful methods for molecular cloning of DNA sequences include in vitro ligation of DNA segments that have been fragmented from sources of high molecular weight genomic DNA into independently replicable vector DNA molecules. Cloning vectors include plasmid DNA (see Cohen et al., 1973), phage DNA (see Thomas et al., 1974), SV40 DNA (see Nussbaum et al., 1976), yeast DNA, E. coli. coli DNA, and most importantly, plant DNA.
[0110]
Various processes are known that may be useful for performing transformations (ie, inserting a heterologous DNA sequence into a host cell, which allows the host to efficiently express the inserted sequence).
[0111]
((Ii) regulatory element)
In one embodiment of the invention, the construct is a plant promoter, for example, the CaMV @ 35S promoter (Odell et al., 1985), or others such as CaMV @ 19S (Lawton et al., 1987), nos (Ebert et al., 1987). , Adh (Walker et al., 1987), sucrose synthase (Yang and Russell, 1990), a-tubulin, actin (Wang et al., 1992), cab (Sullivan et al., 1989), PEPCase (Hudspeth and Grula, 1989) or And those associated with the R gene complex (Chandler et al., 1989). Tissue-specific promoters such as the root cell promoter (Conkling et al., 1990) and tissue-specific enhancers (Fromm et al., 1989) are also intended to be useful, such as ABA-inducible promoters and Trugol-inducible promoters. The same applies to various inducible promoters. In certain embodiments of the invention, the Lat52 promoter may be used (Twell et al., 1991). A particularly useful tissue-specific promoter is the SCARECROW (Scr) root-specific promoter (DiLaurenzio et al., 1996).
[0112]
DNA sequences between the transcription initiation site and the start of the coding sequence (ie, the untranslated leader sequence) can affect gene expression. Thus, it may be desirable to use a specific leader sequence.
[0113]
The functional gene may be a novel promoter or enhancer, or possibly homologous or tissue-specific (eg, root-specific, collar / sheath-specific, ring-organ-specific, stem-specific, earshank-specific, or Pod, kernel (kernel) -specific or leaf-specific) promoter elements or control elements can be introduced. In certain embodiments of the invention, the functional gene may be in an antisense orientation with respect to the promoter.
[0114]
((Ii) Terminator)
It may also be desirable to link the functional gene to a 3 'terminal DNA sequence that terminates transcription and acts as a signal that allows polyadenylation of the mRNA produced by the coding sequence. Such a terminator may be the native terminator of the functional gene or may be a heterologous 3 'end. Examples of terminators that can be used with the present invention are the terminator from the nopaline synthase gene of Agrobacterium tumefaciens (nos 3 'end) (Bevan et al., 1983), the terminator for T7 transcription and the terminator from the octopine synthase gene of Agrobacterium tumefaciens. Or the 3 'end of a protease inhibitor I or II derived from tomato.
[0115]
((Iii) marker gene)
It may be desirable to use one or more marker genes according to the present invention. Such markers may be adapted for use in prokaryotic, lower eukaryotic or higher eukaryotic systems, or may be possible for use in any combination of the above classes of organisms. By using a selectable marker protein or a screening marker protein, the ability to identify transformants can be provided or enhanced. A “marker gene” is a gene that confers another phenotype to a cell that expresses the marker protein, such that such transformed cells can be distinguished from cells without the marker. Such a gene indicates whether the marker confers a property that can be “selected” by chemical means (ie, the use of selective reagents (eg, herbicides, antibiotics, etc.)), or simply Depending on whether or not the property can be identified by a test (ie, a “screen” (eg, green fluorescent protein)), it can encode either a selectable marker or a screening marker. Of course, many examples of suitable marker proteins are known in the art and can be used in the practice of the present invention.
[0116]
Genes encoding “secretable markers” whose secretion can be detected as a means of identifying or selecting for transformed cells are also included in the term selectable or screening markers. Examples include markers that are secretable antigens that can be identified by antibody interaction or markers that are secretable enzymes that can be detected by catalytic activity. Secretable proteins fall into many classes, including, for example, small diffusible proteins that can be detected by ELISA; small active enzymes that can be detected in extracellular solutions (eg, α-amylase, β-lactamase). , Phosphinothricin acetyltransferase); and proteins inserted or captured in the cell wall (e.g., proteins containing a leader sequence as found in the expression unit of extensin or tobacco PR-S).
[0117]
With respect to the selectable secretable marker, the use of a gene that encodes a protein that is sequestered in the cell wall and that protein contains a unique epitope would be particularly advantageous. Such secreted antigen markers ideally utilize a promoter-leader sequence that confers an epitope sequence that provides low background in plant tissue, efficient expression and targeting across the plasma membrane, and enhances cell wall expression. It produces a protein that is bound and still accessible to the antibody. Normally secreted wall proteins that are modified to contain unique epitopes meet all such requirements.
[0118]
(1. Selectable marker)
A number of selectable marker genes can be used in accordance with the present invention, including neo (Potrykus et al., 1985) (which provides kanamycin resistance and can be selected to use kanamycin, G418, paromomycin, etc.); bar Mutated EPSP synthase protein (confers glyphosate resistance); nitrilase such as bxn from Klebsiella ozaenae conferring resistance to bromoxynil (Stalker et al., 1988) (confers bialaphos or phosphinothricin resistance); Mutant acetolactate synthase (ALS), which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (European Patent Application 154,204,1985) Methotrexate resistant DHFR (Thillet et al., 1988), (confers resistance to the herbicide dalapon) dalapon dehalogenase; or mutated anthranilate (give 5-methyl resistance to tryptophan) anthranilate synthase but are exemplified, but not limited to. When a mutant EPSP synthase is used, integration and modification of the appropriate chloroplast transit peptide, CTP (US Pat. No. 5,188,642) or OTP (US Pat. No. 5,633,448) Further advantages from the use of EPSPS (PCT application WO 97/04103) can be seen.
[0119]
An exemplary embodiment of a selectable marker that can be used in a system for selecting transformants encodes the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces ｒｉviridochromogenes. It is a selectable marker. The enzyme phosphinothricin acetyltransferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthase (Murakami et al., 1986; Twell et al., 1989), causing rapid ammonia accumulation and cell death. The use of bar as a selectable marker gene for the production of herbicide-tolerant rice from protoplasts has been described by Ratore et al., (1993).
[0120]
Many S.A. cerevisiae markers are also known and can be used with the present invention, including, for example, the HIS4 gene (Donahue et al., 1982; GenBank number J01331). E. coli that can be cloned, sequenced and used in accordance with the present invention An example of an E. coli marker gene is the Ap gene, which confers resistance to β-lactam antibiotics such as ampacillin (nucleotides 4618-5478 of GenBank Accession No. U66885).
[0121]
(2. Screening marker)
Screening markers that may be used include the β-glucuronidase (GUS) or uidA gene (various chromogenic substrates encode known enzymes); the R locus gene (a product that regulates the production of anthocyanin pigment (red) in plant tissue). (Delaporta et al., 1988); β-lactamase gene (Sutcliffe, 1978) (encoding an enzyme for which various chromogenic substrates are known (eg, PADAC, chromogenic cephalosporin)); xylE gene ( Zukowsky et al., 1983) (encoding a catechol dioxygenase capable of converting pigmented catechol); the α-amylase gene (Ikuta et al., 1990); the tyrosinase gene (Katz et al., 1983) (acid tyrosine to DOPA and dopaquinone). , Which in turn concentrates to form the easily detectable compound melanin); the β-galactosidase gene (encoding the enzyme for which a chromogenic substrate is present); the luciferase (lux) gene (Ow). Et al., 1986) (allows bioluminescence detection); the aequorin gene (Prasher et al., 1985) (which can be used for calcium-sensitive bioluminescence detection); or the gene encoding green fluorescent protein (Sheen et al., 1995; Haseloff et al., 1997; Reichel et al., 1996; Tian et al., 1997; WO 97/41228).
[0122]
Genes from the corn R gene complex can also be used as screenable markers. The corn R gene complex encodes a protein that acts to regulate the production of anthocyanin pigments in most seed and plant tissues. Maize strains can have as many as one or as many as four R alleles, which are tailored to regulate pigmentation in a developmental and tissue-specific manner. Thus, the R gene introduced into such cells causes the expression of a red pigment and, when stably integrated, can be visually scored as a red sector. If the corn strain carries a dominant allele for the gene encoding the enzyme intermediate (C2, A1, A2, Bzl and Bz2) in the anthocyanin biosynthetic pathway, but carries a recessive allele at the R locus, Transformation of any cells from that strain with E. coli results in red pigment formation. Exemplary strains include Wisconsin # 22 (including the rg-Stadler allele and TR112), a K55 derivative (which is rg, b, Pl). Alternatively, if the C1 and R alleles are introduced together, any maize genotype may be utilized.
[0123]
Another screening marker contemplated for use in the present invention is firefly luciferase, encoded by the lux gene. The presence of the lux gene in the transformed cells is detected using, for example, X-ray film, scintillation counting, fluorescence spectroscopy, low-light video camera, photon counting camera or multiwell luminometer. obtain. It is also envisioned that this system can be developed for populational screening for bioluminescence, such as tissue culture plates, or for whole plant screening. The gene encoding green fluorescent protein (GFP) is contemplated as a particular useful reporter gene (Shen et al., 1995; Haseloff et al., 1997; Reichel et al., 1996; Tian et al., 1997; WO97 / 41228). The expression of green fluorescent protein can be visualized in cells or plants as fluorescence following illumination with light of a particular wavelength.
[0124]
(3. Negative selectable marker)
Introduction of a gene encoding a property that can be selected can be useful for removing minichromosomes from the cell or for selecting cells that contain a particular minichromosome. An example of a negative selectable marker that has been investigated is the enzyme cytosine deaminase (Stougard, 1993). In the presence of this enzyme, the compound 5-fluorocytosine is converted to 5-fluorouracil, which is toxic to plant and animal cells. Thus, cells containing the minichromosome with this gene can be directly selected. Other genes encoding proteins that confer sensitivities on plants to particular compounds are also useful in this context. For example, the T-DNA gene 2 from Agrobacterium tumefaciens encodes a protein that catalyzes the conversion of α-naphthaleneacetamide (NAM) to α-naphthaleneacetic acid (NAA), rendering the plant sensitive to high concentrations of NAM ( Depicker et al., 1988).
[0125]
V. Isolation of Centromeres from Plants
The present inventors have provided, for the first time, a nucleic acid sequence of a plant centromere. This allows one skilled in the art to obtain centromere sequences from potentially any species. We provide herein below a number of methods that can be used to isolate such centromeres.
[0126]
((I) Use of conserved sequences)
A number of centromere sequences identified by the inventors have also been shown to be highly conserved by the inventors (see, eg, Example 5B, Tables 3 and 4). Thus, our novel discovery that many genes are present within Arabidopsis centromeres can be used to discover synthetic genes in other organisms (ie, evolutionary in species-to-species gene order). Relationship stored in). For example, the sequence of each Arabidopsis gene can be used for searching with sequence databases from other plants. An exemplary list of such sequences that can be used is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, Given by SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 Array. The genes listed in Tables 3 and 4 are also useful. By finding the same or similar genes, candidates that may be present at or near the centromere region are identified. Mapping of these genes using linked markers identifies potential centromere regions.
[0127]
Where hybridization is used to obtain centromere sequences, it may be desirable to use less stringent hybridization conditions to allow for the formation of heteroduplexes. In these situations, it may be desirable to use conditions such as about 0.15 M to about 0.9 M salt, a temperature ranging from about 20C to about 55C. This allows the cross-hybridizing species to be easily identified as a positive hybridization signal with respect to the control hybridization. In any case, it is common that conditions can be made more stringent by the addition of increased amounts of formamide, which helps to destabilize the hybrid duplex in the same manner as increased temperature or reduced salt. Will be understood. Thus, the hybridization conditions can be easily manipulated, and there are generally selection methods that depend on the desired result.
[0128]
((Ii) Identification of centromere-related features)
The second method takes advantage of the unique DNA properties we have discovered in the region of the Arabidopsis centromere and the adjacent pericentromere. Centromeres are composed of long arrays of 180 bp repeats flanked by regions that are 10-70% retroelements, up to 15% pseudogenes and up to 29% transposons (see FIGS. 12A-T). . This is unique about Centromere. This is because retroelements, transposons and pseudogenes are very rare outside the centromere and pericentromere regions. Furthermore, the gene density decreases from the average of one gene every 4.5 kb on the chromosome arm to one gene per 150 kb in the centromere. This unique centromeric composition can be exploited in a number of ways, and centromeric regions in other species can be found, for example, as follows:
1) Markers specific for retroelements, transposons, repetitive DNA elements and pseudogenes can be devised to genetically map dense regions with similar elements.
[0129]
2) The second method involves in situ hybridization, preferably fluorescence in situ hybridization (FISH). Fluorescently labeled DNA probes, consisting of retroelements, transposons and / or repetitive DNA native to a particular species, can be combined with a microscope and have a% DNA similar to that found in Arabidopsis centromeres. The portion of the chromosome that has the element can be identified.
[0130]
3) Using the sequence database, regions of the genome with an increased number of repetitive DNAs, pseudogenes, retroelements and transposons, similar to the composition of Arabidopsis identified by the present inventors, can be used in centromeric organisms. It can be used to identify chromosomal regions.
[0131]
((Iii) Use of centromere-related protein)
A third method involves immunoprecipitating a known centromere protein or centromere protein and analyzing the bound DNA. Antibodies specific for centromere proteins can be incubated with proteins extracted from cells. The extract may be native or previously processed to crosslink DNA to protein. Antibodies and binding proteins can be purified from protein extracts and DNA can be isolated. The DNA can then be used as a probe for FISH (as discussed above), or used to probe a library to find flanking centromere sequences.
[0132]
(1. Centromere-related protein-specific antibody)
By identifying centromere-associated genes for the first time, we have enabled the production of antibodies against the proteins encoded by such centromere-associated genes. Antibodies can be either monoclonal or polyclonal, which bind to the centromere-related proteins of the invention. Centromere-associated protein targets of antibodies include proteins that bind to the centromere region. In addition, it is specifically contemplated that centromere-related protein-specific antibodies allow for further isolation and characterization of centromere-related proteins. For example, the protein encoded by the centromere can be isolated. Recombinant production of such proteins provides a source of antigen for the production of antibodies.
[0133]
Alternatively, centromeres can be used as ligands to isolate centromere binding proteins using affinity methods. Once isolated, these proteins can be used as antigens for the production of polyclonal and monoclonal antibodies. A variation on this technique has been demonstrated by Rattner (1991) by cloning centromere-related proteins by using antibodies that bind in the vicinity of the centromere.
[0134]
Means for preparing and characterizing antibodies are well known in the art (see, for example, Antibodies: A Laboratory Laboratory Manual, Cold Spring Laboratories, 1988; which is incorporated herein by reference). . Methods for producing monoclonal antibodies (mAbs) generally begin with the same strain as for preparing polyclonal antibodies. Briefly, polyclonal antibodies are prepared according to the present invention by immunizing an animal with an immunogenic composition and collecting antisera from the immunized animal. A wide range of animal species can be used for the production of antisera. Typically, the animal used for the production of antisera is a rabbit, mouse, rat, hamster, guinea pig or goat. Rabbits are a preferred choice for polyclonal antibody production because of ease of operation, maintenance and relatively large blood volumes.
[0135]
As is well known to those skilled in the art, a given composition may vary in its immunogenicity. Thus, it is often necessary to boost the host immune system, which can be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for coupling the polypeptide to the carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazonated benzidine.
[0136]
It is also well known in the art that the immunogenicity of a particular immunogenic composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a nonspecific stimulator of the immune response, including killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.
[0137]
The amount of the immunogenic composition used to produce the polyclonal antibodies will vary depending on the nature of the immunogen and the animal used for immunization. Various routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal). Production of polyclonal antibodies can be monitored by sampling blood of the immunized animal at various time points following immunization. A second, boost injection may be given. The boost and titration process is repeated until a proper titration is achieved. If the desired level of immunogenicity is obtained, the immunized animal can be exsanguinated, serum isolated and stored, and / or the animal can be used to produce a mAb.
[0138]
Monoclonal antibodies can be readily prepared by the use of well-known techniques, such as exemplified in US Pat. No. 4,196,265, which is incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogenic composition (eg, a purified or partially purified minichromosome-related protein, polypeptide or peptide). Is included. The immunizing composition is administered in a manner effective to stimulate the antibody-producing cells. Rodents such as mice and rats are preferred animals, but the use of rabbit, sheep or frog cells is also possible. Although the use of rats may provide certain advantages (Goding $ 1986), mice are preferred, and BALB / c mice are most preferred. This is because the BALB / c mice are routinely used and give a high percentage of stable fusions.
[0139]
Following immunization, somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb production protocol. These cells can be obtained from a biopsy spleen, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells at the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. . Often, a panel of animals is immunized and the spleen of the animal with the highest antibody titer is removed, and spleen lymphocytes are obtained by homogenizing the spleen with a syringe. Typically, spleens from immunized mice are approximately 5 x 10⁷~ 2 × 10⁸Including lymphocytes.
[0140]
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortalized myeloma cell, generally one of the same species as the immunized animal. Myeloma cell lines suitable for use in the hybridoma-producing fusion procedure are preferably non-antibody-producing, have high fusion efficiency, and grow in a particular selective medium that supports growth of only the desired fusion cells (hybridomas). It is an enzyme deficiency that makes it impossible.
[0141]
Any one of a number of myeloma cells can be used as is known to those of skill in the art (Goding 1986; Campbell 1984). For example, when the immunized animal is a mouse, P3-X63 / Ag8, X63-Ag8.653, NS1 / 1.1. Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7 and S194 / 5XX0 Bul; for rats, R210. RCY3, Y3-Ag@1.2.3, IR983F, and 4B210 may be used; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusion .
[0142]
One preferred mouse myeloma cell line is the NS-1 myeloma cell line (also referred to as P3-NS-1-Ag4-1), which, by requesting cell line storage number GM3573, can be used for NIGMS \ Human \ Genetic \ Mutant \ Cell \ Repository. It is readily available from. Another mouse myeloma cell line that can be used is the 8-azaguanine resistant mouse (murine) myeloma SP2 / 0 non-producer cell line.
[0143]
Methods for producing hybrids of antibody-producing spleen or lymph node cells and myeloma cells typically involve somatic cells and myeloma cells in a 2: 1 ratio (where the ratio is about 20: 1 to about 1: 1 respectively). And mixing in the presence of an agent (chemical or electrical) that promotes cell membrane fusion. Fusion methods using the Sendai virus are described in (Kohler et al., 1975; 1976), and methods using polyethylene glycol (PEG) (eg, 37% (v / v) PEG) are described in (Getter et al., 1977). be written. The use of electrically induced fusion methods is also appropriate (Goding $ 1986).
[0144]
Fusion procedures are generally infrequent (about 1 × 10^-6~ 1 × 10^-8) Produces viable hybrids. However, this does not create a problem. Because, this viable, fusion hybrid can be transformed from the original, unfused cells, especially unfused myeloma cells, which usually divide indefinitely, by culturing in selective media. This is because they are distinguished. A selective medium is generally a medium that contains an agent that blocks neosynthesis of nucleotides in tissue culture medium. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block the de novo synthesis of both purines and pyrimidines, while azaserine blocks only purine synthesis. If aminopterin or methotrexate is used, this medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). If azaserine is used, the medium is supplemented with hypoxanthine.
[0145]
A preferred selection medium is HAT. Only cells that can operate the nucleotide recovery pathway can survive in HAT medium. Myeloma cells lack key enzymes of the recovery pathway, such as hypoxanthine phosphoribosyltransferase (HPRT), which cannot survive. B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Thus, only those hybrids formed from myeloma and B cells are viable cells in selective media.
[0146]
This culture provides a population of hybridomas from which a particular hybridoma is selected. Typically, selection of hybridomas is performed by culturing cells by single clone dilution in microtiter plates and then testing individual clone supernatants (after about 2-3 weeks) for the desired reactivity. . The assay should be sensitive, simple, and rapid (eg, radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunobinding assay, etc.).
[0147]
The selected hybridomas are then serially diluted and cloned into individual antibody-producing cell lines, which are then expanded indefinitely to provide mAbs. This cell line can be utilized for mAb production in two basic ways. A sample of the hybridoma can be injected (often intraperitoneally) into a histocompatible animal of the type used to provide the somatic and myeloma cells for the initial fusion. Injected animals develop tumors that secrete specific monoclonal antibodies produced by the fusion cell hybrids. The body fluid of the animal (eg, serum or ascites) can then be withdrawn to provide mAbs at high concentrations. Although individual cell lines can also be cultured in vitro, mAbs are naturally secreted in culture media from which they can be readily obtained at high concentrations. MAbs produced by either means can be further purified, if desired, using filtration, centrifugation and various chromatographic methods (eg, HPLC or affinity chromatography).
[0148]
(2. ELISA and immunoprecipitation)
ELISA can be used with the present invention, for example, in identifying the expression of centromere-related proteins in candidate centromere sequences. Thus, such an assay may facilitate the isolation of centromeres from species other than Arabidopsis. By identifying conserved centromere-related coding sequences, we have provided an important tool for such screening.
[0149]
In an ELISA assay, a protein or peptide comprising a minichromosome-encoding protein antigen sequence is immobilized on a selected surface, preferably a surface exhibiting protein affinity (eg, a well of a polystyrene microtiter plate). After washing to remove incompletely adsorbed material, non-specific proteins known to be antigenically neutral for the test antiserum (eg, bovine serum albumin (BSA), Casein or powdered milk solution) to bind or coat with them. This allows to block non-specific adsorption sites on the immobilized surface, thus reducing the background caused by non-specific binding of antisera to the surface.
[0150]
After binding of the antigenic material to the wells, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilized surface contains the immune complex (antigen / antibody) It is contacted with the antiserum or clinical or biological extract to be tested in a manner that will lead to formation. Such conditions preferably include diluting the antiserum with a diluent (eg, BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS) / Tween®). I do. These added agents also tend to help reduce non-specific background. The layered antiserum is then incubated for about 2 to about 4 hours, preferably at a temperature on the order of about 25C to about 27C. After incubation, the antiserum contact surface is washed to remove non-immune complexed material. A preferred washing procedure involves washing with a solution such as PBS / Tween® or borate buffer.
[0151]
After the formation of a specific immune complex between the test sample and the bound antigen, and subsequent washing, development and equal formation of the immune complex results in the formation of a second antibody having specificity for the first antibody. It can be determined by subjecting to the same procedure. To provide a means of detection, the second antibody preferably has an associated enzyme that produces a color or light upon incubation with a suitable chromogenic substrate. Thus, for example, an antiserum binding surface is contacted with urease or peroxidase conjugated anti-human IgG for a time and under conditions that favor the occurrence of immune complex formation and incubated (eg, at room temperature in a PBS containing solution at room temperature). Time incubation).
[0152]
After incubation with a second enzyme-labeled antibody and subsequent washing to remove unbound material, the amount of label may be reduced to a chromogenic substrate (eg, urea and bromocresol purple, in the case of peroxidase as the enzyme label, Or 2,2'-azino-di- (3-ethyl-benzthiazoline) -6-sulfonic acid (ABTS) and H₂O₂) And quantified by incubation with Quantification is then achieved by measuring the degree of color development (eg, using a visible spectrum spectrometer).
[0153]
(3. Western blot)
Centromeric binding antibodies may find use in immunoblot or western blot analysis, such as for identification of proteins immobilized on a solid support matrix such as nitrocellulose, nylon or a combination thereof. After gel electrophoresis, together with immunoprecipitation, they can be used as single-step reagents for use in the detection of antigens, and a second reagent for this antigen used in the detection of antigens, Cause the ground. This is particularly useful when the antigen being studied is an immunoglobulin (except for the use of immunoglobulin-binding bacterial cell wall components), where the antigen being studied cross-reacts with the detection agent, or It moves at the same relative molecular weight as the reaction signal.
[0154]
Immunologically based detection methods for use with Western blots (including enzymatically labeled, radiolabeled, or fluorescently labeled secondary antibodies to protein moieties) are considered to be of particular use in this regard Can be
[0155]
((Iv) Approach based on gene mapping)
The gene mapping techniques outlined herein for the identification of centromeres in Arabidopsis may find use in other species. In one aspect, this encompasses the actual use of the mapping data provided herein based on synteny between the Arabidopsis chromosome and chromosomes of other species. In addition, new mapping data may be obtained using the techniques described herein. For example, in any plant that makes tetrads, the detailed procedures described herein for tetrad analysis can be used for centromere isolation. Briefly, tetrad analysis measures the frequency of recombination between genetic markers and centromeres by analyzing all four products of individual meiosis. A particular advantage arises from the quartet (qrt 1) mutation in Arabidopsis, which causes four products of meiosis of pollen mother cells in Arabidopsis that remain attached.
[0156]
In addition to Arabidopsis, some naturally occurring plant species are known to release pollen clusters, such as water lilies, cattails, heathers (Ericaceae and Epacridceae), pine primrose (Onagraceae), Droseraceae, Orchidaceae, and Acacia (Mimosaceae) (Preuss @ 1994, Smyth @ 1994). However, these species have not been developed in experimental systems and their use is limited to genetic analysis. However, it is anticipated by the inventors that the cloning and introduction of a quartet mutation, or an antisense copy of the unmutated quartet gene, could potentially enable the use of tetrad analysis in any species.
[0157]
Southern genomic DNA blots combined with RFLP analysis can be used to map centromeres with high resolution. Stock inoculum provides the required amount of DNA for restriction fragment analysis. Southern blots are hybridized with probes labeled by radioactive or non-radioactive methods.
[0158]
In many cases, it may be desirable to identify new polymorphic DNA markers that are linked near the target region. In some cases, this can be easily done. For example, in many plant genomes, polymorphic Sau3A sites can be found for measurements about every 8-20 kB. Subtraction methods are available to identify such polymorphisms (Rosenberg et al., 1994), and these subtractions are performed using DNA from selected centromeric YAC or BAC clones. obtain. Screening for RFLP markers potentially linked to the centromere was also performed using DNA fragments from the centromere-linked YAC clone to probe a blot of genomic DNA from the target organism digested with a panel of restriction enzymes. obtain.
[0159]
To confirm that the entire centromere region has been cloned, a clone or set of clones that hybridize to a marker at either end of each centromere is identified. These attempts can be complicated by the presence of repetitive DNA in the centromere and the potential instability of the centromere clone. Thus, the identification of large clones with unique sequences that serve as useful probes simplifies chromosome walking strategies.
[0160]
Blot hybridization allows comparison of the structure of the clone to the structure of genomic DNA, and thus determines whether the clone has undergone deletion or rearrangement. The identified centromere clones will determine if they share the same sequence, whether they will be localized in situ to cytologically defined centromere regions, and if mapped near Arabidopsis centromeres. Useful for hybridization experiments, used to determine whether they contain repetitive sequences (Richards et al., 1991; Maluszynska et al., 1991).
[0161]
Exemplary methods for performing PFGE and YAC genomic analysis have been described (Ecker, 1990). A large insert YAC library for genome mapping in Arabidopsis thaliana was described in Creusot et al. (1995). The analysis of clones with repetitive DNA sequences in two YAC libraries of Arabidopsis thaliana DNA was discussed by Schmidt et al. (1994). The construction and characterization of the Arabidopsis yeast artificial chromosome library was described by Grill and Somerville (1991).
[0162]
A particularly useful type of clone is the bacterial artificial chromosome (BAC). Because the data suggested that YAC clones often could not spread to the centromere (Willard, 1997). For example, the construction and characterization of a bacterial artificial chromosome library from Arabidopsis thaliana has been described (Choi et al., 1995). Complementation of plant mutants with large genomic DNA fragments can be achieved using transforming mini-chromosomal vectors, thereby speeding up the position cloning (Liu et al., 1999). The construction and characterization of the IGF @ Arabidopsis @ BAC library was described by Mozo et al. (1998). A physical mapping of the Arabidopsis thaliana genome based on the complete BAC has been described (Mozo et al., 1998).
[0163]
(VI. Site-specific integration and excision of nucleic acid segments)
It is specifically anticipated by the inventors that techniques for site-specific integration or excision of nucleic acid segments may be utilized for minichromosome construction (see, eg, Example 8B below). thing). Such techniques can also be used for site-specific integration or excision of transgenes (including mini-chromosomal vectors) that are introduced into plants.
[0164]
Site-specific integration or excision of nucleic acid molecules can be achieved by means of homologous recombination (see, eg, US Pat. No. 5,527,695, which is specifically incorporated by reference herein in its entirety). Incorporated))). Homologous recombination is the reaction between any pair of DNA sequences having similar nucleotide sequences, and the two sequences interact (recombine) to form a new recombinant DNA species. The frequency of homologous recombination increases as the length of the shared nucleotide DNA sequence increases, and is higher for linearized plasmid molecules than for circular plasmid molecules. Homologous recombination can occur between two DNA sequences that are never identical, but the recombination frequency decreases as the differences between the two sequences increase.
[0165]
The introduced DNA sequence can be targeted via homologous recombination by linking the DNA molecule of interest to a sequence that shares homology with the endogenous sequence of the host cell. Once the DNA has entered the cell, the two homologous sequences can interact, inserting the introduced DNA at the site where the homologous genomic DNA sequence is located. Therefore, selection of a homologous sequence contained in the introduced DNA determines the site where the introduced DNA is integrated via homologous recombination. For example, if the DNA sequence of interest is ligated to a DNA sequence that shares homology with a single copy gene of the host plant cell, the DNA sequence of interest will be homologously recombined only at that single specific site. Is inserted through. However, if the DNA sequence of interest is ligated to a DNA sequence that shares homology with the multi-copy gene of the host eukaryotic cell, the DNA sequence of interest will be at each specific site where a copy of the gene is located. It can be inserted via homologous recombination.
[0166]
The DNA can be inserted into the host chromosome or vector by a homologous recombination reaction, which includes a single recombination (which results in the insertion of the full length of the introduced DNA) or two recombinations (two sets). (Which results in the insertion of only DNA located during the recombination event). For example, if it is desired to insert a foreign gene into the genomic site where the selected gene is located, the introduced DNA should include sequences homologous to the selected gene. A single homologous recombination event then results in the entire transgene sequence being inserted into the selected gene. Alternatively, two recombination events are achieved by flanking each end of the DNA sequence of interest, which sequence is intended to be inserted into the genome, with a DNA sequence homologous to the selected gene. obtain. Homologous recombination events involving each of the homologous flanking regions result in the insertion of foreign DNA. Thus, only those DNA sequences located between two regions that share genomic homology become integrated into the genome.
[0167]
Although the introduced sequence can be targeted for insertion at a particular site via homologous recombination, homologous recombination in higher eukaryotes is a relatively rare event as compared to a random insertion event. In plant cells, foreign DNA molecules find homologous sequences in the genome of the cell, and^-4~ 4.2 × 10^-4Recombine at the frequency of Thus, any transformed cell that contains a introduced DNA sequence integrated via homologous recombination will also likely contain many copies of the randomly integrated introduced DNA sequence. Thus, it may be desirable to use a more precise mechanism for site-specific recombination. A preferred mode for performing site-specific recombination involves the use of a site-specific recombinase system. Generally, a site-specific recombinase system consists of three components: two pairs of DNA sequences (first and second site-specific recombination sequences) and a specific enzyme (site-specific recombinase). Site-specific recombinases catalyze recombination reactions only between two site-specific recombination sequences.
[0168]
Many different site-specific recombinase systems can be used in accordance with the present invention, including, but not limited to, the Cre / lox system of bacteriophage P1 (Hoess et al. 1982; U.S. Patent No. 5,658,772, which is specifically incorporated by reference herein in its entirety), the yeast FLP / FRT system (Golic and Lindquist, 1989), the phage Mu Gin recombinase. (Maeser and Kahmann, 1991); E. coli Pin recombinase (Enomoto et al., 1983), a recombinase encoded by the sre gene (ORF469), which can mediate integration of the R4 phage genome (Matsuura et al., 1996), encoded by the PinD of Shigella dysenteriae. Site-specific recombinase (Tominaga, 1997), a site-specific recombinase (Zhang et al., 1997) encoded in the major "pathogenicity island" of Salmonella typhi, Int-B13 of the bacteriophage P4 integrase family. A site-specific recombinase (Ravatn et al., 1998) and the R / RS system of the pSR1 plasmid (Araki , 1992). The bacteriophage P1 @ Cre / lox and yeast FLP / FRT systems constitute two systems that are particularly useful for site-specific recombination. In these systems, the recombinase (Cre or FLP) interacts specifically with its respective site-specific recombination sequence (lox or FRT, respectively) to invert or excise the intervening sequence. The sequence for each of these two systems is relatively short (34 bp for lox and 47 bp for FRT) and is therefore convenient for use with transformation vectors.
[0169]
The FLP / FRT recombinase system has been shown to work efficiently in plant cells, but can also be used, for example, in bacterial cells or in vitro. The efficiency of the FLP / FRT system indicates the structure of the FRT site, and the amount of FLP protein present affects excision activity. Generally, short incomplete FRT sites lead to higher excision product accumulation than full-length full-length FRT sites. This system can catalyze both intra- and inter-molecular reactions and show their use for DNA excision and integration reactions. The recombination reaction is reversible, and this reversibility can reduce the efficiency of the reaction in each direction. Altering the structure of the site-specific recombination sequence is one approach to remedy this situation. The site-specific recombination sequence can be mutated in such a way that the product of the recombination reaction is no longer recognized as a substrate for the reverse reaction, thereby stabilizing the integration or excision event.
[0170]
In the Cre-lox system (found in bacteriophage P1), recombination between loxPs occurs in the presence of Cre recombinase (see, for example, US Pat. No. 5,658,772, which is incorporated herein in its entirety). Is specifically incorporated by reference)). This system has been used to excise genes located between two lox sites that have been introduced into the yeast genome (Sauer, 1987). Cre was expressed from the inducible yeast GAL1 promoter, and the Cre gene was located in a self-replicating yeast vector.
[0171]
Since lox sites are asymmetric nucleotide sequences, the lox sites of the same DNA molecule may have the same or opposite orientations with respect to each other. Recombination between lox sites in the same orientation results in deletion of the DNA segment located between the two lox sites, and a connection between the resulting ends of the first DNA molecule. The deleted DNA segment forms a circular molecule of DNA. The initial DNA molecule and the resulting circular molecule each contain a single lox site. Recombination between lox sites in opposite orientations of the same DNA molecule results in inversion of the DNA segment nucleotide sequence located between the two lox sites. In addition, reverse exchange of DNA segments adjacent to lox sites located on two different DNA molecules can occur. All of these recombination events are catalyzed by the products of the Cre coding region.
[0172]
(VII. Transformed host cells and transgenic plants)
Methods and compositions for transforming bacteria, yeast cells, plant cells or whole plants having one or more minichromosomes are further aspects of the present disclosure. Transgenic bacteria, yeast cells, plant cells or plants, or progeny and seeds derived from such transgenic plants, derived from such a transformation process are also further embodiments of the present invention.
[0173]
Means for transforming bacteria and yeast cells are well-known in the art. Typically, the means of transformation is similar to those well-known means used to transform other bacteria or yeasts (eg, E. coli or Saccharomyces cerevisiae). Methods for DNA transformation of plant cells include Agrobacterium-mediated plant transformation, protoplast transformation (as used herein, “protoplast transformation” includes PEG-mediated transformation, electroporation and electroporation. Protoplast fusion transformation), gene transfer into pollen, injection into reproductive organs, injection into immature embryos and biolistics. Each of these methods has different advantages and disadvantages. Thus, one particular method of introducing a gene into a particular plant line may not always be the most effective for another plant line, but which method is useful for a particular plant line. Are well known in the art.
[0174]
There are many methods for introducing transforming DNA segments into cells, but not all are suitable for delivering DNA to plant cells. Suitable methods include virtually any method by which DNA can be introduced into cells (eg, Agrobacterium infection, direct delivery of DNA (eg, PEG-mediated transformation of protoplasts (Omirulleh et al., 1993), drying / inhibition-mediated DNA). Incorporation, electroporation, agitation using silicon carbide fibers, acceleration of DNA coated particles, etc.)). In certain embodiments, an acceleration method is preferred, such as a particle gun.
[0175]
Techniques for introducing DNA into cells are well known to those skilled in the art. Four general methods for delivering genes to cells have been described: (1) chemical methods (Graham et al., 1973; Zatlukal et al., 1992); (2) physical methods (e.g., microinjection ( Capecchi, 1980), electroporation (Wong et al., 1982; Fromm et al., 1985; U.S. Pat. No. 5,384,253) and gene guns (Johnston et al., 1994; Fynan et al., 1993); (3) viral vectors ( Clapp @ 1993; Lu et al., 1993; Eglitis et al., 1988a; 1988b); and (4) Receptor-mediated mechanisms (Curier et al., 1991; 1992; Wagner et al., 1992).
[0176]
((I) electroporation)
Application of short, high-pressure electrical pulses to various animal and plant cells leads to the formation of nanometer-sized pores in the plasma membrane. DNA is directly incorporated into the cytoplasm of the cell, either through these pores or as a result of redistribution of membrane components with pore closure. Electroporation can be very efficient and can be used for both transient expression of cloned genes and for establishing cell lines that carry an integrated copy of the gene of interest. In contrast to calcium phosphate-mediated transfection and protoplast fusion, electroporation frequently results in cell lines with one, or at most a few, integrated copies of the foreign DNA.
[0177]
Introduction of DNA by electroporation means is well known to those skilled in the art. In this method, certain cell wall degrading enzymes (eg, pectin degrading enzymes) are used to provide target recipient cells that are more likely to be transformed by electroporation than untreated cells. Alternatively, recipient cells are made more amenable to transformation by mechanical injury. Either fragile tissue (eg, a suspension culture of cells) or embryogenic callus can be used to perform transformation by electroporation, or alternatively, immature embryos or other organisations The transformed tissue can be directly transformed. The cell walls of the selected cells are partially degraded by exposing the selected cells to a pectin-degrading enzyme (pectolyase) or mechanically damaging in a controlled manner. Such cells then become recipients of the DNA transfer by electroporation, which can be performed at this stage, and the transformed cells then depend on the nature of the newly integrated DNA. Identified by an appropriate selection or screening protocol.
[0178]
((Ii) Particle gun)
A further advantageous method for delivering transforming DNA segments to plant cells is by microprojectile bombardment. In this way, the particles can be coated with the nucleic acid and delivered to the cells by a driving force. Exemplary particles include particles composed of tungsten, gold, platinum, and the like.
[0179]
In addition to being an effective means of reproducibly and stably transforming monocotyledonous plants, the advantages of biolistics are that neither protoplast isolation (Cristou et al., 1998) nor susceptibility to Agrobacterium infection is required. It is. An exemplary embodiment of a method for delivering DNA to corn cells by acceleration is a biolistic particle delivery system (Biolistics \ Deployment \ Delivery \ System) that screens DNA-coated particles or cells (e.g., (Stainless steel screen or Nytex screen) can be used to advance to a filter surface covered with plant cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. Intervening screens between the launcher and the bombarded cells reduce the size of the bombardment aggregates and increase the frequency of transformation by reducing the damage done to recipient cells by oversized bombardment It is thought that it can contribute to.
[0180]
For bombardment, the cells in suspension are preferably concentrated on a filter or solid culture medium. Alternatively, immature embryos or other target cells can be placed on a solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the particle barrier. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of the techniques described herein, up to 1,000 or more foci of cells that transiently express a marker gene may be obtained. The number of cells in the lesion 48 hours after impact, expressing foreign gene products, often ranges from 1 to 10, with an average of 1 to 3.
[0181]
In shock transformation, culture conditions and shock parameters before shock can be optimized to obtain the maximum number of stable transformants. Both physical and biological parameters for impact are important in this technology. Physical factors are those involved in manipulating the DNA / particulate sediment, or those that affect the flight and speed of either macroprojectiles or microprojectiles. Biological factors, including all steps, can be used to manipulate cells immediately before and after bombardment, to regulate the osmotic pressure of target cells to help reduce shock-related trauma, and to transform DNA (eg, linearize). DNA or intact supercoiled plasmid) properties are also involved. Pre-impact manipulation is believed to be particularly important for successful transformation of immature embryos.
[0182]
It is therefore anticipated that it may be desirable to adjust the various impact parameters in small-scale studies to fully optimize the conditions. In particular, it is desirable to adjust physical parameters such as gap distance, flight distance, tissue distance and helium pressure. Also, trauma reduction factor (TRF) can be minimized by altering the conditions that affect the physiological state of the recipient cells, and thus can affect transformation and integration efficiencies. For example, osmotic pressure, tissue hydration and subculture stage or cell cycle of the recipient cells can be adjusted for optimal transformation. Performing other conventional adjustments will be known to those of skill in the art in light of the present disclosure.
[0183]
((Iii) Agrobacterium-mediated transfer)
Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells. Because the DNA can be introduced into whole plant tissue, thereby avoiding the need for regeneration of intact plants from protoplasts. The use of Agrobacterium-mediated plant integration vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described in (Fraley et al., 1985; Rogers et al., 1987). Advances in Agrobacterium-mediated transfer now allow the introduction of large segments of DNA (Hamilton, 1997; Hamilton et al., 1996).
[0184]
When using conventional transformation vectors, chromosomal integration is required for stable inheritance of foreign DNA. However, the vectors described herein can be used for transformation with or without integration. This is because the centromere functions required for stable inheritance are contained in minichromosomes. In certain embodiments, a transformation event in which the minichromosome does not integrate into the chromosome may be preferred in that problems with site-specific changes in expression and insertional mutagenesis may be avoided.
[0185]
Incorporation of Ti-DNA is a relatively accurate process with little rearrangement. As described (Spielmann et al., 1986; Jorgensen et al., 1987), the region of DNA to be transferred is defined by its border sequences, and intervening DNA is usually inserted within the plant genome. As described (Klee et al., 1985), recent Agrobacterium transformation vectors have been described in E. coli. It is replicable in E. coli and Agrobacterium, allowing for easy manipulation. In addition, recent technological advances in Agrobacterium-mediated gene transfer vectors have modified the arrangement of genes and restriction sites in the vector to facilitate the construction of vectors capable of expressing genes encoding various polypeptides. The described (Rogers et al., 1987) vector has a convenient multilinker region flanked by a promoter and polyadenylation site for direct expression of the inserted polypeptide-encoding gene, and the present invention Is appropriate for the purpose. In addition, Agrobacterium containing both the armed and disarmed Ti genes can be used for transformation. In plant lines where Agrobacterium-mediated transfer is efficient, this is the method of choice because of the easy and defined nature of gene transfer.
[0186]
Agrobacterium-mediated transformation of the leaf disc and other tissues (eg, cotyledons and hypocotyls) appears to be restricted to plants that are naturally infected with Agrobacterium. Agrobacterium-mediated transformation is most efficient in dicotyledonous plants. As described (Bytebier et al., 1987; U.S. Pat. No. 5,591,616, which is incorporated herein by reference in detail) using Agrobacterium vectors in asparagus and in corn. More significantly, transgenic plants are being produced, but most monocotyledonous plants do not appear to be the natural hosts for Agrobacterium. Thus, commercially important cereals (eg, rice, corn, and wheat) usually must be transformed using alternative methods. However, as described above, transformation of asparagus using Agrobacterium can also be achieved (see, for example, Bytebier et al., 1987). Agrobacterium-mediated transfer can be more efficient by using mutants that are defective in the integration of Agrobacterium @ T-DNA, but are receptive for delivery of that DNA to cells (Mysore et al., 2000a). ). Furthermore, even in Arabidopsis ecotypes and Arabidopsis mutants that are repellent to Agrobacterium root transformation, germline transformation can be performed (Mysore et al., 2000b).
[0187]
Transgenic plants formed using Agrobacterium transformation methods typically contain a single gene on one chromosome. Such transgenic plants can be said to be hemizygous for the added gene. A more accurate name for such a plant is independent @segregation. Because each transformed plant shows only one T-DNA integration event.
[0188]
Transgenic plants that are homozygous for the added foreign DNA (ie, transgenic plants that contain two copies of the transgene and have one gene at the same locus on each chromosome of the chromosome pair) are more preferred. . Homozygous transgenic plants are sexually crossed (self-pollinated) independent transgenic individual transgenic plants containing a single added transgene, causing some of the produced seeds to germinate and the resulting plant Can be obtained by analyzing for enhanced activity compared to a control (native, non-transgenic) plant or an independent segregant transgenic plant.
[0189]
Plants in which the minichromosome is not integrated into the chromosome are even more preferred. Such a plant may be referred to as 2n + x, where 2n is the diploid number of the chromosome and x is the number of mini-chromosomes. Initially, transformants may be 2n + 1 (ie, have one additional minichromosome). In this case, it may be desirable to self-pollinate the plant or to cross the plant with another 2n + 1 plants to produce a 2n + 2 plant. The 2n + 2 plant is preferred in that it is expected that the plant will pass this minichromosome to all its progeny via meiosis.
[0190]
It should be understood that two different transgenic plants can also be crossed to produce progeny that contain two independently segregated added foreign minichromosomes. Self-pollination of appropriate progeny can produce plants that are homozygous for both added foreign minichromosomes, encoding the polypeptide of interest. Backcrossing to parent plants and out-crossing with non-transgenic plants are also contemplated.
[0191]
((Iv) Other transformation methods)
Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and a combination of these treatments (Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1986; Uchimiya et al., 1986; Callis et al., 1987; Marcotte et al., 1988).
[0192]
The application of these lines to different plant lines for the purpose of producing transgenic plants depends on the ability to regenerate that particular plant line from protoplasts. Exemplary methods for cereal regeneration from protoplasts have been described (Fujimura et al., 1985; Toriyama et al., 1986; Yamada et al., 1986; Abdulah et al., 1986).
[0193]
Other methods of introducing DNA into insect cells or tissues can be used to transform plant lines that are not successfully regenerated from protoplasts. For example, cereal regeneration from immature embryos or explants can be effected as described (Vasil 1988). In addition, "particle guns" or high speed particle gun technology may be utilized (Vasil 1992).
[0194]
Using the latter technique, DNA is carried into the cytoplasm through the cell wall, on the surface of small metal particles, as described (Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988). . The metal particles penetrate several layers of cells, thus allowing transformation of cells within a tissue explant.
[0195]
For example, protoplast fusion can be used to integrate the assembled minichromosome into host cells, such as yeast cells, and then fuse these cells to plant protoplasts. Chromosomes that lack plant centromeres (eg, yeast cells in this example) are eliminated by the plant cells, while minichromosomes are stably maintained. Many examples of protocols for protoplast fusion that can be used with the present invention have been described (see, eg, Negrutiu et al., 1992, and Peterson).
[0196]
Liposomal fusion involves, for example, packaging the recombinant construct into lipid droplets (liposomes) and then fusing these liposomes to plant protoplasts, thus delivering AC to the plant cells, resulting in a centromere ( For example, mini-chromosomes) can be used to introduce recombinant constructs (see Lurqui and Rollo, 1993).
[0197]
(VIII. Foreign genes for expression in plants)
One particular important advance of the present invention is that it provides methods and compositions for the expression of foreign genes in plant cells. One advance in the constructs of the present invention is that they allow the introduction of multiple genes, potentially representing an entire biochemical pathway. Importantly, the present invention allows for the transformation of plant cells with mini-chromosomes containing many structural genes. Another advantage is that more than one minichromosome can be introduced, allowing the genes of the combination to be moved and shuffled. In addition, the ability to eliminate minichromosomes from plants provides additional flexibility, which allows to modify the set of genes contained within the plant. Furthermore, by using a site-specific recombinase, it is possible to add a gene to an existing mini-chromosome existing in a plant.
[0198]
The added genes are often genes that direct the expression of a particular protein or polypeptide product, but they may also be non-expressed DNA segments (eg, transposons that do not direct their own transposition (eg, For example, Ds)). As used herein, an “expressed gene” can be transcribed into RNA (eg, mRNA, antisense RNA, etc.) or translated into protein, expressed as a trait of interest, and the like. Which is not limited to a selectable marker gene, a screening marker gene or a non-screening marker gene. We also note that when two expressed genes (not necessarily marker genes) are used in combination with a marker gene, they can be separated on either the same or different DNA segments for transformation. It is contemplated that the gene may be used. In the latter case, different vectors are delivered simultaneously to the recipient cells to maximize co-transformation.
[0199]
The choice of a particular DNA segment to be delivered to a recipient cell depends on the purpose of the transformation. One of the main goals of crop transformation is to add some commercially desirable agriculturally important traits to plants. Such traits include herbicide resistance or resistance; insect resistance or resistance; disease resistance or resistance (viral, bacterial, fungal, nematode); stress resistance and / or resistance (eg, (As exemplified by resistance or resistance to drying, heat, cold, freezing, excessive humidity, salt stress); oxidative stress; increased yield; food content and properties; physical appearance; female sterility; ); Standability; prolificity; starch content and quality; oil content and quality; protein quality and content; amino acid composition; and the like. One of skill in the art may desire to incorporate one or more genes that confer any such desired trait (s), for example, the gene (s) encoding herbicide tolerance.
[0200]
In certain embodiments, the present invention contemplates the transformation of a recipient cell having a minichromosome containing more than one exogenous gene. As used herein, an “exogenous gene” is a gene that is not normally found in the host's genome in the same context. This allows the gene to be isolated from a different species than the host genomic gene, or one or more regulatory regions that are different from the regulatory regions found in the host genome but unaltered native gene. It is intended to be isolated from a host genome operably linked to the region. Two or more exogenous genes can also be supplied in one transformation event, either using different transgene-encoding vectors, or using one vector incorporating two or more gene-encoding sequences. . For example, a plasmid carrying a bar expression unit and an aroA expression unit, either at the position of convergence, at the divergent position, or at the collinear position, may be particularly useful. A further preferred combination is a combination of an insect resistance gene (eg, the Bt gene) together with a protease inhibitor gene (eg, pinII) or the use of bar in combination with any of the above genes. Of course, any two or more transgenes of any type (eg, herbicide resistance, insect resistance, disease (virus, bacterial, fungal, nematode) or drought resistance, male sterility, dry down) (Drydown), standability, fertility, starch properties, a transgene that provides oil quantity and oil quality, or a transgene that enhances yield or nutrient quality) is desired. Can be used as
[0201]
((I) herbicide tolerance)
Genes encoding phosphinothricin acetyltransferase (bar and pat), glyphosate resistant EPSP synthase gene, glyphosate degrading enzyme gene encoding glyphosate oxidoreductase gox, deh gene (dehalogenase dehalogenase enzyme inactivating darapon) ), Herbicide resistance (eg, sulfonylureas and imidazolinones) acetolactase synthase, and the bxn gene (encoding a nitrilase enzyme that degrades bromoxynil) are resistant to herbicides for use in transformation. A good example of a gene. The bar and pat genes encode the enzyme phosphinothricin acetyltransferase (PAT), which inactivates the herbicide phosphinothricin, and that the compound inhibits the glutamine synthase enzyme. Hinder. The enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSP synthase) is usually inhibited by the herbicide N- (phosphonomethyl) glycine (glyphosate). However, genes encoding glyphosate resistant EPSP synthase enzymes are known. These genes are specifically contemplated for use in plant transformation. The deh gene encodes the enzyme dalapone dehalogenase and confers resistance to the herbicide dalapone. The bxn gene encodes a specific enzyme, nitrilase, that converts bromoxynil to a non-herbicide degradation product.
[0202]
((Ii) insect resistance)
Potential insect resistance genes that can be introduced include the Bacillus thuringiensis crystal toxin gene or the Bt gene (Watrud et al., 1985). The Bt gene may provide resistance to lepidopteran pests or beetle plagues (eg, European Corn Borer (ECB)). Preferred Bt toxin genes for use in such embodiments include the CryIA (b) and CryI (c) genes. B. Influencing insect growth or development Endotoxin genes from other species of thuringiensis can also be used in this regard.
[0203]
Preferred Bt genes for use in the transformation protocols disclosed herein have been modified such that the coding sequence results in increased expression in plants, and more particularly in monocotyledonous plants. It is intended to be the Bt gene. Means for preparing synthetic genes are known in the art and are disclosed, for example, in US Patent Nos. 5,500,365 and 5,689,052 (each of these disclosures Their entirety is specifically incorporated herein by reference). Examples of such modified Bt toxin genes include the synthetic Bt @ CryIA (b) gene (Perlak et al., 1991) and the synthetic CryIA (c) gene called 1800b (PCT application WO 95/06128). Some examples of other Bt toxin genes known to those of skill in the art are provided in Table 1 below.
[0204]
[Table 1]

Protease inhibitors may also provide insect resistance (Johnson et al., 1989) and thus have utility in transformation in plants. The use of pin II, a protease inhibitor-II gene from tomato or potato, is considered to be particularly useful. The use of the pinII gene in combination with the Bt toxin gene has been found to be even more advantageous, the combined effect of which produces synergistic insecticide activity. Other genes encoding inhibitors of the digestive system of insects, or genes encoding enzymes or cofactors that facilitate the production of inhibitors, may also be useful. This group includes oryzacystatin inhibitors and It may be exemplified by amylase inhibitors, such as those inhibitors from wheat and barley.
[0205]
Also, the gene encoding the lectin may provide additional or alternative pesticidal properties. Lectins, originally called phytohemagglutinins, are carbohydrate binding proteins that have the ability to agglutinate red blood cells from a range of species. Lectins have recently been identified as insecticides with activity against weevil, ECB and rootworm (Murdock et al., 1990; Czapla and Lang, 1990). Lectin genes intended to be useful include, for example, barley pathogen agglutinin and wheat pathogen agglutinin (WGA) and rice lectin (Gatehouse et al., 1984), with WGA being preferred.
[0206]
When introduced into insect plague, genes that control the production of large or small polypeptides that are active against insects (such as lytic peptides, peptide hormones and toxicants and venoms) are: It forms another aspect of the present invention. For example, expression of juvenile hormonal esterases directed against certain insect plagues is also intended to result in insecticidal activity, or possibly to arrest metamorphosis (Hammock et al., 1990).
[0207]
Transgenic plants expressing a gene encoding an enzyme that affects insect epidermal integrity form yet another aspect of the present invention. Such genes also include, for example, genes encoding chitinases, proteases, lipases and genes for the production of nicomycin (a compound that inhibits chitin synthesis). Intended to produce resistant plants. Genes encoding activities that affect insect molting (eg, genes that affect the production of ecdysteroid UDP glucosyltransferase) also fall within the useful transgenes of the present invention.
[0208]
Genes encoding enzymes that facilitate the production of compounds that reduce the nutritional quality of the host plant against insect plague are also encompassed by the present invention. For example, altering the sterol composition may make it possible to confer plant insecticidal activity. Sterols are derived from insect foods, are obtained from insects, and are used for hormone synthesis and membrane stability. Thus, altering a plant sterol composition by expression of a novel gene (eg, a gene that directly promotes the production of an undesired sterol, or a gene that converts a desired sterol to an undesired form), can result in insect growth and / or It has a negative effect on development and thus imparts insecticidal activity to plants. Lipoxygenases are naturally occurring plant enzymes that have shown antinutritional effects in insects and have been shown to reduce the nutritional quality of insect food. Thus, further embodiments of the invention relate to transgenic plants with enhanced lipoxygenase activity that can be resistant to insect feeding.
[0209]
Tripsacum dactylides are a type of grass family that is resistant to certain insects, including corn rootworms. Genes encoding proteins that are toxic to insects, or involved in the biosynthesis of compounds that are toxic to insects, have been isolated from Tripsacum, and these new genes have become resistant to insects. It is expected to be useful to give. It is known that the basis of insect resistance in Tripsacum is genetic. Because the resistance was transmitted to Zeamays through sexual crossing (Branson and Guss, 1972). It is further noted that other cereal, monocotyledonous or dicotyledonous plant species may have genes encoding proteins that are toxic to insects, which are useful for producing insect resistant plants. is expected.
[0210]
Additional genes encoding proteins characterized as having potential insecticidal activity can also be used consistently as transgenes. Such genes include, for example, cowpea trypsin inhibitor (CpTI; Hilder et al., 1987), which can be used as an inhibitor of root-knotworm; a gene encoding avermectin, which can prove to be particularly useful as an inhibitor of corn root-knotworm (Avermectin and) Abamectin., Campbell, W.C., Ed., 1989; Ikeda et al., 1987); ribosome-inactivating protein genes; and even genes that regulate plant structure. Transgenic plant comprising an anti-insect antibody gene and a gene encoding an enzyme capable of converting a non-toxic insecticide (pro-insecticide) applied outside the plant to a pesticide inside the plant Is also contemplated.
[0211]
((Iii) environment or stress tolerance)
Improving a plant's ability to withstand various environmental stresses, including, but not limited to, drought stress, excessive water stress, cold stress, freezing stress, high temperature stress, salt stress, and oxidative stress, This can be done through the expression of new genes. Benefits may be realized through increased tolerance to freezing temperatures through the introduction of "antifreeze" proteins such as Winter @ Flounder (Cutler et al., 1989) or synthetic gene derivatives thereof. Improved cold tolerance can also be provided through increased expression of glycerol-3-phosphate acetyltransferase in chloroplasts (Wolter et al., 1992). Resistance to oxidative stress (often exacerbated by conditions such as cold temperatures, in combination with high light intensity) can be conferred by expression of superoxide dismutase (Gupta et al., 1993) and improved by glutathione reductase (Bowler Et al., 1992). Such a strategy may take into account resistance to freezing in newly emerging regions, as well as spreading slower maturation, higher yielding species to relatively earlier maturation zones.
[0212]
It is expected that expression of novel genes that advantageously provide water content, total water potential, osmotic potential and turgor pressure of the plant will enhance the plant's ability to withstand drought. As used herein, the terms "drought resistance" and "drought tolerance" refer to the stress induced by a decrease in water availability when compared to a normal environment. It is used to refer to the increased resistance or tolerance of a plant, and the ability of the plant to function and survive in a less watery environment. In this aspect of the invention, it is proposed, for example, that expression of a gene encoding the biosynthesis of an osmotically active solute (eg, a polyol compound) provides protection against drought. In this class there are genes encoding mannitol-L-phosphate dehydrogenase (Lee and Saier, 1982) and genes encoding trehalose-6-phosphate synthase (Kaasen et al., 1992). Through the subsequent action of the native phosphatase in the cells or through the introduction and co-expression of specific phosphatases, these introduced genes each result in the accumulation of either mannitol or trehalose, both of which are stress-induced. Are sufficiently shown as protective compounds that can mitigate the effects of Mannitol accumulation in transgenic tobacco has been demonstrated, and preliminary results indicate that plants expressing high levels of this metabolite can tolerate the applied osmotic stress (Tarczynski et al., 1992, 1993).
[0213]
Similarly, the efficacy of either other metabolites (eg, alanopine or propionic acid) in protecting enzyme function or other metabolites (eg, alanopaine) in protecting membrane integrity is (Loomis et al., 1989), and therefore, expression of the genes encoding the biosynthesis of these compounds may confer drought resistance in a manner similar or complementary to mannitol. Other examples of naturally occurring metabolites that are osmotically active and / or provide some direct protective effect during drought and / or desiccation include fructose. Erythritol (Coxson et al., 1992), sorbitol, dulcitol (Karsten et al., 1992), glucosylglycerol (Reed et al., 1984; Erdmann et al., 1992), sucrose, stachyose (Koster and Leopold, 1988; Blackman et al., North Carolina), La. (Bernal-Lugo and Leopold, 1992), proline (Rensburg et al., 1993), glycine, betaine, ononitol and pinitol (Ve). non and Bohnert, 1992), and the like. Increased reproductive fitness during periods of continuous canopy growth and stress is enhanced by the introduction and expression of genes (eg, the osmotically active compounds described above and genes controlling other such compounds). Is done. Currently, preferred genes that promote the synthesis of osmotically active polyol compounds are those encoding enzymes (mannitol-1-phosphate dehydrogenase, trehalose-6-phosphate synthase and myo-inositol @ 0-methyltransferase). .
[0214]
It is expected that expression of certain proteins will also increase drought tolerance. Three classes of late embryogenic proteins have been assigned based on structural similarity (see Dure et al., 1989). All three classes of LEA have been demonstrated to be present in mature (ie, dried) seeds. In these three classes of LEA proteins, type II (dehydrin type) is generally associated with drought and / or drought tolerance in vegetative plant parts (ie, Mundy and Chua, 1988; Piatkowski et al., 1990; Yamaguchi-Shinozaki et al., 1992). Recently, type III LEA (HVA-1) in tobacco has been found to affect plant height, maturity and drought tolerance (Fitzpatrick, 1993). In rice, HVA-1 gene expression affected tolerance to water deficiency and salinity (Xu et al., 1996). Thus, expression of structural genes from all three LEA groups conferred drought tolerance. Other types of proteins induced during water stress include thiol proteases, aldolases, and transmembrane transporters (Guerrero et al., 1990), which provide various protections and / or during drought stress. It may provide a repairable function. It is also expected that genes that achieve lipid biosynthesis, and thereby membrane composition, will also be useful in conferring drought tolerance on plants.
[0215]
Many of these genes for improving drought resistance have complementary modes of action. Thus, it is recognized that combinations of these genes may have additive and / or synergistic effects in improving drought resistance in plants. Many of these genes also improve freezing tolerance (or resistance); the physical stresses experienced during freezing and drought are virtually similar and can be mitigated in a similar manner. Although the benefits may be conferred through the constitutive expression of these genes, preferred means of expressing these novel genes include turgor inducible promoters, such as Guerrero et al., 1990 and Shagan et al., 1993 (see, ), Which is incorporated herein by reference). The spatial and temporal expression patterns of these genes allow plants to better tolerate stress.
[0216]
It is proposed that expression of genes associated with certain morphological traits that would allow increased water uptake from dry soil would be beneficial. For example, the introduction and expression of a gene that alters root characteristics can enhance water uptake. It is expected that expression of genes that enhance reproductive fitness during hours of stress is of significant value. For example, expression of a gene that improves pollen shedding and receptive tune synchrony of the female flower part (ie, silk) can be beneficial. In addition, it is proposed that expression of genes that minimize grain stunting during times of stress can increase the amount of grain harvested and is therefore of value.
[0217]
Given the overall role of water in determining yield, enabling plants to use water more efficiently through the introduction and expression of new genes is not limited by the availability of soil moisture But it is expected to improve overall efficiency. By introducing genes that improve the plant's ability to maximize water use over the full range of stresses on water availability, harvest stability or yield consistency can be realized.
[0218]
(Vi) Disease resistance
It is proposed that increased resistance to disease can be realized through the introduction of genes into plants (eg, monocotyledonous plants (eg, corn)). It is possible to generate resistance to diseases caused by viruses, bacteria, fungi and nematodes. It is also anticipated that control of the mycotoxin producing organism can be achieved through expression of the introduced gene.
[0219]
Resistance to the virus can occur through the expression of new genes. For example, it has been demonstrated that expression of a viral coat protein in transgenic plants can confer resistance to infection of the plant by viruses and possibly other closely related viruses (Cuozzo et al., 1988; Hemenway et al., 1988; Abel et al., 1986). It is expected that expression of antisense genes targeted to essential viral functions may also confer resistance to the virus. For example, an antisense gene targeted to a gene responsive to viral nucleic acid replication can inhibit replication and induce resistance to the virus. It is believed that interference with other viral functions through the use of antisense genes may also increase resistance to the virus. It is further proposed that through other attempts, including but not limited to the use of satellite viruses, it may be possible to achieve resistance to the virus.
[0220]
It is proposed that increased resistance to diseases caused by bacteria and fungi can be achieved through the introduction of new genes. It is expected that genes encoding so-called "peptide antibiotics", proteins that affect pathogenesis-related (PR) proteins, toxin resistance, and host-pathogen interactions (eg, morphological characteristics) will be useful. Peptide antibiotics are polypeptide sequences that are inhibitory to the growth of bacteria and other microorganisms. For example, a class of peptides called cecropin and magainin inhibit the growth of many species of bacteria and fungi. It is proposed that expression of the PR protein in monocotyledonous plants (eg, corn) may be useful in conferring resistance to bacterial diseases. These genes are induced after pathogen attack on host plants and fall into at least five protein classes (Bol, Linthorst and Cornelissen, 1990). β-1′3-glucanases, chitinases, and proteins thought to function in plants as resistance to osmotin and diseased organisms are included in PR proteins. Other genes with antibacterial properties have been identified (e.g., UDA (Lepidoptera lectin) and hevein (Broakaert et al., 1989; Barkai-Golan et al., 1978). It is known that certain plant diseases are caused by the production of phytotoxins. It is proposed that resistance to these diseases is achieved through the expression of novel genes encoding enzymes that can degrade or otherwise inactivate phytotoxins. Expression of a novel gene that alters the interaction between the host plant and the pathogen may increase the ability of the diseased organism to invade host plant tissue (eg, increase leaf cuticular waxiness or other morphology). It is also anticipated that it may be useful in reducing various characteristics.
[0221]
(V) Agricultural characteristics of plants
Two factors that determine where crop plants can be grown are the average daytime temperature during the growing season and the length of time between frosts. In areas where a particular crop can be grown, various restrictions exist as to the maximum time to grow to maturity and allow harvesting. For example, varieties grown in a particular area are selected for their ability to mature in the required time, with the maximum possible harvest, and dry down to a harvestable moisture content. Thus, crops of varying maturity are developed for different habitats. It is desirable to have maximum drying in the field to minimize the amount of energy required for further drying after harvesting, except for the need to dry down sufficiently to allow harvesting. Also, the more easily the product (eg, grain) can dry down, the longer the time available for growth and grain filling. Genes that affect maturation and / or drydown can be identified and introduced into plant lines using transformation techniques, applied to different or same habitats, but harvesting for moisture during harvesting It is conceivable to create new varieties with improved high ratios. Expression of genes involved in the regulation of plant development can be particularly useful.
[0222]
It is anticipated that genes that can improve standability and other plant growth characteristics can be introduced into plants. Expression in plants of new genes that give stronger stalks, improved root systems, or prevent or reduce ear drop, is of great value to farmers. For example, it is proposed that the introduction and expression of genes that increase the total amount of photosynthetic products that can be obtained by increasing light distribution and / or interruption be advantageous. In addition, expression of genes that increase photosynthetic efficiency and / or canopy further increases productivity. It is expected that expression of the phytochrome gene in crop plants may be advantageous. Expression of such genes can reduce apical dominance, render plants semi-dwarf, and increase negativity (US Pat. No. 5,268,526). Such attempts allow for an increased plant population in the field.
[0223]
(Vi) Use of nutrition
The ability to utilize available nutrients can be a limiting factor in the growth of crop plants. It is proposed that the introduction of new genes can alter the availability of nutrient uptake, tolerance pH extremes, flow across plants, storage pools, and metabolic activity. These modifications allow plants (eg, corn) to use available nutrients more efficiently. For example, it is expected that increased activity of enzymes normally present in plants and involved in nutrient utilization will increase nutrient availability. An example of such an enzyme is phytase. It is further expected that enhanced utilization of nitrogen by plants is desirable. Expression of the glutamate dehydrogenase gene (eg, the E. coli gdhA gene) in plants can induce increased immobilization of nitrogen in organic compounds. Furthermore, the expression of gdhA in plants can induce enhanced resistance to the herbicide glufosinate by incorporation of excess ammonia of glutamate, thus detoxifying ammonia. It is also anticipated that expression of novel genes may make available previously inaccessible nutrient sources, such as enzymes that release nutritious components from more complex molecules (possibly macromolecules). Is done.
[0224]
(Vii) Male sterility
Male sterility is useful in producing hybrid seed. It is proposed that male sterility can be produced via expression of a novel gene. For example, it has been shown that expression of genes encoding proteins that interfere with male inflorescence and / or gamete development results in male sterility. Chimeric ribonuclease genes in anthers of transgenic tobacco and oilseed rape have been demonstrated to induce male sterility (Mariani et al., 1990).
[0225]
Many mutations conferring cytoplasmic male sterility have been found in maize. In particular, one mutation, referred to as T cytoplasm, also correlates with susceptibility to Southern corn summer blight. A DNA sequence termed TURF-13 (Levings, 1990) that correlates with T cytoplasm was identified. It is proposed that it is possible to distinguish male sterility from disease susceptibility through the introduction of TURF-13 via transformation. If it is necessary to be able to restore male sterility for breeding purposes and grain production, it is proposed that a gene encoding male fertility restoration can also be introduced.
[0226]
(Viii) improved nutritional content
Genes can be introduced into plants to improve the nutritional quality or content of a particular crop. Introduction of genes that alter the nutritional composition of crops can greatly enhance feed or food prices. For example, many grain proteins may be suboptimal for feed and food purposes, especially when given to pigs, poultry, and humans. This protein lacks some amino acids that are essential in the diet of these species and requires the addition of supplements to the grain. Restricted essential amino acids can include lysine, methionine, tryptophan, threonine, valine, arginine and histidine. Some amino acids are restricted only after the corn has been supplemented with other inputs for feed formulations. The level of these essential amino acids in seeds and grains is due to the introduction of genes to increase amino acid biosynthesis, to introduce genes to reduce amino acid degradation, and to increase the storage of amino acids in proteins Or the introduction of a gene to increase the transport of amino acids to the seed or grain, but is not limited thereto.
[0227]
The protein composition of a crop can be varied in various ways to improve the amino acid balance, including increasing the expression of native protein, reducing the expression of crops with poor composition, Changing the composition of a novel protein, and introducing genes encoding entirely novel proteins possessing superior composition.
[0228]
Introduction of genes that alter the oil content of crop plants may also have value. Increased oil content can result in increased metabolizable energy content and density of seeds for diet and food use. The introduced gene may encode an enzyme that eliminates or reduces the rate-limiting or controlled steps in fatty acid or lipid biosynthesis. Such genes include, but are not limited to, genes encoding acetyl-CoA carboxylase, ACP-acetyltransferase, β-ketoacyl-ACP synthase, and other well-known fatty acid biosynthetic activities. Another possibility is a gene encoding a protein without enzymatic activity, such as an acyl-carrying protein. Genes that alter the balance of fatty acids present in the oil can be introduced, which provide a healthier or nutritious feed. The introduced DNA may also encode a sequence that blocks the expression of enzymes involved in fatty acid biosynthesis, which alters the proportion of fatty acids present in the crop.
[0229]
For example, by increasing the degree of branching, a gene can be introduced that enhances the nutritional value of the starch component of the crop, resulting in improved utilization of starch in livestock by slowing its metabolism. In addition, other major components of the crop can be varied, including genes that affect various other nutritional, processing, or other quality aspects. For example, pigmentation can be increased or decreased.
[0230]
Feed or food crops can also have insufficient amounts of vitamins and require supplementation to provide sufficient nutritional value. Introduction of genes that enhance vitamin biosynthesis includes, for example, vitamin A, E, B₁₂, And choline. The mineral content can also be suboptimal. Thus, inter alia, genes that affect the accumulation or availability of compounds including phosphorus, sulfur, calcium, manganese, zinc and iron are of value.
[0231]
Many other examples of crop improvement can be used with the present invention. This improvement does not need to be on grain, but may improve crop value, for example, for silage. The introduction of DNA to accomplish this can include sequences that alter lignin production, such as those that result in a "brown central vein" phenotype associated with a predominant feed value for cattle.
[0232]
In addition to direct improvements in feed and food values, genes that improve crop processing and improve the value of the products resulting from processing can also be introduced. If through a wet mill, use the crop. Thus, for example, novel genes that increase efficiency by reducing immersion time and reduce the cost of such processing may also find use. Improving the value of the wet milled product may include changing the amount or quality of the components of the starch, oil, corn gluten flour or gluten diet. Elevating starch can be achieved through the identification and elimination of rate-limiting steps in starch biosynthesis, or by reducing the levels of other components of the crop that result in an increase in the percentage of starch.
[0233]
Oil is another product of wet mills, the value of which can be improved by gene transfer and expression. The properties of the oil improve its effectiveness in producing and using products derived from cooking oils, shortenings, lubricants or other oils, and improving its health-related properties when used in food-related applications Can be changed to Upon extraction, new fatty acids that can function as starting materials for chemical synthesis can also be synthesized. Changes in the properties of the oil may be achieved by altering the type, level, or regulation of the fatty acids present in the oil. In turn, this may be due to the addition of genes encoding enzymes that catalyze the synthesis of new fatty acids and the lipids containing them, or to increase the levels of native fatty acids, while presumably reducing the levels of precursors Can be achieved by Alternatively, DNA sequences that delay or block steps in fatty acid biosynthesis can be introduced, resulting in an increase in precursor fatty acid intermediates. Genes that can be added include desaturases, epoxidases, hydratases, dehydratases and other enzymes that catalyze reactions involving fatty acid intermediates. Representative examples of catalytic steps that can be blocked include desaturation of stearic acid to oleic acid and oleic acid to linolenic acid, which results in the accumulation of stearic acid and oleic acid, respectively. . Another example is the interruption of the extension step, which is a C₈~ C₁₂This produces an accumulation of saturated fatty acids.
[0234]
(Ix) Production or assimilation of chemicals and biologicals
It is contemplated that transgenic plants prepared according to the present invention may be used to produce or produce useful biological compounds that are either not produced at all or not produced at the same level as prior corn plants. And more. Alternatively, plants produced according to the present invention can be made to metabolize certain compounds (eg, hazardous wastes), thereby allowing bioremediation of these compounds.
[0235]
New plants that produce these compounds are enabled by the introduction and expression of one or potentially many genes using the constructs provided by the present invention. Many possible arrays include any biological compound currently produced by any organism (eg, proteins, nucleic acids, early and intermediate metabolites, carbohydrate polymers, enzymes for use in bioremediation, Enzymes to modify pathways that produce secondary plant metabolites (eg, flavonoids or vitamins), enzymes that can produce drugs, and compounds of interest for manufacturers (eg, specialized chemicals and plastics) ), But is not limited thereto. These compounds can be produced by plants, extracted during harvest and / or processing, and of any currently recognized useful purpose (eg, drugs, aromas and some named industrial enzymes). Can be used for
[0236]
(X) Non-protein expression sequence
DNA can be introduced into plants for the purpose of expressing RNA transcripts whose function to affect the plant phenotype has not yet been translated into protein. Two examples are antisense RNA and RNA with ribozyme activity. Both can provide a potential function in reducing or eliminating expression of a native or introduced plant gene. However, as described in detail below, DNA is not necessarily expressed to produce a plant phenotype.
[0237]
(1. Antisense RNA)
The gene can be constructed or isolated, which, when transcribed, produces an antisense RNA that is complementary to all or a portion of the target messenger RNA. This antisense RNA reduces the production of messenger RNA polypeptide products. The polypeptide product can be any protein encoded by the plant genome. The above genes are called antisense genes. Thus, antisense genes can be introduced into plants by transformation methods to produce new transgenic plants with reduced expression of the selected protein of interest. For example, the protein can be an enzyme that catalyzes a reaction in plants. A decrease in enzymatic activity can reduce or eliminate the products of a reaction involving any enzymatically synthesized compound in a plant (eg, fatty acids, amino acids, carbohydrates, nucleic acids, etc.). Alternatively, the protein may be a storage protein (eg, zein) or a structural protein, and reduced expression of these proteins may induce a change in the amino acid composition of the seed or a morphological change of the plant, respectively. The above possibilities are provided by way of example only and do not represent the full scope of application.
[0238]
(2. Ribosome)
Genes can also be constructed or isolated, produce an RNA enzyme (ribozyme) that when transcribed can act as an endoribonuclease, and catalyze the cleavage of an RNA molecule with a selected sequence. Cleavage of selected messenger RNAs can reduce the production of their encoded polypeptide products. These genes can be used to prepare new transgenic plants carrying them. The transgenic plant may possess a reduced level of the polypeptide, including but not limited to the polypeptides listed above.
[0239]
Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes have a specific catalytic domain that retains endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987; Forster and Symons, 1987). For example, many ribozymes promote the transesterification with a high degree of specificity, often cleaving only one of several phosphates in an oligonucleotide substrate (Cech et al., 1981; Michel and Westof, 1990; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the need for the substrate to bind to the internal guide sequence ("IGS") of the ribozyme via a specific base pair interaction prior to chemical reaction.
[0240]
Ribozyme catalysts have been first observed as part of sequence-specific cleavage / ligation reactions involving nucleic acids (Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855 states that certain ribozymes can act as endonucleases with better sequence specificity than known ribonucleases and approach the sequence specificity of DNA restriction enzymes. Report that it can.
[0241]
Several different ribozyme motifs have been described as having RNA cleavage activity (Symons, 1992). Examples include Tobacco @ Ringspot @ Virus (Prody et al., 1986), Avocado @ Sunblot @ Viroid (Palukatis et al., 1979; Symons, 1981) and the sequences from Lucerne @ Transient @ Strak @ Virus, Inc. Including. Sequences from these and related viruses are referred to as hammerhead ribozymes based on the predicted folded secondary structure.
[0242]
Other suitable ribozymes include RNaseＲP-derived sequences having RNA cleavage activity (Yuan et al., 1992, Yuan and Altman, 1994, US Pat. Nos. 5,168,053 and 5,624,824), hairpins. Ribozyme structures (Berzal-Herranz et al., 1992; Chowrira et al., 1993) and Hepatitis @ Deita virus-based ribozymes (US Pat. No. 5,625,047). The general design and optimization of ribozymes directed to RNA cleavage activity has been discussed in detail (Haseloff and Gerlach, 1988, Symons, 1992, Chowrira et al., 1994; Thompson et al., 1995).
[0243]
Another variable for ribozyme design is the choice of cleavage site on a given target RNA. Ribozymes are targeted to a given sequence by virtue of annealing to a site by complimentary base pair interaction. Two extensions of homology are required for this targeting. These extensions of homologous sequences flank the catalytic ribozyme structure defined above. Each extension of the homologous sequence can vary from 7 to 15 nucleotides in length. The only need to define homologous sequences is that in the target TNA they are separated by specific sequences that are cleavage sites. For hammerhead ribozymes, the cleavage site is a dinucleotide sequence for the target RNA, either uracil (U) followed by adenine or cytosine, or uracil (A, C, or U) (Perrimann et al., 1992; Thompson et al., 1995). The frequency of occurrence of this dinucleotide in any given RNA is statistically 3/16. Thus, for a given target messenger RNA of 1,000 bases, a 187 dinucleotide cleavage site is statistically possible.
[0244]
The design and testing of ribozymes for efficient cleavage of target RNA is a process well known to those skilled in the art. Examples of scientific methods for designing and testing ribozymes are described by Chowrira et al. (1994) and Lieber and Strauss (1995), each of which is incorporated by reference. The identification of operational and preferred sequences for use in down-preparing a given gene is a simple matter of preparing and testing a given sequence and is routinely performed and known to those skilled in the art. This is a "screening" method.
[0245]
(3. Induction of gene silencing)
It is also possible that genes can be introduced to produce novel transgenic plants with reduced expression of negative gene products by a mechanism of co-suppression. In tobacco, tomato, and petunia, it has been demonstrated that the expression of the sense transcript of the negative gene reduces or eliminates the expression of the negative gene in a manner similar to that observed with the antisense gene (Goring). Et al., 1991; Smith et al., 1990; Napoli et al., 1990; van der Kroll et al., 1990). The introduced gene may encode all or part of the targeted negative protein, but its translation may not be required for reduced levels of the negative protein.
[0246]
(4. Non-RNA-expressing sequence)
DNA elements, including those that are transposable elements such as Ds, Ac, or Mu, can be inserted into genes and cause mutations. These DNA elements can be inserted to inactivate (or activate) a gene, and thereby "tag" a particular trait. In this example, the transposable element does not cause instability of the tagged mutation. Because the use of this element does not depend on its potential to move in the genome. Once the desired trait has been tagged, the introduced DNA sequence is used to clone the corresponding gene, for example, using the introduced DNA sequence as a PCR primer in conjunction with PCR gene cloning techniques. (Shapiro, 1983; Dellaporta et al., 1988). Once identified, all genes for a particular trait, including the desired control or regulatory regions, can be isolated, cloned, and manipulated as desired. For the purpose of gene tagging, the availability of a DNA element introduced into an organism is independent of the DNA sequence and may be used for any biological activity of the DNA sequence, ie, transcription into RNA or translation into protein. Not dependent. The sole function of this DNA element is to divide the DNA sequence of the gene.
[0247]
It is anticipated that unexpressed DNA sequences, including novel synthetic sequences, can be introduced into cells as an exclusive "label" of those cells and plants and their seeding. It is not necessary that the labeled DNA element disrupts the function endogenous to the host organism. Because the sole function of this DNA must identify the origin of the organism. For example, a unique DNA sequence can be introduced into a plant, and the DNA element identifies all cells, plants, and progeny of these cells, as derived from the labeled source. It is proposed that the inclusion of labeled DNA makes it possible to distinguish between proprietary germplasm or germplasm from such unlabeled germplasm.
[0248]
Another possible element that can be introduced is the matrix attachment region element (MAR), such as the chicken lysozyme A element (Stief, 1989), which can be positioned around the expressible gene of interest. May increase the overall expression of and reduce position-dependent effects upon integration into the plant genome (Stief et al., 1989; Phi-Van et al., 1990).
[0249]
(5. Others)
Other examples of non-protein-expressed sequences specifically envisioned for use with the present invention include, for example, tRNA sequences to alter codon usage, and, for example, resistance to various agents (eg, antibodies). RRNA variants that can be given are included.
[0250]
(IX. Biologically functional equivalents)
Improvements and modifications can be made in the centromeric DNA segment of the present invention, and still obtain a functional molecule with the desired characterization. The following is a discussion based on altering the centromere nucleic acid to create the equivalent, ie, an improved, secondary product molecule.
[0251]
In certain embodiments of the invention, the mutated centromere sequence is expected to be useful for increasing the utilization of the centromere. It is specifically anticipated that the function of the centromere of the present invention may be based on the secondary structure of the DNA sequence of the centromere and / or the protein that interacts with the centromere. By altering the DNA sequence of the centromere, the affinity of one or more centromere-related proteins for the secondary structure of the centromere and / or the centromere sequence can be altered, thereby altering the activity of the centromere. Alternatively, alterations can be made in the centromeres of the present invention that do not carry out the activity of this centromere. Alterations in the centromere sequence that reduce the size of the DNA segment required to confer centromere activity are expected to be particularly useful in the present invention. The alteration is because the centromere increases the fidelity transferred during mitosis and meiosis.
[0252]
(X. plant)
The term "plant" as used herein refers to any type of plant. We have provided below an illustrative description of some plants that can be used in the present invention. However, this listing is not in any limiting fashion. This is because other types of plants are known to those skilled in the art and can be used in the present invention.
[0253]
Common categories of plants used in agriculture include vegetable gardening (Korean thistle, kohlrabi, Kibanasushiro, western leek, asparagus, lettuce (eg, head, leaf, romaine), Chinese cabbage, taro, broccoli, melon ( For example, muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brassels bud, cabbage, cardoni, carrot, Chinese cabbage, cauliflower, okra, onion, celery, parsley, chickpea, American bowfish, chicory, Chinese cabbage , Pepper, collard, potato, cucumber plants (green beans, cucumber), squash, cucumber, radish, dried bulb onion, kabuha button, eggplant, salsify, escalor, shallot, endive, garlic, spinach, leek, pumpkin, green Things (green), sugar beet, is (sugar beet and fodder beet), including sweet potatoes, Swiss chard, horseradish, tomatoes, kale, turnip, and spices).
[0254]
Other types of plants that often find commercial use include fruits and vine crops (e.g., apples, apricots, cherries, nectarines, peaches, pears, plums, quince almonds, chestnuts, hazelnuts, becans, pistachios, walnuts, Citris, blueberries, boysenberries, cranberries, raisins, loganberries, raspberries, strawberries, black strawberries, grapes, avocados, bananas, kiwis, oysters, pomegranates, pineapples, tropical fruits, pears, melons, mangos, papayas and litchis) Including.
[0255]
Many of the most widely amplified plants are crop plants (eg, evening primrose, meadowfoam, cereals (field, sweet, popcorn), hops, jojoba, peanuts, rice, safflower, small grains (barley, oats, Rye, wheat, etc.), sorghum, tobacco, kabok, legume plants (bean, lentils, peas, soybeans), oil plants (oilseed rape, mustard, opium, olives, sunflowers, coconut, castor oil plants, cacao) Nuts, peanuts), fiber plants (cotton, flax, hemp, jute), camphoraceae (cinnamon, camphor), or plants (eg, coffee, sugar cane, tea, and natural rubber plants).
[0256]
Still other examples of plants include flower bed plants (eg, flowers, cacti, succulent multiplex plants, and decorative plants) and trees (eg, forests (evergreens such as hardwoods and conifers), fruits, decorative trees, and Tree-bearing trees, as well as shrubs and other seedlings).
[0257]
(XI. Definition)
As used herein, the term "autonomous folding sequence" or "ARS" or "origin of replication" refers to the origin of DNA replication recognized by proteins that initiate DNA replication.
[0258]
As used herein, the term “binary BAC” or “binary bacterial artificial chromosome” is required for Agrobacterium-mediated transformation (eg, Hamilton et al., 1996; Hamilton, 1997; and Liu et al., 1999). Bacterial vector containing a unique T-DNA border sequence.
[0259]
As used herein, the term "candidate centromere sequence" refers to a nucleic acid sequence that is intended to be assayed for potential centromere function.
[0260]
As used herein, "centromere" refers to any DNA sequence that confers the ability to separate into daughter cells via cell division. In one circumstance, this sequence is from about 1% to about 100% (about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the daughter cells). , Or about 95). Such alterations in separation efficiency find important application within the scope of the present invention. For example, a mini-chromosome transport centromere that provides 100% stability may be maintained on all daughter cells in the selection, while a mini-chromosome transport centromere that provides 1% stability may be transiently present in the transgenic organism. But can be eliminated if desired. In certain embodiments of the invention, the centromere provides a stable separation of nucleic acid sequences and, via mitosis or meiosis (including both mitosis and meiosis), a recombinant construct comprising the centromere including. Plant centromeres are not necessarily plant-derived, but have the ability to promote DNA separation in plant cells.
[0261]
As used herein, the term "centromere-associated protein" refers to a protein encoded by the sequence of the centromere or a protein encoded by host DNA and which binds to the centromere with relatively high affinity.
[0262]
As used herein, "eukaryote" refers to a living organism whose cells contain a nucleus. Eukaryotes can be distinguished from "prokaryotes", which are organisms that lack nuclei. Eukaryotes and prokaryotes essentially differ in the way their genetic information is organized and in their pattern of RNA and protein synthesis.
[0263]
As used herein, the term "expression" refers to the process by which a structural gene produces an RNA molecule, typically referred to as messenger RNA (mRNA). This mRNA is typically, but not always, transferred to a polypeptide.
[0264]
As used herein, "genome" refers to a DNA sequence that contains all of the genes and genetic information within a given cell of an organism. Usually, this means information contained within the nucleus, but is taken to include organelles as well.
[0265]
As used herein, the term "higher eukaryote" refers to a multicellular eukaryote, typically characterized by its more complex physiological mechanisms and relatively large size. In general, complex organisms such as plants and animals fall into this category. Preferred higher eukaryotes transformed by the present invention include, for example, monocotyledonous and dicotyledonous angiosperm species, gymnosperm species, fern plant species, plant tissue culture cells of these species, animal cells and algae Cells. It is of course understood that eukaryotes and prokaryotes can be transformed by the methods of the present invention as well.
[0266]
As used herein, the term "host" refers to an expression vector containing any organism or plant chromosome that is a replica of a replicable plasmid. Ideally, the host strain used for cloning experiments should be free of any restriction enzyme activity that can degrade the foreign DNA used. Preferred examples of host cells for cloning useful in the present invention include bacteria (eg, Escherichia coli, Bacillus subtilis, Pseudomonas, Streptomyces, Salmonella) and yeast cells (eg, S. cerevisiae). Host cells that can be targeted for minichromosome expression can be plant cells of any source, and specifically Arabidopsis, corn, rice, sugar cane, sugar corn, barley, soybean, tobacco, wheat, tomato. , Potatoes, citris, or any other agriculturally or scientifically important species.
[0267]
As used herein, “hybridization” refers to complementary RNA and DNA strand pairs that produce RNA-DNA hybrids, or generally different to produce double-stranded DNA molecules. Or a pair of two DNA single strands from the same source.
[0268]
As used herein, the term "linker" generally refers to a DNA molecule that is up to 50 or 60 nucleotides in length and that is chemically synthesized or cloned from another vector. In a preferred embodiment, the fragment contains one, and preferably more than one, restriction enzyme sites for blunt and twisted cleavage enzymes (eg, Bam @ HI). One end of the linker fragment is adapted to be connectable to one end of the linear molecule, and the other end is adapted to be connectable to the other end of the linear molecule. Is done.
[0269]
As used herein, a "library" is a pool of random DNA fragments to be cloned. In principle, any gene can be isolated by screening the library with specific hybridization probes (see, eg, Young et al., 1977). Each library may contain the DNA of a given organism inserted as fragments produced with dispersed restriction enzymes or as randomly oriented fragments in thousands of plasmid vectors. For the purposes of the present invention, E. coli, yeast and Salmonella plasmids are particularly useful when the genomic insert is from another organism.
[0270]
As used herein, the term "lower eukaryote" refers to eukaryotes characterized by relatively simple physiology and compositions, and most often single cells.
[0271]
As used herein, a "mini-chromosome" is a recombinant DNA construct that contains a centromere and is capable of transferring to daughter cells. The stability of this construct through cell division is about 1% to about 100% (5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95%). The minichromosome construct can be a cyclic or linear molecule. It may contain one or more elements such as telomeres, ARS sequences and genes. The number of such sequences included is limited only by the physical size limitations of the construct body. Although it may include DNA from natural chromosomes, it may be preferable to limit the amount of DNA to the minimum required to obtain a separation efficiency in the range of 1-100%. The minichromosome can be inherited through mitosis or meiosis or through both mitosis and meiosis. As used herein, the term mini-chromosome refers to the term "plant artificial chromosome" or "PLAC" and to PLACs or plant artificial chromosomes that specifically apply to constructs within the meaning of the term mini-chromosome Include and include all technologies that
[0272]
As used herein, "mini-chromosome-encoded protein" means the polypeptide encoded by the mini-chromosome sequence of the present invention. This includes sequences (eg, selectable markers, telomeres, etc.), as well as those proteins encoded by any other selected functional gene on this minichromosome.
[0273]
"180 base pair repeats" is defined as any one of the specific repeats disclosed in SEQ ID NOs: 184-212, or a "continuous" sequence derived therefrom. Thus, a given "repeat of 180 base pairs" may include more or less than 180 base pairs and may reflect sequences not represented by any of the specific sequences provided therein.
[0274]
As shown herein, the term "plant" includes plant cells, plant protoplasts, plant calli, and the like, as well as whole plants reproduced therefrom.
[0275]
As used herein, the term “plasmid” or “cloning vector” refers to covalently closed circular extrachromosomal or linear DNA (which can repeat itself autonomously in a host cell; And are usually non-essential for cell survival). A wide variety of plasmids and other vectors are known and commonly used in the art (eg, Cohen et al., US Pat. No. 4,468,464, which discloses examples of DNA plasmids). And specifically incorporated herein by reference)).
[0276]
As used herein, a “probe” is any biological reagent (usually tagged in the same way for ease of identification), a gene, gene product, DNA segment or Used to identify or isolate a protein.
[0277]
As used herein, the term "recombinant" refers to any gene exchange, including DNA strand breaks and recombination.
[0278]
As used herein, "regulatory sequence" refers to any DNA sequence that affects the efficiency of transcription or translation of any gene. The term includes, but is not limited to, promoters, enhancers and terminators.
[0279]
As used herein, a "selectable marker" is a gene whose presence most often results in a defined phenotype and growth advantage for cells containing this marker. This growth advantage may be present under standard conditions, altered conditions such as elevated temperatures, or in the presence of certain chemicals such as herbicides or antibodies. The use of selectable markers is disclosed, for example, in Broach et al. (1979). Examples of selectable markers include the thymidine kinase gene, the cellular adenine phosphoribosyltransferase gene and the dihydrylfolate reductase gene, the hygromycin phosphotransferase gene, the bar gene and the neomycin phosphotransferase gene, among others. Preferred selectable markers in the present invention include genes whose expression confers antimicrobial or herbicide resistance to host cells. This gene is sufficient to allow for the maintenance of the vector in the host cell, and to facilitate manipulation of the plasmid in new host cells. Particular objects of the invention are, for example, proteins that confer cellular resistance to ampicillin, chloramphenicol, tetracycline, G-418, bialaphos, and glyphosate.
[0280]
As used herein, a "screening marker" is a gene whose presence results in an identifiable phenotype. This phenotype may be observable under standard conditions, altered conditions such as elevated temperature, or in the presence of the particular chemical used to detect this phenotype.
[0281]
As used herein, the term "site-specific recombination" refers to any gene exchange involving disruption and recombination of DNA strands at a particular DNA sequence.
[0282]
As used herein, a "structural gene" is a sequence that encodes a polypeptide or RNA, including the 5 'and 3' ends. The structural gene is from the host or another species into which the structural gene is transformed into the host. Structural genes are preferred, but not required, and include one or more regulatory sequences (eg, promoters, terminators or enhancers) that regulate the expression of the structural gene. Structural genes are preferred, but not required, and confer some useful phenotype (eg, herbicide resistance) for the organism containing the structural gene. In one embodiment of the invention, the structural gene may encode an RNA sequence that is not translated into a protein (eg, a tRNA or rRNA gene).
[0283]
As used herein, the term "telomere" refers to capping the ends of a chromosome, thereby preventing chromosome end degradation, ensuring replication, and preventing fusion to other chromosomal sequences. Means the sequence to be obtained.
[0284]
As used herein, the term "transformation" or "transfection" refers to the acquisition of new DNA in a cell via chromosomal or extrachromosomal addition of DNA. This is the process by which naked DNA, protein-coated DNA, or whole minichromosomes are introduced into cells by the process, resulting in potential genetic changes.
[0285]
(XII. Example)
The following example includes demonstrating a preferred embodiment of the present invention. The present technology is disclosed in embodiments in accordance with the exemplary technology disclosed by the present inventor and provides sufficient functionality in the practice of the present invention, and thus may be considered to construct a preferred mode for its practice. It should be understood by those skilled in the art. However, one of ordinary skill in the art, in light of the present disclosure, will appreciate that many modifications may be made in the specific embodiments disclosed and similar or similar results without departing from the spirit, scope and scope of the invention. It should be understood that can still be obtained. More specifically, it is clear that certain agents that are chemically and physiologically relevant can be substituted for the agents described herein, while achieving the same or similar results. . All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
[0286]
(Example 1)
(Production of Arabidopsis thaliana mapping population)
To generate a pollen donor plant, the two parental transport qrls are crossed with each other. The qrtl-1 allele was on the Landsberg ecotype background, and the qrtl-2 allele was on the Columbia ecotype background. The Landsberg ecotype is easily identifiable from the Columbia ecotype. Because it transports a recessive mutation, erecta, resulting in stems to chickens, more compact flowering, and more round and smaller leaves than wild type. To utilize this as a marker for the donor plant, the qrtl-2 allele was crossed to the artl-1 female stigma. F₁Progeny are heterozygous for all molecular markers, but this progeny maintain the quartet phenotype of the fused allele grain quadruple. In addition, the ERECTA phenotype of the Columbia plant is displayed. This visible marker serves as an identification that the crossover is successful in generating plants that segregate ecotype-specific markers. Further testing was performed on donor plants by performing PCR analysis to ensure that the progeny were heterozygous at the molecular locus.
[0287]
Due to the fact that pollen grains cannot be assayed directly for marker separation, and because of the desire to create a long-term source that can be used for multiple marker assays, the It was necessary to cross the molecules. This created a set of progeny plants that gave rise to both large amounts of tissue and seeds. These crosses were effectively achieved by generating recipient plants homozygous for male sterility (ms1). Recessive mutant ms1 was selected to prevent recipient plants from self-fertilization and progeny being mistaken for tetrad plants. Due to the fact that homozygous plants do not self-fertilize, the progeny produced by heterozygous male sterility 1 plants, from which sterile recipient plants can be selected, are maintained. Need to be done.
[0288]
(Example 2)
(Tetrad pollination)
Tetrad pollination was performed as follows. Mature flowers were removed from the donor plants and tapped on glass microscope slides to release mature tetrad pollen grains. The slide was then placed under a 20-40X Zeiss dissecting microscope. A small wooden dowel was used to isolate individual tetrad pollen grains, and this dowel was fitted with rubber glue and eyebrows. Using a light microscope, a tetrad pollen unit was selected and brought into contact with the eyebrows. The tetrad attaches preferentially to the eyebrows and is therefore picked up from the microscope slide and transported to the stigma surface of the recipient plant. This transfer was performed without the use of a microscope, then the tetrad attached eyebrows were placed against the stigma surface of the recipient, and the hair was dragged by hand across the stigma surface. The tetrad then preferentially adhered to the stigma of the recipient and completed cross-pollination.
[0289]
Initially, a set of 57 tetrad seeds, each consisting of 3-4 seeds, was collected. Plants were grown from these sets of tetrad seeds and tissues were harvested. DNA was extracted from a small amount of stored tissue for PCR-based separation analysis. In addition, a visible separation of the erecta phenotype was recorded. If the plant gives rise to seeds, the seeds were collected as a source of the large amounts of DNA required to analyze RFLP isolation by Southern blotting.
[0290]
(Example 3)
(Preparation and analysis of centromere-spanning-contig)
Previously, DNA fingerprinting and hybridization analysis of two bacterial artificial chromosome (BAC) libraries led to the construction of a physical map covering almost all of a single copy portion of the Arabidopsis genome (Marra et al., 1999). . However, the presence of repetitive DNA (including 180 bp repeats, retroelements, and intermediate repeats) near the Arabidopsis centromere complicated efforts to anchor the centromere BAC contig to specific chromosomes (Murata et al.). Helop-Harrison et al., 1999; Brandes et al., 1997; Franz et al., 1998; Wright et al., 1996; Konniecznyt et al., 1991; Pelissier et al., 1995; Voytas and Ausubel, 1988; Chye et al., 1997; Richards et al., 1991; Simens et al., 1988; Thompson et al., 1996; Pelissier et al., 1996). We used gene mapping to unambiguously assign these non-fixed contigs to specific centromeres, and used crossovers (Copenhaver et al., 1998) to give information about the entire genome, and used 48 plants. The polymorphic markers were recorded in. In this way, several centromere contigs were associated with a physical map of chromosome arms (see Example 6 and Table 4), and a large set of DNA markers defining centromere boundaries was generated. DNA sequence analysis confirmed the structure of the contig for chromosomes II and IV (Lin et al., 1999).
[0291]
CEN2 and CEN4 were specifically selected for analysis. Both are on structurally similar chromosomes, have a 3.5 Mb rDNA array on their distal end, have regions measuring 3 Mb and 2 Mb, respectively, between rDNA and centromere, and It has 16 Mb and 13 Mb regions on their long arms (Copenhaver and Pikaard, 1996).
[0292]
Centromere analysis was performed at the nucleotide level using substantially complete and annotated sequences on chromosomes II and IV (http://www.ncbi.nlm.nih.gov/Entrez/nucleotides). .Html). Sequence composition was analyzed within the genetically defined centromere boundaries and compared to adjacent peri-centromeric regions (FIGS. 12A-T). Analysis of the two centromeres facilitated comparison of sequence patterns and identification of conserved sequence elements.
[0293]
The centromere sequence was found to have a 180 bp repeat. These sequences were found to be present in the gap of each centromere contig (FIGS. 3, 12B, 12L), with few repeats in other parts of the genome and no long arrays. . BAC clones near these gaps have terminal sequences corresponding to repetitive elements that are likely to make up the majority of the DNA between the contigs (including 180 bp repeats, 5SΔrDNA, or 160 bp repeats) (FIG. 3). Fluorescence in situ hybridization indicated that these repeats were abundant components of Arabidopsis centromeres (Murata et al., 1997; Heslop-Harrison et al., 1999; Brandes et al., 1997). Gene mapping and pulsed-field gel electrophoresis show that many 180 bp repeats are present in long arrays measuring between 0.4 and 1.4 Mb in the centromere region (Round et al., 1997); sequence analysis Revealed more scattered copies near the gap. We specifically contemplate the use of such 180 bp repeats for the construction of minichromosomes. The annotated sequences on chromosomes II and IV identify regions with homology to intermediate repeat DNA, both present within the functional centromere and in adjacent regions (FIGS. 12B-12E and 12L-12O). .
[0294]
In the 4.3 kb sequencing region, including CEN2 and the 2.8 Mb sequencing region (including CEN4), retrotransposon homology was found to account for more than 10% of the DNA sequence. The maximum is 62% and 70%, respectively (FIG. 12C, 12M). Sequences with similarities to the transposon or intermediate repeat elements were found to occupy a similar band, but were less common (up to 29% and 11% for chromosome II, IV, respectively). % Density (FIGS. 12D-12E and 12B-12O). Finally, unlike in the case of Drosophila and Neurospora centromeres (Sun et al., 1997; Cambareri et al., 1998), in Arabidopsis centromeres, low complexity DNA (microsatellites, homopolymeric areas, and AT-rich isocores). ) Was not found to be enriched. Near CEN2, the density of simple repeats is comparable to that on the distal chromosome arm, accounting for 1.5% of the sequence in the centromere, 3.2% in the flanking region, and 20% in length. ３319 bp (average 71 bp). Except for the insertion of mitochondrial DNA at CEN2, the DNA in and around the centromere did not contain large regions that deviated significantly from the genomic mean of about 64% A + T (FIGS. 12F, 12P) (Bevan et al., 1999).
[0295]
Unlike the 180 bp repeat, in the vicinity of CEN2 and CEN4, all other repetitive elements were less abundant than the flanking regions within the genetically defined centromere. High concentrations of repetitive elements outside of the functional centromere domain suggest that centromere activity may be insufficient. Thus, identifying segments of the Arabidopsis genome that were enriched in these repeats did not pinpoint regions that provide centromere function; a similar situation exists in the genomes of other higher eukaryotes. It can happen.
[0296]
The repetitive DNA adjacent to the centromere can play an important role, forming an altered chromatin structure that acts to aggregate or stabilize the centromere structure. Alternatively, other mechanisms may result in the accumulation of repetitive elements near the centromere. Although evolutionary models predict that repetitive DNA accumulates in regions of low recombination (Charlesworth et al., 1986; Charlesworth et al., 1994), many Arabidopsis repetitive elements are more active in recombination than the centromere itself. Abundant in the centromere area. Alternatively, retroelements and other transposons can be inserted preferentially into regions adjacent to the centromere, or can be removed from the rest of the genome at a higher rate.
[0297]
(Example 4)
(Gene mapping of centromere)
To map centromeres, F that is heterozygous for hundreds of polymorphic DNA markers₁Plants were generated by crossing quartet mutants from the Landsberg and Columbia ecotypes (Chang et al., 1988; Ecker, 1994; Konieczy and Ausubel, 1993). In the tetrads from these plants, the genetic markers segregate in a 2: 2 ratio (FIG. 6; Preuss et al., 1994). Separation of the markers was then performed on Landsberg homozygotes using F₁It was determined in plants produced by crossing four pollen molecules from plants. The genotype of pollen grains in the tetrad was inferred from the genotype of the progeny. First, seeds were produced from more than 100 successful tetrad pollinations, and tissues and seeds were collected from 57 of these. This provided enough material for PCR as well as the seeds needed to generate the large amounts of tissue needed for Southern hybridization and RFLP mapping. To obtain a more accurate positioning of the centromere, the original tetrad pollination was increased from 57 tetrads to more than 1000 tetrads.
[0298]
PCR analysis was performed to determine marker separation. To account for the contribution of the Landsberg background from the dams, the Landsberg complement of each of the four tetrad plants or one was subtracted. As shown in FIG. 5, markers from sites throughout the genome were used for comparison with all other marker pairs. The tetra-form indicates an intersection between one or both markers and their centromeres, while the dimorphism indicates that there was no intersection (or the presence of a double intersection).
[0299]
Thus, at every locus, the resulting diploid progeny was either L / C or C / C. The maps generated in these plants, unlike existing maps (representing the average of recombination in both males and females), are based solely on male meiosis. Therefore, some well-established genetic distances were recalculated. This determines whether the recombination frequency has changed significantly.
[0300]
The large amount of genetic data obtained from the analysis must be compared in pairs to perform a tetrad analysis. All of these data were maintained in the Microsoft @ Excel spreadsheet format, assigning the Landsberg allele to a value of "1" and the Columbia allele to a value of "0". In the tetrad, the segregation of markers on one chromosome was compared to a centromere-linked reference locus on a different chromosome (see Table 2 below). The PD, NPD, and TT tetrads were easily distinguished by values of 2, 0, and 1, respectively, by multiplying the value of each locus by the appropriate reference and adding the results for each quad.
[0301]
Monitoring the location of crossings in this population identified regions of the chromosome that could be recombinantly isolated from the centromere (tetratype) and regions that always cosegregate with the centromere (type 2) (Copenhaver et al., 1988; Copenhaver). Et al., 1999). In the centromere, the tetratype frequency was reduced to zero; consequently, the centromere boundary was defined as a position exhibiting a small but detectable number of tetratype patterns. By recording the segregation of centromere linkage markers in about 400 tetrads, centromeres 1-5 were located in regions on the physical map corresponding to 550 kb, 1445 kb, 1600 kb, 1790 kb, and 1770 kb contigs, respectively (FIG. 3). In addition, a number of useful recombinants were identified for each centromere interval. The results of the analysis indicate that centromeres are present within large domains that limit recombination machinery activity, and that the transfer between these domains and surrounding recombination-proficient DNA is significantly more rapid. It was shown that.
[0302]
[Table 2]

Analysis of polymorphisms corresponding to the 180 bp repeats (RCEN marker, Round et al., 1997) confirmed that these repeats were mapped within a genetically defined centromere. Polymorphisms associated with the 180 bp repeat were analyzed by pulsed-field gel electrophoresis as previously described (Round et al., 1997). Separation of these polymorphs in tetrads with informative crosses confirmed the complete linkage of the 180 bp repeat array at each centromere. In genetic units, the centromere interval averaged 0.44 cM (% recombination = 1/2 tetra-type frequency). This reflects that the recombination rate was at least 10 to 30 times lower than the genome average of 221 kb / cM (Somerville and Somerville, 1999; http://nasc.nott.ac.uk/new_ri_map.html). .
[0303]
The low recombination frequencies typically observed near higher eukaryotic centromeres may be due to DNA alterations or abnormal chromatin status (Choo, 1998; Puechbury, 1999; Mahtani and Willard, 1998; Charlesworth et al., 1986; Charlesworth et al., 1994). To alter these conditions, and thus to improve the centromere mapping resolution by increasing the frequency of recombination, cause the F1 Landsberg / Columbia plant to cause DNA damage, alter the chromatin structure, Or treated with one of a series of compounds known to alter DNA modification. F1 Landsberg qrt1 / Columbia qrt1 plants are grown in 1 ″ square pots under light for 24 hours and methanesulfonic acid ethyl ester (0.05%), 5-aza-2′-deoxycytidine (25 or 100 mg / 1), Zeocin (1 μg / ml), methanesulfonic acid methyl ester (75 ppm), cis-diaminedichloroplatinum (20 μg / ml), mitomycin C (10 mg / l), n-nitroso-n-ethylurea ( 100 μM), n-butyric acid (20 μM), trichostatin A (10 μM), or 3-methoxybenzamide (2 mM). The plants were watered and the stems with the flowers were immersed in these solutions. Alternatively, plants were exposed to 350 nm UV (7 or 10 seconds) or heat shock (38 or 42 ° C. for 2 hours). Landsberg stigmas were pollinated 3-5 days after each treatment using pollen tetrads from these plants; subsequently, F1 plants were subjected to further treatment (3-5, up to 5 times per plant). Every day).
[0304]
Tetrads from treated plants were crossed to Landsberg stigma and progeny from 8-107 tetrads subjected to each treatment were collected and analyzed, yielding over 600 additional tetrads. Although the sample size was insufficient to determine whether individual treatments had a significant effect, these tetrads showed higher recombination in the region immediately adjacent to the centromere ( (1.6 vs. 3.4% recombination in untreated and treated plants, respectively). The centromere map location was purified on chromosome 2 to chromosome 5 (FIG. 1). This resulted in intervals spanning contigs of 880 kb, 1150 kb, 1260 kb, and 1070 kb, respectively, and all four molecules consistently mapped centromere function to the same region (Copenhaver et al., 1999).
[0305]
Attempts to increase recombination have resulted in a large number of tetrads with crossovers near the centromere; these crossings were clustered within a narrow region of the centromere border. Five intersections occurred over a 70 kb region near CEN2 and seven occurred over a 200 kb region near CEN1, and no intersections were detected at close centromere intervals of 880 kb and 550 kb, respectively (FIG. 3). Thus, centromeres were found within large domains that limit recombination machinery activity; the transition between these domains and surrounding recombination-skilled DNA is significantly more rapid (FIGS. 12A and K). . Although analysis of more tetrads resulted in additional recombination events, the observed cross-distribution indicates that centromere positions were not significantly purified.
[0306]
(Example 5)
(Sequence analysis of Arabidopsis centromere)
(A. Amount of gene in centromere region)
S. In cerevisiae, the expressed gene is located within a 1 kb essential centromere sequence and In pombe, multiple copies of the tRNA gene are present in an 80 kb fragment required for centromere function (Kuhn et al., 1991). In contrast, in higher eukaryotic centromeres, genes are considered to be relatively rare, with significant exceptions. The Drosophila light, concertina, responder, and rolled loci all map to the centromere region of chromosome 2, and translocations that remove light from its native heterochromatin background inhibit gene expression. In contrast, many Drosophila genes and human genes normally present in euchromatin become inactive when they are inserted near the centromere. Thus, genes located near the centromere are thought to have special regulatory elements that allow expression (Karpen, 1994; Lohe and Hilliker, 1995). The Arabidopsis CEN2 and CEN4 sequences provided herein provide a powerful source for understanding how gene density and expression correlate with centromere location and related chromatin.
[0307]
Annotations on chromosomes II and IV (http://www.ncbi.nlm.nih.gov/Entrez/nucleotide.html) have identified many genes within and close to CEN2 and CEN4 (FIG. 8, FIGS. 12A-12T). The putative gene density on the Arabidopsis chromosome arm averages 25 per 100 kb, and in the repetitive rich region adjacent to CEN2 and CEN4, respectively, this is reduced to 9 and 7 genes per 100 kb (Bevan Et al., 1999). Many putative genes are also present within the recombination-definitive genetically defined centromere. Within CEN2, there were 5 putative genes per 100 kb; while CEN4 was significantly different, 12 genes per 100 kb.
[0308]
There was strong evidence that some of the putative centromere genes were transcribed. The phosphoenolpyruvate gene (CUE1) defines one CEN5 border; and mutations in this gene cause defects in light-regulated gene expression (Li et al., 1995). Within the sequenced portions of CEN2 and CEN4, 17% (27/160) of the putative genes have greater than 95% identity with the cloned cDNA (EST), and are more likely in CEN4 than in CEN2. Matching was three times larger (http://www.tigr.org/tdb/at/agad/). Twenty-four of these genes have multiple exons, and four correspond to single-copy genes with known functions. A list of the putative genes identified is shown in Table 3 below. A list of additional genes encoded within the boundaries of CEN4 is listed in Table 4. The identification of these genes is that the genes may themselves contain unique regulatory elements or may be located at genomic locations adjacent to unique regulatory or regulatory elements involved in centromere function or gene expression. Is significant. In particular, we contemplate the use of these genes, or DNA sequences 0-5 kb upstream or downstream of these sequences, for insertion into selected genes on the minichromosome. It is anticipated that such elements, even when in the unique environment of the centromere, could possibly result in beneficial regulatory control of the expression of these genes.
[0309]
A search was performed in a database of annotated genomic Arabidopsis sequences to determine if the remaining 23 genes were uniquely encoded in the centromere. With the exception of two genes, homologs with greater than 95% identity were not found anywhere in 80% of the sequenced genome. The number of independent cDNA clones corresponding to a single copy gene provides an estimate of the level of gene expression. On chromosome II, a putative gene with high quality matching (greater than 95% identity) to the cDNA database matched on average four independent cDNA clones (range 1-78). Within CEN2 and CEN4, 11 out of 27 genes exceed this average (Table 3). Finally, the genes encoded by CEN2 and CEN4 are not members of a single gene family, and they do not correspond to genes that are predicted to play a role in centromere function, but instead Has a role.
[0310]
Many genes in the Arabidopsis centromere region are not functional due to early stop codons or disrupted open reading frames, but few pseudogenes were found on the chromosomal arms. Most of these pseudogenes have homology to mobile elements, but many typically correspond to genes that are not mobile (FIGS. 12I-J and 12S-T). Within the genetically defined centromere, there were 1.0 (CEN2) and 0.7 (CEN4) of these immobile pseudogenes per 100 kb; the repetitive rich regions adjacent to the centromere were each 100 kb Has 1.5 and 0.9. The distribution of pseudogenes and transposable elements is overlapping, indicating that DNA insertion in these regions contributed to gene disruption.
[0311]
[Table 3]

[0312]
[Table 4]

(B. Storage of Centromere DNA)
PCR primer pairs were designed to study the conservation of CEN2 and CEN4 sequences. These primer pairs correspond to unique regions within the Columbia sequence and are used to probe the centromere region of Landsberg and Colombia at approximately 20 kb intervals (FIGS. 14A, B). The primers used for the analysis are listed in FIGS. Amplified products of appropriate length were obtained in both ecotypes for the majority of primer pairs (85%). This indicates the following. Indicates that the amplified regions are highly similar. In the remaining cases, the primer pairs amplified Columbia DNA but not Landsberg DNA (even at very low stringency). In these regions, additional primers were designed to determine the degree of heterology. In addition to the large insertion of mitochondrial DNA in CEN2, two other non-conserved regions were identified (FIGS. 14A, B). Since this DNA does not contain the Landsberg centromere, it appears necessary for centromere function; therefore, the relevant portion of the centromere sequence was reduced to 577 kb (CEN2) and 1250 kb (CEN4). A high degree of sequence conservation between Landsberg and Columbia centromeres indicated that: It was shown that the suppression of recombination frequency was not due to large regions of heterology but instead was due to the properties of the centromere itself.
[0313]
(C. Sequence similarity between CEN2 and CEN4)
A search was performed for a novel sequence motif between CEN2 and CEN4 to identify centromere function. Searches were performed except for comparisons of retroelements, transposons, characterized centromere repeats, and coding sequences resembling mobile genes. After covering simple repeats (including homopolymer tracts and microsatellites), contigs of unique sequences measuring 417 kb and 851 kb for CEN2 and CEN4, respectively, were compared using BLAST (http: // blast.wustl.edu).
[0314]
This comparison indicated the following: The composite DNA within the centromere region indicated that it was not homologous over the entire sequence length. However, 16 DNA segments in CEN2 were shown to match 11 regions of CEN4 with greater than 60% identity (FIG. 16). The sequences were grouped into families of related sequences, and AtCCS1-7 (Arabidipsis thaliana centromere conserved sequences 1-7) were designed. These sequences were not previously known when repeated in the Arabidopsis genome. This sequence contained a total of 17 kb (4%) of CEN2Δ DNA and had an average length of 1017 bp. And the content of A + T was 65%. Based on similarity, matching sequences were identified as 8 sequences each (AtCCS1 and AtCCS2; SEQ ID NOS: 1-14), 3 sequences (from a small family encoding a putative open reading frame) (AtCCS3; SEQ ID NOs: 21-21). 22), and 4 sequences (found once in the centromere) (AtCCS-AtCCS7; SEQ ID NOs: 15-20), one of the sequences corresponding to the predicted CEN2 and CEN4 proteins (across exons and introns). (AtCCS6; SEQ ID NO: 17) were classified into a group containing two families (FIG. 16).
[0315]
A search of the Arabidopsis genomic sequence database demonstrated the following. It was demonstrated that the AtCCS1 to AtCCS5 appearing in the centromere and the region around the centromere were moderately repeated sequences. This remaining sequence is present only in genetically defined centromeres. All 16 S.D. A similar comparison of cerevisiae centromeres defined the following. A consensus was defined consisting of a conserved 8 bp CDEI motif, an AT-rich 85 bp CDEII element, and a 26 bp CDEII region with 7 highly conserved nucleotides (Fleig et al., 1995). In contrast, three S.D. Examination of the pombe centromere revealed conservation of the overall centromere structure but did not reveal a widely conserved motif (Clark, 1998).
[0316]
(Example 6: Results of mapping: Arabidopsis chromosomes 1 to 5) Centromeres on chromosome 1 were mapped between mi343 (56.7 cM) and T27K12 (59.1 cM). A more sophisticated location places the centromere between markers T22C23-t7 (about 58.3 cM) and T3P8-sp6 (about 59.1 cM). Included in this interval were the previously described markers EKRIV and RCEN1.
[0317]
The centromere on chromosome 2 was mapped between mi310 (18.6 cM) and g4133 (23.8 cM). A more sophisticated location places the centromere between markers F5J15-sp6 (about 19.1 cM) and T15D9 (about 19.3 cM). The following sequenced (http://www.ncbi.nlm.nih.gov/Entrez/nucleotide.html) BAC (bacterial artificial chromosome) clones are known for: It is known to measure the region between the markers F5J15-sp6 and T15D9 (T13E11, F27C21, F9A16, T5M2, T17H1, T18C6, T5E7, T12J2, F27B22, T6C20, T14C8, F7B19 and T15D19).
[0318]
There is a gap in BAC coverage between T12J and F27B22. RARE cleavage, pulsed? Eld gel or DNA sequence tiling is used to isolate DNA in gaps for sequencing.
[0319]
Centromeres on chromosome 3 were mapped between atpox (48.6 cM) and ATA (53.8 cM). A more sophisticated location placed the centromere between the markers T9G9-sp6 (about 53.1 cM) and T5M14-sp6 (about 53.3 cM). Included within this interval is the previously described marker RCEN3.
[0320]
Centromeres on chromosome 4 were mapped between mi233 (18.8 cM) and mi167 (21.5 cM). A more sophisticated location places the centromere between markers T24H24.30k3 (about 20.3 cM) and F13H14-t7 (about 21.0 cM). The following sequenced (hppt: //www.ncbi.nlm.nih.gov/Entrez/nucleotide.html) BAC (bacterial artificial chromosome) clones have the markers F5J15-sp6 and T6A13-sp6 (T27D20, T19B17, T26N6). , F4H6, T19J18, T4B21, T1J1, T32N4, C17L7, C6L9, F6H8, F2I12, F14G16, and F28D6).
[0321]
There is a gap in the BAC cleavage between F2I12 and F14G16. RARE cleavage, pulsed field gel or DNA sequence tiling is used to isolate the DNA in the gap for sequencing.
[0322]
The centromere on chromosome 5 was mapped between nga76 (71.6 cM) and PhyC (74.3 cM). A more sophisticated location places the centromere between markers F13K20-t7 (about 69.4 cM) and CUE1 (about 69.5 cM). Included in this interval are the publicly available markers, um579D, mi291b, CMs1.
[0323]
Tables showing BAC clones known to be located within the centromere on a given chromosome 1-5, and Genbank inventory numbers for the sequences of these clones are shown in Tables 5 and 6 below.
[0324]
The genetic location (ie, cM value) corresponding to Lister and Dean Recombinant Inbled Genetic map is http: // nasc. nott. ac. uk / new_ri_map. Available online at html. Markers are available at http: // genome-www. stanford. edu / Arabidopsis / aboutoutcaps. html.
[0325]
[Table 5]

[0326]
[Table 6]

Example 7 Construction of BAC Vector to Test Centromere Function
BAC clones can be retrofitted with one or more plant telomeres and a selectable marker, together with the DNA elements required for Agrobacterium transformation (FIG. 9). This method provides a means to deliver any BAC clone to plant cells and tests for centromere function.
[0327]
This method works in the following way. The conversion vector includes an inverted root canal filling cassette. This reverse root canal filling cassette is adjacent to Tn10, Tn5, Tn7, Mu or other transposable elements and contains an origin of replication and a selectable marker for Agrobacterium, a plant telomere array followed by a T-DNA right. And a left border, followed by a second plant telomere array, and a plant selectable marker (FIG. 9). This conversion vector contains the E. coli carrying the target BAC. E. coli strains. The transposable elements adjacent to the inverted root canal filling cassette then randomly mediate translocation of the cassette to the BAC clone. This reverse root canal-filled BAC clone can now be transformed into an appropriate Agrobacterium strain and then transformed into a plant re-transformation. Here, high fidelity meiotic and mitotic transmission can be tested, indicating that the clone contained a fully functional plant centromere.
[0328]
Example 8 Construction of Plant Mini Chromosome
The mini chromosome is constructed as follows. Constructed by combining essential chromosomal elements previously isolated. Exemplary mini-chromosome vectors include vectors designed to be "shuttle vectors." The shuttle vector can be maintained in a convenient host, eg, E. coli, Agrobacterium, or yeast, as well as in plant cells.
[0329]
(A. General techniques for mini-chromosome construction)
Minichromosomes are E. coli. In E. coli or other bacterial cells, it can be maintained as a cyclic molecule. It can be maintained by placing with mobile stuffer fragments between telomere sequence blocks. This stuffer fragment is an unwanted DNA sequence, flanked by unique restriction sites. This fragment is removed by restriction digestion of the circular DNA, creating a linear molecule with telomere ends. The linear minichromosome can then be isolated, for example, by gel electrophoresis. In addition to stuffer fragments and plant telomeres, minichromosomes contain an origin of replication and a selectable marker. Selectable markers can function in plants, allowing cyclic molecules to be maintained in bacterial cells. Minichromosomes also contain plant selectable markers, plant centromeres, and plant ARSs, which allow replication and maintenance of DNA molecules in plant cells. Finally, the minichromosome contains several unique restriction sites, where additional DNA sequence inserts can be cloned. For example, the quickest method of physical construction of minichromosomes, ie, the ligation of various essential elements, is collectively clear, for example, to those skilled in the art.
[0330]
A number of minichromosome vectors have been designed by the present inventors and are disclosed herein for illustrative purposes (FIGS. 7A-7H). However, these vectors are not limiting and many changes and modifications can be made, and further functional vectors may be obtained, as will be apparent to those skilled in the art.
[0331]
(B. Altered technique for mini-chromosome construction)
A two-step process was developed for minichromosome construction. This method allows the addition of essential elements to BAC clones containing centromere DNA. These procedures can occur in vivo and eliminate the problem of chromosome breakage. This problem of chromosome breakage often occurs in vitro. The details and advantages of this technique are as follows:
1) One plasmid can be made. This plasmid contains a marker, an origin for Agrobacterium translocation and border sequences in plants, a marker for selection and screening, plant telomeres, and loxP sites or other sites useful for site-specific recombination in vivo or in vitro. Including. The second plasmid can be a BAC clone and is isolated from available genomic libraries (FIG. 11A).
[0332]
2) Single E.C. The two plasmids are mixed, either in E. coli or in vitro, and a site-specific recombinant cre is introduced. This causes the two plasmids to fuse at the loxP site (FIG. 11B).
[0333]
3) If deemed necessary, useful restriction sites (AseI / PacI or NotII) are included to remove excess material (eg, other selectable markers or origins of replication).
[0334]
4) Mutation is Kan^RGenes (with or without) (FIGS. 11B, 11C), including the LAT52ΔGUS gene (with or without), the LAT52GFP gene, and including the GUS gene (FIGS. 11C, 11D and 11E). Under the control of other plant promoters.
[0335]
(C. Method for preparing stable non-integrated minichromosome)
Techniques have been developed to confirm that mini-chromosomes do not integrate into the host genome (FIG. 11F). In particular, the minichromosome must be maintained as a separate element separate from the host chromosome. To confirm that the introduced minichromosome does not integrate, we envision the following diversity. This diversity encodes a lethal plant gene (eg, diphtheria toxin or any other gene product that, when expressed, causes lethality in the plant). This gene may be located between the right Agrobacterium border and the telomere. Minichromosomes that enter the plant nucleus and integrate into the host chromosome result in lethality. However, if the minichromosome remains separate, and further if the ends of the construct are degraded to telomeres, the lethal gene is removed and the cell survives.
[0336]
Example 9 In Vivo Screening of Centromere Activity by Analysis of the Centromere Chromosome
The method was designed for screening for centromere activity (FIG. 10). In this method, a plant is first transformed with a binary BAC clone, which contains DNA for a genetically defined centromere region. By allowing the DNA to integrate into the host chromosome, it is expected that this integration will result in a chromosome with two centromeres. This is an unstable situation. This situation often leads to chromosome breakage. During mitosis and meiosis reduction, a single chromosome carrying two or more functional centromeres frequently breaks at the junction between two centromeres when pulled in opposite poles , Leading to chromosomal damage. This can cause severe growth deletions and non-viable progeny. When genes important or essential for cellular and developmental processes are disrupted by breakage phenomena. Thus, regions with centromere function can be identified by searching for clones. Here, when this clone is introduced into a host plant, it exhibits any of the following expected properties: reduced efficiency of transformation; transformed plants show abnormal sectors and increased lethality Consequences of genetic instability when integrated into natural chromosomes; particularly, difficulty in maintaining transformed plants when grown under conditions that do not select for the maintenance of the transgene; A tendency to integrate into the genome at the tip or in the centromere region. In contrast, clones containing non-centromere DNA are expected to incorporate a more random pattern. Confirmation of the distribution and pattern of the resulting integration can be determined by sequencing the ends of the inserted DNA.
[0337]
Screening was performed by identification of a clone larger than 100 kb encoding centromere DNA in a BiBAC library (a binary bacterial artificial chromosome) (Hamilton, 1997). This is done by a screening filter containing the BiBAC genomic library for clones encoding DNA from centromeres (FIG. 10, step 1). BiBAC vectors are used because they can contain large inserts of Arabidopsis genomic material and encode two-component sequences required for Agrobacterium-mediated transformation. The centromere sequence containing the BiBAC vector was then integrated directly into the chromosome by Agrobacterium-mediated transformation (FIG. 10, step 2). As a control, a BiBAC construct containing non-centromere DNA was also used for transformation. BiBACs carrying sequences with centromere function result in the formation of a centromere chromosome. Progeny from the transformed plants are analyzed for: Analyzes are made for stunting and overall morphological differences that contribute to chromosome destruction due to the formation of the centromere chromosome (FIG. 10, step 3). Non-centromere sequences are expected to show little phenotypic difference from wild-type plants.
[0338]
Example 10 Sophisticated Centromere Mapping with Process for Increased Recombination
To achieve a more sophisticated map location for centromeres in Arabidopsis thaliana, various chemical and environmental treatments were used to stimulate recombination. These treatments were used on cross pollen donors achieved to produce a tetrad set of plants (see Example 2). Pollen donor plants were individually planted in 1 inch square pots and grown in a growth room with 24 hours light until flowering. The flowering plants were then pickled in one of the following solutions and irrigated with 50 ml of the same solution.
[0339]
[Table 7]

After treatment, the plants were then returned to the growth room and grown under standard conditions for 2-5 days. Pollen was then collected from the newly opened flowers and used and pollinated as described in Example 2. The pollen donor plants were then treated again as above and used for another round of pollination. Pollen donor plants were typically subjected to 5-10 treatments and pollen collection.
[0340]
Processing was also performed using non-chemical reagents. As described above, using the treatment, a more sophisticated map location for centromeres in Arabidopsis was achieved by stimulating recombination in additional pollen donor plants. The treatment was as follows.
[0341]
[Table 8]

Heat shock treatment was performed by placing a pot containing pollen donor plants in a shallow dish filled with water (to prevent drying). Then, the dishes containing the plants were placed on a growing machine at an appropriate temperature. The pollen donor plants were subjected to UV irradiation by placing them in a BioRad @ UV chamber, and the plants were irradiated at the appropriate wavelength for different amounts of time. Both UV and heat shock plants were subjected to several treatments and pollen collection. Plants exposed to a gamma radiation source (Cobalt-60) were treated only once and then disposed of to prevent accumulation of harmful chromosomal rearrangements.
[0342]
After treatment, the plants were then returned to the growth room and grown under standard conditions for 2-5 days. Pollen was then collected from the newly opened flowers and used to pollinate susceptible stigmas as described in Example 2. The pollen donor plants were then treated again as described above and used for another round of fertilization. Pollen donor plants were typically subjected to 5-10 treatments and pollen collection. The results are shown in Table 9 below.
[0343]
[Table 9]

Example 11 Promotion of Hereditary Gene Transfer
The following is also contemplated by the inventor. It is also contemplated that recombination stimulating techniques or processes may be used to facilitate gene transfer. Introgression describes breeding techniques whereby one or more desired traits are transferred from another line (B) to one line (A). This trait is then isolated in the genetic background of the desired line (A) by a series of backcrosses to the same line (A). The number of backcrosses required to isolate the desired trait in the desired genetic background depends on the frequency of recombination in each backcross.
[0344]
Backcrossing transfers certain desired traits from one source to inbred lines or other plants that do not have that trait. This can be achieved, for example, by first crossing as follows. By crossing a dominant inbred line (A) (recurrent parent) with a donor inbred line (nonrecurrent parent). They carry the appropriate gene for the trait in question (eg, a construct prepared according to the present invention). The progeny of this cross are first selected in the recurrent offspring for the desired property (transferred from the non-repetitive parent), and the selected progeny is then backcrossed to the predominant repetitive parent (A). Is done. After five or more selected backcross generations for the desired property, the progeny are hemizygous for the locus that modulates the transferred trait, but dominant for most or almost all other genes. Similar to parents. Self-pollinate the last backcross generation to obtain offspring. The progeny are purely bred for the transferred gene (ie, one or more transformation events).
[0345]
Thus, a transgene selected through a series of breeding operations can be transferred from one line to a completely different line without the need for further recombination operations. Transgenes are useful in: Typically, it is useful when acting genetically as any other gene. And can be manipulated by breeding techniques in the same manner as any other corn gene. Thus, inbred plants that truly breed one or more transgenes can be produced. Crossing with different inbred plants can produce many different hybrids with different transgene combinations. In this manner, plants can be produced that have the desired agronomic characteristics often associated with hybrids ("hybrid efficacy") as well as the desired characteristics conferred by one or more transgenes.
[0346]
Breeding can also be used to transfer whole mini-chromosomes from one plant to another. For example, by crossing a first plant with a minichromosome to a second plant without a minichromosome, progeny of any generation of the cross can be obtained. The progeny have minichromosomes or any additional number of desired minichromosomes. Through a series of backcrosses, plants can be obtained. This plant has the genetic background of the second plant, but has the minichromosome from the first plant.
[0347]
All compositions and methods disclosed and claimed herein can be made and made without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention are described in terms of preferred embodiments, the following will be apparent to one of ordinary skill in the art. It will be apparent that modifications may be made below without departing from the concept, spirit and scope of the invention. It will be apparent that the compositions and methods described herein can be applied to and adapted to the steps or sequence of steps of the methods described herein. More specifically, the following is clear. It is clear that certain agents, both chemically and physiologically related, can be substituted for the agents described herein, but that the same or similar results can be achieved. All such similar substitutes and modifications apparent to those skilled in the art are within the spirit, scope and concept of the invention as defined by the appended claims.
[0348]
(References)
The following references are incorporated herein by reference to the extent that they provide the exemplary procedures or other detailed supplements set forth herein.
[0349]
[Table 10]

[Brief description of the drawings]
FIG.
FIG. 1 is a centromere chromosome map for unaligned quadrants: a cross of two parents (AABB × aabb), where “A” is present on the centromere of one chromosome and “B Is linked to the centromere of the second chromosome. In meiosis, the A and B chromosomes associate independently and have the same number of parental ditype (PD) and non-parental ditype (NPD) tetrads (recombinant progeny are gray) Shown). Tetratype (TT) results only from the transfer between "B" and its centromere.
FIG. 2
FIG. 2 is a low resolution map of the location of the Arabidopsis centromere. Trisomic mapping was used to determine the location of a map of four centromeres of the five Arabidopsis chromosomes (Koorneef, 1983; Sears et al., 1970). For chromosome 4, no useful trisomal species was obtained. Using the method of Koorneef and Sears et al., 1983 (which relies on low-resolution deletion mapping), two visible markers (ttl and chl) whose centromeres on chromosome 1 are 5 cM apart. It was found to be located between Centromere locations on other chromosomes are mapped for low resolution.
FIG. 3
FIG. 3 is a physical map of the locations of Arabidopsis centromeres that are genetically defined. Each centromere region is drawn to scale; physical size is derived from DNA sequencing (chromosomes II and IV) or from assessment based on BAC fingerprinting (Marra et al., 1999; Mozo et al., 1999). (Chromosomes I, III, and V). For each chromosome, the location of the markers (top), the number of tetratypes / all quadrants in those markers (bottom), the centromere boundaries (from the fingerprint analysis (Marra et al., 1999; Mozo et al., 1999)) (Thick black bar) and the name of the contig. For each contig, more than two genetic markers (developed from a database of BAC terminal sequences) (http://www.tigr.org/tdb/at/abe/bac)　end　search. html). PCR primers corresponding to these sequences were used to identify size or restriction site polymorphisms in the Columbia and Landberg ecotypes (Bell and Ecker, 1994; Konieczny and Ausubel, 1993); primer sequences are available ( http://genome-www.stanford.edu/Arabidopsis/aboutcaps.html). Tetratype quadrants (boxes) resulting from treatments that stimulate crossing; locations of centimorgan (cM) markers distributed on the recombinant inbred line (RI) map (oval) (http: //nasc.nott) .Ac.uk / new　ri　map. html; Somerville and Somerville, 1999); and sequence boundary gaps (open circles) in the physical map corresponding to the 180 bp repeat (Round et al., 1997), 5S rDNA (black circles) or 160 bp repeats (grey circles) are indicated. Copenhaver et al., 1999).
FIG. 4
FIG. 4 is an exemplary list of seed stocks used for tetrad chromosome analysis in Arabidopsis thaliana. Individual strains are identified by strain number (column B). The quadrant chromosome member number (column A) indicates the source of the quadrant chromosome (ie, T1 indicates the seed from quadrant 1 and its number -1, -2, -3 or -4. Indicates individual members of the quadrants). The listed strains have been deposited with the Arabidopsis Biological Resources Resources Center (ABRC) at Ohio State University under the name Daphne Preuss.
FIG. 5
FIG. 5 shows marker information for creating a centromere map. The DNA polymorphism used to map the centromere is indicated by the chromosome (Column 1). The name of each marker is shown in column 2, the name of the marker used by Copenhaver et al., 1999 to locate the centromere is given in column 3, and the marker type is shown in column 4. CAPS (co-dominant amplification polymorphism site) is a marker that can be amplified by PCR and detected by digestion with appropriate restriction enzymes (also shown in column 3). SSLP (Simple Sequence Length Polymorphism) detects polymorphism by amplifying PCR products of different length. Column 5 specifically mentions when the marker is available on a public website (eg, http://genome-www.sranford.edu/Arabiopsis). For those markers not available on the public website, the sequences of the forward and reverse primers used to amplify the marker are listed in columns 6 and 7, respectively.
FIG. 6
FIG. 6 is a scoring PCR-based marker for quadrant analysis. The progeny genotype from one pollen tetrad chromosome (T2) was determined for two genetic markers (SO392 and nga76). Analysis of four progeny plants using PCR (T2-1 to T2-4) and gel electrophoresis allow the genotype of the plant to be determined and the genotype of the parent pollen to be inferred .
FIG. 7A
FIG. 7A is an exemplary minichromosome vector. The vector shown in FIG. 7A can be high copy number, low copy number or one copy. coli origin of replication. In FIG. 7A, the vector contains a recognition sequence for a conventional restriction endonuclease with a specificity of 4-8 bp as well as very rare cleavage enzymes (eg, I-PpoｐI, I-Cue I, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7A shows E. coli with plant ARS. 1 shows an E. coli plant circular shuttle vector.
FIG. 7B
FIG. 7B is an exemplary minichromosome vector. The vector shown in FIG. 7B may be high copy number, low copy number or one copy. coli origin of replication. In FIG. 7B, the vector contains a conventional restriction endonuclease recognition sequence with 4-8 bp specificity and very rare cleavage enzymes (eg, I-PpoII, I-CueII, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7B shows a plant circle vector without a plant ARS. The vector relies on the plant origin of replication function being found in other plant DNA sequences (eg, selectable or screenable markers).
FIG. 7C
FIG. 7C is an exemplary minichromosome vector. In FIG. 7C, the vector contains a conventional restriction endonuclease recognition sequence with 4-8 bp specificity and very rare cleavage enzymes (eg, I-PpoＰI, I-Cue I, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7C shows a yeast plant circular shuttle vector with a plant ARS. This yeast ARS is contained twice, ensuring that once the large insert is stable on either side of the multiple cloning site.
FIG. 7D
FIG. 7D is an exemplary minichromosome vector. In FIG. 7D, the vector contains a recognition sequence for a conventional restriction endonuclease with a specificity of 4-8 bp as well as very rare cleavage enzymes (eg, I-PpoｐI, I-Cue I, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7D shows a yeast-plant circular shuttle vector without a plant ARS. This vector relies on plant origin for replication functions found in other plant DNA sequences (eg, selectable markers). This yeast ARS is contained twice, ensuring that once the large insert is stable on either side of the multiple cloning site.
FIG. 7E
FIG. 7E is an exemplary minichromosome vector. The vector shown in FIG. 7E can be high copy number, low copy number or one copy. coli origin of replication. In FIG. 7E, the vector contains a conventional restriction endonuclease recognition sequence with 4-8 bp specificity as well as very rare cleavage enzymes (eg, I-PpoＩI, I-Cue I, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7E shows E. coli with plant ARS. 1 shows an E. coli-Agrobacterium plant circular shuttle vector. Vir function for T-DNA transfer is provided in trans, using an appropriate Agrobacterium strain.
FIG. 7F
FIG. 7F is an exemplary minichromosome vector. The vector shown in FIG. 7F may be high copy number, low copy number or one copy. coli origin of replication. In FIG. 7F, the vector contains a conventional restriction endonuclease recognition sequence with 4-8 bp specificity and very rare cleavage enzymes (eg, I-PpoＰI, I-Cue I, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7F shows E. coli without plant ARS. 1 shows an E. coli-Agrobacterium plant circular shuttle vector. This vector relies on plant origin for replication functions found in other plant DNA sequences (eg, selectable markers). Vir function for T-DNA transfer is provided in trans, using an appropriate Agrobacterium strain.
FIG. 7G
FIG. 7G is an exemplary minichromosome vector. In FIG. 7G, the vector contains a conventional restriction endonuclease recognition sequence with 4-8 bp specificity as well as very rare cleavage enzymes (eg, I-PpoｐI, I-Cue I, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7G shows a linear plant vector having a plant ARS. The linear vector can be constructed in vitro and then transferred to the plant, for example, by mechanical means (eg, microprojectile bombardment, electroporation or PEG-mediated transformation).
FIG. 7H
FIG. 7H is an exemplary minichromosome vector. In FIG. 7H, the vector contains a recognition sequence for a conventional restriction endonuclease with a specificity of 4-8 bp as well as very rare cleavage enzymes (eg, I-PpoｐI, I-Cue I, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7H shows a linear plant vector without a plant ARS. The linear vector can be constructed in vitro and then transferred to the plant, for example, by mechanical means (eg, microprojectile bombardment, electroporation or PEG-mediated transformation).
FIG. 7I
FIG. 7AI is an exemplary minichromosome vector. The vector shown in FIG. 7I can be of high copy number, low copy number or one copy. coli origin of replication. In FIG. 7I, the vector contains a conventional restriction endonuclease recognition sequence with 4-8 bp specificity and very rare cleavage enzymes (eg, I-PpoＰI, I-Cue I, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7I. This figure is identical to FIGS. 7A-7F, respectively, except that they do not contain plant telomeres. These vectors remain circular once delivered to plant cells, and therefore do not require telomeres to stabilize their ends.
FIG. 7J
FIG. 7J is an exemplary minichromosome vector. The vector shown in FIG. 7J may be high copy number, low copy number or one copy. coli origin of replication. In FIG. 7J, the vector contains a conventional restriction endonuclease recognition sequence with 4-8 bp specificity and very rare cleavage enzymes (eg, I-PpoＰI, I-Cue I, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7J. This figure is identical to FIGS. 7A-7F, respectively, except that they do not contain plant telomeres. These vectors remain circular once delivered to plant cells, and therefore do not require telomeres to stabilize their ends.
FIG. 7K
FIG. 7K is an exemplary minichromosome vector. In FIG. 7K, the vector contains a conventional restriction endonuclease recognition sequence with 4-8 bp specificity as well as very rare cleavage enzymes (eg, I-Ppo−I, I-Cue I, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7K. This figure is identical to FIGS. 7A-7F, respectively, except that they do not contain plant telomeres. These vectors remain circular once delivered to plant cells, and therefore do not require telomeres to stabilize their ends.
FIG. 7L
FIG. 7L is an exemplary minichromosome vector. In FIG. 7L, the vector contains a conventional restriction endonuclease recognition sequence with 4-8 bp specificity as well as very rare cleavage enzymes (eg, I-PpoＩI, I-Cue I, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7L. These figures are identical to FIGS. 7A-7F, respectively, except that they do not contain plant telomeres. These vectors remain circular once delivered to plant cells, and therefore do not require telomeres to stabilize their ends.
FIG. 7M
FIG. 7M is an exemplary minichromosome vector. In FIG. 7M, the vector contains a conventional restriction endonuclease recognition sequence with 4-8 bp specificity as well as very rare cleavage enzymes (eg, I-PpoＰI, I-Cue I, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7M. These figures are identical to FIGS. 7A-7F, respectively, except that they do not contain plant telomeres. These vectors remain circular once delivered to plant cells, and therefore do not require telomeres to stabilize their ends.
FIG. 7N
FIG. 7N is an exemplary minichromosome vector. In FIG. 7N, the vector contains a conventional restriction endonuclease recognition sequence with 4-8 bp specificity and very rare cleavage enzymes (eg, I-PpoＩI, I-Cue I, PI-Tli, PI- It contains a multiple cloning site, which may contain recognition sequences for Psp I, Not I, and PI Sce I), with the centromere flanked by Lox sites that can serve as targets for the site-specific recombinase Cre. FIG. 7N. These figures are identical to FIGS. 7A-7F, respectively, except that they do not contain plant telomeres. These vectors remain circular once delivered to plant cells, and therefore do not require telomeres to stabilize their ends.
FIG. 8
FIG. 8 is a feature of the sequences in CEN2 (A) and CEN4 (B). The center bar indicates the annotated genomic sequence of the indicated BAC clone; black is the genetically defined centromere; white is the region adjacent to the centromere. Sequences corresponding to genes and repetitive features (occupied by boxes (top and bottom bars, respectively)) are defined as FIGS. 12A-T; putative non-migrating genes, red; genes carried by mobile elements, Black; non-migrating pseudogene, pink; pseudogene carried by mobile element, grey; retro element, yellow; transposon, green; centromere repeat as previously defined, navy blue; 180 bp repeat, light blue. Chromosome-specific centromere features include large mitochondrial DNA insertions (orange; CEN2) and novel arrays of tandem repeats (purple; CEN4). Gaps (//) in the physical map, unannotated regions (hatch boxes) and expressed genes (filled circles) are indicated.
FIG. 9
FIG. 9 is a method for converting a BAC clone (or any other bacterial clone) to a minichromosome. Some conversion vectors use a BAC clone (or a target) via non-homologous recombination (interchangeable element-mediated) or by the action of a site-specific recombinase system (eg, Cre-Lox or FLP-FRT). Other bacterial clones).
FIG. 10
FIG. 10 is a method for analysis of the centromere chromosome in Arabidopsis. The BiBAC vector-containing centromere fragment (〜100 kb) is integrated into the Arabidopsis genome using an Agrobacterium-mediated transformation procedure and studied for adverse effects due to the formation of the dicentromeric chromosome. 1) BiBAC containing centromere fragments are identified using standard protocols. 2) Plant transformation. 3) Analysis of defects in growth and development of the plant-containing centromere chromosome.
FIG. 11A
FIG. 11A is a method for converting a BAC clone (or any other bacterial clone) to a minichromosome. The required selectable marker and E. coli. The origin of replication for propagation of the genetic material in E. coli, Agrobacterium and Arabidopsis and the loci required for Agrobacterium-mediated transformation into Arabidopsis are cloned into a transformation vector. Using Cre / loxP recombination, the conversion vector is recombined into a BAC-containing centromere fragment to form a minichromosome.
FIG. 11B
FIG. 11B is a method for converting a BAC clone (or any other bacterial clone) into a minichromosome. The required selectable marker and E. coli. The origin of replication for propagation of the genetic material in E. coli, Agrobacterium and Arabidopsis and the loci required for Agrobacterium-mediated transformation into Arabidopsis are cloned into a transformation vector. Using Cre / loxP recombination, the conversion vector is recombined into a BAC-containing centromere fragment to form a minichromosome.
FIG. 11C
FIG. 11C is a method for converting a BAC clone (or any other bacterial clone) into a minichromosome. The required selectable marker and E. coli. The origin of replication for propagation of the genetic material in E. coli, Agrobacterium and Arabidopsis and the loci required for Agrobacterium-mediated transformation into Arabidopsis are cloned into a transformation vector. Using Cre / loxP recombination, the conversion vector is recombined into a BAC-containing centromere fragment to form a minichromosome.
FIG. 11D
FIG. 11D is a method for converting a BAC clone (or any other bacterial clone) into a minichromosome. The required selectable marker and E. coli. The origin of replication for propagation of the genetic material in E. coli, Agrobacterium and Arabidopsis and the loci required for Agrobacterium-mediated transformation into Arabidopsis are cloned into a transformation vector. Using Cre / loxP recombination, the conversion vector is recombined into a BAC-containing centromere fragment to form a minichromosome.
FIG. 11E
FIG. 11E is a method for converting a BAC clone (or any other bacterial clone) into a minichromosome. The required selectable marker and E. coli. The origin of replication for propagation of the genetic material in E. coli, Agrobacterium and Arabidopsis and the loci required for Agrobacterium-mediated transformation into Arabidopsis are cloned into a transformation vector. Using Cre / loxP recombination, the conversion vector is recombined into a BAC-containing centromere fragment to form a minichromosome.
FIG. 11F
FIG. 11F is a method for converting a BAC clone (or any other bacterial clone) into a minichromosome. The required selectable marker and E. coli. The origin of replication for propagation of the genetic material in E. coli, Agrobacterium and Arabidopsis and the loci required for Agrobacterium-mediated transformation into Arabidopsis are cloned into a transformation vector. Using Cre / loxP recombination, the conversion vector is recombined into a BAC-containing centromere fragment to form a minichromosome.
FIG. 11G
FIG. 11G is a method for converting a BAC clone (or any other bacterial clone) into a minichromosome. The required selectable marker and E. coli. The origin of replication for propagation of the genetic material in E. coli, Agrobacterium and Arabidopsis and the loci required for Agrobacterium-mediated transformation into Arabidopsis are cloned into a transformation vector. Using Cre / loxP recombination, the conversion vector is recombined into a BAC-containing centromere fragment to form a minichromosome.
FIG.
FIGS. 12A-T are characteristics of centromere regions on chromosomes II and IV. (Top) Drawing of genetically defined centromeres (shaded grey, CEN2, left; CEN4, right), adjacent peripheral centromere DNA, and distal segments of each chromosome are determined by DNA sequencing As measured in Mb (shaded gray gaps corresponding to gaps in the physical map). Position in cM on the RI map (http://nasc.nott.ac.uk/new　ri　map. html) and physical distance in Mb (onset in Northern telomeres and in centromere gap). (Bottom) The density of each feature (FIGS. 12A-12T) is plotted with respect to chromosomal location in Mb. (FIGS. 12A, 12K) Curves (dashed lines) representing cM position (filled squares) and genomic average of 1 cM / 221 kb for markers on the RI map. One crossover in CEN4 in the RI mapping population (http://nasc.nott.ac.uk/new　ri　map. html; Somerville and Somerville, 1999) may reflect the differences between male and female meiotic recombination monitored herein. (FIGS. 12B-12E and 12L-12O) The percentage of DNA occupied by repetitive elements was calculated for a 100 kb window using a 10 kb sliding interval. (FIGS. 12B, 12L) 180 bp repeats; (FIGS. 12C, 12M) Sequences with similarity to retro elements (del, Tall, Tall, copia, Athila, LINE, Ty3, TSCL, 106B (Athila-like), Tatl, LTR and (Including Cinful); (FIGS. 12D, 12N) Sequences with similarity to transposons (including Tagl, En / Spm, Ac / Dc, Taml @ MuDR, Limpet, MITES, and Mariner); (FIGS. 12E, 120) Centromeres as described above. Repeats (163A, 164A, 164B, 278A, 11B7RE, mi167, pAT27, 160 bp, 180 bp and 500 bp repeats and include telomere sequences (Murata et al., 1997; Heslop-Harrison et al., 1999) Brandes et al., 1997; Franz et al., 1998; Wright et al., 1996; Konieczny et al., 1991; Pelissier et al., 1995; Voytas and Ausubel, 1988; Chiye et al., 1997; Tsay et al., 1993; Richard et al., 1991; Thompson et al., 1996; Pelissier et al., 1996 Franz et al., 1998; Pelissier et al., 1995; Voytas and Ausubel, 1988; Thompson et al., 1996. (FIG. 12F, 12P)% (adenosine and thymidine) with a 25 kb sliding interval. Calculated for a 50 kb window (FIGS. 12G-12J, 12Q-12T). The number of offspring or pseudogenes was plotted over a 100 kb window using a 10 kb sliding interval (FIGS. 12G, 12I, 12Q, 12S) The estimated gene (FIGS. 12G, 12Q) and the pseudogene (FIG. 12I, 12S) are typically not found on mobile DNA elements; (FIGS. 12H, 12J, 12R, 12T) putative genes (FIGS. 12H, 12R) and pseudogenes (FIGS. 12J, 12T) Often carried on mobile DNA (including reverse transcriptase, transposase, and retroviral polyprotein), dashed lines indicate regions in which sequencing or annotation is in progress, Annotations are in GenBank records (http: //www.ncbi.nlm.nih. gov / Entrez / nucleotide. html), the AGAD database (http://www.tigr.org/tdb/at/agad/.) and a BLAST comparison with a database of repeated Arabidopsis sequences (http: ///nucleus.cshl.org/protarab/AtRepBase.htm). ); The latest annotation record may change individual entries, but the overall structure of the area is not significantly changed.
FIG. 13
FIG. 13 shows a method for converting centromeric DNA containing BAC clones into minichromosomes for introduction into plant cells. The specific elements described are provided for purposes of illustration and not limitation. A) Referring to BAC clone diagram, centromere DNA location (red), site-specific recombination sites (eg, loxP), and F origin of replication. B) Conversion vector containing selectable and color markers (eg, 35S-Bar, nptII, LAT52-GUS, Scarecrow-GFP), telomeres, site-specific recombination sites (eg, loxP), antibiotic resistance markers (eg, amp) Or spc / str), Agrobacterium @ T-DNA border (Agro @ Left and Right) and origin of replication (RiA4). C) The product of site-specific recombination using Cre recombinase at the loxP site yields a circular product with centromere DNA and telomere flanking markers. D) Minichromosome immediately after transformation into a plant; the left and right borders are then probably removed by the plant cells and additional telomere sequences are added by plant telomerase.
FIG. 14
Figures 14A-B are centromere DNA conversions. BAC clones (bars) used for sequences CEN2 (FIG. 14A) and CEN4 (FIG. 14B) are shown; arrows indicate genetically defined centromere boundaries. PCR primer pairs yielding products from Columbia alone (filled circles) or from both Landsberg and Columbia (open circles); BAC-encoding DNA with homology to the mitochondrial genome (grey bars); 180 bp repeats (grey boxes); unsequencing DNA (dashed lines); as well as gaps (double slashes) in the physical map are indicated.
FIG. 15A
FIG. The primers used to analyze centromere sequence conversion in the thaliana @ Columbia and Landsberg ecotypes. FIG. 15A: Primers used for amplification of chromosome 2 sequence.
FIG. 15B
FIG. The primers used to analyze centromere sequence conversion in the thaliana @ Columbia and Landsberg ecotypes. FIG. 15B: Primers used for amplification of chromosome 4 sequence.
FIG.
FIG. 16 shows an arrangement common to CEN2 and CEN4. Genetically defined centromeres (thick line), sequenced BAC clones (thin line), and unannotated BAC clones (dash line) are shown in FIGS. 14A, B. Repeated AtCCS1 (A. thaliana centromere conserved sequences) and AtCCS2 (closed and open circles, respectively), AtCCS3 (triangles), and AtCCS4-7 (4-7, respectively) are shown (GenBank accession numbers AF204874-AF204880); It was identified using BLAST 2.0 (http://blast.wustl.edu).
FIG.
Sequenced BAC clone from Centromere 2. The sequenced BAC clone is indicated by a horizontal line near the top of the figure (see, for example, T14A4). The red box indicates the centromere 2 border, and contains the centromere for that BAC clone, providing the GenBank accession number in the lower right panel. The contiguous sequences in the red boxes are provided in SEQ ID NO: 209 and SEQ ID NO: 210. The horizontal line at the bottom of the sequenced clones indicates additional BAC clones; the sequenced endpoints of these BACs are indicated by closed circles. Clones with one or more undetermined endpoints are shown in red text documents.
FIG.
Sequenced BAC clone from Centromere 4. The sequenced BAC clone from Centromere 4 is indicated by a horizontal line near the top of the figure (see, for example, T24M8). The red box indicates the centromere 4 border, and contains the centromere for that BAC clone, providing the GenBank accession number in the lower right panel. The contiguous sequences in the red boxes are provided in SEQ ID NO: 211 and SEQ ID NO: 212. The horizontal line at the bottom of the sequenced clones indicates additional BAC clones; the sequenced endpoints of these BACs are indicated by closed circles. Clones with one or more undetermined endpoints are shown in red text documents.
FIG.
Sequence tiling strategy for centromeres 1, 3, and 5 (tiling @ path). The boundaries of these centromeres were determined as described in Copenhaver et al. (1999). The contig number refers to the fingerprint contig compiled by Marra et al. (1999). Some of these clones have been sequenced and provided with accession numbers (see attached table). In other cases, sequencing was completed by the Arabidopsis Genome Project.
FIG.
Centromere 2 derived DNA location in BiBAB vector. Clones were placed on a physical map by fingerprint and PCR analysis and compared to sequenced BAC clones.
FIG. 21
An exemplary method for adding a selectable or screening marker to a BiBAC clone. The desired marker was flanked by transposon boundaries and incubated with BiBAC in the presence of transposase. Thereafter, the BiBAC is introduced into a plant. Frequently, these BiBACs can integrate into the native chromosome, create a centromere chromosome that can be altered in stability and cause chromosome breakage, generating new chromosomal fragments.
FIG. 22
Chromosome stability assay. The stability of a native chromosome, assembled mini-chromosome, or dicentromeric chromosome can be assessed by monitoring the combination of colored markers through cell division. These markers are linked to the centromere in a modified BAC or BiBAC vector and introduced into the plant. Adjustment of the marker gene with the appropriate promoter will determine which tissues will be assayed. For example, a root-specific promoter (eg, SCARECROW) allows one to monitor the combination in a file in root cells; a post-meiotic pollen-specific promoter (eg, LAT52) provides for monitoring of the combination through meiosis. Enables, and common promoters (eg, the 35S cauliflower mosaic virus promoter) make it possible to monitor combinations in many other plant tissues. Quantitative assays assess the general pattern of stability and determine the size of the section corresponding to the loss of the marker. Qualitative assays, on the other hand, require insight into the cell lineage and make it possible to calculate the number of chromosomal loss events during mitosis and meiosis.
FIG. 23A
Sequence alignment for 180 bp repeats from centromeres 1-4. The column on the left shows the source of the repeated copy of BAC and any assigned number given to the sequence. For example, the name f12g6-1 indicates a repeat copy from BAC number f12g6 and any given repeat number 1. The nucleic acid sequences of BACs containing repetitive copies, designated as f12g6, f5a13, t25f15, t12j2, t14c8, t6c20, f21i2 and f6h8, are represented by SEQ ID NO: 184, SEQ ID NO: 191, SEQ ID NO: 189, SEQ ID NO: 205, SEQ ID NO: 205, respectively. 206, SEQ ID NO: 186, SEQ ID NO: 208, and SEQ ID NO: 207. (FIG. 23A) Alignment of 180 bp repeats from Centromere 1.
FIG. 23B
Sequence alignment for 180 bp repeats from centromeres 1-4. The column on the left shows the source of the repeated copy of BAC and any assigned number given to the sequence. For example, the name f12g6-1 indicates a repeat copy from BAC number f12g6 and any given repeat number 1. The nucleic acid sequences of BACs containing repetitive copies, called f12g6, f5a13, t25f5, t12j2, t14c8, t6c20, f21i2 and f6h8, are shown in SEQ ID NO: 184, SEQ ID NO: 191, SEQ ID NO: 189, SEQ ID NO: 205, SEQ ID NO: 205, respectively. 206, SEQ ID NO: 186, SEQ ID NO: 208, and SEQ ID NO: 207. (FIG. 23B) Alignment of 180 bp repeats from Centromere 2.
FIG. 23C
Sequence alignment for 180 bp repeats from centromeres 1-4. The column on the left shows the source of the repeated copy of BAC and any assigned number given to the sequence. For example, the name f12g6-1 indicates a repeat copy from BAC number f12g6 and any given repeat number 1. The nucleic acid sequences of BACs containing repetitive copies, called f12g6, f5a13, t25f5, t12j2, t14c8, t6c20, f21i2 and f6h8, are shown in SEQ ID NO: 184, SEQ ID NO: 191, SEQ ID NO: 189, SEQ ID NO: 205, SEQ ID NO: 205, respectively. 206, SEQ ID NO: 186, SEQ ID NO: 208, and SEQ ID NO: 207. (FIG. 23C) Alignment of 180 bp repeats from Centromere 3.
FIG. 23D
Sequence alignment for 180 bp repeats from centromeres 1-4. The column on the left shows the source of the repeated copy of BAC and any assigned number given to the sequence. For example, the name f12g6-1 indicates a repeat copy from BAC number f12g6 and any given repeat number 1. The nucleic acid sequences of BACs containing repetitive copies, called f12g6, f5a13, t25f5, t12j2, t14c8, t6c20, f21i2 and f6h8, are shown in SEQ ID NO: 184, SEQ ID NO: 191, SEQ ID NO: 189, SEQ ID NO: 205, SEQ ID NO: 205, respectively. 206, SEQ ID NO: 186, SEQ ID NO: 208, and SEQ ID NO: 207. (FIG. 23D) Alignment of 180 bp repeats from Centromere 4.

Claims (231)

A recombinant DNA construct comprising a plant centromere. 2. The recombinant DNA construct of claim 1, further comprising a telomere. 3. The recombinant DNA construct according to claim 2, wherein said telomere is a plant telomere. The recombinant DNA construct according to claim 3, wherein the plant telomere is Arabidopsis thaliana telomere. 3. The recombinant DNA construct according to claim 2, wherein said telomeres are yeast telomeres. 2. The recombinant DNA construct according to claim 1, further comprising an autonomously replicating sequence (ARS). The recombinant DNA construct according to claim 6, wherein the ARS is a plant ARS. The recombinant DNA construct according to claim 6, wherein the plant ARS is Arabidopsis thaliana ARS. The recombinant DNA construct according to claim 1, further comprising a structural gene. The recombinant DNA construct according to claim 9, wherein the structural gene comprises a selection marker gene or a screening marker gene. The recombinant DNA construct according to claim 9, further comprising a second structural gene. The structural gene is selected from the group consisting of antibiotic resistance gene, herbicide resistance gene, nitrogen fixation gene, plant pathogen defense gene, plant stress inducible gene, toxin gene, receptor gene, ligand gene and seed storage gene, A recombinant DNA construct according to claim 9. 13. The recombinant DNA construct according to claim 12, wherein said construct is capable of expressing said structural gene. 14. The recombinant DNA construct according to claim 13, wherein said construct is capable of expressing said structural gene in prokaryotes. 14. The recombinant DNA construct according to claim 13, wherein said construct is capable of expressing said structural gene in eukaryotes. 16. The recombinant DNA construct according to claim 15, wherein said eukaryote is a higher eukaryote. 17. The recombinant DNA construct according to claim 16, wherein said higher eukaryote is a plant. The recombinant DNA construct according to claim 9, wherein the structural gene is selected from the group consisting of a hormone gene, an enzyme gene, an interleukin gene, a coagulation factor gene, a cytokine gene, an antibody gene, and a growth factor gene. 19. The recombinant DNA construct according to claim 18, wherein said construct is capable of expressing said structural gene. 20. The recombinant DNA construct of claim 19, wherein said construct is capable of expressing said structural gene in a prokaryote. 20. The recombinant DNA construct of claim 19, wherein said construct is capable of expressing said structural gene in eukaryotes. 22. The recombinant DNA construct of claim 21, wherein said eukaryote is a higher eukaryote. 23. The recombinant DNA construct according to claim 22, wherein said higher eukaryote is a plant. 2. The recombinant DNA construct of claim 1, further defined as a plasmid. 25. The recombinant DNA construct of claim 24, wherein said plasmid comprises an origin of replication. 26. The recombinant DNA construct of claim 25, wherein said origin of replication functions in bacteria. The origin of replication is E.C. 27. The recombinant DNA construct of claim 26, which functions in E. coli. 27. The recombinant DNA construct of claim 26, wherein said origin of replication functions in Agrobacterium. 26. The recombinant DNA construct of claim 25, wherein said origin of replication functions in plants. 26. The recombinant DNA construct of claim 25, wherein said origin of replication functions in yeast. The yeast is S. 31. The recombinant DNA construct according to claim 30, which is cerevisiae. 25. The recombinant DNA construct according to claim 24, wherein said plasmid comprises a selectable marker. 33. The recombinant DNA construct of claim 32, wherein said selectable marker functions in bacteria. The selection marker is E. coli. 33. The recombinant DNA construct of claim 32, which functions in E. coli. 33. The recombinant DNA construct of claim 32, wherein said selectable marker functions in Agrobacterium. 33. The recombinant DNA construct of claim 32, wherein said selectable marker functions in a plant. 33. The recombinant DNA construct of claim 32, wherein said selectable marker functions in yeast. The yeast is S. 38. The recombinant DNA construct according to claim 37, which is cerevisiae. 2. The recombinant DNA construct of claim 1, which can be maintained as a chromosome, wherein the chromosome is transmitted in dividing cells. 2. The recombinant DNA construct according to claim 1, wherein said plant centromere is Arabidopsis thaliana centromere. 41. The recombinant DNA construct according to claim 40, wherein the plant centromere is a chromosome 1 centromere of Arabidopsis thaliana. 42. The recombinant DNA construct according to claim 41, wherein the centromere is adjacent to the genetic markers T22C23-T7 and T3P8-SP6. 43. The method of claim 42, wherein the centromere is further defined as being adjacent to the genetic markers T22C23-T7 and T5D18, T22C23-T7 and T3L4, T5D18 and T3P8-SP6, T5D18 and T3L4, and T3L4 and T3P8-SP6. Recombinant DNA construct. 41. The recombinant DNA construct of claim 40, wherein said plant centromere comprises Arabidopsis thaliana chromosome 2 centromere. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 100 to about 611,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 209. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 500 to about 611,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 209. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 1,000 to about 611,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 209. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 10,000 to about 611,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 209. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 20,000 to about 611,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 209. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 40,000 to about 611,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 209. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 80,000 to about 611,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 209. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 150,000 to about 611,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 209. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 300,000 to about 611,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 209. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises the nucleic acid sequence of SEQ ID NO: 209. 45. The recombinant DNA construct of claim 44, wherein the centromere comprises from about 100 to about 50,959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 210. 45. The recombinant DNA construct of claim 44, wherein the centromere comprises from about 500 to about 50,959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 210. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 1,000 to about 50,959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 210. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 5,000 to about 50,959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 210. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 10,000 to about 50,959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 210. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 20,000 to about 50,959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 210. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 30,000 to about 50,959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 210. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises from about 40,000 to about 50,959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 210. 45. The recombinant DNA construct of claim 44, wherein said centromere comprises the nucleic acid sequence of SEQ ID NO: 210. 41. The recombinant DNA construct according to claim 40, wherein the plant centromere is Arabidopsis thaliana chromosome 3 centromere. 63. The recombinant DNA construct of claim 62, wherein the centromere is further defined as flanking the genetic markers T9G9-SP6 and T5M14-SP6. Said centromeres are T9G9-SP6 and TI4H20, T9G9-SP6 and T7K14, T9G9-SP6 and T21P20, T14H20 and T7K14, T14H20 and T21P20, T14H20 and T5M14-SP6, T7K14 and T5M14-T6, T20K and T20K5 66. The recombinant DNA construct of claim 65, further defined as flanked by a pair of genetic markers selected from the group consisting of SP6. 41. The recombinant DNA construct of claim 40, wherein the plant centromere is Arabidopsis thaliana chromosome 4 centromere. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 100 to about 1,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 211. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 500 to about 1,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 211. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 1,000 to about 1,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 211. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 5,000 to about 1,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 211. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 10,000 to about 1,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 211. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 50,000 to about 1,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 211. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 100,000 to about 1,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 211. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 200,000 to about 1,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 211. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 400,000 to about 1,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 211. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 800,000 to about 1,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 211. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises the nucleic acid sequence of SEQ ID NO: 211. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 100 to about 163,317 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 212. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 500 to about 163,317 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 212. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 1,000 to about 163,317 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 212. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 5,000 to about 163,317 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 212. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 10,000 to about 163,317 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 212. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 30,000 to about 163,317 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 212. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 50,000 to about 163,317 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 212. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 80,000 to about 163,317 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 212. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises from about 120,000 to about 163,317 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 212. 68. The recombinant DNA construct of claim 67, wherein said centromere comprises the nucleic acid sequence of SEQ ID NO: 212. 41. The recombinant DNA construct of claim 40, wherein the plant centromere is Arabidopsis thaliana chromosome 5 centromere. 90. The recombinant DNA construct of claim 89, wherein said centromere is adjacent to the genetic markers F13K20-T7 and CUE1. 90. The recombinant DNA construct of claim 90, wherein the centromere is F13K20-T7 and T18M4, F13K20-T7 and T18F2, F13K20-T7 and T24I20, T18M4 and T18F2, T18M4 and T24I20, T18M4 and CUE1, T18F2 and T24I20. A recombinant DNA construct that is adjacent to a pair of genetic markers selected from the group consisting of T18F2 and CUE1, and T24I20 and CUE1. 2. The recombinant DNA construct according to claim 1, comprising n copies of the repetitive nucleotide sequence, wherein n is at least 2. 93. The recombinant DNA construct of claim 92, wherein n is from about 5 to about 100,000. 93. The recombinant DNA construct of claim 92, wherein n is from about 10 to about 80,000. 93. The recombinant DNA construct of claim 92, wherein n is from about 25 to about 60,000. 93. The recombinant DNA construct of claim 92, wherein n is from about 100 to about 50,000. 93. The recombinant DNA construct of claim 92, wherein n is from about 200 to about 40,000. 93. The recombinant DNA construct of claim 92, wherein n is from about 400 to about 30,000. 93. The recombinant DNA construct of claim 92, wherein n is from about 1,000 to about 30,000. 93. The recombinant DNA construct of claim 92, wherein n is from about 5,000 to about 20,000. 93. The recombinant DNA construct of claim 92, wherein n is between about 10,000 and about 15,000. The repeating nucleotide sequence is SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 93. The recombinant DNA construct of claim 92, which can be isolated from the nucleic acid sequence provided by 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211 or SEQ ID NO: 212. A mini chromosome vector containing plant centromere and telomere sequences. 104. The minichromosome vector of claim 103, comprising an autonomously replicating sequence. 114. The minichromosome vector of claim 103, comprising a second telomere sequence. 114. The minichromosome vector of claim 103, comprising a structural gene. 114. The minichromosome vector of claim 103, further defined as comprising a second constructed gene. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21 further defined as comprising a nucleic acid sequence selected from the group consisting of: A mini-chromosome vector according to claim 103. Cells transformed with a recombinant DNA construct comprising a plant centromere. 110. The cell of claim 109, wherein said cell is a prokaryotic cell. 110. The cell of claim 109, wherein said cell is a eukaryotic cell. 112. The cell of claim 111, wherein said cell is a yeast cell. 110. The cell of claim 109, wherein said cell is a higher eukaryotic cell. 114. The cell of claim 113, wherein said higher eukaryote is a plant cell. 115. The cell of claim 114, wherein the plant cell is from a dicotyledon. 115. The cell of claim 115, wherein said dicotyledonous plant is selected from the group consisting of tobacco, tomato, potato, sugar beet, pea, carrot, cauliflower, broccoli, soybean, canola, sunflower, alfalfa, cotton and Arabidopsis. 117. The cell of claim 116, wherein said dicot is Arabidopsis thaliana. 115. The cell of claim 114, wherein said plant cell is derived from a monocotyledonous plant. 119. The cell of claim 118, wherein said monocotyledonous plant is selected from the group consisting of wheat, corn, rye, rice, turf, oats, barley, sorghum, millet and sugarcane. 110. The cell of claim 109, wherein said plant centromere is Arabidopsis thaliana centromere. 121. The cell of claim 120, further defined as an Arabidopsis thaliana cell. 110. The cell of claim 109, wherein said recombinant DNA construct comprises a telomere. 110. The cell of claim 109, wherein said recombinant DNA construct comprises an autonomously replicating sequence (ARS). 110. The cell of claim 109, wherein said recombinant DNA construct comprises a structural gene. 125. The cell of claim 124, wherein said structural gene comprises a selection marker gene or a screening marker gene. 125. The cell of claim 124, wherein said recombinant DNA construct comprises a second structural gene. 125. The cell of claim 124, further defined as being capable of expressing said structural gene. A plant comprising the cell of claim 109. A method of preparing a transgenic plant cell, comprising contacting a starting plant cell with a recombinant DNA construct comprising a plant centromere, whereby the starting plant cell is transformed with the recombinant DNA construct. how to. 130. The method of claim 129, wherein said recombinant DNA construct comprises a structural gene. 130. The method of claim 130, wherein said recombinant DNA construct comprises a second structural gene. 130. The method of claim 129, wherein said plant centromere is Arabidopsis thaliana centromere. 133. The method of claim 132, wherein said starting plant cell is an Arabidopsis thaliana cell. A transgenic plant comprising a minichromosome vector, said vector comprising plant centromere and telomere sequences. 135. The transgenic plant of claim 134, wherein said minichromosome vector comprises an autonomously replicating sequence. 135. The transgenic plant of claim 134, wherein said minichromosome vector comprises a second telomere sequence. 135. The transgenic plant of claim 134, wherein said minichromosome vector comprises a structural gene. The structural gene is selected from the group consisting of antibiotic resistance gene, herbicide resistance gene, nitrogen fixation gene, plant pathogen defense gene, plant stress inducible gene, toxin gene, receptor gene, ligand gene and seed storage gene, 138. The transgenic plant of claim 137. 138. The transgenic plant of claim 137, wherein the first exogenous structural gene is selected from the group consisting of a hormone gene, an enzyme gene, an interleukin gene, a coagulation factor gene, a cytokine gene, an antibody gene, and a growth factor gene. . 135. The transgenic plant of claim 134, wherein said mini-chromosome vector comprises a second structural gene. The mini-chromosome vector has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, A nucleic acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 135. The transgenic plant of claim 134, comprising. 135. The transgenic plant of claim 134, further defined as a dicotyledon. 143. The transgenic plant of claim 143, wherein the dicotyledonous plant is selected from the group consisting of tobacco, tomato, potato, sugar beet, pea, carrot, cauliflower, broccoli, soybean, canola, sunflower, alfalfa, cotton and Arabidopsis. . 145. The transgenic plant of claim 143, wherein said dicotyledonous plant is Arabidopsis thaliana. 135. The transgenic plant of claim 134, further defined as a monocotyledonous plant. 146. The transgenic plant of claim 145, wherein said monocotyledonous plant is selected from the group consisting of wheat, corn, rye, rice, turf, oats, barley, sorghum, millet and sugarcane. A method for producing a mini-chromosome vector, comprising:
(A) obtaining a first vector and a second vector, wherein the first vector or the second vector comprises a selection or screening marker, an origin of replication, a telomere, and a plant centromere; Wherein said first vector and said second vector comprise a site for site-specific recombination; and (b) contacting said first vector with said second vector to produce said first vector. Causing a site-specific recombination between the site for the site-specific recombination on the vector of the second vector and the site for the site-specific recombination on the second vector, Producing a minichromosome vector comprising a screening marker, said origin of replication, said telomere and said plant centromere. 148. The method of claim 147, wherein said contacting is performed in vitro. 149. The process of claim 148, wherein said contacting occurs in vivo. 150. The method of claim 149, wherein said contacting is performed in a prokaryotic cell. 151. The method of claim 150, wherein said prokaryotic cell is an Agrobacterium cell. The prokaryotic cell is E. coli. 150. The method of claim 150, wherein the method is an E. coli cell. 150. The method of claim 149, wherein said contacting is performed in a lower eukaryotic cell. 153. The method of claim 153, wherein said lower eukaryotic cell is a yeast cell. 150. The method of claim 149, wherein said contacting is performed in a higher eukaryotic cell. 160. The method of claim 155, wherein said higher eukaryotic cell is a plant cell. 157. The method of claim 156, wherein said plant cells are Arabidopsis thaliana cells. 148. The method of claim 147, wherein said contacting is performed in the presence of a recombinase. 160. The method of claim 158, wherein said recombinase is selected from the group consisting of Cre, Flp, Gin, Pin, Sre, pinD, Int-B13, and R. 148. The method of claim 147, wherein said first vector or said second vector comprises a border sequence for Agrobacterium-mediated transformation. 148. The method of claim 147, wherein said plant cell centromere is Arabidopsis thaliana centromere. 147. The method of claim 147, wherein said telomeres are plant telomeres. 148. The method of claim 147, wherein said plant selection marker or screening marker is selected from the group consisting of GFP, GUS, BAR, PAT, HPT or NPTII. A method for screening candidate centromere sequences for plant centromere activity, the method comprising the following steps:
(A) obtaining an isolated nucleic acid sequence comprising the candidate centromere sequence;
(B) transforming a plant cell to be integrated with the isolated nucleic acid; and (c) screening for the centromere activity of the candidate centromere sequence;
A method comprising: The screening step comprises observing a phenotypic effect present in the plant cell transformed to integrate or the plant containing the plant cell, wherein the phenotypic effect is 165. The method of claim 164, wherein the method is absent in a control plant cell that has not been transformed to incorporate the nucleic acid sequence or in a plant containing the control plant cell. The effects of the phenotype include reduced viability, reduced efficiency of the transformation, genetic instability in the nucleic acid transformed to integrate, abnormal plant area, increased ploidy, aneuploidy and 166. The method of claim 165, wherein the method is selected from the group consisting of increased integrated transformation in a distal chromosomal region or a centromeric chromosomal region. 165. The method of claim 164, wherein the isolated nucleic acid sequence comprises a bacterial artificial chromosome. 168. The method of claim 167, wherein said bacterial artificial chromosome is further defined as a binary bacterial artificial chromosome. 169. The method of claim 164, wherein said transforming to integrate comprises using Agrobacterium-mediated transformation. 169. The method of claim 164, wherein said control plant cells have been transformed to integrate with a nucleic acid sequence other than the candidate centromere sequence. A recombinant DNA construct comprising an Arabidopsis polyubiquitin 11 promoter, wherein the promoter comprises about 25 to 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 180. 172. The recombinant DNA construct of claim 171, wherein said promoter comprises from about 75 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 180. 172. The recombinant DNA construct of claim 171, wherein said promoter comprises about 125 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 180. 172. The recombinant DNA construct of claim 171, wherein said promoter comprises from about 200 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 180. 172. The recombinant DNA construct of claim 171, wherein said promoter comprises from about 400 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 180. 172. The recombinant DNA construct of claim 171, wherein said promoter comprises about 800 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 180. 172. The recombinant DNA construct of claim 171, wherein said promoter comprises from about 1,000 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 180. 172. The recombinant DNA construct of claim 171, wherein said promoter comprises the nucleic acid sequence in SEQ ID NO: 180. 172. The recombinant DNA construct of claim 171, further comprising an enhancer. 172. The recombinant DNA construct of claim 171, further comprising a telomere sequence. 172. The recombinant DNA construct of claim 171, further comprising a plant centromere sequence. 172. The recombinant DNA construct of claim 171, further comprising an ARS. 172. The recombinant DNA construct of claim 171, wherein said promoter is operably linked to a structural gene. The structural gene is selected from the group consisting of antibiotic resistance gene, herbicide resistance gene, nitrogen fixation gene, plant pathogen defense gene, plant stress inducible gene, toxin gene, receptor gene, ligand gene and seed storage gene, 183. A recombinant DNA construct according to claim 183. 183. The recombinant DNA construct of claim 183, wherein said structural gene is selected from the group consisting of a hormone gene, an enzyme gene, an interleukin gene, a coagulation factor gene, a cytokine gene, an antibody gene, and a growth factor gene. A recombinant DNA construct comprising an Arabidopsis @ 40S ribosomal protein S16 promoter, wherein the promoter comprises about 25 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 182. 189. The recombinant DNA construct of claim 186, wherein the promoter comprises about 75 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 182. 189. The recombinant DNA construct of claim 186, wherein the promoter comprises about 125 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 182. 189. The recombinant DNA construct of claim 186, wherein said promoter comprises about 200 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 182. 189. The recombinant DNA construct of claim 186, wherein the promoter comprises about 400 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 182. 189. The recombinant DNA construct of claim 186, wherein the promoter comprises about 800 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 182. 189. The recombinant DNA construct of claim 186, wherein said promoter comprises from about 1,000 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 182. 189. The recombinant DNA construct of claim 186, wherein said promoter comprises the nucleic acid sequence of SEQ ID NO: 182. 189. The recombinant DNA construct of claim 186, further comprising an enhancer. 189. The recombinant DNA construct of claim 186, further comprising a telomere sequence. 189. The recombinant DNA construct of claim 186, further comprising a plant centromere sequence. 189. The recombinant DNA construct of claim 186, further comprising an ARS. 189. The recombinant DNA construct of claim 186, wherein said promoter is operably linked to a structural gene. The structural gene is selected from the group consisting of antibiotic resistance gene, herbicide resistance gene, nitrogen fixation gene, plant pathogen defense gene, plant stress inducible gene, toxin gene, receptor gene, ligand gene and seed storage gene, 198. A recombinant DNA construct according to claim 198. 210. The recombinant DNA construct of claim 198, wherein said structural gene is selected from the group consisting of a hormone gene, an enzyme gene, an interleukin gene, a coagulation factor gene, a cytokine gene, an antibody gene, and a growth factor gene. A recombinant DNA construct comprising an Arabidopsis polyubiquitin 11'3 'regulatory sequence, wherein the 3' regulatory sequence comprises from about 25 to about 2001 contiguous nucleotides of the nucleic acid sequence set forth in SEQ ID NO: 181. Construct. 220. The recombinant DNA construct of claim 201, wherein said 3 'regulatory sequence comprises from about 75 to about 2001 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 181. 220. The recombinant DNA construct of claim 201, wherein said 3 'regulatory sequence comprises from about 125 to about 2001 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 181. 220. The recombinant DNA construct of claim 201, wherein said 3 'regulatory sequence comprises from about 200 to about 2001 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 181. 220. The recombinant DNA construct of claim 201, wherein said 3 'regulatory sequence comprises from about 400 to about 2001 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 181. 220. The recombinant DNA construct of claim 201, wherein said 3 'regulatory sequence comprises from about 800 to about 2001 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 181. 220. The recombinant DNA construct of claim 201, wherein said 3 'regulatory sequence comprises from about 1,000 to about 2001 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 181. 220. The recombinant DNA construct of claim 201, wherein said 3 'regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 181. 220. The recombinant DNA construct of claim 201, further comprising an enhancer. 220. The recombinant DNA construct of claim 201, further comprising a telomere sequence. 220. The recombinant DNA construct of claim 201, further comprising a plant centromere sequence. 220. The recombinant DNA construct of claim 201, further comprising an ARS. 220. The recombinant DNA construct of claim 201, wherein said 3 'regulatory sequence is operably linked to a structural gene. The structural gene is selected from the group consisting of antibiotic resistance gene, herbicide resistance gene, nitrogen fixation gene, plant pathogen defense gene, plant stress inducible gene, toxin gene, receptor gene, ligand gene and seed storage gene, 213. A recombinant DNA construct according to claim 213. 213. The recombinant DNA construct of claim 213, wherein said structural gene is selected from the group consisting of a hormone gene, an enzyme gene, an interleukin gene, a coagulation factor gene, a cytokine gene, an antibody gene, and a growth factor gene. A recombinant DNA construct comprising an Arabidopsis 40S ribosomal protein S16 3 'regulatory sequence, wherein the 3' regulatory sequence comprises from about 25 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 183. . 220. The recombinant DNA construct of claim 216, wherein said 3 'regulatory sequence comprises from about 75 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 183. 218. The recombinant DNA construct of claim 216, wherein said 3 'regulatory sequence comprises from about 125 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 183. 218. The recombinant DNA construct of claim 216, wherein said 3 'regulatory sequence comprises from about 200 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 183. 218. The recombinant DNA construct of claim 216, wherein said 3 'regulatory sequence comprises from about 400 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 183. 220. The recombinant DNA construct of claim 216, wherein said 3 'regulatory sequence comprises from about 800 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 183. 220. The recombinant DNA construct of claim 216, wherein said 3 'regulatory sequence comprises from about 1,000 to about 2,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 183. 223. The recombinant DNA construct of claim 216, wherein said 3 'regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 183. 220. The recombinant DNA construct of claim 216, further comprising an enhancer. 220. The recombinant DNA construct of claim 216, further comprising a telomere sequence. 220. The recombinant DNA construct of claim 216, further comprising a plant centromere sequence. 220. The recombinant DNA construct of claim 216, further comprising an ARS. 220. The recombinant DNA construct of claim 216, wherein said 3 'regulatory sequence is operably linked to a structural gene. The structural gene is selected from the group consisting of an antibiotic resistance gene, a herbicide resistance gene, a nitrogen fixation gene, a plant pathogen defense gene, a plant stress inducible gene, a toxin gene, a receptor gene, a ligand gene and a seed storage gene, 229. A recombinant DNA construct according to claim 228. 229. The recombinant DNA construct of claim 228, wherein said structural gene is selected from the group consisting of a hormone gene, an enzyme gene, an interleukin gene, a coagulation factor gene, a cytokine gene, an antibody gene, and a growth factor gene. The centromere is SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, 68. The recombinant DNA construct of claim 67, comprising at least about 100 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 208.

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