EP1165792A2 - Compositions de chromosomes vegetaux et methodes - Google Patents

Compositions de chromosomes vegetaux et methodes

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Publication number
EP1165792A2
EP1165792A2 EP00916559A EP00916559A EP1165792A2 EP 1165792 A2 EP1165792 A2 EP 1165792A2 EP 00916559 A EP00916559 A EP 00916559A EP 00916559 A EP00916559 A EP 00916559A EP 1165792 A2 EP1165792 A2 EP 1165792A2
Authority
EP
European Patent Office
Prior art keywords
dna
gene
plant
centromere
sequences
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP00916559A
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German (de)
English (en)
Inventor
Daphne Preuss
Gregory Copenhaver
Kevin Keith
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University of Chicago
Original Assignee
University of Chicago
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Filing date
Publication date
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Publication of EP1165792A2 publication Critical patent/EP1165792A2/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation

Definitions

  • the other general method of genetic transformation involves integration of introduced DNA sequences into the recipient cell's chromosomes, permitting the new information to be replicated and partitioned to the cell's progeny as a part of the natural chromosomes
  • the most common form of integrative transformation is called "transfection" and is frequently used in mammalian cell culture systems
  • Transfection involves introduction of relatively large quantities of deproteinized DNA into cells
  • the introduced DNA usually is broken and joined together in various combinations before it is integrated at random sites into the cell's chromosome (see.
  • a third drawback of the Agrobacterium T-DNA system is the reliance on a "gene addition" mechanism: the new genetic information is added to the genome (i.e., all the genetic information a cell possesses) but does not replace information already present in the genome.
  • Artificial chromosomes are man-made linear or circular DNA molecules constructed from cis-acting DNA sequence elements that are responsible for the proper replication and partitioning of natural chromosomes (see Murray et al., 1983).
  • Desired elements include: ( 1 ) Autonomous Replication Sequences (ARS) (these have properties of replication origins, which are the sites for initiation of DNA replication), (2) Centromeres (site of kinetochore assembly and responsible for proper distribution of replicated chromosomes at mitosis or meiosis), and (3) Telomeres (specialized DNA structures at the ends of linear chromosomes that function to stabilize the ends and facilitate the complete replication of the extreme termini of the DNA molecule).
  • ARS Autonomous Replication Sequences
  • Centromeres site of kinetochore assembly and responsible for proper distribution of replicated chromosomes at mitosis or meiosis
  • Telomeres specialized DNA structures at the ends of linear chromosomes that function to stabilize the ends and facilitate the complete replication of the extreme termini of the DNA molecule.
  • ARSs have been isolated from unicellular fungi, including Saccharomyces cerevisiae (brewer's yeast) and Schizosaccharomyces pombe (see Stinchcomb et al., 1979 and Hsiao et al., 1979).
  • An ARS behaves like a replication origin allowing DNA molecules that contain the ARS to be replicated as an episome after introduction into the cell nuclei of these fungi. Plasmids containing these sequences replicate, but in the absence of a centromere they are partitioned randomly into daughter cells.
  • chromosomes have been constructed in yeast using the thiee cloned essential chromosomal elements Murray et al 1983. disclose a cloning system based on the in vitro constiuction of linear DNA molecules that can be transformed into yeast where they are maintained as artificial chromosomes
  • yeast artificial chromosomes contain cloned genes, origins of replication centromeres and telomeres and are segregated in daughter cells with high fidelity when the YAC is at least 100 kB in length Smaller CEN containing vectors may be stably segregated, however, when in circular form
  • yeast CEN sequence will not confer stable inheritance upon vectors transformed into higher eukaryotes While such DNA fragments can be readily introduced they do not stably exist as episomes in the host cell This has seriously hampered efforts to produce artificial chromosomes in higher organisms
  • telomeres are important in maintaining the stability of chromosomal termini they do not encode the information needed to ensure stable inheritance of an artificial chromosome
  • centromere function is crucial for stable chromosomal inheritance in almost all eukaryotic organisms (reviewed in Nicklas 1988)
  • broken chromosomes that lack a centromere are rapidly lost from cell lines while fragments that have a centromere are faithfully segregated
  • the centromere accomplishes this by attaching via centromere binding proteins to the spindle fibers during mitosis and meiosis, thus ensuring proper gene segregation during cell divisions
  • a method for the identification of plant centromeres may comprise tetrad analysis
  • tetrad analysis measures the recombination frequency between genetic makers and a centromere by analyzing all four products of individual meiosis
  • qi t 1 c ⁇ iat tet
  • the c ⁇ iaitet mutation may also find use in accordance with the invention in species other than Aiahidopsis
  • several naturally occurring plant species are also known to release pollen clusters including water lilies cattails heath (E ⁇ caceae and Epaci idceae) evening primrose (Onaeiaceae) sundew s (Dioseiaceae) orchids (Orchidaceae) and acacias (Mimosace
  • the invention provides a recombinant DNA construct comprising a plant centromere
  • the recombinant DNA construct may additionally comprise any other desired sequences, for example, a telomere. including a plant telomere such as an Arabidopsis thaliana telomere. or alternatively, a yeast or any other type of telomere
  • a telomere including a plant telomere such as an Arabidopsis thaliana telomere. or alternatively, a yeast or any other type of telomere
  • ARS autonomous replicating sequence
  • one may wish to include a structural gene on the construct, or multiple genes (for example, two. three, four. five. six. seven, eight, nine, ten.
  • the construct is capable of expressing the structural gene, for example, in a prokaryote oi eukaryote, including a lower eukaryote. or a higher eukaryote such as a plant
  • the invention provides a recombinant DNA construct comprising a plant centromere and which is a plasmid
  • the plasmid may contain any desired sequences, such as an origin of replication, including an origin of replication functions in bacteria, such as E. coli and Agrobactermm, or in plants or yeast, for example, such as S cerevisiae
  • the plasmid may also comprises a selection marker, which may function in bacteria, including E. coli and Agrobactermm. as well as a selection marker that functions in plants or yeast, such as 5 cerevisiae.
  • the invention provides a recombinant DNA construct comprising a plant centromere and which is capable of being maintained as a chromosome, wherein the chromosome is transmitted in dividing cells.
  • the plant centromere may be from any plant.
  • the invention provides a plant centromere which is further defined as an Arabidopsis thaliana centromere.
  • the plant centromere is an Arabidopsis thaliana chromosome 1 centromere. and may still further be defined as flanked by the genetic markers T22C23-T7 and T3P8-SP6, or still further as flanked by the genetic markers T22C23-T7 and T5D18, T22C23-T7 and T3L4, T5D18 and T3P8-SP6, T5D 18 and T3L4, and T3L4 and T3P8-SP6.
  • the plant centromere comprises an Arabidopsis thaliana chromosome 2 centromere
  • the chromosome 2 centromere may comprise, for example, from about 100 to about 61 1.000. about 500 to about 61 1.000, about 1.000 to about 61 1.000, about 10.000 to about 61 1.000. about 20.000 to about 61 1.000. about 40.000 to about 61 1.000, about 80.000 to about 61 1.000, about 150.000 to about 61 1 ,000. or about 300,000 to about 61 1.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:209.
  • centromere may also be defined as comprising from 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 10.000 to about 50.959. 20.000 to about 50.959. about 30.000 to about 50.959. or about 40,000 to about 50.959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:210. and may comprise the nucleic acid sequence of SEQ ID NO:210.
  • the centromere may comprise sequences from both SEQ ID NOS.209 and 210. including the aforementioned fragments, or the entirety of SEQ ID NOS 209 and 210 In particular embodimemnts. the inventors contemplate a 3' fragment of SEQ ID NO 209 can be fused to a 5' fragment of SEQ ID NO 210. optionally including one or more 180 bp repeat sequence disposed therebetween
  • the invention provides an Arabidopsis thaliana chromosome 3 centromere
  • the centromere may be further defined as flanked by the genetic markers T9G9-SP6 and T5M 14-SP6. and still further defined as flanked by a pair of genetic markeis selected from the group consisting of T9G9-SP6 and TI4H20. T9G9-SP6 and T7K14. T9G9-SP6 and T21P20. T14H20 and T7K14, T14H20 and T21P20, T 14H20 and T5M 14-SP6. T7K14 and T5M 14-SP6. T7K14 and T5M 14-SP6. T7K14 and T21P20. and T21P20 and T5M14-SP6
  • the invention provides an Arabidopsis thaliana chromosome 4 centromere
  • the centromere may comprise from about 100 to about 1.082.000. about 500 to about 1.082,000. about 1.000 to about 1.082,000. 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, about 200.000 to about 1.082.000, about 400.000 to about 1.082,000, or about 800,000 to about 1 ,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 21 1. including comprising the nucleic acid sequence of SEQ ID NO 21 1
  • the centromere may also be defined as comprising from about 100 to about 163.317.
  • centromere may comprise sequences from both SEQ ID NOS 21 1 and 212. including the aforementioned fragments, or the entirety of SEQ ID NOS 21 1 and 212 In particular embodimemnts. the inventors contemplate a 3 ' fragment of SEQ ID NO:21 1 can be fused to a 5' fragment of SEQ ID NO:212. optionally including one or more 180 bp repeat sequence disposed therebetween.
  • a Arabidopsis thaliana chromosome 1 , 3 or 5 centromere selected from the nucleic acid sequence given by SEQ ID NO: 184.
  • SEQ ID NO: 198. SEQ ID NO: 199.
  • the construct comprises at least 100 base pairs, up to an including the full length, of one of the preceding sequences.
  • the construct may include 1 or more 180 base pair repeats.
  • the invention provides an Arabidopsis thaliana chromosome 5 centromere.
  • the centromere may be further defined as flanked by the genetic markers F13K20-T7 and CUE 1. and still further defined as flanked by a pair of genetic markers selected from the group consisting of F13K20-T7 and T 18M4.
  • T18M4 and T24I20 T18M4 and CUE1.
  • the invention provides a recombinant DNA construct comprising a plant centromere, and further defined as comprising n copies of a repeated nucleotide sequence, wherein n is at least 2
  • Potentially any number of repeat copies capable of physically being placed on the recombinant construct could be included on the construct, including about 5. 10. 15. 20. 30. 50. 75. 100. 150. 200. 300. 400. 500. 750. 1.000. 1.500. 2,000. 3.000. 5.000. 7.500. 10.000. 20.000. 30.000. 40.000. 50.000. 60.000. 70.000. 80.000. 90,000 and about 100.000.
  • the repeated nucleotide sequence may be isolatable from the nucleic acid sequence given by 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. SEQ ID NO:208. SEQ ID NO:209, SEQ ID NO:210. SEQ ID NO:21 1 or SEQ ID NO:212. Examples of such sequences that could be used are given in FIGs. 23A-23D.
  • the length of the repeat used may vary, but will preferably range from about 20 bp to about 250 bp, from about 50 bp to about 225 bp, from about 75 bp to about 210 bp. from about 100 bp to about 205 bp, from about 125 bp to about 200 bp. from about 150 bp to about 195 bp. from about 160 bp to about 190 and from about 170 bp to about 185 bp including about 180 bp.
  • the repeats may be included as part of centromeric structures.
  • the number of repeats may vary and include 1. 2, 3. 4. 5. 6. 7. 8. 9. 10, 1 1. 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.
  • the invention provides a minichromosome vector comprising a plant centromere and a telomere sequence. Any additional desired sequences may be added to the minichromosome. such as an autonomous replicating sequence, a second telomere sequence and a structural gene. One or more of the foregoing sequences may be added . up to the maximum number of such sequences that can physically be placed on the minichromosome.
  • the minichromosome may comprise any of the centromere compositions disclosed herein.
  • the minichromosome may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1. SEQ ID NO:2, SEQ ID NO:3. SEQ ID NO:4. SEQ ID NO:
  • SEQ ID NO:5. SEQ ID NO:6.
  • SEQ ID NO:7 SEQ ID NO:8.
  • the minichromosome also may contain "negative " selectable markers which confer susceptibility to an antibiotic, herbicide 01 other agent, thereby allowing tor selection against plants, plant cells or cells of any othei organism of interest containing a minichromosome
  • the minichromosome also mav include genes which control the copy number of the minichromosome within a cell
  • One or more structural genes also may be included in the minichromosome Specifically contemplated as being useful will be as many structural genes as may be inserted into the minichromosome while still maintaining a functional vector. This may include one. two. three, four, five. six. seven, eight, nine or more structural genes
  • the invention provides a recombinant DNA construct comprising a plant centromere.
  • the cell may be of any type, including a prokaryotic cell or eukaryotic cell Where the cell is a eukaryotic cell, the cell may be. foi example, a yeast cell or a higher eukaryotic cell, such as plant cell
  • the plant cell may be from a dicotyledonous plant, such as tobacco, tomato, potato, soybean, canola. sunflower, alfalfa. cotton and Arabidopsis. or may be a monocotyledonous plant cell, such as wheat, maize, rye. rice, turfgrass. oat.
  • the plant centromere is an Arabidopsis thaliana centromere
  • the cell may be an Arabidopsis thaliana cell.
  • the recombinant DNA construct may comprise additional sequences, such as a telomere. an autonomous replicating sequence (ARS). a structural gene, or a selectable or screenable marker gene, including as many of such sequences as may physically be placed on said recombinant DNA construct.
  • the cell is further defined as capable of expressing said
  • a plant comprising the aforementioned cells
  • the invention provides a method of preparing a transgenic plant cell compnsmg contacting a starting plant cell with a recombinant DNA construct comprising a plant centromere, whereby said starting plant cell is transformed with said recombinant DNA construct
  • the recombinant DNA construct may comprise any desired sequences such as manv structuial genes as can physically be placed on said lecombinant DNA construct
  • the centromere is an Aiabidopsis thaliana centiomeie.
  • the plant cell may be an Aiabidopsis thaliana cell
  • the invention provides a tiansgenic plant comprising a minichromosome vectoi , wherein the v ector comprises a plant centromere and a telomere sequence
  • the minichromosome vector may further comprise an autonomous replicating sequence, second telomere sequence, or a structural gene, such as an antibiotic resistance gene, a herbicide resistance gene, a nitrogen fixation gene, a plant pathogen defense gene.
  • the minichromosome vector may further comprise a nucleic acid sequence selected from the group consisting of 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 1 1. SEQ ID NO 12, SEQ ID NO 13.
  • the transgenic plant may be any type of plant such as a dicotyledonous plant for example, tobacco, tomato, potato pea, carrot, cauliflower, broccoli, soybean canola sunflower, alfalta cotton and Aiabidopsis or may be a monocotyledonous plant such as wheat maize, rye. rice turfgrass, oat barlev- sorghum, millet, and sugarcane
  • the invention provides a method of producing a minichromosome vector comprising (a) obtaining a first vector and a second vector wherein said first vector oi said second vector comprises a selectable or screenable marker, an origin of replication a telomere, and a plant centromere and wherein said first vector and said second vector comprises a site for site-specific recombination and (b) contacting said first v ector with said second v ectoi to allow site-specific recombination to occur between said site for site-specific recombination on said first vector and said site for site-specific recombination on said second vector to create a minichromosome vector comprising said selectable or screenable marker, said origin of replication, said telomere and said plant centromere.
  • the contacting may be done in vitro or /; vivo, including wherein the contacting is carried out in a prokaryotic cell such as an Agrobcicterium or E. coli cell, or in a lower eukaryotic cell, such as a yeast cell.
  • the contacting may still further be carried out in a higher eukaryotic cell, such as a plant cell, including an Arabidopsis thaliana cell.
  • the contacting may be done in the presence of potentially any recombinase. including Cre, Flp, Gin, Pin, Sre, pinD, Int-B 13. and R.
  • the first vector or second vector may comprise border sequences for Agrobacterium-mediated transformation.
  • the plant centromere is an Arabidopsis thaliana centromere.
  • the telomere may be a plant telomere. Any plant selectable or screenable marker could be used, including GFP. GUS, BAR, PAT, HPT or NPTII.
  • a method of screening a candidate centromere sequence for plant centromere activity, said method comprising the steps of: (a) obtaining an isolated nucleic acid sequence comprising a candidate centromere sequence: (b) integratively transforming plant cells with said isolated nucleic acid: and (c) screening for centromere activity of said candidate centromere sequence.
  • the screening may comprise ob.serving a phenotypic effect present in the integratively transformed plant cells or plants comprising the plant cells, wherein the phenotypic effect is absent in a control plant cell not integratively transformed with said isolated nucleic acid sequence, or a plant comprising said control plant cell.
  • Types of phenotypic effects that could be screened for include reduced viability, reduced efficiency of said transforming, genetic instability in the integratively transformed nucleic acid, aberrant plant sectors, increased ploidy, aneuploidy, and increased integrative transformation in distal or centromeric chromosome regions.
  • the isolated nucleic acid sequence may comprise a bacterial artificial chromosome, which may be further defined as a binary bacterial artificial chromosome.
  • the integratively transforming may comprise use of any type of transformation, such as Agrohacterimn- edinted transformation.
  • the control plant cell has been integratively transformed with a nucleic acid sequence other than a candidate centromere sequence
  • the invention provides a iecombinant DNA construct comprising an Arabidopsis polyubiquitin 1 1 promoter, wherein the promotei comprises from about 25 to about 2.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 180
  • the promoter may comprise 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. from about 800 to about 2.000, from about 1 ,000 to about 2.000, or from about 1 ,500 to about 200 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 180, or may comprise the nucleic acid sequence of SEQ ID NO.180.
  • the promoter containing construct may comprise any additional desired sequences, for example, that of an enhancer, a telomere sequence, a plant centromere sequence, an ARS, or a structural gene, including an antibiotic resistance gene, a herbicide resistance gene, a nitrogen fixation gene, a plant pathogen defense gene, a plant stress-induced gene, a toxin gene, a receptor gene, a ligand gene, a seed storage gene, a hormone gene, an enzyme gene, an interleukin gene, a clotting factor gene, a cytokine gene, an antibody gene, and a growth factor gene
  • the promoter may be operably linked to the 5 ' end of the structuial gene
  • the invention provides a recombinant DNA construct comprising an Aiabidopsis 40S ⁇ bosomal protein S 16 promoter, wherein said promoter comprises from about 25 to about 2.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 182
  • the promotei may comprise 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. from about 800 to about 2,000. from about 1 ,000 to about 2.000 or from about 1500 to about 2.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 182. or may comprise the nucleic acid sequence of SEQ ID NO 182
  • the promoter containing construct may comprise any additional desiied sequences, for example, that of an enhancer, a telomere sequence, a plant
  • the promoter may be operably linked to the 5 ' end of the structural gene.
  • the invention provides a recombinant DNA construct comprising an Arabidopsis polyubiquitin 1 1 3 ' regulatory sequence including the terminator sequence, wherein the 3 ' regulatory sequence comprises from about 25 to about 2001 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 181.
  • the 3 ' regulatory sequence may be further defined as comprising from about 75 to about 2001. from about 125 to about 2001 , from about 200 to about 2001. from about 400 to about 2001.
  • the recombinant sequence may further comprise any other sequence, for example, an enhancer, a telomere sequence, a plant centromere sequence, an ARS, and a structural gene, including an antibiotic resistance gene, a herbicide resistance gene, a nitrogen fixation gene, a plant pathogen defense gene, a plant stress-induced gene, a toxin gene, a receptor gene, a ligand gene, a seed storage gene, a hormone gene, an enzyme gene, an interleukin gene, a clotting factor gene, a cytokine gene, an antibody gene, and a growth factor gene.
  • the terminator may be operably linked to the 3' end of the structural gene.
  • the invention provides a recombinant DNA construct comprising an Arabidopsis 40S ⁇ bosomal protein S 16 3 ' regulatory sequence including the terminator 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.
  • the 3 ' regulatory sequence may comprise 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. from about 800 to about 2,000, or from about 1.000 to about 2.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 183.
  • the recombinant sequence may further comprise any other sequence, for example, an enhancer, a telomere sequence, a plant centromere sequence, an ARS, and a structural gene, including an antibiotic- resistance gene, a herbicide resistance gene, a nitrogen fixation gene, a plant pathogen defense gene, a plant stress-induced gene, a toxin gene, a receptor gene, a ligand gene, a seed storage gene, a hormone gene, an enzyme gene, an interleukin gene, a clotting factor gene, a cytokine gene, an antibody gene, and a growth factor gene.
  • the terminator may be operably linked to the 3' end of the structural gene.
  • the invention provides methods for expressing foreign genes in plants, plant cells or cells of any other organism of interest.
  • the foreign genes may be from any organism, including plants, animals and bacteria.
  • minichromosomes could be used to simultaneously transfer multiple foreign genes to a plant comprising entire biochemical or regulatory pathways.
  • the minichromosomes can be used as DNA cloning vectors. Such a vector could be used in plant and animal sequencing projects.
  • the current invention may be of particular use in the cloning of sequences which are "unclonable " in yeast and bacteria, but which may be easier to clone in a plant based system.
  • minichromosomes disclosed herein may be used to clone functional segments of DNA such as origins of DNA replication, telomeres, telomere associated genes, nuclear matrix attachment regions (MARs), scaffold attachment regions (SARs), boundary elements, enhancers, silencers, promoters, recombinational hot-spots and centromeres.
  • This embodiment may be carried out by cloning DNA into a defective minichromosome which is deficient for one or more type of functional elements.
  • centromere may be isolated from agriculturally important species such as. for example, vegetable crops, including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce (e g , head, leaf, romaine).
  • agriculturally important species such as. for example, vegetable crops, including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce (e g , head, leaf, romaine).
  • centromeres could be isolated from fruit and vine crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, quince almonds, chestnuts, filberts, pecans, pistachios walnuts, citrus, blueberries, boysenber ⁇ es.
  • centromeres could be isolated in accordance with the invention from field crop plants, such as evening primrose, meadow foam, corn (field, sweet popcorn), hops. jo
  • sorghum tobacco, kapok, leguminous plants (beans, lentils, peas, soybeans), oil plants (rape, mustard, poppy, olives, sunfloweis. coconut. castor oil plants, cocoa beans, groundnuts), fibre plants (cotton flax. hemp.
  • lauraceae cinnamon, camphor
  • plants such as coffee, sugarcane, tea. and natural rubber plants.
  • plants from which centromeres could be isolated include bedding plants such as flowers, cactus, succulents and ornamental plants, as well as trees such as forest (broad-leaved trees and evergreens, such as conifers), fruit. ornamental, and nut-bearing trees, as well as shrubs and other nursery stock.
  • the minichromosome vectors described herein may be used to perform efficient gene replacement studies.
  • gene replacement has been detected on only a few occasions in plant systems and has only been detected at low frequency in mammalian tissue culture systems (see Thomas et al., 1986; Smithies et al., 1985)
  • the reason for this is the high frequency of illegitimate nonhomologous recombination events relative to the frequency of homologous recombination events (the latter are responsible for gene replacement)
  • Artificial chromosomes may participate in homologous recombination preferentially.
  • the artificial chromosome vectors disclosed by the present invention will be maintained in the nucleus through meiosis and available to participate in homology-dependent meiotic recombination
  • the vectors could be used to introduce extremely long stretches of DNA from the same or any other organism into cells.
  • Specifically contemplated inserts include those from about several base pans to one hundred megabase pans, including about 1 kb, 25 kB.
  • the present invention provides methods for the construction of minichromosome vectors for the genetic transformation of plant cells. uses of the vectors, and organisms transformed by them. Standard reference works setting forth the general principles of recombinant DNA technology include Lewin 1985 Other works describe methods and products of genetic engineering See, e g Maniatis et al 1982 Watson et al 1983 Setlow et al 1979 and Dillon et al 1985
  • the inv ention provides a method of preparing a transgenic cell
  • the method comprises the steps of a ) obtaining a nucleic acid molecule comprising Aiabidopsis thaliana centromere DNA having the following characteristics 1 ) mapping to a location on an Arabidopsis thaliana chromosome defined by a pair of genetic markers selected from the group consisting of m ⁇ 342 and T27K 12 m ⁇ 310 and g4133, atpox and ATA m ⁇ 233 and m ⁇ l67, and F13K20- t7 and CUE1 , and 2 ) sorts DNA to the spindle poles in meiosis 1 in a pattern indicating the disjunction of homologous chromosomes b) preparing a recombinant construct comprising said nucleic acid molecule and c) transforming a recipient cell with said recombinant construct
  • the cell may be, for example a lower eukaryotic cell including a yeast cell, or may be a higher eukaryotic cell Where the cell is a higher eukaryotic cell, the cell may 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 invention, the Arabidopsis thaliana centromere is defined by the marker pair m ⁇ 342 and T27K12 which may be further defined by the genetic marker pair T22C23-t7 and T3P8-sp6, and / or is defined by the marker pair m ⁇ 310 and g4133 which may be fuithei defined by the genetic marker pair F5J 15-sp6 and T15D9 and / or is defined by the marker pair atpox and ATA which may be further defined by the genetic marker pair T9G9-sp6 and T5M 14-sp6.
  • the transforming may comprise use of a method selected from the group consisting of Agrobacteriiim- ediated transformation, protoplast transfoimation.
  • the recombinant construct may comprise desired elements, including a telomere. such as an Arabidopsis thaliana or yeast telomere.
  • the recombinant construct may also comprise an autonomous replicating sequence (ARS). for example, an Arabidopsis thaliana ARS
  • the recombinant construct may also comprise a prokaryotic or eukaryotic selectable or screenable marker gene.
  • Also desired to include with a recombinant construct may be one or more structural genes
  • Exemplary structural genes include a gene 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-induced gene, a toxin gene, a seed storage gene, a hormone gene, an enzyme gene, an interleukin gene, a clotting factor gene, a cytokine gene, an antibody gene, and a growth factor gene.
  • the method may further comprise the step of regenerating a transgenic plant from said cell
  • the invention provides a method of 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 the Arabidopsis thaliana centromere is defined by a pair of genetic markers selected from the group consisting of m ⁇ 342 and T27K 12. m ⁇ 310 and g4133, atpox and ATA. m ⁇ 233 and m ⁇ l67. and F13K20-t7 and T17M l l-sp6.
  • the ability to demonstrate a stable inheritance pattern may be determined by preparing a recombinant cell that comprises the recombinant construct.
  • the Arabidopsis thaliana centromere is defined by the marker pair m ⁇ 342 and T27K12. which may be further defined by the genetic marker pair T22C23-t7 and T3P8-sp6. and / or is defined by the marker pair m ⁇ 310 and g4133, which may be further defined by the genetic marker pair F5J 15-sp6 and T 15D9.
  • the marker pair atpox and ATA which may be further defined by the genetic marker pair T9G9-sp6 and T5M 14-sp6. and / or is defined by the markei pair m ⁇ 233 and mi 167, which may be further defined by the genetic marker pan T24H24 30k3 and F13H !4-t7. and / or is defined by the genetic marker pair F13K20-t7 and CUE1. which may be further defined bv a genetic marker pair selected from the group consisting of F13K20-T7 and T18M4. F13K20-T7 and T18F2, F13K20-T7 and T24I20. T18M4 and T18F2. T18M4 and T24I20, T 18M4 and CUE1 , T18F2 and T24I20, T18F2 and CUE 1. and T24I20 and CUEl .
  • the recombinant construct is not chromosomally integrated
  • Said obtaining may comprise obtaining a BAC or YAC clone comprising said Arabidopsis thaliana centromere DNA
  • the DNA may be obtained by a method that includes the use of pulsed-field gel electrophoresis. and may be obtained by a method that includes positional cloning.
  • the positional cloning may comprise identifying a contiguous set of clones comprising said Arabidopsis thaliana centromere DNA. wherein said set of clones is flanked by a pair of genetic markers selected from the group consisting of m ⁇ 342 and T27K12. m ⁇ 310 and g4133. atpox and ATA. m ⁇ 233 and mi 167. and F13K20-t7 and T17M 1 l-sp6
  • the contiguous set of clones may span the Arabidopsis thaliana centromeie
  • the recombinant construct may comprise a selectable or screenable marker and said step of determining may comprise determining a phenotype conferred by the selectable oi screenable marker
  • the determining may comprise, for example, determining the ability of the recombinant construct to demonstrate a stable inheritance pattern in mitosis and / or meiosis.
  • the invention provides a transgenic cell prepared by a method provided by the invention Also provided by the invention aie a transgenic plant, plant parts and tissue cultures comprising the transgenic cell
  • the Aiabidopsis thaliana centromere is defined by the marker pair m ⁇ 342 and T27K12. which may be further defined by the genetic maiker pair T22C23-t7 and T3P8-sp6. and / or is defined by the marker pair m ⁇ 310 and g4133.
  • -09- may be further defined by the genetic marker pair F5J 15-sp6 and T15D9: and / or is defined by the marker pair atpox and ATA. which may be further defined by the genetic marker pair T9G9-sp6 and T5M 14-sp6: and / or is defined by the marker pair mi233 and mi 167. which may be further defined by the genetic marker pair T24H24.30k3 and F13H 14-t7: and / or is defined by the genetic marker pair F13K20-t7 and CUE 1. which may be further defined by a genetic marker pair selected from the group consisting of F13K20-T7 and T18M4, F13K20-T7 and T 18F2.
  • T18M4 and T18F2 T18M4 and T24I20, T18M4 and CUE 1 , T18F2 and T24I20.
  • T18F2 and CUE1, and T24I20 and CUEl T18F2 and CUE1, and T24I20 and CUEl .
  • a centromere used in accordance with the invention is not from Arabidopsis, for example, from Arabidopsis thaliana.
  • a plant or plant cell comprising a centromere composition in accordance with the invention may also be from a plant other than Arabidopsis.
  • FIG. 1 Centromere mapping with unordered tetrads: A cross of two parents (AABB x aabb). in which "A” is on the centromere of one chromosome, and "B” is linked to the centromere of a second chromosome. At meiosis, the A and B chromosomes assort independently, resulting in equivalent numbers of parental ditype (PD) and nonparental ditype (NPD) tetrads (recombinant progeny are shown in gray). Tetratype tetrads (TT) result only from a crossover between "B " and the centromere.
  • PD parental ditype
  • NPD nonparental ditype
  • TT Tetratype tetrads
  • FIG. 2 Low resolution map location of Arabidopsis centromeres. Trisomic mapping was used to determine the map position of centromeres on four of the five Arabidopsis chromosomes (Koornneef, 1983: Sears et al.. 1970). For chromosome 4. useful trisomic strains were not obtained. With the methods of Koornneef and Sears et al, 1983. (which rely on low-resolution deletion mapping) the centromere on chromosome 1 was found to lie between the two visible markers, ttl and chl, that are separated by 5 cM. Centromere positions on the other chromosomes are mapped to a lower resolution.
  • FIG. 3 Physical maps of the genetically-defined Arabidopsis centromeres. Each centromeric region is drawn to scale; physical sizes are derived from DNA sequencing (chromosomes II and IV) or from estimates based on BAC fingerprinting (Marra er /., 1999; Mozo et al, 1999) (chromosomes I, III. and V). Indicated for each chromosome are positions of markers (above), the number of tetratype / total tetrads at those markers (below), the boundaries of the centromere (thick black bars), and the name of contigs derived from fingerprint analysis (Marra et al, 1999; Mozo et al, 1999).
  • BAC-end 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 Landsberg ecotypes (Bell and Ecker, 1994: Konieczny and Ausubel. 1993); primer sequences are available (http://genome-wwvv.stanford.edu/Arabidopsis/aboutcaps.html).
  • Tetratype tetrads resulting from treatments that stimulate crossing over boxes
  • positions of markers in centimorgans (cM) shared with the recombinant inbred (RI) map (ovals) http://nasc.nott.ac.uk/new_ri_map.html: Somerville and Somerville, 1999
  • sequences bordering gaps in the physical map that correspond to 180 bp repeats open circles
  • 5S rDNA black circles
  • 160 bp repeats gray cucles
  • uie indicated Copenhaver et al , 1999
  • FIG. 4 Exemplary list of seed stock used foi tetiad analysis in Aiabidopsis thaliana
  • the individual strains are identified by the strain number (column B)
  • the tetrad member number (column A) indicates the tetrad souice (i e .
  • T l indicates seeds from tetrad number 1 , and the numbers - 1. -2, -3 or -4 indicate individual membeis of the tetrad)
  • the strains listed have been deposited with the Aiabidopsis Biological Resources Centei (ABRC) at Ohio State University undei the name of Daphne Preuss
  • FIG. 5 Marker information for centromere mapping DNA polymorphisms used to localize the centromeres are indicated by chromosome (Column 1 ) The name of each marker is shown in Column 2 the name of the markers used by Copenhaver et al 1999 to position centromeres is given in Column 3 and marker type is indicated in Column 4 CAPS (Co-dominant Amplified Polymorphic Sites) are markers that can be amplified with PCR and detected by digesting with the appropriate restriction enzyme (also indicated in Column 3) SSLPs (Simple Sequence Length Polymorphisms) detect polymorphisms by amplifying different length PCR products Column 5 notes if the marker is available on public web sites (e http //genome-www Stanford edu/ Arabidopsis) For those markers that are not available on public web sites the sequences of the foiwaid and reverse primers used to amplify the marker aie listed in columns 6 and 7 respectively
  • FIG. 6 Scoring PCR-based markers for tetrad analysis
  • Exemplary Minichromosome vectors The vectors shown in FIG. 7A, FIG. 7B, FIG. 7E, FIG. 7F, FIG. 71 and FIG. 7J have an E. coli origin of replication which can be high copy number, low copy number or single copy. In FIGS. 7A-7N.
  • the vectors include a multiple cloning site which can contain recognition sequences for conventional restriction endonucleases with 4-8 bp specificity as well as recognition sequences for very rare cutting enzymes such as, for example.
  • the centromere is flanked by Lox sites which can act as targets for the site specific recombinase Cre.
  • FIG. 7A Shows an E. coli plant circular shuttle vector with a plant ARS.
  • FIG. 7B Shows a plant circular vector without a plant ARS.
  • FIG. 7C Shows a yeast-plant circular shuttle vector with a plant ARS.
  • the yeast ARS is included twice, once on either side of multiple cloning site to ensure that large inserts are stable.
  • FIG. 7D Shows a yeast-plant circular shuttle vector without a plant ARS.
  • the vector relies on a plant origin of replication function found in other plant DNA sequences such as selectable markers.
  • the yeast ARS is included twice, once on either side of the multiple cloning site to ensure that large inserts are stable.
  • FIG. 7E Shows an E. coli-Agrobacteriitm-plani circular shuttle vector with a plant ARS.
  • FIG. 7F Shows an E. coli-Agroh ⁇ cteriitm-planl circular shuttle vector without a plant ARS. The vector relies on a plant origin of replication function found in other plant DNA sequences such as selectable markers. Vir functions for T-DNA transfer would be provided in trans by a using the appropriate Agrob ⁇ cterium strain.
  • FIG. 7G Shows a linear plant vector with a plant ARS. The linear vector could be assembled in vitro and then transferred into the plant by, for example, mechanical means such as micro projectile bombardment, electroporation, or PEG-mediated transformation.
  • FIG. 7H Shows a linear plant vector with a plant ARS. The linear vector could be assembled in vitro and then transferred into the plant by, for example, mechanical means such as micro projectile bombardment, electroporation, or PEG-mediated transformation.
  • FIGs. 7I-7N Shows a linear plant vector without a plant ARS.
  • the linear vector could be assembled in vitro and then transferred into the plant by, for example, mechanical means such as micro projectile bombardment, electroporation. or PEG-mediated transformation.
  • FIGs. 7I-7N The figures are identical to FIGs. 7A-7F, respectively, with the exception that they do not contain plant telomeres These vectors will remain circular once delivered into the plant cell and therefore do not require telomeres to stabilize their ends
  • FIG. 8 Sequence teatuies at CEN2 (A) and CEN 4 (B) Central bars depict annotated genomic sequence of indicated BAC clones, black, genetically-defined centromeres, white regions flanking the centromeres Sequences corresponding to genes and repetitive features, filled boxes (above and below the bars, respectively), are defined as in FIG 12A-T, predicted nonmobile genes, red genes carried by mobile elements, black, nonmobile pseudogenes, pink, pseudogenes carried by mobile elements, gray. retroelements, yellow, transposons, green, previously defined centromenc repeats, dark blue 180 bp repeats, pale blue Chromosome-specific centromere features include a large mitochond ⁇ al DNA insertion (orange CEN2). and a novel array of tandem repeats (purple, CEN4) Gaps in the physical maps (//). unannotated regions (hatched boxes), and expressed genes (filled circles) are shown
  • FIG. 9 Method for converting a BAC clone (or any other bacterial clone) into a minichromosome
  • a portion of the conversion vector will integrate into the BAC clone (or other bacterial clone of interest) either through non-homologous recombination (transposable element mediated) or by the action of a site specific recombinase system such as Cre-Lox or FLP-FRT
  • FIG. 10 Method for analysis of diccnt ⁇ c chromosomes in Aiabidopsis BiBAC vectors containing centromeie fragments (- 100 kb) are integrated into the Aiabidopsis genome using A robactermm-medialed transformation procedures and studied for adverse affects due to formation of dicent ⁇ c chromosomes 1) BiBACs containing centromere fragments are identified using standard protocols 2) Plant transformation 3) Analysis of detects in growth and development of plants containing dicent ⁇ c chromosomes FIG. 11A-G. Method for converting a BAC clone (or any other bacterial clone) into a minichromosome. The necessary selectable markers and origins of replication for propagation of genetic material in E. coli.
  • Agrohacterium and Arabidopsis as well as the necessary genetic loci for Agrohacterium mediated transformation into Arabidopsis are cloned into a conversion vector.
  • Cre/loxP recombination the conversion vectors are recombined into BACs containing centromere fragments to form minichromosomes.
  • FIG. 12A-T Properties of centromeric regions on chromosomes II and IV.
  • Top Drawing of genetically-defined centromeres (gray shading, CEN2, left: CEN4, right), adjacent pericentromeric DNA. and a distal segment of each chromosome, scaled in Mb as determined by DNA sequencing (gaps in the grey shading correspond to gaps in the physical maps). Positions in cM on the RI map (http://nasc.nott.ac.uk/new_ri_map.html) and physical distances in Mb, beginning at the northern telomere and at the centromeric gap, are shown. (Bottom) The density of each feature (FIGs.
  • FIG. 12A-12T is plotted relative to the position on the chromosome in Mb.
  • FIG. 12A, 12K cM positions for markers on the RI map (solid squares) and a curve representing the genomic average of 1 cM/221 kb (dashed line).
  • a single crossover within CEN4 in the RI mapping population http://nasc.nott.ac.uk/new_ri_map.html: Somerville and Somerville, 1999) may reflect a difference between male meiotic recombination monitored here and recombination in female meiosis.
  • FIGs. 12B, 12L 180 bp repeats: (FIGs. 12C. 12M) sequences with similarity to retroelements, including del, Tal , Tal l , copia, Athila, LINE, Ty3, TSCL, 106B (Athila-like), Tat l .
  • centromeric repeats including 163 A. 164A, 164B. 278A, 1 1 B7RE, m ⁇ l 67. pAT27, 160-. 180- and 500-bp repeats, and telomeric sequences
  • telomeric sequences e.g., telomere sequences
  • FIGs. 12F, 12P % adenosine + thymidine was calculated for a 50 kb window with a sliding interval of 25 kb (FIGs. 12G-12J, 12Q-12T). The number of predicted genes or pseudogenes was plotted over a window of 100 kb with a sliding interval of 10 kb.
  • FIG. 12G, 12Q and pseudogenes (FIGs. 121, 12S) typically not found on mobile DNA elements: (FIGs. 12H, 12J, 12R, 12T) predicted genes (FIGs. 12H, 12R) and pseudogenes (FIGs. 12,1, 12T) often carried on mobile DNA, including reverse transcriptase, transposase. and retroviral polyproteins. Dashed lines indicate regions in which sequencing or annotation is in progress, annotation was obtained from GenBank records
  • FIG. 13 Methods for converting a BAC clone containing centromere DNA into a minichromosome for introduction into plant cells.
  • the specific elements described are provided for exemplary purposes and are not limiting.
  • FIG. 14A-B Conservation of centromere DNA BAC clones (bars) used to sequence CEN2 (FIG. 14A) and CEN4 (FIG. 14B) are indicated, arrows denote the boundaries of the genetically-defined centromeres PCR primer pans yielding products from only Columbia (filled circles) or from both Landsberg and Columbia (open circles). BACs encoding DNA with homology to the mitochond ⁇ al genome (gray bars), 180 bp repeats (gray boxes), unsequenced DNA (dashed lines), and gaps in the physical map (double slashes) are shown
  • FIG. 15A-B Primers used to analyze conservation of centromere sequences in the A thaliana Columbia and Landsberg ecotvpes
  • FIG. 15A Primers used for amplification of chromosome 2 sequences
  • FIG. 15B Primers used for amplification of chromosome 4 sequences
  • FIG. 16 Sequences common to CEN2 and CEN4 Genetically-defined centromeres (bold lines), sequenced (thin lines), and unannotated (dashed lines) BAC clones are displayed as in FIG 14A, B Repeats AtCCS l (A thaliana centromere conserved sequence) and AtCCS2 (closed and open circles, respectively), AtCCS3 (triangles), and AtCCS4-7 (4-7. respectively) are indicated (GenBank Accession numbers AF204874 to AF204880). and were identified using BLAST 2 0 (http //blast wustl edu)
  • FIG. 17 Sequenced BAC clones from centromere 2
  • the sequenced BAC clones are indicated by the horizontal lines near the top of the figure (see for example T14A4)
  • the red box denotes the boundaries of centromere 2. and for the BAC clones that comprise the centromere.
  • GenBank Accession numbers are given in the lower right panel
  • the contiguous sequences within the red box aie given by SEQ ID NO 209 and SEQ ID NO 210 Horizontal lines below the sequenced clones indicate additional BAC clones.
  • sequenced end points of these BACs are indicated with a closed circle Clones with one or more endpoints that aie undetermined are indicated by led text
  • FIG. 18 Sequenced BAC clones from centiomere 4
  • the sequenced BAC clones from centromeie 4 are indicated by the horizontal lines near the top of the figure (see for example T24M8)
  • the red box denotes the boundaries of centromere 4 and for the BAC clones that comprise the centromere GenBank Accession numbers are given in the lower right panel
  • the contiguous sequences w ithin the red box are given by SEQ ID NO 21 1 and SEQ ID NO 212
  • Horizontal lines below the sequenced clones indicate additional BAC clones, sequenced end points of these BACs are indicated with a closed circle Clones with one or more endpoints that are undetermined are indicated by red text
  • FIG. 19 Sequence tiling path of centromeres 1 3 and 5 The boundaries of these centromeres was determined as described in Copenhaver et al ( 1999) Contig numbers refer to the fingerprint contigs assembled by Marra et al ( 1999) Some of these clones have been sequenced and accession numbers are provided (see attached list) In other cases sequencing will be finished bv the Aiabidopsis genome project
  • FIG. 20 Position of DNA from centiomere 2 earned in BiBAC vectors Clones were placed on the physical map by fingerprint and PCR analysis and comparison with the sequenced BAC clones
  • FIG. 21 Exemplary methods tor adding selectable oi screenable markers to BiBAC clones
  • the desired marker is flanked by transposon borders and incubated with the BiBAC in the presence of transposase Subsequently the BiBAC is introduced into plants Often these BiBACs may integrate into natural chromosome, creating a dicent ⁇ c chromosome which may have altered stability and mav cause chromosome breakage resulting in novel chromosome fiagments
  • FIG. 22 Assay of chromosome stability The stability of natural chromosomes, constructed minichromosome.
  • chromosomes can be assessed by monitoring the assoitment of coloi maikers thiough cell div ision
  • the markei s are linked to the centromere in modified BAC or BiBAC vectors and introduced into plants Regulation of the marker gene by an appropriate promoter determines which tissues will be assayed
  • root-specific promotei s such as SCARECROW make it possible to monitor assortment in files of root cells
  • post-meiotic pollen-specific promoters such as LAT52 allow monitoring of assortment through meiosis
  • general promoters such as the 35S Cauliflower mosaic virus promoter make it possible to monitor assortment in many other plant tissues
  • Qualitative assays assess the general pattern of stability and measure the size of sectors corresponding to marker loss, while quantitative assays require knowledge of cell lineage and allow the number of chromosome loss events to be calculated during mitosis and meiosis
  • FIG. 23A-D Sequence alignments for 180 bp repeats from centromeres 1 -4
  • the left hand column indicates the BAC source of the repeat copy and an arbitrarily assigned number given to the sequence
  • the designation fl 2g6-l indicates a repeat copy from BAC number f 12g6 and arbitrarily given a repeat number of 1
  • the nucleic acid sequences of the BACs containing the repeat copies designated fl 2g6, f5al 3, t25fl5. t l2j2, tl4c8, t6c20. f21 ⁇ 2. and f6h8 are given by SEQ ID NO 184.
  • SEQ ID NO 206 are given by SEQ ID NO 191.
  • FIG. 23A Alignment of 180 bp repeats from centromere 1
  • FIG. 23B Alignment of 180 bp repeats from centromere 2
  • FIG. 23C Alignment of 180 bp repeats from centromere 3
  • FIG. 23D Alignment of 180 bp repeats from centromere 4
  • the inventors have overcome the deficiencies in the prior art by providing, for the first time, the nucleic acid sequence of a plant chromosome
  • the significance of this achievement relative to the prior art is exemplified by the general lack of detailed information in the art regarding the centromeres of multicellular organisms in geneial
  • the most extensiv e and reliable charactenzation of centromere sequences has come from studies of low er eukaryotes such as S. cerevisiae and S. pombe. wheie the ability to analyze centromere functions has provided a clear picture of the desired DNA sequences
  • the S. cere ⁇ ⁇ s ⁇ ae centromere consists of three essential regions, CDEI.
  • pombe centromeres are between 40 and 100 kB in length and consist of repetitive elements that comprise 1 to 3% of each chromosome (Baum et al, 1994). Subsequent studies, using tetrad analysis to follow the segregation of artificial chromosomes, demonstrated that less than 1/5 of the naturally occurring S. pombe centromere is sufficient for centromere function (Baum et al , 1994)
  • centromeres of mammals and other higher eukaryotes are poorly defined Although DNA fragments that hybridize to centromeric regions in higher eukaryotes have been identified, little is known regarding the functionality of these sequences (see Tyler-Smith et al, 1993) In many cases centromere repeats correlate with centromere location, with probes to the repeats mapping both cytologically and genetically to centromere regions Many of these sequences are tandemly-repeated satellite elements and dispersed repeated sequences in arrays ranging from 300 kB to 5000 kB in length (Willard 1990) To date, only one of these repeats, a 171 bp element known as the alphoid satellite, has been shown by in situ hybridization to be present at each human centromere (Tyler-Smith et al , 1993) Whether repeats themselves repiesent functional centromeres remains controversial, as othei genomic DNA is requned to confer inheritance upon a region of DNA (Willard.
  • centromeres have been estimated by analyzing the segregation of chromosome fragments. This approach is imprecise, however, because a limited set of fragments can be obtained, and because normal centromere function is influenced by surrounding chromosomal sequences (for example, see Koornneef. 1983: FIG 2)
  • a more precise method for mapping centromeres that can be used in intact chromosomes is tetrad analysis (Mortimer et al. 1981 ). which provides a functional definition of a centromere in its nativ e chromosomal context. At present, the only centromeres that have been mapped in this manner are from lower eukaryotes.
  • novel chiomosomes can have alphoid DNA spread throughout their length yet have only a single centromeric constriction, indicating that a block of alphoid DNA alone may be insufficient for centromere function (Tyler-Smith et al. 1993)
  • Cytology in Aiabidopsis thaliana has served to coi relate centromere structuie with repeat sequences
  • a fluorescent dye DAPI allows visualization of centromeric chromatin domains in metaphase chromosomes
  • FISH fluorescence //; situ hv bndization (FISH) probe based on 180 bp pALl repeat sequences colocahzed with the DAPI signature near the centromeres of all five Aiabidopsis chromosomes (Maluszynska et al , 1991 , Martinez-Zapater et al , 1986)
  • FISH fluorescence //; situ hv bndization
  • centromeres of Arabidopsis thaliana have been mapped using trisomic strains where the segregation of chromosome fragments (Koornneef 1983 ) oi whole chromosomes (Sears et al 1970) w as used to localize four of the centromeres to within 5 12 17 and 38 cM respectively (FIG 2)
  • These positions have not been refined by moie iecent studies because the method is limited the difficulty of obtaining v iable trisomic strains (Koornneef 1983)
  • These factors introduce significant error into the calculated position of the centromere and in Aiabidopsis where 1 cM coriesponds roughly to 200 kB (Koornneef 1987 Hwang et al 1991 ), this method did not map any of the centromeres with sufficient precision to make chromosome walking stiategies practical Mapping of the Arabidopsis genome was also discussed by (Hauge et al ,
  • tetrads such as those pioduced by S DCevtsiae or Aiabidopsis genetic mapping using tetrad analysis requires that two markers be scored simultaneously (Whitehouse 1950) Tetrads fall into different classes depending on whether the markers are in a parental (nonrecombinant) or nonparental (recombinant) configuration (FIG 1 )
  • a tetrad with only nonrecombinant members is referred to as a parental ditype (PD) one with only recombinant members as a nonpendedal ditype (NPD) and a tetrad w ith two recombinant and two nonrecombinant members as a tetratype (TT) (Perkins 1953)
  • PD parental ditype
  • NPD nonpendedal ditype
  • TT tetratype
  • Tetratype tetrads arise only when a crossover has occurred between a marker in question and its centromere. Thus, to identify genes that are closely linked to the centromere, markers are examined in a pair-wise fashion until the TT frequency approaches zero.
  • the genetic distance (in centimorgans, cM) between the markers and their respective centromeres is defined by the function [( 1/2)TT]/100 (Mortimer et al, 1981 ). Because positional information obtained by tetrad analysis is a representation of physical distance between two points, as one approaches the centromere the chance of a recombination event declines.
  • Tetrad analysis has been used to genetically track centromeres in yeasts and other fungi in which products of a single meioses can be collected.
  • the budding yeast Saccharomyces cerevisiae lacks mitotic condensation and thus cytogenetics (Hegemann et al, 1993), yet due to tetrad analysis, has served as the vehicle of discovery for centromere function. Meiosis is followed by the generation of four spores held within an ascus and these can be directly assayed for gene segregation.
  • the recessive qrtl mutation makes it possible to perform tetrad analysis in
  • Centromere mapping with tetrad analysis requires simultaneous analysis of two markers, one of which must be centromere-linked (FIG. 1 ) To identify these centromere-linked markers, markers distributed across all 5 chromosomes were scored and compared in a pairwise fashion.
  • a collection of Arabidopsis tetrad sets was prepared by the inventors for use in tetrad analysis.
  • progeny plants from > 1.000 isolated tetrad seed sets have been germinated and leaf tissue collected and stored from each of the tetrad piogeny plants
  • the leaf tissue from individual plants was used to make DNA for PCR based marker analysis.
  • the plants also were allowed to self-fertilize and the seed they produced was collected.
  • seedlings can be germinated and their tissues utilized for making genomic DNA Tissue pooled from multiple seedlings is useful for making Southern genomic DNA blots for the analysis of restriction fragment length polymo ⁇ hisms (RFLPs).
  • RFLPs restriction fragment length polymo ⁇ hisms
  • the inventors used genetic mapping to unambiguously assign these unanchored contigs to specific centromeres, scoring polymo ⁇ hic markers in 48 plants with crossovers informative for the entire genome (Copenhaver et al, 1998). In this manner. several centromeric contigs were connected to the physical maps of " the chromosome arms (see EXAMPLE 6). and a large set of DNA markers defining centromere boundaries were generated DNA sequence analysis confirmed the structure of the contigs for chromosomes II and IV (Lin et al. 1999)
  • CEN2 and CEN4 were selected in particular for analysis Both reside on structurally similar chromosomes with a 3 5 Mb rDNA arrays on their distal tips, with regions measuring 3 and 2 Mb. respectivelv . between the rDNA and centromeres, and 16 and 13 Mb regions on their long arms (Copenhaver and Pikaard. 1996)
  • centromere sequences were found to harbour 180 bp repeat sequences These sequences were found to reside in the gaps of each centromeric contig (FIG 3. FIGs 12B. 12L). with few repeats and no long arrays elsewhere in the genome BAC clones near these gaps have end sequences corresponding to repetitive elements that likely constitute the bulk of the DNA between the contigs, including 180 bp repeats, 5S rDNA or 160 bp repeats (FIG 3) Fluorescent in situ hybridization has shown these repetitive sequences are abundant components of Arabidopsis centromeres (Murata et al 1997. Heslop-Har ⁇ son et al , 1999.
  • the repetitive DNA flanking the centromeies may play an important role, forming an altered chromatin conformation that serves to nucleate or stabilize centromere structure Alternatively, other mechanisms could result in the accumulation of repetitive elements near centromeres
  • evolutionary models predict lepetitive DNA accumulates in regions of low recombination (Charlesworth et al 1986, Charlesworth et al , 1994) many Arabidopsis repetitive elements are more abundant in the recombinationally active pe ⁇ centrome ⁇ c regions than in the centromeres themselves
  • retroelements and other transposons may preferentially insert into regions flanking the centromeres or be eliminated from the rest of the genome at a higher rate
  • Centromere Compositions Certain aspects of the present invention concern isolated nucleic acid segments and recombinant vectors comprising a plant centromere
  • the plant centromere is an Aiabidopsis thaliana centromere
  • nucleic acid sequences comprising an ,4 thaliana chromosome 2 centromere are provided
  • the sequence of the Aiabidopsis thaliana chromosome 2 centromere 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 flank a series of 180 bp repeats in centromere 2 of A thaliana
  • the chromosome 2 centromeie may tuithei be defined as compnsmg n number of repeats linked to a nucleic acid sequence included in SEQ ID NO.209 or SEQ ID NO:210. or sequences isolated from both of those sequences
  • the number of repeats (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. 8000. 10,000. 30.000. 50.000 or about 100,000.
  • the actual repeat sequence used may vary.
  • repeat sequences that could be used are given in FIGs. 23A-23D and included in the nucleic acid sequences given by SEQ ID NOs 184-208
  • the length of the repeat used may also vary, and may include repeats of. 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 larger or a repeat sequence, for example, as listed in FIG 23A-FIG 23D and included in the nucleic acid sequences given by SEQ ID NOs 184-208
  • Isolated segments of the nucleic acid sequences of SEQ ID NO:209 and SEQ ID NO:210 are also contemplated to be of use with the invention, either with or without being linked to a series of repeats.
  • contiguous nucleic acid segments of about 100. 200, 400. 800, 1 ,500. 3.000. 5.000, 7.500, 10.000, 15.000. 25.000. 40.000. 75.000. 100,000. 125.000. 150.000. 250.000. 350.000. 450.000. 600.000.
  • nucleic acid sequences of SEQ ID NO 209 or SEQ ID NO.210 specifically form part of the instant inv ention
  • such nucleic acid sequences may be linked to n number of repeated sequences, for example, 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. 8000, 10.000. 50.000 or about 100.000
  • the repeat sequence may comprise, 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.
  • nucleic acid sequences comprising an A thaliana chromosome 4 centromere are provided The sequence of the Aiabidopsis thaliana chromosome 4 centromere is exemplif ied by the nucleic acid sequences of SEQ ID NO 21 1 and SEQ ID NO 212 As shown in FIG 18, the nucleic acid sequences of SEQ ID NO 21 1 and SEQ ID NO 212 in Arabidopsis flank a series of repeated sequences As such, the chromosome 4 centromere may further be defined as comprising n number of repeats linked to a nucleic acid sequence included in SEQ ID NO 21 1 or SEQ ID NO 212, or sequences from both SEQ ID NO.21 1 and SEQ ID NO 212 In particular embodiments of the invention, the number of repeats (n), is about 2.
  • the actual repeat sequence used may vary Representative samples of repeat sequences that could be used are given in FIGs 23A-23D wherein these sequences are included in the nucleic acid sequences given by SEQ ID NOs 184- 208
  • the length of the repeat used may also vary, and may include repeats of, tor 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 larger
  • nucleic acid sequences of SEQ ID NO 21 1 and SEQ ID NO 212 are also contemplated to be of use with the invention, either with or without being linked to a series of repeated sequences
  • contiguous nucleic acid segments of about 100, 200 400. 800. 1.500. 3,000, 5.000, 7.500. 10.000. 15.000 25.000 40.000, 75.000. 100.000. 125.000. 150.000, 250.000. 350.000. 450.000 600 000, 700.00 bp of the nucleic acid sequences of SEQ ID NO 21 1 or SEQ ID NO 212 specifically form part of the instant invention
  • such nucleic acid sequences may be linked to n number of repeated sequences for example, where n is 2. 4. 8. 15. 25.
  • the repeat sequence may comprise, 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.
  • 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 Also included with such sequences aie contiguous stretch of from about 10 15. 20, 25. 30 40. 50. 75. 100. 125. 150. 200. 300. 500, 750. 1 ,000. 1.500.
  • nucleic acid sequence of SEQ ID NO 180 and SEQ ID NO 181 it may be desirable to operably link the Arabidopsis polyubiquitin 1 1 promoter sequences to the 5' end of a coding sequence It may also be desiiable to operably link the Aiabidopsis polyubiquitin 1 1 terminator sequence to the 3 ' end of a coding sequence
  • regulatory regions from the Aiabidopsis 40S ⁇ bosomal protein S 16 gene including promoter and terminator sequences thereof
  • 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 Also included with such sequences are contiguous stretch of fiom about 10 15. 20. 25 30 40. 50 75. 100. 125, 150. 200. 300. 500 750. 1.000. 1.500.
  • the invention includes the centromeie sequences giv en by 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 1 SEQ ID NO 12 SEQ ID NO 1 .
  • 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. as well as lengths of about 5.
  • nucleic acid segment comprising an isolated or purified centromeric sequence refers to a nucleic acid segment including centromere sequences and. in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring sequences, or other nucleic acid sequences
  • gene is used for simplicity to refer to a functional nucleic acid segment, protein, polypeptide or peptide encoding unit As will be understood by those in the art.
  • genomic sequences cDNA sequences and smaller engineered gene segments that may express, or may be adapted to express, proteins, polypeptides or peptides
  • isolated substantially away from other sequences means that the sequences of interest, in this case centromere sequences, are included within the genomic nucleic acid clones provided herein. Of course, this refers to the nucleic acid segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
  • the invention concerns isolated nucleic acid segments and recombinant vectors inco ⁇ orating nucleic acid sequences that encode a centromere functional sequence that includes a contiguous sequence from the centromeres of the current invention.
  • the invention concerns isolated nucleic acid segments and recombinant vectors that include within their sequence a contiguous nucleic acid sequence from an A. thaliana centromere. Again, nucleic acid segments that exhibit centromere function activity will be most preferred.
  • nucleic acid segments of the present invention may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of " almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • nucleic acid sequences disclosed herein may find a variety of other uses.
  • the centromere sequences described herein may find use as probes or primers in nucleic acid hybridization embodiments.
  • nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 14 nucleotide long contiguous DNA segment of a centromere sequence of the current invention, for example, of the sequences given by SEQ ID NOS 1-212. and particularly.
  • SEQ ID NOS 1 -21 and SEQ ID NOS' 180-212 will find particular utility Longer contiguous identical or complementaiy sequences, e.g . those of about 20. 30. 40. 50. 100, 200. 500, 1.000. 2.000. 5.000 bp. etc . including all intermediate lengths and up to and including the full-length sequence of the sequences given in SEQ ID NOS 1-212. also will be of use in certain embodiments.
  • nucleic acid probes As described in detail herein, the ability of such nucleic acid probes to specifically hybridize to centromeric sequences will enable them to be of use in detecting the presence of similar, partially complementary sequences from other plants or animals
  • centromeres for the preparation of mutant species primers, or primers for use in preparing other genetic constructions
  • fragments may also be obtained by other techniques such as. e g . by mechanical shearing or by restriction enzyme digestion
  • Small nucleic acid segments or fragments may be readily prepared by. for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.
  • fragments may be obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of U. S. Patents 4,683, 195 and 4.683,202 (each inco ⁇ orated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
  • centromere sequences of the current invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments.
  • one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence.
  • relatively stringent conditions e.g., one will elect relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C.
  • Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating centromeric DNA segments.
  • nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization
  • an appropriate means such as a label
  • appropriate indicator means include fluorescent, radioactive, enzymatic or other ligands such as avidin/biotin. which are capable of giving a detectable signal
  • colo ⁇ met ⁇ c indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotomet ⁇ cally to identify specific hybridization with complementary nucleic acid-containing samples
  • v ery large DNA tiagments up to the size of an entne chromosome are prepared by embedding Aiabidopsis tissues in agarose using toi example, the method described by Copenhaver et al ( 1995) Single stranded DNA ohgomers with sequences homologous to sites flanking the region of DNA to be purified are made to form triple stranded complexes with the agarose embedded DNA using the recombinase enzyme RecA The DNA is then treated with a site specific methylase such as, for example.
  • RARE RecA-Assisted Restriction Endonuclease
  • PFGE pulsed-field gel electrophoresis
  • CHEF Contour-clamped Homogeneous Electric Field electrophoresis
  • Large DNA fragments resolved on CHEF gels can then be analyzed using standard Southern hybridization techniques to identify and measure the size of those fragments which contain both centromere flanking markers and therefor, the centromere After determining the size of the centromere containing fragment by comparison with known size standaids, the region from the gel that contains the centromere fragment can be cut out of a duplicate gel This centromeric DNA can then be analyzed, sequenced.
  • minichromosomes can be constructed by attaching telomeres and selectable markers to the centromere fragment cut from the agarose gel using standard techniques which allow DNA gation within the gel slice Plant cells can then be transformed with this hybrid DNA molecule using the techniques described herein below IV. ##Recombinant Constructs Comprising Centromere Sequences ##
  • the minichromosome will preferably include an autonomous replication sequence (ARS) functional in plants, a centromere functional in plants, and a telomere functional in plants
  • ARS autonomous replication sequence
  • telomere sequence that could be used is an Arabidopsis telomere, which consists of head to tail arrays of the monomer repeat CCCTAAA totaling a few (for example 3-4) kb in length
  • the telomeres of Arabidopsis like those of other organisms, vary in length and do not appear to have a strict length requirement
  • GenBank accession number M20158 Richards and Ausubel. 1988
  • Yeast telomere sequences have also been described (see. e.g , Louis. 1994. Genbank accession number S70807) Additionally, a method for isolating a higher eukaryotic telomere from Arabidopsis thaliana was described by Richards and Ausubel ( 1988)
  • ARS function may be provided to the vector. Additionally, many 5. cerevisiae autonomous replicating sequences have been sequenced and could be used to fulfill the ARS function.
  • Saccharomyces cerevisiae autonomously replicating sequence ARS 131 A GenBank number L25319
  • origins of replications have been also been sequenced and cloned from E. coli and could be used with the invention, for example, the Col E l origin of replication (Ohmori and Tomizawa, 1979; GenBank number V00270).
  • One Agrohacterium origin that could be used is RiA4. The localization of origins of replication in the plasmids of Agrobacterium rhizogenes strain A4 was described by Jouanin et al (1985).
  • DNA may be included.
  • a visible marker such as green fluorescent protein
  • Inclusion of an E. coli plasmid replication origin and selectable marker also may be preferred.
  • the inventors have described a number of exemplary minichromosome constructs in FIGs. 7A-7H. although it will be apparent to those in skill art that many changes may be made in the order and types of elements present in these constructs and still obtain a functional minichromosome within the scope of the instant invention.
  • Artificial plant chromosomes which replicate in yeast also may be constructed to take advantage of the large insert capacity and stability of " repetitive DNA inserts afforded by this system (see Burke et al, 1987).
  • yeast ARS and CEN sequences may be added to the vector.
  • the artificial chromosome is maintained in yeast as a circular molecule using a stuffer fragment to separate the telomeres.
  • a fragment of DNA. from any source whatsoever, may be purified and inserted into a minichromosome at any appropriate restriction endonuclease cleavage site.
  • DNA segment usually w ill include various regulatory signals foi the expression of pioteins encoded by the fragment Alternatively, regulatory signals resident in the minichromosome may be utilized
  • restriction enzymes Restriction endonucleases generally internally cleave DNA molecules at specific recognition sites, making breaks within "recognition" sequences that in many, but not all. cases exhibit two-fold symmetry around a given point Such enzymes typically create double-stranded breaks
  • Some endonucleases create fragments that have blunt ends, that is. that lack any protruding single strands
  • An alternative way to create blunt ends is to use a restriction enzyme that leaves overhangs, but to fill in the overhangs with a polymerase. such as kleno . thereby resulting in blunt ends
  • blunt end ligation can be used to join the fragments directly together
  • any pair of ends may be
  • exonucleases that preferentially break off terminal nucleotides are referred to as exonucleases
  • small deletions can be produced in any DNA molecule by treatment with an exonuclease which starts from each 3' end of the DNA and chews away single strands in a 3' to 5' direction, creating a population of DNA molecules with single-stranded fragments at each end.
  • exonucleases that digest DNA from the 5' end or enzymes that remove nucleotides from both strands have often been used.
  • Some exonucleases which may be particularly useful in the present invention include Bal3 l. SI.
  • Phosphatases and kinases also may be used to control which fragments have ends which can be joined Examples of useful phosphatases include shrimp alkaline phosphatase and calf intestinal alkaline phosphatase An example of a useful kinase is T4 polynucleotide kinase.
  • the termini of the linearized plasmid and the termini of the DNA fragment being inserted must be complementary or blunt in order tor the ligation reaction to be successful Suitable complementarity can be achieved by choosing appropriate restriction endonucleases (i.e., if the fragment is produced by the same restriction endonuclease or one that generates the same overhang as that used to linearize the plasmid. then the termini of both molecules w ill be complementary).
  • at least two classes of the vectors used in the present invention are adapted to receive the foreign oligonucleotide fragments in only one orientation. After joining the DNA segment to the vector, the resulting hybrid DNA can then be selected from among the large population of clones or libraries.
  • a method useful for the molecular cloning of DNA sequences includes in vitro joining of DNA segments, fragmented from a source of high molecular weight genomic DNA, to vector DNA molecules capable of independent replication.
  • the cloning vector may 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 DNA and most significantly, plant DNA.
  • a variety of processes are known which may be utilized to effect transformation; i.e., the inserting of a heterologous DNA sequences into a host cell, whereby the host becomes capable of efficient expression of the inserted sequences.
  • constructs may include 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 & Russell. 1990), a-tubulin, actin (Wang et al, 1992), cab (Sullivan et al, 1989). PEPCase (Hudspeth & Grula, 1989) or those associated with the R gene complex (Chandler et al, 1989).
  • the DNA sequence between the transcription initiation site and the start of the coding sequence i.e., the untranslated leader sequence
  • the untranslated leader sequence can influence gene expression. Therefore, one may also wish to employ a particular leader sequence.
  • a functional gene could be introduced under the control of novel promoters or enhancers, etc., or perhaps even homologous or tissue specific (for example, root-, collar/sheath-, whorl-, stalk-, earshank-, kernel- or leaf-specific) promoters or control elements.
  • the functional gene may be in an antisense orientation relative to the promoter.
  • Terminators It may also be desirable to link a functional gene to a 3' end DNA sequence that acts as a signal to terminate transcription and allow for the poly-adenylation of the mRNA produced by coding sequences.
  • a terminator may be the native terminator of the functional gene or, alternatively, may be a heterologous 3' end.
  • terminators that could be used with the invention are those from the nopaline synthase gene of Agrobacterium tumefaciens (nos 3" end) (Bevan et al, 1983), the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato.
  • marker genes may be desirable to use one or more marker genes in accordance with the invention. Such markers may be adapted for use in prokaryotic, lower eukaryotic or higher eukaryotic systems, or may be capable of use in any combination of the foregoing classes of organisms. By employing a selectable or screenable marker protein, one can provide or enhance the ability to identify transformants. "Marker genes” are genes that impart a distinct phenotype to cells expressing the marker protein and thus allow such transformed cells to be distinguished from cells that do not have the marker Such genes may encode either a selectable or screenable marker, depending on whether the maiker confers a trait which one can "select "" for by chemical means. ; e .
  • a selective agent e g , a herbicide, antibiotic, or the like
  • screening e g., the green fluorescent protein
  • suitable marker proteins are known to the art and can be employed in the practice of the invention.
  • selectable or screenable markers also are genes which encode a "secretable marker" whose secretion can be detected as a means of identifying or selecting for transformed cells
  • markers which are secretable antigens that can be identified by antibody interaction, or even secretable enzymes which can be detected by their catalytic activity
  • Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e g . by ELISA. small active enzymes detectable in extracellular solution (e g .
  • proteins that are inserted or trapped in the cell wall e g , proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S
  • a gene that encodes a protein that becomes sequestered in the cell wall, and which protein includes a unique epitope is considered to be particularly advantageous
  • Such a secreted antigen markei would ideally employ an epitope sequence that would provide low background in plant tissue, a promoter-leader sequence that would impart efficient expression and targeting across the plasma membrane, and would produce protein that is bound in the cell wall and yet accessible to antibodies
  • a normally secreted wall protein modified to include a unique epitope would satisfy all such requirements
  • selectable marker genes may be used in accordance with invention including but not limited to neo (Potrvkus t al 1985) w hich provides kanamvcin resistance and can be selected for using kanamycin G418 paiomomycin etc bai which confers bialaphos oi phosphinothncm resistance a mutant EPSP synthase protein (Hinchee et al 1988) conferring glyphosate resistance, a nit ⁇ lase such as bxn from Klehsiella o aenae which confers resistance to bromoxynil (Stalker ef al 1988), a mutant acetolactate synthase (ALS) which confers resistance to lmidazohnone, sulfonylurea or other ALS inhibiting chemicals (European Patent Application 154 204, 1985), a methotrexate resistant DHFR (Thillet et al 1988), a dalapon dehalogenas
  • selectable marker capable of being used in systems to select transformants are those that encode the enzyme phosphinothncm acetyltransferase such as the bar gene from Stieptoi ces I gi oscopicus or the pat gene from Strepto nces xti idochi omoqenes
  • the enzyme phosphinothncm acetyl transferase (PAT) inactivates the active ingredient in the herbicide bialaphos phosphinothncm (PPT) PPT inhibits glutamine synthetase (Murakami et al 1986 Twell et al 1989) causing rapid accumulation of ammonia and cell death
  • PPT phosphinothncm acetyl transferase
  • a number of 5 barreniae maiker genes are also know n and could be used with the invention, such as foi example the HIS4 gene (Donahue et al 1982 GenBank number JO 1331 )
  • An example of an £ coli marker gene which has been cloned and sequenced and could be used in accordance with the invention is the Ap gene w hich confers resistance to beta-lactam antibiotics such as ampacillin (nucleotides 4618 to 5478 of GenBank accession number U66885 )
  • Screenable Markers that may be employed include a ⁇ -glucuronidase (GUS) or uidA gene w hich encodes an enzyme for which various chromogenic substrates are known, an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al , 1988), a ⁇ -lactamase gene (Sutchffe, 1978), which encodes an enzyme for which various chromogenic substrates are known (e g , PADAC a chromogenic cephalospo ⁇ n), a vv/E gene (Zukowsky et al , 1983) which encodes a catechol dioxygenase that can convert chromogenic catechols an ⁇ -amylase gene (Ikuta et al , 1990) a tyrosinase gene (Katz et al , 1983) which encodes an enzyme capable of oxidizing
  • R gene complex Genes from the maize R gene complex can also be used as screenable markers
  • the R gene complex in maize encodes a protein that acts to regulate the production of anthocyanin pigments in most seed and plant tissue
  • Maize strains can have one or as many as four R alleles which combine to regulate pigmentation in a developmental and tissue specific manner
  • an R gene introduced into such cells will cause the expression of a red pigment and.
  • a maize line carries dominant alleles for genes encoding for the enzymatic intermediates in the anthocvanin biosynthetic pathway (C2 A l A2 Bz l and Bz2), but carries a recessive allele at the R locus transformation of any cell from that line with R will result in red pigment formation
  • Exemplary lines include Wisconsin 22 which contains the rg-Stadler allele and TR1 12.
  • a K55 derivative which is r-g. b, PI
  • any genotype of maize can be utilized if the C I and R alleles are introduced together
  • Another screenable marker contemplated for use in the present invention is fiiefly luciferase, encoded by the lux gene
  • the presence of the lux gene in transformed cells may be detected using, for example. X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry. It also is envisioned that this system may be developed for populational screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening
  • the gene which encodes green fluorescent protein (GFP) is contemplated as a particularly useful reporter gene (Sheen et al . 1995, Haseloff et al , 1997, Reichel et al , 1996; Tian et al. 1997, WO 97/41228). Expression of green fluorescent protein may be visualized in a cell or plant as fluorescence following illumination by particular wavelengths of light
  • genes encoding traits that can be selected against may be useful for eliminating minichromosomes from a cell or for selecting against cells which comprise a particular minichromosome.
  • An example of a negative selectable marker which has been investigated is the enzyme cytosine deaminase (Stouggard, 1993). In the presence of this enzyme the compound 5-fluorocytos ⁇ ne is converted to 5-fluorourac ⁇ l which is toxic to plant and animal cells. Therefore, cells comprising a minichromosome with this gene could be directly selected against.
  • Other genes that encode proteins that render the plant sensitive to a certain compound will also be useful in this context. For example.
  • T-DNA gene 2 from Agrobacter m tumefaciens encodes a protein that catalyzes the conversion of ⁇ -naphthalene acetamide (NAM) to ⁇ -naphthalene acetic acid (NAA) renders plant cells sensitive to high concentrations of NAM (Depicker et al , 1988) V. Isolation of Centromeres From Plants
  • centromere The inventors have provided, for the first time, the nucleic acid sequence of a plant centromere This will allow one of skill in the art to obtain centromere sequences from potentially any species The inventors specifically provide herein below a number of methods which may be employed to isolate such centromeres
  • centromere sequences identified by the inventors were also shown by the inventors to be highly conserved (see e g , Example 5B, Table 3, and Table 4)
  • the novel finding of the inventors that a number of genes reside within the Arabidopsis centromere can therefore be used to find syntenic genes in other organisms (; e , evolutionary conserved relationships in gene order from species to species)
  • sequence of each Arabidopsis gene can be used to search through sequence databases from other plants
  • An exemplary list of such sequences that could be used is a sequence given by 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 1 1 , 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 Also useful would be the genes listed in Tables 3 and 4 Finding identical or similar genes would identify candidates that may reside within or near centromeric regions Mapping these genes using linked markers would identify potential centromeric regions
  • hybridization is used to obtain centromere sequences, it may be desirable to use less stringent hybridization conditions to allow formation of a heteroduplex
  • Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations
  • conditions can be rendered more stringent by the addition of increasing amounts of formamide which serves to destabilize the hybrid duplex in the same manner as increased temperature or decreased salt
  • hybridization conditions can be readily manipulated and thus will generally be a method of choice depending on the desired results
  • the second method takes advantage of the unique DNA properties that the inventors hav e discovered at the Aiabidopsis centromere and adjacent pe ⁇ centromere regions
  • the 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 to the centromere since retroelements, transposons and pseudogenes are very rare outside the centromere and pe ⁇ centromere region
  • gene density decreases from an average of a gene every 4 5 kb on the chromosomal arm down to one in 150 kb at the centromere
  • This unique centromere composition could be exploited in a number of ways to find centromere legions in other species, for example
  • the second method involves in situ hybridization and preferablv fluorescent 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 microscopy to identify parts of a chromosome with a similar percentage of DNA elements as that found at the Arabidopsis centromere
  • FISH fluorescent in situ hybridization
  • the third method involves lmmunoprecipitating known centromere proteins or kinetochore proteins and analyzing bound DNA Antibodies specific to centromere proteins can be incubated with proteins extracted from cells Extiacts can be native or previously treated to cross-link DNA to proteins The antibodies and bound proteins can be purified away from the protein extracts and the DNA isolated The DNA can then be used as a probe for FISH (as talked about above) or to probe libraries to find neighboring centromere sequences
  • centromere-associated genes By identifying, for the first time, centromere-associated genes, the inventors have enabled the production of antibodies to the proteins encoded by such centromere- associated genes
  • the antibodies may be either monoclonal or polyclonal which bind to centromere-associated proteins of the current invention
  • the centromere-associated protein targets of the antibodies include proteins which bind to the centromere region Further, it is specifically contemplated that these centromere-associated protein specific antibodies would allow for the further isolation and characterization of the centromere-associated proteins For example proteins may be isolated which are encoded by the centromeres Recombinant production of such proteins provides a source of antigen for production of antibodies
  • centromere may be used as a ligand to isolate using affinity methods, centromere binding proteins Once isolated these protein can be used as antigens for the production polyclonal and monoclonal antibodies A variation on this technique has been demonstrated by Rattner ( 1991 ), by cloning of centromere-associated proteins through the use of antibodies which bind in the vicinity of the centromere
  • mAbs monoclonal antibodies
  • a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal
  • an immunogenic composition in accordance with the present invention
  • antisera from that immunized animal
  • the animal used for production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat
  • a rabbit is a preferred choice for production of polyclonal antibodies because of the ease of handling, maintenance and relatively large blood volume
  • a given composition may vary in its immunogenicity It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a car ⁇ ei
  • exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA)
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin also can be used as car ⁇ eis
  • Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde. /n-maleimidobencoyl-N-hydroxysuccinimide ester, carbodimide and bis-biazotized benzidine
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants
  • adjuvants include complete Freund s adjuvant (a non-specific stimulator of the immune response containing killed M cobacter m tubeiciilosis). incomplete Freund's adjuvants and aluminum hydroxide adjuvant
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization
  • a variety of routes can be used to administer the immunogen (subcutaneous, intramuscular intradermal. intravenous and lntrape ⁇ toneal )
  • the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization
  • a second booster miection also may be giv en
  • the piocess of boosting and tite ⁇ ng is repeated until a suitable titer is achieved
  • the immunized animal can be bled and the serum isolated and stored and/or the animal can be used to generate mAbs
  • Monoclonal antibodies may be readily prepared through use of well-known techniques, such as those exemplified in U S Patent 4, 196 265, inco ⁇ orated herein by reference Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e g , a purified or partially purified minichromosome -associated protein, polypeptide or peptide The immunizing composition is administered in a manner effective to stimulate antibody producing cells Rodents such as mice and rats are preferred animals however the use of rabbit, sheep or frog cells also is possible The use of rats may provide certain advantages (Goding 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions
  • somatic cells with the potential for producing antibodies specifically B lymphocytes (B cells) are selected for use in the mAb generating protocol
  • B cells may be obtained from biopsied spleens 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 that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible
  • a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe
  • a spleen from an immunized mouse contains approximately 5 x 10 7 to 2 x 10 8 lymphocytes
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell generally one of the same species as the animal that w as immunized Myeloma cell lines
  • any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding 1986 Campbell 1984)
  • the immunized animal is a mouse
  • P3-X63/Ag8, X63-Ag8 653 NS l/1 Ag 4 1 , Sp210-Agl4 FO, NSO/U, MPC- 1 1 MPC1 1-X45-GTG 1 7 and S 194/5XX0 Bui for rats
  • one may use R210 RCY3, Y3-Ag 1 2 3 IR983F and 4B210 and U-266, GM 1500-GRG2 LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions
  • NS- 1 myeloma cell line (also termed
  • P3-NS-l-Ag4- l which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573
  • Another mouse myeloma cell line that may be used is the 8-azaguan ⁇ ne-res ⁇ stant mouse mu ⁇ ne myeloma SP2/0 non-producer cell line
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2 1 ratio, though the ratio may vary from about 20 1 to about 1 1 respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes Fusion methods using Sendai virus have been described
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media
  • agents are aminopte ⁇ n, methotrexate, and azase ⁇ ne Aminopte ⁇ n and methotrexate block de novo synthesis of both pu ⁇ nes and pynmidmes.
  • azase ⁇ ne blocks only pu ⁇ ne synthesis
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium)
  • HAT medium Hypoxanthine
  • the preferred selection medium is HAT Only cells capable of opeiating nucleotide salvage pathways are able to survive in HAT medium
  • the myeloma cells are defective in key enzymes of the salvage pathway, e g . hypoxanthine phospho ⁇ bosyl transferase (HPRT), and they cannot survive
  • the B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells
  • This cultu ⁇ ng provides a population of hyb ⁇ domas from which specific hyb ⁇ domas are selected Typically selection of hyb ⁇ domas is performed by cultu ⁇ ng the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays enzyme immunoassays, cytotoxicity assays plaque assays, dot lmmunobinding assays, and the
  • the selected hyb ⁇ domas would then be serially diluted and cloned into individual antibody-producing cell lines which clones can then be propagated indefinitely to provide mAbs
  • the cell lines may be exploited for mAb production in two basic ways
  • a sample of the hybridoma can be injected (often into the peritoneal cav ity) into a histocompatible animal of the type that w s used to provide the somatic and myeloma cells for the original fusion
  • the injected animal develops tumois secreting the specific monoclonal antibody produced by the fused cell hybrid
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration
  • the individual cell lines also could be cultured in vitio where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations mAbs produced by either means may be further purified, if desired using filtration, cent
  • ELISAs may be used in conjunction with the invention, for example, in identifying expression of a centromere-associated protein in a candidate centromere sequence Such an assay could thereby facilitate the isolation of centromeres from species other than Arabidopsis By identifying conserved centromere-associated coding sequences, the inventors have provided the essential tools for such a screen
  • proteins or peptides comprising minichromosome-encoded protein antigen sequences are immobilized onto a selected surface, preferablv a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate
  • a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of milk powdei
  • BSA bovine serum albumin
  • casein casein
  • milk powdei milk powdei
  • the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first
  • the second antibody will preferably have an associated enzyme that will generate color or light development upon incubating with an appropriate chromogenic substrate
  • a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e g . incubation for 2 hours at room temperature in a PBS-containing solution)
  • the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2.2'-az ⁇ no-d ⁇ -(3-ethyl-benzth ⁇ azol ⁇ ne)-6-sulfon ⁇ c acid (ABTS) and H 2 0 2 .
  • a chromogenic substrate such as urea and bromocresol purple or 2.2'-az ⁇ no-d ⁇ -(3-ethyl-benzth ⁇ azol ⁇ ne)-6-sulfon ⁇ c acid (ABTS) and H 2 0 2 .
  • peroxidase as the enzyme label Quantitation is then achieved by measuring the degree of color generation, e g . using a visible spectra spectrophotometer 3 Western Blots
  • Centromere- associated antibodies may find use in immunoblot or western blot analysis, for example, for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof In conjunction with immunoprecipitation followed by gel electrophoresis. these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adveise background This is especially useful when the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent. or they migrate at the same relative molecular weight as a cross-reacting signal
  • Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel- or fluorescently-tagged secondary antibodies against the protein moiety are considered to be of particular use in this regard
  • centromeres in Arabidopsis may find use in other species In one aspect, this may comprise actual use of the mapping data piovided herein based on synteny between Arabidopsis chromosomes and those of other species Further new mapping data may be obtained using the techniques described herein
  • the detailed methodology described herein for tetrad analysis could be used for the isolation of centromeres
  • tetrad analysis measures the recombination frequency between genetic makers and a centromere by analyzing all four pioducts of individual meiosis
  • tetrad analysis measures the recombination frequency between genetic makers and a centromere by analyzing all four pioducts of individual meiosis
  • qi t 1 mutation in Aiabidopsis which causes the four products of pollen mother cell meiosis in Aiabidopsis to remain attached
  • Sev eral naturally occurring plant species in addition to Aiabidopsis are known to release pollen clusters, including water lilies
  • centromeres or a series of clones are identified that hybridize to markers on either side of each centromere.
  • These efforts can be complicated by the presence of repetitive DNA in the centromere as well as by the potential instability of centromere clones
  • identification of large clones with unique sequences that will sei ve as useful probes simplifies a chromosome walking strategy Blot hybridization allows comparison of the structure of the clones ith that of genomic DNA and thus determines whether the clones have suffered deletions oi rearrangements
  • the centromenc clones identified aie useful for hybridization experiments that can be used to deteimine whether they share common sequences whether they localize /// situ to the cytologically defined centromeric region, and whethei they contain repetitive sequences thought to map near Aiabidopsis centromeres (Richards et al 1991. Maluszynska et al 1991 )
  • a particularly useful type of clone is the bacterial artificial chromosome (BAC) as data has suggested that YAC clones may sometimes not span centromeres (Willard, 1997)
  • BAC bacterial artificial chromosome
  • the construction and characterization of a bacterial artificial chromosome library from, for example Aiabidopsis thaliana has been described (Choi et al 1995)
  • the complementation of plant mutants with large genomic DNA fragments can be achieved using transformation-competent minichromosome vectoi s, thereby speeding positional cloning (Liu et al .
  • Homologous recombination is a reaction between any pair of DNA sequences having a similar sequence of nucleotides where 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 sequences increases and is higher with linearized plasmid molecules than with circularized plasmid molecules
  • Homologous recombination can occur between two DNA sequences that are less than identical but the recombination frequency declines as the di ergence between the two sequences increases
  • Introduced DNA sequences can be targeted via homologous recombination by linking a DNA molecule of interest to sequences sharing homology with endogenous sequences of the host cell Once the DNA enters the cell the two homologous sequences can interact to insert the introduced DNA at the site where the homologous genomic DNA sequences were located Therefore, the choice of homologous sequences contained on the introduced DNA will determine the site where the introduced DNA is integrated via homologous recombination For example if the DNA sequence of interest is linked to DNA sequences sharing homology to a single copy gene ot a host plant cell the DNA sequence of interest will be inserted via homologous recombination at only that single specific site However if the DNA sequence ot interest is linked to DNA sequences sharing homology to a multicopy gene of the host eukaryotic cell, then the DNA sequence of interest can be inserted via homologous iecombination at each of the specific sites where a copy of the gene is located
  • DNA can be inserted into a host chromosome or vector by a homologous recombination reaction involv ing either a single reciprocal recombination (resulting in the insertion of the entire length of the introduced DNA) or through a double reciprocal recombination (resulting in the insertion of only the DNA located between the two iecombination events )
  • a homologous recombination reaction involv ing either a single reciprocal recombination (resulting in the insertion of the entire length of the introduced DNA) or through a double reciprocal recombination (resulting in the insertion of only the DNA located between the two iecombination events )
  • the introduced DNA should contain sequences homologous to the selected gene
  • a single homologous recombination event would then result in the entire introduced DNA sequence being inserted into the selected gene
  • a double recombination event can be achieved by flanking each end of the DNA sequence of interest (the sequence intended to be inserted into the genome)
  • homologous recombination is a relatively rare event compared to random insertion events
  • foreign DNA molecules find homologous sequences in the cell's genome and recombine at a frequency of approximately 0 5-4.2X 10 "4
  • a preferred manner for carrying out site-specific recombination comprises use of " a site-specific recombinase system
  • a site specific recombinase system consists of three elements: two pairs of DNA sequence (first and second site-specific recombination sequences) and a specific enzyme (the site-specific recombinase)
  • the site-specific recombinase will catalyze a recombination reaction only between two site-specific recombination sequences
  • a number of different site specific recombinase systems could be employed in accordance with the instant invention, including, but not limited to. the Cre/lox s tem of bactenophage P I (Hoess et al . 1982. U S Patent No 5.658.772, specifically inco ⁇ orated herein by reference in its entirety), the FLP FRT system of yeast (Gohc and Lindquist. 1989). the Gin recombinase of phage Mu (Maeser and Kahmann. 1991 ). the Pin recombinase of E coli ( ⁇ nomoto et al 1983).
  • the bactenophage PI Cre/lox and the yeast FLP/FRT systems constitute two particularly useful systems for site specific recombination
  • a recombinase (Cre or FLP) will interact specifically with its respective site-specific recombination sequence (lox oi FRT. respectively) to invert or excise the intervening sequences
  • the sequence for each of these two systems is relatively short (34 bp for lox and 47 bp for FRT) and therefore, convenient for use with transformation vectors
  • the FLP/FRT recombinase system has been demonstrated to function efficiently in plant cells, but could also be used in. for example, a bacterial cell or /;; vitro
  • the performance of the FLP/FRT system indicates that FRT site structure, and amount of the FLP protein present affect excision activity In general, short incomplete FRT sites lead to higher accumulation of excision products than the complete full-length FRT sites
  • the systems can catalyze both intra- and intermolecular reactions, indicating their utility foi DNA excision as well as integration reactions
  • the recombination reaction is reversible and this reversibility can compromise the efficiency of the reaction in each direction Altering the structure of the site-specific recombination sequences is one approach to remedying this situation
  • the site-specific recombination sequence can be mutated in a manner that the product of the recombination reaction is no longer recognized as a substrate for the reverse reaction, thereby stabilizing the integration oi excision event In the Cre-lox system, discovered in
  • Cre recombination between loxP sites occurs in the presence of the Cre recombinase (see. e.g . U S Patent No 5.658.772. specifically inco ⁇ orated herein by refeience in its entuety)
  • This sy stem has been utilized to excise a gene located between two lox sites which had been introduced into a yeast genome (Sauer. 1987) Cre was expressed from an mducible yeast GAL1 promoter and this Cre gene was located on an autonomously lephcating yeast vector
  • lox sites on the same DNA molecule can have the same or opposite orientation with respect to each other Recombination between lox sites in the same orientation results in a deletion of the DNA Segment located between the two lox sites and a connection between the resulting ends of the original DNA molecule
  • the deleted DNA segment forms a circular molecule of DNA
  • the original DNA molecule and the resulting circular molecule each contain a single lox site Recombination between lox sites in opposite orientations on the same DNA molecule result in an inversion of the nucleotide sequence of the DNA segment located between the two lox sites
  • reciprocal exchange of DNA segments proximate to lox sites located on two different DNA molecules can occur. All of these recombination events are catalyzed by the product of the Cre coding region
  • transgenic bacterium, yeast cell, plant cell or plant derived from such a transformation process or the progeny and seeds from such a transgenic plant also are further embodiments of the invention
  • means ot transformation are similar to those well known means used to transform other bacteria or yeast such as E coli or Saccharoimces cerevisiae
  • Methods for DNA transformation of plant cells include plant transformation protoplast transformation (as used herein protoplast transformation includes PEG-mediated transformation electroporation and protoplast fusion tiansformation) gene transfer into pollen injection into reproductiv e organs injection into immature embryos and particle bombaidment
  • protoplast transformation includes PEG-mediated transformation electroporation and protoplast fusion tiansformation
  • Suitable methods are believed to include virtually any method by which DNA can be introduced into a cell such as by Agrobactermm infection direct delivery of DNA such as, foi example by PEG-mediated transformation ot protoplasts (Omirulleh et al 1993), by desiccation/inhibition-mediated DNA uptake by electroporation, by agitation with silicon carbide fibers by acceleration of DNA coated particles, etc
  • acceleration methods are preferred and include, for example, microprojectile bombardment and the like
  • Electroporation can be extremely efficient and can be used both for transient expression of cloned genes and for establishment of cell lines that carry integrated copies of the gene of interest Electroporation in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few integrated copies of the foreign DNA
  • a further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombai dment
  • particles may be coated with nucleic acids and delivered into cells by a propelling force
  • Exemplary particles include those comprised of tungsten gold platinum and the like
  • An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biohstics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto 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. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing damage inflicted on the recipient cells by projectiles that are too large.
  • cells in suspension are preferably concentrated on filters or solid culture medium Alternatively, immature embryos or other target cells may be arranged on solid culture medium
  • the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.
  • one or more screens also are positioned between the acceleration device and the cells to be bombarded
  • TRFs trauma reduction factors
  • a robactermm-mediaied transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration ot an intact plant from a protoplast
  • a ⁇ ioh ⁇ cterium-mcdialed plant integrating vectors to introduce DNA into plant cells is well known in the art See tor example the methods described (Fralev et ⁇ l 1985, Rogers et ⁇ l 1987) Advances in A,i,'/ofr ⁇ ./ctet ⁇ //?;-med ⁇ ated transfer now allow introduction of large segments of DNA (Hamilton 1997 Hamilton et ⁇ l 1996)
  • chiomosomal integration is required for stable inheritance of the foreign DNA
  • the vector described herein may be used for transformation with or without integration as the centromere function required for stable inheritance is encoded within the minichromosome
  • transformation events in which the minichromosome is not chromosomally integrated may be preferred in that problems w ith site-specific v ariations in expression and insertional mutagenesis mav be avoided
  • the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements
  • the region of DNA to be transferred is defined by the border sequences and intervening DNA is usually inserted into the plant genome as described (Shmann et al , 1986, Jorgensen et al , 1987)
  • Modem Agrobacterium transformation vectors are capable of replication in E coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et ⁇ / 1985)
  • recent technological advances in vectors for Agiobacteiium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expiessing various polypeptide coding genes
  • the vectors described (Rogers et al 1987) have convenient multi-linker regions flanked by a promoter and a polyadenvlation site for direct expression of inserted polypeptide coding genes and are suitable for present pu ⁇ oses
  • Agro/w te ⁇ -fw-mediated transformation ot leaf disks and other tissues such as cotyledons and hypocotvls appears to be limited to plants that Agiob ⁇ cte ⁇ um naturally infects Agiob ⁇ ctenum-mediated transformation is most efficient in dicotyledonous plants
  • the transformation of asparagus using Ai ⁇ i obacteriiim also can be achieved (sec, for example Bytebier et al 1987) transfer may be made more efficient through the use of " a mutant that is defective in integration of the Agrobactermm T-DNA but competent for delivery of the DNA into the cell (Mysor
  • a transgenic plant formed using Agrobacterium transformation methods typically contains a single gene on one chromosome Such transgenic plants can be referred to as being hemizygous for the added gene.
  • a more accurate name for such a plant is an independent segregant. because each transformed plant represents a unique T-DNA integration event.
  • transgenic plant that is homozygous for the added foreign DNA, i.e . a transgenic plant that contains two copies of a transgene. one gene at the same locus on each chromosome of " a chromosome pair
  • a homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single added transgene. germinating some of the seed produced and analyzing the resulting plants produced for enhanced activ ity relative to a control (native, non-transgenic) or an independent segregant transgenic plant
  • a plant in which the minichromosome has not been chromosomally integrated Such a plant may be termed 2n + x. wheie 2n is the diploid number of chromosomes and where x is the number of minichromosomes Initially, transformants may be 2n+l . i.e. having 1 additional minichromosome In this case, it may be desirable to self the plant or to cross the plant with another 2n + 1 plant to yield a plant which is 2n + 2 The 2n + 2 plant is preferred in that it is expected to pass the minichromosome through meiosis to all its offspring
  • transgenic plants also can be mated to produce offspring that contain two independently segregating added, exogenous minichromosomes Selfing of appropriate progeny can produce plants that are homozygous tor both added, exogenous minichromosomes that encode a poly peptide of interest Back-crossing to a parental plant and out-crossing with a non-transgenic plant also are contemplated
  • Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see. e.g , Potrykus et al, 1985. Lorz et al , 1985, Fromm et al , 1986; Uchimiya et al, 1986. Calhs et al, 1987: Marcotte et al, 1988)
  • DNA is carried through the cell wall and into the cytoplasm 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 through several layers of cells and thus allow the transformation of cells within tissue explants
  • Protoplast fusion could be used to integrate a minichromosome constructed in a host cell, such as a yeast cell, and then fuse those cells to plant protoplasts
  • the chromosomes lacking plant centromeres such as yeast chromosomes in this example
  • Numerous examples of protocols foi protoplast fusion that could be used with the invention have been described (see. e g . Negrutiu et al . 1992. and Peterson)
  • Liposome fusion could be used to intioduce a recombinant construct comprising a centromere, such as a minichromosome, by. for example, packaging the recombinant construct into small droplets of lipids (hposomes) and then fusing these hposomes to plant protoplasts thus delivering the AC into the plant cell (see Lurqui and Rollo, 1993).
  • a centromere such as a minichromosome
  • One particularly important advance of the present invention is that it provides methods and compositions for expression of exogenous genes in plant cells
  • One advance of the constructs of the current invention is that they enable the introduction of multiple genes, potentially representing an entire biochemical pathway
  • the current invention allows for the transformation of plant cells with a minichromosome comprising a number of structural genes
  • Another advantage is that more than one minichromosome could be introduced, allowing combinations of genes to be moved and shuffled
  • the ability to eliminate a minichromosome from a plant would provide additional flexibility, making it possible to alter the set of genes contained within a plant Further, by using site-specific recombinases. it should be possible to add genes to an existing minichromosome once it is in a plant
  • an ' expressible gene is any gene that is capable of being transcribed into RNA (e g . mRNA. antisense RNA. etc ) or translated into a protein, expressed as a trait of interest. or the like, etc .
  • One of the major pu ⁇ oses of transformation of crop plants is to add some commercially desirable, agronomically important traits to the plant
  • Such traits include but are not limited to. herbicide resistance or tolerance, insect resistance or tolerance, disease resistance or tolerance (viral, bacterial fungal nematode) stress tolerance and/or resistance, as exemplified by resistance or tolerance to diought.
  • an ' exogenous gene is a gene not normally found in the host genome in an identical context By this it is meant that the gene may be isolated from a different species than that of the host genome or alternatively isolated from the host genome but operably linked to one or more regulatory regions which differ from those found in the unaltered, native gene
  • Two or more exogenous genes also can be supplied in a single transformation event using either distinct tiansgene-encoding vectors or using a single vector inco ⁇ orating two or more gene coding sequences
  • plasmids bearing the bai and aroA expression units in either convergent divergent or cohnear orientation are considered to be particularly useful
  • Further preferred combinations arc those of an insect resistance gene such as a Bt gene, along with a protease inhibitoi gene such as pinll.
  • any two or more transgenes ot any description such as those conferring herbicide insect disease (vital, bacterial, fungal nematode) oi drought lesistance male sterility drydown standabihty piohficacy. starch properties oil quantity and quality or those incieasing yield or nutritional quality may be employed as desired
  • the bai and pat genes code lor an enzyme phosphinothncm acetyltransferase (PAT) which inactivates the herbicide phosphinothncm and prevents this compound from inhibiting glutamine synthetase enzymes
  • PAT phosphinothncm ace
  • preferred Bt genes foi use in the transformation protocols disclosed herein will be those in which the coding sequence has been modified to effect increased expiession in plants and more particularly, in monocot plants
  • Means foi preparing synthetic genes are well known in the art and are disclosed in. for example.
  • Protease inhibitors also may provide insect resistance (Johnson et al, 1989). and will thus hav e utility in plant transformation
  • the use ot a protease inhibitor II gene, pinll. from tomato or potato is envisioned to be particularly useful Even more advantageous is the use of a pinll gene in combination with a Bt toxin gene, the combined effect of which has been discovered to produce synergistic insecticidal activity
  • Other genes which encode inhibitors of the insect ' s digestive system, or those that encode enzymes or co-factors that facilitate the production of inhibitoi s. also may be useful This group may be exemplified by oryzacystatin and amylase inhibitors such as those from wheat and barley.
  • genes encoding lectins may confer additional or alternative insecticide properties
  • Lectins (originally termed phytohemagglutinins) arc multivalent carbohydrate-binding proteins which have the ability to agglutinate red blood cells from a range of species. Lectins have been identified recently as insecticidal agents with activity against weevils. ECB and rootworm (Murdock et al , 1990. Czapla & Lang. 1990)
  • Lectin genes contemplated to be useful include, for example, barley and wheat germ agglutinin (WGA) and rice lectins (Gatehouse et al, 1984). with WGA being preferred
  • Genes controlling the production of large or small polypeptides active against insects when introduced into the insect pests form another aspect of the invention.
  • insect pests such as, e g . lytic peptides. peptide hormones and toxins and venoms
  • the expression of juvenile hormone esterase. directed towards specific insect pests also may result in insecticidal activity, or perhaps cause cessation of metamo ⁇ hosis (Hammock et al , 1990)
  • Transgenic plants expressing genes which encode enzymes that affect the integrity of the insect cuticle form yet another aspect of the invention
  • genes include those encoding, e.g.. chitinasc. proteases, hpases and also genes for the production of nikkomycin.
  • a compound that inhibits chitin synthesis the introduction of any of which is contemplated to produce insect resistant plants
  • Genes that code for activities that aflect insect molting, such as those affecting the production of ecdysteroid UDP-glucosyl transferase also fall within the scope of the useful transgenes of the present invention
  • Genes that code for enzymes that facilitate the pioduction of compounds that reduce the nutritional quality of the host plant to insect pests also are encompassed by the present invention It may be possible, for instance to confer insecticidal activity on a plant by altering its sterol composition Sterols aie obtained by insects from their diet and are used for hormone synthesis and membrane stability Therefore alterations in plant sterol composition by expression of novel genes e g , those that directly promote the production of undesirable sterols or those that conv ert desirable sterols into undesirable forms, could have a negative effect on insect grow th and/or development and hence endow the plant with insecticidal activity Lipoxygenases arc naturally occurring plant enzymes that have been shown to exhibit anti-nut ⁇ tional effects on insects and to i educe the nutritional quality of their diet Therefore further embodiments of the invention concern transgenic plants with enhanced hpoxygenase activity which may be resistant to insect feeding
  • Ti ipsacum dactxloides is a species of grass that is resistant to certain insects including corn root worm It is anticipated that genes encoding proteins that are toxic to insects or are involved in the biosynthesis of compounds toxic to insects w ill be isolated from Tiipsacum and that these novel genes w ill be useful in conferring resistance to insects It is known that the basis of insect resistance in Ti ipsacum is genetic because said resistance has been transferred to Zea nun s v ia sexual crosses (Branson and Guss 1972) It is furthei anticipated that other cereal monocot or dicot plant species may have genes encoding proteins that are toxic to insects which would be useful for producing insect resistant plants
  • genes encoding proteins characterized as having potential insecticidal activity also may be used as tiansgenes in accoidance herewith
  • Such genes include foi example, the covvpea trypsin inhibitor (CpTI. Hilder et al 1987) which may be used as a iootworm deterrent, genes encoding avermectin (Aveimecttn and Abamectin . Campbell. W C .
  • Late Embryogenic Proteins Three classes have been assigned based on structural similarities (see Dure et al. 1989). All three clas.ses of " LEAs have been demonstrated in maturing (i.e. desiccating) seeds. Within these 3 types of LEA proteins. the Type-II (dehydrin-type) have generally been implicated in drought and/or desiccation tolerance in vegetative plant parts (i.e. Mundy and Chua, 1988; Piatkowski et al, 1990; Yamaguchi-Shinozaki et al, 1992). Recently, expression of a Type-Ill LEA (HVA- 1) in tobacco was found to influence plant height, maturity and drought tolerance (Fitzpatrick, 1993).
  • HVA-1 Type-Ill LEA
  • HVA-1 HVA-1 gene influenced tolerance to water deficit and salinity (Xu et al, 1996). Expression of structural genes from all three LEA groups may therefore confer drought tolerance. Other types of proteins induced during water stress include thiol proteases, aldolases and transmembrane transporters (Guerrero et al, 1990), which may confer various protective and/or repair-type functions during drought stress. It also is contemplated that genes that effect lipid biosynthesis and hence membrane composition might also be useful in conferring drought resistance on the plant.
  • genes that are involved with specific mo ⁇ hological traits that allow for increased water extractions from drying soil would be of benefit
  • introduction and expiession of genes that alter root characteristics may enhance water uptake
  • expression of genes that enhance reproductive fitness during times of stress would be of signif icant v alue Foi example expiession of genes that improve the synchiony of pollen shed and receptiveness of the female flower parts ; e silks would be of benefit
  • expression of genes that minimize kernel abortion during times of stress increase the amount of grain to be harvested and hence be of value
  • Resistance to viruses may be produced thiough expression of novel genes
  • expression of a viral coat protein in a transgenic plant can impart resistance to infection of the plant by that virus and perhaps other closely related viruses (Cuozzo et al 1988 Hemenway et al 1988 Abel et al 1986)
  • expression of antisense genes targeted at essential viral functions mav also impart resistance to viruses
  • an antisense gene targeted at the gene responsible for replication of viral nucleic acid may inhibit replication and lead to resistance to the virus
  • lnteiference with other viral functions through the use of antisense genes also may increase resistance to viruses.
  • Peptide antibiotics are polypeptide sequences which are inhibitory to growth of bacteria and other microorganisms.
  • the classes of peptides referred to as cecropins and magainins inhibit growth of many species of bacteria and fungi.
  • expression of PR proteins in monocotyledonous plants such as maize may be useful in conferring resistance to bacterial disease.
  • genes are induced following pathogen attack on a host plant and have been divided into at least five classes of proteins (Bol, Linthorst, and Cornelissen, 1990). Included amongst the PR proteins are ⁇ -1 , 3-glucanases, chitinases, and osmotin and other proteins that are believed to function in plant resistance to disease organisms. Other genes have been identified that have antifungal properties, e.g., UDA (stinging nettle lectin) and hevein (Broakaert et a , 1989; Barkai-Golan et al, 1978). It is known that certain plant diseases are caused by the production of phytotoxins.
  • UDA stinging nettle lectin
  • hevein Broakaert et a , 1989; Barkai-Golan et al, 1978. It is known that certain plant diseases are caused by the production of phytotoxins.
  • resistance to these diseases would be achieved through expression of a novel gene that encodes an enzyme capable of degrading or otherwise inactivating the phytotoxin. It also is contemplated that expression of novel genes that alter the interactions between the host plant and pathogen may be useful in reducing the ability of the disease organism to invade the tissues of the host plant, e.g., an increase in the waxiness of the leaf cuticle or other mo ⁇ hological characteristics.
  • genes may be introduced into plants that would improve standabihty and other plant growth characteristics Expression of novel genes in plants which confer stronger stalks, improved root systems, or prev ent or reduce ear droppage would be of great value to the farmer It is proposed that introduction and expression of genes that increase the total amount of photoassimilate available by, for example, increasing light distribution and/or interception would be advantageous In addition the expression of genes that increase the efficiency of photosynthesis and/or the leaf canopy would further increase gains in productivity It is contemplated that expression of a phytochrome gene in crop plants may be advantageous Expression of such a gene may reduce apical dominance, confer semidwarfism on a plant, and increase shade tolerance (U S Patent No 5 268,526) Such approaches would allow for increased plant populations in the field
  • E coli gdhA genes may lead to increased fixation of nitrogen in organic compounds
  • expression of gdhA in plants may lead to enhanced resistance to the herbicide glufosinate by incorporation of excess ammonia into glutamate. thereby detoxifying the ammonia
  • expression of a novel gene may make a nutrient source available that was previously not accessible, e g.. an enzyme that releases a component of nutrient value from a more complex molecule, perhaps a macromolecule
  • male Sterihtx Male sterility is useful in the production of hybrid seed It is proposed that male sterility may be produced through expression of novel genes For example, it has been shown that expression of genes that encode proteins that interfere with development of the male inflorescence and/or gametophyte result in male sterility Chimeric ⁇ bonuclease genes that express in the anthers of transgenic tobacco and oilseed rape have been demonstrated to lead to male sterility (Ma ⁇ ani et al , 1990)
  • T cytoplasm A DNA sequence, designated TURF- 13 (Levings. 1990). was identified that correlates with T cytoplasm It is proposed that it would be possible through the introduction of TURF- 13 via transformation, to separate male sterility from disease sensitivity As it is necessary to be able to restore male fertility for breeding purposes and for grain production, it is proposed that genes encoding restoration of male fertility also may be introduced
  • Genes may be introduced into plants to improve the nutrient quality or content of a particular crop
  • Introduction of genes that alter the nutrient composition of a crop may greatly enhance the feed or food value.
  • the protein of " many grains is suboptimal for feed and food pu ⁇ oses. especially when fed to pigs, poultry, and humans.
  • the protein is deficient in several amino acids that are essential in the diet of these species, requiring the addition of supplements to the grain.
  • Limiting essential amino acids may include lysine, methionine. tryptophan. threonine, vahne. argimne. and histidine Some amino acids become limiting only after corn is supplemented with other inputs for feed formulations.
  • the levels of these essential amino acids in seeds and grain may be elevated by mechanisms which include, but are not limited to. the introduction of genes to increase the biosynthesis of the amino acids, decrease the degradation of the amino acids, increase the storage of the amino acids in proteins, or increase transport of the amino acids to the seeds or grain
  • the protein composition of a crop may be altered to improve the balance of amino acids in a variety of ways including elevating expression of native proteins, decreasing expression of those with poor composition, changing the composition of native proteins, or introducing genes encoding entirely new proteins possessing superior composition
  • genes that alter the oil content of a crop plant may also be of value Increases in oil content may result in increases in metabohzable-energy-content and density of the seeds for use in feed and food
  • the introduced genes may encode enzymes that remove or reduce rate-limitations or regulated steps in fatty acid or hpid biosynthesis.
  • Such genes may include, but are not limited to. those that encode acetyl- CoA carboxylase. ACP-acyltransferase. ⁇ -ketoacyl-ACP synthase. plus other well known fatty acid biosynthetic activities.
  • genes that encode proteins that do not possess enzymatic activity such as acyl carrier protein Genes may be introduced that alter the balance of fatty acids present in the oil providing a more healthful or nutritive feedstuff
  • the introduced DNA also may encode sequences that block expression of enzymes involved in fatty acid biosynthesis, altering the proportions of fatty acids present in crops.
  • Genes may be introduced that enhance the nutritive value of the starch component of crops, for example by increasing the degree of branching, resulting in improved utilization of the starch in livestock by delaying its metabolism
  • other major constituents of a crop may be altered, including genes that affect a variety ot other nutritive, processing, or other quality aspects
  • pigmentation may be increased or decreased
  • Feed or food crops may also possesses insufficient quantities of vitamins, requiring supplementation to provide adequate nutritive value
  • Introduction of genes that enhance vitamin biosynthesis may be envisioned including, for example, vitamins A. E, B
  • genes that affect the accumulation or availability of compounds containing phosphorus, sulfur, calcium, manganese, zinc, and iron among others would be valuable
  • improvements of crops may not necessarily involve grain, but may, for example. improve the value of a crop for silage Introduction of DNA to accomplish this might include sequences that alter lignin production such as those that result in the ' brown midrib" phenotype associated with supe ⁇ or feed value foi cattle
  • genes also may be introduced which improve the processing of " crops and improve the value ot the products resulting from the piocessing One use of crops if via wetmilling
  • novel genes that increase the efficiency and reduce the cost of such processing, foi example by deci easing steeping time may also find use
  • Oil is another product of wetmilling, the value of which may be improved by introduction and expiession of genes
  • Oil properties may be altered to improve its performance in the production and use of cooking oil. shortenings, lubricants oi other oil- de ⁇ ved products or improvement of its health attributes when used in the food-related applications
  • Novel fatty acids also may be synthesized which upon extraction can serve as starting materials tor chemical syntheses
  • the changes in oil properties may be achieved by altering the type level, or hpid arrangement of the fatty acids present in the oil This in turn may be accomplished by the addition of genes that encode enzymes that catalyze the synthesis of novel fatty acids and the lipids possessing them or by increasing levels of nativ e fatty acids while possibly reducing levels of precursors
  • DNA sequences may be introduced which slow or block steps in fatty acid biosynthesis resulting in the increase in precursor fatty acid intermediates Genes that might be added include desaturases.
  • hydratases dehydratases and othei enzymes that catalyze reactions involving tatty acid intermediates
  • catalytic steps that might be blocked include the desaturations from stea ⁇ c to oleic acid and oleic to hnolenic acid resulting in the respective accumulations ot stea ⁇ c and oleic acids
  • Another example is the blockage of elongation steps resulting in the accumulation of C 8 to Ci: saturated fatty acids
  • transgenic plant prepared in accordance with the invention may be used for the production or manufacturing of useful biological compounds that were either not produced at all, or not produced at the same level, in the corn plant previously.
  • plants produced in accordance with the invention may be made to metabolize certain compounds, such as hazardous wastes, thereby allowing bioremediation of these compounds.
  • novel plants producing these compounds are made possible by the introduction and expression of one or potentially many genes with the constructs provided by the invention.
  • the vast array of possibilities include but are not limited to any biological compound which is presently produced by any organism such as proteins, nucleic acids, primary and intermediary metabolites, carbohydrate polymers, enzymes for uses in bioremediation, enzymes for modifying pathways that produce secondary plant metabolites such as flavonoids or vitamins, enzymes that could produce pharmaceuticals. and for introducing enzymes that could produce compounds of interest to the manufacturing industry such as specialty chemicals and plastics.
  • the compounds may be produced by the plant, extracted upon harvest and/or processing, and used for any presently recognized useful purpose such as pharmaceuticals, fragrances, and industrial enzymes to name a few.
  • DNA may be introduced into plants for the pu ⁇ ose of expressing RNA transcripts that function to affect plant phenotype yet are not translated into protein.
  • Two examples are antisense RNA and RNA with ribozyme activity. Both may serve possible functions in reducing or eliminating expression of native or introduced plant genes.
  • DNA need not be expressed to effect the phenotype of a plant.
  • Genes may be constructed oi isolated which when transcribed, produce antisense RNA that is complementary to all or part(s) of a targeted messenger RNA(s)
  • the antisense RNA leduces pioduction of the polypeptide product of the messenger RNA
  • the polypeptide pioduct may be any piotein encoded by the plant genome
  • An antisense gene may thus be introduced into a plant by transformation methods to produce a novel transgenic plant with reduced expression of a selected protein of interest
  • the protein may be an enzyme that catalyzes a reaction in the plant Reduction of the enzyme activity may reduce or eliminate products of the reaction which include any enzymatically synthesized compound in the plant such as fatty acids, amino acids, carbohydrates, nucleic acids and the like
  • the protein may be a storage protein such as a zein or a structural protein, the decreased expression of which may lead to changes in seed amino acid composition or plant morphological changes respectively
  • the possibilities cited above are provided only by
  • Genes also may be constructed or isolated which when transcribed produce RNA enzymes (ribozymes) which can act as endonbonucleases and catalyze the cleavage of RNA molecules with selected sequences The cleavage of selected messenger RNAs can result in the reduced production of their encoded polypeptide products
  • ribozymes RNA enzymes
  • These genes may be used to prepare novel transgenic plants which possess them
  • the transgenic plants may possess reduced levels of polypeptides including, but not limited to the polypeptides cited above
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion Ribozymes have specific catalytic domains that possess endonuclease activity
  • Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce. 1989, Cech et al . 1981 )
  • U. S. Patent 5.354.855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ⁇ bonucleases and approaching that of the DNA restriction enzymes
  • RNA cleavage activity examples include sequences from the Group I self splicing introns including Tobacco Ringspot Virus (Prody et al , 1986), Avocado Sunblotch Viroid (Palukaitis et al , 1979. Symons, 1981). and Lucerne Transient Streak Virus (Forster and Symons. 1987) Sequences from these and related viruses are referred to as hammerhead ribozyme based on a predicted folded secondary structure
  • ribozymes include sequences from RNase P with RNA cleavage activity (Yuan et al , 1992. Yuan and Altman, 1994, U S Patents 5, 168,053 and 5.624,824). hai ⁇ in ribozyme structures (Berzal-Hcrranz et al . 1992.
  • the other variable on ribozyme design is the selection of a 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 interactions Two stretches of homology are required for this targeting These stretches of homologous sequences flank the catalytic ribozyme structure defined above Each stretch of homologous sequence can vary in length from 7 to 15 nucleotides The only requirement foi defining the homologous sequences is that, on the target RNA. they are separated by a specific sequence which is the cleavage site For hammerhead ribozyme.
  • the cleavage site is a dinucleotide sequence on the target RNA is a uracil (U) followed by either an adenine. cytosine or uracil (A.C or U) (Per ⁇ man et al , 1992, Thompson et al , 1995)
  • the frequency of this dinucleotide occurring in any given RNA is statistically 3 out of 16 Therefore, for a given target messenger RNA of 1.000 bases. 187 dinucleotide cleavage sites are statistically possible
  • Designing and testing ribozymes for efficient cleavage of a 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 Chownra et al , ( 1994) and Lieber and Strauss ( 1995). each inco ⁇ orated by reference The identification of operative and preferred sequences for use in down regulating a given gene is simply a matter of preparing and testing a given sequence, and is a routinely practiced "screening" method known to those of skill in the art
  • genes may be introduced to produce novel transgenic plants which have reduced expression of a native gene product by the mechanism of co-suppression It has been demonstrated in tobacco, tomato, and petunia (Goring et al , 1991. Smith et al , 1990. Napoli et al , 1990, van der Krol et al , 1990) that expression of the sense transcript of a native gene ill reduce or eliminate expression of the native gene in a manner similar to that observed for antisense genes
  • the introduced gene may encode all or part of the targeted native protein but its translation may not be required for reduction of levels of that native protein
  • DNA elements including those of transposable elements such as Ds. Ac. or Mu. may be inserted into a gene to cause mutations These DNA elements may be inserted in order to inactivate (or activate) a gene and thereby tag" a particular trait In this instance the transposable element does not cause instability of the tagged mutation because the utility of the element does not depend on its ability to move in the genome
  • the introduced DNA sequence may be used to clone the corresponding gene, e g , using the introduced DNA sequence as a PCR primer together with PCR gene cloning techniques (Shapuo 1983 Dellaporta et al , 1988) Once identified, the entire gene(s) for the particular trait, including control or regulatory regions where desired, may be isolated, cloned and manipulated as desired
  • the utility of DNA elements introduced into an organism for pu ⁇ oses of gene tagging is independent of the DNA sequence and does not depend on any biological activity of the DNA sequence, z e , transcription into RNA or translation into protein The sole
  • unexpressed DNA sequences including novel synthetic sequences, could be introduced into cells as proprietary ' labels" of those cells and plants and seeds thereof It would not be necessary for a label DNA element to disrupt the function of a gene endogenous to the host organism, as the sole function of this DNA would be to identify the origin of the organism For example, one could introduce a unique DNA sequence into a plant and this DNA element would identify all cells, plants. and progeny of these cells as having arisen from that labeled source It is proposed that inclusion of label DNAs would enable one to distinguish proprietary gcrmplasm or germplasm derived from such from unlabelled germplasm
  • MAR matrix attachment region element
  • Stief chicken l sozyme A element
  • non-protein expressing sequences specifically envisioned for use with the invention include tRiNA sequences, for example, to alter codon usage, and rRNA val iants, for example which may confer resistance to v arious agents such as antibiotics
  • mutated centromeric sequences are contemplated to be useful for increasing the utility of the centromere
  • the function of the centromeres of the current invention may be based upon the secondary structure of the DNA sequences of the centromere and / or the proteins which interact with the centromere
  • the DNA sequence ot the centromere one may alter the affinity of one or more centromere-associated prote ⁇ n(s) for the centromere and / or the secondary structuie of the centromeric sequences thereby changing the activity of the centromere
  • changes may be made in the centromeres of the invention which do not effect the activity of the centromere
  • Changes in the centromeric sequences which reduce the size of the DNA segment needed to confer centiomere activity are contemplated to be particularly useful in the current invention, as would changes which increased the fidelity with which the centromere was transmitted during mitosis and meiosis
  • plant refers to any type of plant
  • the inventois have provided below an exemplary description ot some plants that may be used with the invention However, the list is not in any way limiting as other types of plants will be known to those of skill in the art and could be used with the invention
  • a common class of plants exploited in agriculture are vegetable crops, including artichokes, kohlrabi, arugula. leeks, asparagus, lettuce (e g . head. leaf, romaine).
  • fruit and vine crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, quince almonds, chestnuts, filberts, pecans, pistachios, walnuts, citrus, blueberries. boysenber ⁇ es. cranberries, currants, loganberries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwi, persimmons, pomegranate, pineapple, tropical fruits, pomes, melon, mango, papaya, and lychee
  • plants include bedding plants such as flowers, cactus, succulents and ornamental plants, as well as tiees such as forest (broad-leaved trees and evergreens, such as conifers), fruit, ornamental, and nut-bearing trees, as well as shrubs and other nursery stock XI. Definitions
  • ARS ' or “ongin of replication” refer to an origin of DNA replication recognized by pioteins that initiate DNA replication
  • binary BAC oi "binary bacterial artificial chromosome” refer to a bacterial vector that contains the T-DNA border sequences necessary for Agrobactermm mediated transformation (see. for example. Hamilton et al . 1996, Hamilton. 1997, and Liu et al , 1999
  • centromere sequence refers to a nucleic acid sequence which one wishes to assay for potential centromere function
  • centromere is any DNA sequence that confers an ability to segregate to daughter cells through cell division.
  • this sequence may produce a segregation efficiency to daughter cells ranging from about 1 % to about 100%:. including to about 5%, 10%, 20%, 30%. 40%, 50%. 60%, 70%, 80%, 90% or about 95% of daughter cells Variations in such a segregation efficiency may find important applications within the scope of the invention, for example, mini-chromosomes carrying centromeres that confer 100% stability could be maintained in all daughter cells without selection, while those that confer 1 % stability could be temporarily introduced into a transgenic organism, but be eliminated when desired In particular embodiments of the invention, the centromere may confer stable segregation of a nucleic acid sequence.
  • centromere-associated protein refers to a protein encoded by a sequence of the centromere or a protein which is encoded by host DNA and binds with relatively high affinity to the centromere.
  • eukaryote refers to living organisms whose cells contain nuclei.
  • a eukaryote may be distinguished from a "prokaryote" which is an organism which lacks nuclei. Prokaryotes and eukaryotes differ fundamentally in the way their genetic information is organized, as well as their patterns of RNA and protein synthesis.
  • RNA molecules typically termed messenger RNA (mRNA).
  • mRNA messenger RNA
  • the mRNA is typically, but not always, translated into polypeptide(s).
  • the term "genome” refers to all of the genes and DNA sequences that comprise the genetic information within a given cell of an organism. Usually, this is taken to mean the information contained within the nucleus, but also includes the organelles.
  • higher eukaryote means a multicellular eukaryote, typically characterized by its greater complex physiological mechanisms and relatively large size. Generally, complex organisms such as plants and animals are included in this category. Preferred higher eukaryotes to be transformed by the present invention include, for example, monocot and dicot angiosperm species, gymnosperm species, fern species, plant tissue culture cells of these species, animal cells and algal cells. It will of course be understood that prokaryotes and eukaryotes alike may be transformed by the methods of this invention.
  • host refers to any organism that is the recipient of a replicable plasmid. or expression vector comprising a plant chromosome.
  • host strains used for cloning experiments should be free of any restriction enzyme activity that might degrade the foreign DNA used.
  • Preferred examples of host cells for cloning, useful in the present invention are bacteria such as Escherichia coli. Bacillus subtilis, Pseiidomonas, Streptomxces. Salmonella, and yeast cells such as S. cerevisiae.
  • Host cells which can be targeted for expression of a minichromosome may be plant cells of any source and specifically include Arabidopsis. maize, rice, sugarcane, sorghum, barley. soybeans, tobacco, wheat, tomato, potato, citrus, or any other agronomically or scientifically important species.
  • hybridization refers to the pairing of complementary RNA and DNA strands to produce an RNA-DNA hybrid, or alternatively, the pairing of two DNA single strands from genetically different or the same sources to produce a double stranded DNA molecule.
  • linker refers to a DNA molecule, generally up to 50 or 60 nucleotides long and synthesized chemically, or cloned from other vectors.
  • this fragment contains one, or preferably more than one, restriction enzyme site for a blunt-cutting enzyme and a staggered-cutting enzyme, such as BainWl.
  • One end of the linker fragment is adapted to be ligatable to one end of the linear molecule and the other end is adapted to be ligatable to the other end of the linear molecule.
  • a "library” is a pool of random DNA fragments which are cloned. In principle, any gene can be isolated by screening the library with a specific hybridization probe (see, for example, Young et al, 1977).
  • Each library may contain the DNA of a given organism inserted as discrete restriction enzyme-generated fragments or as randomly sheered fragments into many thousands of plasmid vectors.
  • E. coli, yeast, and Salmonella plasmids are particularly useful when the genome inserts come from other organisms.
  • lower eukaryote refers to a eukaryote characterized by a comparatively simple physiology and composition, and most often unicellularity. Examples of lower eukaryotes include flagellates, ciliates, and yeast.
  • a "minichromosome” is a recombinant DNA construct including a centromere and capable of transmission to daughter cells.
  • the stability of this construct through cell division could range between from about 1 % to about 100%. including about 5%, 10%, 20%. 30%>, 40%, 50%, 60%, 70%, 80%, 90% and about 95%.
  • the minichromosome construct may be a circular or linear molecule. It may include elements such as one or more telomeres, ARS sequences, and genes. The number of " such sequences included is only limited by the physical size limitations of the construct itself " .
  • minichromosome may be inherited through mitosis or meiosis, or through both meiosis and mitosis.
  • minichromosome specifically encompasses and includes the terms "plant artificial chromosome " or "PLAC,” and all teachings relevant to a PLAC or plant artificial chromosome specifically apply to constructs within the meaning of the term minichromosome.
  • minichromosome-encoded protein it is meant a polypeptide which is encoded by a sequence of a minichromosome of the current invention. This includes sequences such as selectable markers, telomeres, etc.. as well as those proteins encoded by any other selected functional genes on the minichromosome.
  • a “ 180 base pair repeat” is defined as any one of the specific repeats disclosed in SEQ ID NOS: 184-212, or a “consensus” sequence derived therefrom. Thus, a given “180 base pair repeat” may include more or less than 180 base pairs, and may reflect a sequence not represented by any of the specific sequences provided herein.
  • the term "plant” includes plant cells, plant protoplasts, plant calh. and the like, as well as whole plants regenerated therefrom
  • plasmid ' oi cloning vectoi refeis to a closed covalently circular extrachromosomal DNA or linear DNA which is able to autonomously replicate in a host cell and which is normally nonessential to the survival of the cell
  • plasmids and other vectors are known and commonly used in the art (see, for example. Cohen et al, U.S. Patent No. 4.468.464. which discloses examples of DNA plasmids, and which is specifically inco ⁇ orated herein by reference).
  • a "probe” is any biochemical reagent (usually tagged in some way for ease of identification), used to identify or isolate a gene, a gene product, a DNA segment or a protein
  • recombination refers to any genetic exchange that involves breaking and rejoining of DNA strands.
  • regulatory sequence refers to any DNA sequence that influences the efficiency of transcription or translation of any gene.
  • the term includes, but is not limited to. sequences comprising promoters, enhancers and terminators.
  • a "selectable marker” is a gene whose presence results in a clear phenotype, and most often a growth advantage for cells that contain the marker This growth advantage may be present under standard conditions, altered conditions such as elevated temperatuie. or in the presence of " certain chemicals such as herbicides or antibiotics Use of selectable markers is described, for example, in Broach et al ( 1979) Examples of selectable markers include the thymidine kinase gene, the cellular adenine-phosphonbosyltransferase gene and the dihydrylfolate reductase gene.
  • hygromycin phosphotransferase genes the bar gene and neomycin phosphotransferase genes, among othei s
  • Prefened selectable markers in the present invention include genes whose expression confer antibiotic or herbicide resistance to the host cell, sufficient to enable the maintenance of a vector within the host cell, and which facilitate the manipulation of the plasmid into new host cells
  • a "screenable 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 certain chemicals used to detect the phenotype.
  • site-specific recombination refers to any genetic exchange that involves breaking and rejoining of DNA strands at a specific DNA sequence
  • a "structural gene” is a sequence which codes for a polypeptide or
  • RNA and includes 5' and 3' ends.
  • the structural gene may be from the host into which the structural gene is transformed or from another species.
  • a structural gene will preferably, but not necessarily, include one or more regulatory sequences which modulate the expression of the structural gene, such as a promoter, terminator or enhancer.
  • a structural gene will preferably, but not necessauly, confer some useful phenotype upon an organism comprising the structural gene, for example, herbicide resistance
  • a structural gene may encode an RNA sequence which is not translated into a protein, for example a tRNA or rRNA gene.
  • telomere refers to a sequence capable of capping the ends of a chromosome, thereby preventing degradation of the chromosome end. ensuring replication and preventing fusion to other chromosome sequences.
  • transformation or “transfection” refer to the acquisition in cells of new DNA sequences through the chromosomal or extra- chromosomal addition of DNA This is the process by which naked DNA DNA coated with protein, or whole minichromosomes are introduced into a cell, resulting in a potentially heritable change
  • Tetrad pollinations were carried out as follows A mature flower was removed from the donor plant and tapped upon a glass microscope slide to release mature tetrad pollen grains This slide was then placed under a 20-40x Zeiss dissecting microscope To isolate individual tetrad pollen grains, a small wooden dowel was used to which an eyebrow hair with rubber cement was mounted Using the light microscope, a tetrad pollen unit was chosen and touched to the eyebrow hair The tetrad preferentially adhered to the eyebrow hair and was thus lifted from the microscope slide and transported the recipient plant stigmatic surface The transfer was carried out without the use of the microscope, and the eyebrow hair with adhering tetrad was then placed against the recipient stigmatic surface and the hair was manually dragged across the stigma surface The tetrad then preferentially adhered to the stigma of the recipient and the cross pollination was completed Initially.
  • CEN2 and CEN4 were selected in particular for analysis Both reside on structurally similar chromosomes with a 3 5 Mb rDNA arrays on their distal tips, with regions measuring 3 and 2 Mb, respectively between the rDNA and centromeres, and 16 and 13 Mb regions on their long arms (Copenhaver and Pikaard, 1996)
  • the virtually complete and annotated sequence of chromosomes II and IV was used to conduct an analysis of centromeres at the nucleotide level (http //www ncbi nlm nih gov/Entrez/nucleotide html)
  • the sequence composition was analyzed within the genetically-defined centromere boundaries and compared to the adjacent pencentromenc regions (FIGs 12A-T) Analysis of the two centromeres facilitated comparisons of sequence patterns and identification of conserved sequence elements
  • centromere sequences were found to harbour 180 bp repeat sequences These sequences were found to reside in the gaps of each centromeric contig (FIG 3, FIGs 12B, 12L). With few repeats and no long arrays elsewhere in the genome BAC clones near these gaps have end sequences corresponding to repetitive elements that likely constitute the bulk of the DNA between the contigs, including 180 bp repeats, 5S rDNa oi 160-bp repeats (FIG 3) Fluorescent in situ hybridization has shown these repetitive sequences are abundant components of Arabidopsis centromeres (Murata et al , 1997, Heslop-Hamson et al , 1999, Brandes et al , 1997) Genetic mapping and pulsed-field gel electrophoresis indicate that many 180 bp repeats reside in long arrays measuring between 0 4 and 1 4 Mb in the centromeric regions (Round et al , 1997).
  • the resulting diploid progeny was either L/C or C/C
  • the map generated with these plants is based solely on male meioses, unlike the existing map, which represents an average of recombination's in both males and females Therefore, several well-established genetic distances were recalculated and thus will determine whether recombination frequencies are significantly altered
  • the large quantities of genetic data generated by the analysis must be compared pair-wise to perform tetrad analysis All of the data was managed in a Microsoft Excel spread-sheet format, assigning Landsberg alleles a value of " 1 " and Columbia alleles a value of "0"' Within a tetrad, the segregation of markers on one chromosome was compared to centromere-linked reference loci on a different chromosome (see Table 2 below) Multiplying the values of each locus by an appropriate reference, and adding the results for each tetrad easily distinguished PD, NPD, and TT tetrads with values of 2. 0, and 1 , respectively
  • centromeres chromosomal regions that could be separated by recombination from centromeres (tetratype). as well as regions that always cosegregated with centromeres (ditype) (Copenhaver et al , 1998, Copenhaver et al 1999) Tetratype frequencies decrease to zero at the centromere, consequently, centromere boundaries were defined as the positions that exhibited small but detectable numbers of tetratype patterns
  • centromeres 1-5 were localized to regions on the physical map corresponding to contigs of 550, 1445, 1600, 1790 and 1770 kb, respectively (FIG 3) Additionally, for each centromeric interval, a number of useful recombtnants were identified The results of the analysis indicated that centromeres reside within large domains that restrict recombination machinery activity and that the transition between these domains and the surrounding recombination-proficient DNA
  • Table 2 Scoring protocol for tetratypes.
  • the low recombination frequencies typically observed near higher eukaryotic centromeres may be due to DNA modifications or unusual chromatin states (Choo. 1998. Puechberty, 1999, Mahtani and Willard, 1998, Charlesworth et al , 1986, Charlesworth et al, 1994) To modify these states, and thus improve centromere mapping resolution by raising recombination frequencies.
  • FI Landsberg/Columbia plants were treated with one of a series of compounds known to cause DNA damage, modify chromatin structure, or alter DNA modifications.
  • centromeres were refined on chromosomes 2 to 5 (FIG 1 ), yielding intervals spanned by contigs of 880. 1 150. 1260. and 1070 kb. respectively, with all tetrads consistently localizing centromere functions to the same region (Copenhaver et al , 1999)
  • the phosphoenolpyruvate gene (CUEl) defines one CEN 5 border; 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 predicted genes shared
  • CEN2 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 predicted genes identified is given in Table 3. below. A list of additional genes encoded within the boundaries of CEN4 are listed in Table 4. The identification of these genes is significant in that the genes may themselves contain unique regulatory elements or may reside in genomic locations flanking unique control or regulatory elements involved in centromere function or gene expression.
  • the current inventors contemplate use of these genes, or DNA sequences 0 to 5 kb upstream or downstream of these sequences, for insertion into a gene of choice in a minichromosome It is expected that such elements could potentially yield beneficial regulatory controls of the expression of these genes, even when in the unique environment of a centromere
  • Table 3 Predicted genes within CEN2 and CEN4 that correspond to the cDNA database.
  • PPM2 Peroxisomal membrane protein
  • ATP synthase gamma chain 1 (APCI) ⁇ AAD48955 3 protein kinase and EF hand AAD03453 3
  • Table 4 List of additional genes encoded within the boundaries of CEN4.
  • centromere on chromosome 4 was mapped between m ⁇ 233 ( 18 8 cM) and mi 167 (21.5 cM). A more refined position places the centromere between the markers T24H24.30k3 (-20.3 cM) and F13H 14-t7 (-21.0 cM).
  • the following sequenced http://www.ncbi.nlm.nih.gov/Entrez/nucleotide.html
  • BAC (bacterial artificial chromosome) clones are known to span the region between the markers F5J 15-sp6 and T6A13-sp6: T27D20, T19B 17, T26N6.
  • centromere on chromosome 5 was mapped between nga76 (71 6 cM) and PhyC (74.3 cM). A more refined position places the centromere between the markers F13K20-t7 (-69.4 cM) and CUE l (-69 5 cM) Contained within this interval arc the publicly available markers: um579D. m ⁇ 291 b. CMs l .
  • centromeres 1 3 and 5 are given in SEQ ID NOS 184- 208 Sequences foi contigs including the centiomere 2 and 4 clones are given by SEQ ID NOS 209-212 BAC clone number designations are given The centiomcie number origin of the clone is as indicated Where a second date is given the second date indicates the date for the lev ised sequence
  • a BAC clone may be retrofitting with one or more plant telomeres and selectable markers together with the DNA elements necessary for Agiobactermm transformation (FIG 9) This method ill provide a means to delivei any BAC clone into plant cells and to test it for centromere function
  • the conversion vector contains a retrofitting cassette
  • the retrofitting cassette is flanked by Tn 10 Tn5 Tn7 Mu or other transposable elements and contains an origin of replication and a selectable marker for Agiobactermm, a plant telomere array followed by T-DNA right and left borders followed by a second plant telomere array and a plant selectable marker (FIG 9)
  • the conversion vector is transformed into an E coli strain carrying the target BAC
  • the transposable elements flanking the retrofitting cassette then mediate transposition of the cassette iandomly into the BAC clone
  • the retrofitted BAC clone can now be transformed into an appropriate stiain of Agi obaclei mm and then into plant cells vvheie it can be tested for high fidelity meiotic and mitotic transmission which would indicate that the clone contained a complete functional plant centromere
  • Minichromosomes are constructed by combining the prev iously isolated essential chiomosomal elements
  • Exemplary minichromosome vectors include those designed to be shuttle vectors ; e thev can be maintained in a convenient host (such as E coli A obacteuum or yeast) as well as plant cells
  • a minichromosome can be maintained in £ coli or other bacterial cells as a circulai molecule by placing a removable stuffer fragment between the telomeric sequence blocks
  • the stuffer fragment is a dispensable DNA sequence bordered by unique restriction sites, which can be lemoved b> restriction digestion of the circular DNAs to create linear molecules with telomeric ends
  • the linear minichromosome can then be isolated by for example, gel electrophoiesis In addition to the stuffer fragment and the plant telomeres.
  • the minichromosome contains a replication origin and selectable marker that can function in plants to allow the circular molecules to be maintained in bacterial cells
  • the minichromosomes also include a plant selectable marker a plant centromere, and a plant ARS to allow replication and maintenance of the DNA molecules in plant cells
  • the minichromosome includes several unique restriction sites where additional DNA sequence inserts can be cloned The most expeditious method of physically constructing such a minichromosome * e hgating the various essential elements together for example, will be apparent to those of ordinary skill in this art
  • FIGs 7A-7H A number of minichromosome vectoi s have been designed by the current inventors and are disclosed herein for the purpose of illustration (FIGs 7A-7H) These vectors are not limiting however as it will be apparent to those of skill in the art that many changes and alterations may be made and still obtain a functional vector
  • a two step method was developed tor construction of minichromosomes which allows adding essential elements to BAC clones containing centromeric DNA These procedures can take place in vn o eliminating problems of chromosome breakage that often happen in the test tube
  • the details and adv antages of the techniques are as follows 1.)
  • One plasmid can be created that contains markers, origins and bordei sequences for Agrobacterium transfer, markers for selection and screening in plants, plant telomeres. and a loxP site 01 other site useful for site-specific recombination in vivo or //; i ttro
  • the second plasmid can be an existing BAC clone, isolated from the available genomic libraries (FIG. 1 1 A).
  • the two plasmids are mixed, either within a single E. coli cell, or in a test tube, and the site-specific recombinase cre is introduced. This will cause the two plasmids to fuse at the loxP sites (FIG. 1 IB).
  • Variations include vectors with or without a Kan R gene (FIGs 1 IB. 1 IC). with or without a LAT52 GUS gene, with a LAT52 GFP gene, and with a GUS gene under the control of other plant promoters (FIGs. 1 IC, 1 ID and 1 IE).
  • minichromosomes do no integrate into the host genome (FIG. 1 IF).
  • minichromosomes must be maintained as distinct elements separate from the host chromosomes
  • the inventors envision a variety that would encode a lethal plant gene (such as dipthena toxin or any other gene product that, when expressed. causes lethality in plants). This gene could be located between the right Agrobactermm border and the telomere.
  • a method for the screening of centromere activity (FIG 10) In the method plants are first transformed w ith binaiy BAC clones that contain DNA from the genetically-defined centromeric regions By allowing the DNA to integrate into the host chromosomes, it is expected that this integration will lesult in a chromosome with two centromeres This is an unstable situation which often leads to chromosome breakage, as single chromosomes harboring two or more functional centromeres will often times break at junctions between the two centromeres when pulled towards opposite poles during mitotic and meiotic events This can lead to severe growth defects and inviable progeny when genes important or essentially for cellular and developmental processes are disrupted by the breakage event Therefore regions having centromere function could be identified by looking for clones that exhibit upon introduction into a host plant any of the following predicted properties reduced efficiencies of transformation causation of genetic instability when integrated into natural chromosomes such that the transformed plants show aberrant sectors and increased lethality a difficulty to maintain
  • the screen is performed by identifying clones of greater than 100 kb that encode centromere DNA in a BiBAC library (binary bacterial artificial chromosomes) (Hamilton 1997) This is done by screening filters comprising a BiBAC genomic hbraiy for clones that encode DNA from the centromeres (FIG 10 step 1 )
  • the BiBAC v ector is used because it can contain large inserts of Arabidopsis genomic material and also encodes the binary sequences needed for A ⁇ i obactenum-mediated transformation
  • the centromere sequence containing BiBAC v ectors aie then directly integrated into chromosomes by Agrobactermm-mediated transformation (FIG. 10. step 2 ) As a control.
  • BiBAC constructs containing non-centromenc DNA also are used for transformation
  • BiBACs harboring sequences with centromere function will result in forming dicent ⁇ c chromosomes.
  • Progeny from transformed plants will be analyzed for nonviabihty and gross morphological differences that can be attributed to chromosomal breaks due to the formation of dicentnc chromosomes (FIG. 10. step 3)
  • Non-centromere sequences are expected to show little phenotypic differences from wildtype plants
  • pollen donor plants were again treated as described above and used in another round ot pollination. Pollen donor plants were typically subjected to 5-10 rounds of treatment and pollen collection.
  • Treatments were also performed using non-chemical agents As above, the treatments were used to achieve more refined map positions for the centromeres in Arabidopsis by stimulating recombination in additional pollen donor plants The treatments were as follows:
  • Heat shock treatments were performed by placing the pot containing the pollen donor plants in shallow dishes filled with water (to prevent desiccation), and placing the plant-containing dishes in incubators of the appropriate temperature UV exposure was performed by placing the pollen donor plants in a BioRad UV chamber and illuminating the plants at the appropriate wave length for varying amounts of time. Both the UV and heat shock plants were subjected to several rounds of treatment and pollen collection Plants exposed to a gamma radiation source (Cobalt-60) were treated only once and then discarded to prevent the accumulation of deleterious chromosomal rearrangements. Following treatment, plants were then returned to the growth room and grown under standard conditions for 2-5 days.
  • gamma radiation source Cobalt-60
  • Introgression describes a breeding technique whereby one or more desired traits is transferred into one strain (A) from another (B). the trait is then isolated in the genetic background of the desired strain (A) by a series of backcrosses to the same strain (A).
  • the number of backcrosses required to isolate the desired trait in the desired genetic background is dependent on the frequency of recombination in each backcross Backcrossing transfers a specific desirable trait from one source to an inbred or other plant that lacks that trait This can be accomplished, for example, by first crossing a supenoi inbred (A) (recurrent parent) to a donor inbred (non-iecurrent parent), which carries the appropriate gene(s) for the trait in question, for example, a construct prepared in accordance with the current invention
  • the progeny of this cross first are selected in the resultant progeny for the desired trait to be transferred fiom the non-recurrent parent, then the selected progeny are mated back to the superior recurrent parent (A)
  • the progeny are hemizygous for loci controlling the characteristic being transferred, but are like the superior parent for most or almost all other genes
  • the last backcross generation would be selfed to give progeny which are pure breeding for the
  • transgenes are valuable in that they typically behave genetically as any other gene and can be manipulated by breeding techniques in a manner identical to any other corn gene Therefore, one may produce inbred plants which are true breeding for one or more transgenes. By crossing different inbred plants, one may produce a large number of different hybrids with different combinations of transgenes In this way. plants may be produced which have the desirable agronomic properties frequently associated with hybrids ( " hybrid vigor *' ), as well as the desirable characteristics imparted by one or more ⁇ ransgene(s).
  • Breeding also can be used to transfer an entire minichromosome from one plant to another plant For example, by crossing a first plant having a minichromosome to a second plant lacking the minichromosome. progeny of any generation of this cross may be obtained having the minichromosome. or any additional number of desired minichromosomes Through a series of backcrosses.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure While the compositions and methods of this invention have been descnbed in terms of preferred embodiments, it will be apparent to those of skill in the art that variations mav be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved 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
  • Copenhaver and Pikaard "RFLP and physical mapping with an rDNA-specific endonuclease reveals that nucleolus organizer regions of Arabidopsis thaliana adjoin the telomeres on chromosomes 2 and 4," Plant J., 9:259-276, 1996.
  • Copenhaver et al "Use of RFLPs larger than 100 kbp to map position and internal organization of the nucleolus organizer region on chromosome 2 in Arabidopsis thaliana. " Plant J. 7, 273-286. 1995. Copenhaver et al. Proc. Natl. Acad. Sci. 95:247. 1998.
  • Cuozzo et al Biotechnology. 6:549-553. 1988.
  • Curiel et al "Adenovirus enhancement of transferrin-polylysine-mediated gene delivery.” Proc. Natl Acad. Sci. USA 88( 19):8850-8854. 1991.
  • Curiel et al high-efficiency gene transfer mediated by adenovirus coupled to DNA-polylysine complexes.” Hum. Gen. Tlier. 3(2): 147- 154. 1992.
  • Fujimura et al Plant Tissue Culture Letters. 2:74. 1985.
  • Fynan et al "DNA vaccines: protective immunizations by parenteral. mucosal. and gene gun inoculations," Proc: Nat 'I Acad. Sci. USA 90(24): 1 1478-1 1482, 1993.
  • Gatehouse et al J. Sci. Food. Agric, 35:373-380, 1984.
  • Gefter et al Somatic Cell Genet. 3:231-236. 1977.
  • Maps 1987 A compilation of linkage and restriction maps of genetically studied organisms. 724-745. 1987. Koorneef. "The use of telotrisomics for centromere mapping in Arabidopsis thaliana (L. ) Heynh. Genetica. 62:33-40. 1983. Koster and Leopold. Plant PhysioL 88:829-832. 1988.
  • Vasil et al "Herbicide-resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus," Biotechnologx: 10:667-674, 1992. Vasil. Biotechnology, 6:397. 1988. Vernon and Bohnert, The EMBO J. , 1 1 :2077-2085. 1992.
  • a lecombinant DNA constaict comprising a plant centromere
  • telomere is a plant telomere
  • telomeie is an Arabidopsis thaliana telomere
  • telomere is a yeast telomere
  • the recombinant DNA construct of claim 1. which additionally comprises an autonomous replicating sequence (ARS)
  • ARS autonomous replicating sequence
  • said structuial gene is selected from the group consisting of an antibiotic resistance gene, a herbicide lesistance gene, a nitiogen fixation gene, a plant pathogen defense gene, a plant stress-induced gene, a toxin gene, a receptoi gene, a ligand gene and a seed storage gene
  • centromere is further defined as flanked by the genetic markers T22C23-T7 and T5D 18.
  • centromere comprises from about 100 to about 61 1.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:209.
  • centromere comprises from about 500 to about 61 1.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:209
  • centromere comprises from about 40.000 to about 61 1.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 209
  • centromere comprises from about 80,000 to about 611,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 209
  • centromeie comprises from about 150.000 to about 61 1.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 209
  • centromere comprises from about 300.000 to about 61 1.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 209
  • centromere comprises the nucleic acid sequence of SEQ ID NO 209
  • centromere comprises from about 100 to about 50.959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 210
  • centromere comprises from about 500 to about 50,959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 210 57.
  • centromere comprises from about 1.000 to about 50.959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:210.
  • centromere comprises from about 5,000 to about 50,959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:210.
  • centromere comprises from about 10,000 to about 50,959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:210.
  • centromere comprises from about 20,000 to about 50,959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:210.
  • centromere comprises from about 30,000 to about 50,959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:210.
  • centromere comprises from about 40.000 to about 50.959 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:210.
  • centromere comprises the nucleic acid sequence of SEQ ID NO:210.
  • centromere is an Arabidopsis thaliana chromosome 3 centromere.
  • centromere is further defined as flanked by the genetic markers T9G9-SP6 and T5M 14-SP6
  • centromere is still further defined as flanked by a pair of genetic markers selected from the group consisting of T9G9-SP6 and TI4H20. T9G9-SP6 and T7K14. T9G9-SP6 and T21 P20. T14H20 and T7K14, T14H20 and T21P20. T14H20 and T5M 14-SP6. T7K14 and T5M 14-SP6. T7K 14 and T21 P20, and T21 P20 and T5M 14-SP6.
  • centromere comprises from about 100 to about 1.082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:21 1.
  • centromere comprises from about 500 to about 1 ,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO-21 1.
  • centromere comprises from about 1 ,000 to about 1 ,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:21 1
  • centromere comprises from about 5.000 to about 1.082.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO.21 1.
  • centromere comprises from about 10,000 to about 1.082.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:21 1.
  • centromere comprises from about 50,000 to about 1 ,082.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:21 1.
  • centromere comprises from about 100,000 to about 1 ,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:21 1.
  • centromere comprises from about 200,000 to about 1.082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:21 1.
  • centromere comprises from about 400,000 to about 1 ,082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:21 1.
  • centromere comprises from about 800.000 to about 1.082,000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:21 1.
  • centromere comprises the nucleic acid sequence of SEQ ID NO:21 1.
  • centromere comprises from about 100 to about 163,317 contiguous nucleotides of the nucleic acid sequence of " SEQ ID NO:212.
  • centromere comprises from about 500 to about 163 317 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 212
  • centromeie comprises from about 1 000 to about 163.317 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 212
  • centromere comprises from about 5 000 to about 163,317 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 212
  • centromere comprises from about 10.000 to about 163.317 contiguous nucleotides of the nucleic acid sequence of SEQ ID N0 212
  • centromere comprises from about 30.000 to about 163,317 contiguous nucleotides of the nucleic acid sequence of SEQ ID N0 212
  • centromeie compnses from about 50 000 to about 163.317 contiguous nucleotides of the nucleic acid sequence of SEQ ID N0 212
  • centromere comprises from about 80 000 to about 163,317 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 212
  • centromere comprises from about 120 000 to about 163.317 contiguous nucleotides of the nucleic acid sequence ol SEQ ID NO 212
  • centromere is flanked by a pair of genetic markers selected from the group consisting of F13K20-T7 and T18M4, F13K20-T7 and T18F2. F13K20-T7 and T24I20. T18M4 and T 18F2. T 18M4 and T24I20. T18M4 and CUEl . T18F2 and T24I20. T18F2 and CUEl. and T24I20 and CUEl
  • n is from about 10 to about 80.000
  • n is from about 25 to about 60 000 96.
  • n is from about 100 to about 50.000.
  • n is from about 200 to about 40,000.
  • n is from about 400 to about 30.000.
  • n is from about 1.000 to about 30,000.
  • n is from about 5.000 to about 20.000.
  • n is from about 10.000 to about 15,000.
  • SEQ ID NO:203 SEQ ID NO:204. SEQ ID NO:205. SEQ ID NO:206. SEQ ID NO:207. SEQ ID NO:208. SEQ ID NO:209. SEQ ID NO:210. SEQ ID NO:21 1 or SEQ ID NO:212.
  • a minichromosome vector comprising a plant centromere and a telomere sequence.
  • the minichromosome vector of claim 103. comprising an autonomous replicating sequence
  • the minichromosome vector of claim 103. comprising a second telomere sequence.
  • the minichromosome vector of claim 103. comprising a structural gene.
  • the minichromosome vector of claim 103 further defined as comprising a second structural gene.
  • the minichromosome vector of claim 103 further defined as comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: l . 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: 1 1 , 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.
  • the cell of claim 1 1 1 wherein said cell is a yeast cell.
  • dicotyledonous plant is selected from the group consisting of group consisting of tobacco, tomato, potato, sugar beet, pea. carrot, cauliflower, broccoli, soybean, canola, sunflower, alfalfa, cotton and Arabidopsis:
  • the cell of claim 120 further defined as an Arabidopsis thaliana cell.
  • recombinant DNA construct comprises an autonomous replicating sequence (ARS).
  • ARS autonomous replicating sequence
  • the cell of claim 124 further defined as 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 said starting plant cell is transformed with said recombinant DNA construct.
  • the plant centromere is an Arabidopsis thaliana centromere
  • a transgenic plant comprising a minichromosome vector, wherein said vector comprises a plant centromere and a telomere sequence 135
  • transgenic plant of claim 134 wherein said minichromosome vectoi comprises a second telomere sequence
  • transgenic plant of claim 134 wherein said minichromosome vector comprises a structural gene
  • transgenic plant of claim 137 wherein said 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-induced gene a toxin gene, a receptor gene, a ligand gene and a seed storage gene
  • the transgenic plant of claim 137 wherein said first exogenous structural gene is selected from the group consisting of a hormone gene, an enzyme gene, an interleukin gene a clotting factor gene a cytokine gene, an antibody gene, and a growth factor gene
  • transgenic plant of claim 134 wherein said minichromosome vector comprises a nucleic acid sequence selected from the group consisting of 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 13. SEQ ID NO 14
  • SEQ ID NO 17 SEQ ID NO 18.
  • SEQ ID NO 19 SEQ ID NO 20 and SEQ ID NO 21
  • the transgenic plant of claim 134 further defined as a dicotyledonous plant 143
  • the transgenic plant of claim 143 wherein said dicotyledonous plant is selected from the group consisting of tobacco, tomato, potato, sugar beet. pea. canot. cauliflower, broccoli, soybean, canola. sunflowei . alfalfa, cotton and Arabidopsis
  • transgenic plant of claim 143 wherein the dicotyledonous plant is Arabidopsis thaliana
  • transgenic plant of claim 134 further defined as a monocotyledonous plant
  • transgenic plant of claim 145 wherein said monocotyledonous plant is selected from the group consisting of wheat, maize, rye. rice, turfgrass. oat. barley, sorghum, millet, and sugarcane.
  • a method of producing a minichromosome vector comprising
  • said prokaryotic cell is an Agi obactei nun cell
  • prokaryotic cell is an E cob cell
  • said first vector or said second vectoi comprises bordei sequences for Agr ⁇ /? ⁇ -/cre/// ⁇ /;;-med ⁇ ated transformation 161.
  • said plant centromere is an Arabidopsis thaliana centromere.
  • telomere is a plant telomere.
  • plant selectable or screenable marker is selected from the group consisting of GFP, GUS, BAR, PAT, HPT or NPTII.
  • a method of screening a candidate centromere sequence for plant centromere activity comprising the steps of:
  • said screening comprises observing a phenotypic effect present in the integratively transformed plant cells or plants comprising said plant cells, wherein said phenotypic effect is absent in a control plant cell not integratively transformed with said isolated nucleic acid sequence, or a plant comprising said control plant cell.
  • phenotypic effect is selected from the group consisting of: reduced viability, reduced efficiency of " said transforming, genetic instability in the integratively transformed nucleic acid, aberrant plant sectors, increased ploidy, aneuploidy, and increased integrative transformation in distal or centromeric chromosome regions.
  • a recombinant DNA construct compnsmg an Arabidopsis polyubiquitin 1 1 promoter, wherein said promoter comprises from about 25 to about 2 000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 180
  • the recombinant DNA construct of claim 183 wherein said 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 sti ess-induced gene a toxin gene a receptor gene a ligand gene and a seed storage gene 185.
  • said structural gene is selected from the group consisting of a hormone gene, an enzyme gene, an interleukin gene, a clotting factor gene, a cytokine gene, an antibody gene, and a growth factor gene.
  • a recombinant DNA construct comprising an Arabidopsis 40S ribosomal protein S 16 promoter, wherein said promoter comprises from about 25 to about 2.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 182.
  • the recombinant DNA construct of claim 198 wherein said 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-induced gene, a toxin gene, a receptor gene, a ligand gene and a seed storage gene
  • a recombinant DNA construct comprising an Aiabidopsis polyubiquitin 1 1 3 regulatory sequence, wherein said 3 regulatory sequence comprises from about 25 to about 2001 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO 181 202 The recombinant DNA construct of claim 201. wherein said wherein said 3 ' regulatory sequence compnses from about 75 to about 2001 contiguous nucleotides ot the nucleic acid sequence of SEQ ID NO 181
  • the recombinant DNA construct of claim 201 further comprising a telomere sequence 21 1
  • the recombinant DNA construct of claim 201 further comprising a plant centromere sequence.
  • a recombinant DNA construct comprising an Arabidopsis 40S ribosomal protein S 16 3' regulatory sequence, wherein said 3' regulatory comprises from about 25 to about 2.000 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 183.
  • the recombinant DNA construct of claim 216, wherein said 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.

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Abstract

La présente invention concerne l'identification et le clonage de centromères végétaux fonctionnels chez les Arabidopsis. Ceci permet la construction de minichromosomes héréditaires stables pouvant servir de vecteurs dans la construction de cellules transgéniques végétales et animales. De plus, des données sur la structure et la fonction de ces régions se révèleront précieuses pour l'isolation d'éléments génétiques centromériques et liés aux centromères supplémentaires ainsi que des polypeptides d'autres espèces.
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JP2011125354A (ja) 2011-06-30
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BR0009119A (pt) 2001-12-26

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