CA2352464A1 - Methods for transforming plastids - Google Patents

Abstract

An improved method is provided for the transformation of a plant cell plastid.
The improved method allows for the increased efficiency of the foreign DNA
penetrating the plastid membrane. The method generally involves the use of a plant tissue source having an altered plastid morphology in plastid transformation methods. The present invention finds use in plastid transformation methods for a wide variety of plant species.

Description

METHODS FOR TRANSFORMING PLASTIDS
FIELD OF THE INVENTION
This invention relates to the application of genetic engineering techniques to plants. More specifically, the invention relates to methods for the transformation of plant cell plastids.
BACKGROUND
The plastids of higher plants are an attractive target for genetic engineering.
Plant plastids (chloroplasts, amyloplasts, elaioplasts, chromoplasts, etc.) are the major biosynthetic centers that in addition to photosynthesis are responsible for production of industrially important compounds such as amino acids, complex carbohydrates, fatty acids, and pigments. Plastids are derived from a common precursor known as a 2 0 proplastid and thus the plastids present in a given plant species all have the same genetic content. Plant cells contain 500-10,000 copies of a small 120-i60 kilobase circular genome, each molecule of which has a large (approximately 25kb) inverted repeat. Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest which potentially can result in very high levels of foreign 2 5 gene expression.
Current plastid transformation methods are inefficient, as such there is need for constructs and methods which improve plastid transformation.
SUMMARY OF THE INVENTION
By this invention, methods which allow for the improved transformation of a foreign DNA into plant cell plastids are provided. Such methods generally involve SUBSTITUTE SKEET (RULE 26~

utilizing a plant tissue source which contains cells with an altered plastid morphology in the transformation methods. The alteration in the plant plastid morphology includes, inter alia, plastid size and number. By utilizing tissue derived from such plants in plastid transformation methods, efficiency of transformation of a foreign DNA into the plant cell plastid may be increased.
As exemplified herein, constructs useful for genetic engineering of plant cells to provide for a method of increasing plastid transformation efficiency are provided.
The constructs include nucleic acid sequences coding for protein sequences involved in controlling division of plant cell organelles. The expression of such nucleic acid sequences in a plant cell provides for an altered number andlor size of the chloropiasts within the host cell.
DNA sequences, also referred to herein as polynucleotides, for use in transformation contain an expression construct comprising a promoter region which is functional in a plastid, and a DNA sequence encoding a gene involved in controlling the division of plant cell organelles.
Methods for the use of transformed plants with altered plastid morphology are described. Such methods include plant breeding or transformation methods to provide plant cells having both the nuclear and plastid constructs.
The present invention also provides methods for increasing the efficiency of 2 0 chloroplast transformation. The method generally comprises transforming the plastids of a plant tissue which has been modified to have an altered number and/or size of plastids contained within the plant cell.
The present invention also provides a mechanism for enhancing the efficiency of chloroplast transformation in plant species.
2 5 The present invention also provides methods for improving the selectability of plant comprising, transforming a plant cell source having an altered plastid morphology with a construct comprising a promoter functional in a plant cell plastid operably associated with a nucleic acid sequence encoding a selectable marker.
Selectable markers of interest in the present invention include herbicide tolerance 3 0 genes such as glyphosate tolerance genes, and antibiotic resistance genes.
Glyphosate tolerance genes include the CP4 gene from Agrobacterium.

SUBSTITUTE SHEET (RULE 26) Another aspect of the present invention provides methods for preparing a plant cell source with increased plastid transformation efficiency comprising, transforming a plant cell with a construct comprising a promoter functional in plant cell operably associated with a nucleic acid sequence encoding aFtsZ protein.
Also considered part of this invention are the plants and plant cells obtained using the methods described herein.
DESCRIPTION OF THE FIGURES
10 Figure 1 provides an amino acid sequence alignment of the Arabidopsis FtsZl (SEQ ID N0:2), the Brassica FtsZI (SEQ ID NO:b), the tobacco FtsZl (SEQ ID
N0:9), the Soybean FtsZl (SEQ ID N0:72) and the corn FtsZl (SEQ ID N0:73) protein sequences.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the subject invention, methods are provided which allow for the improved transformation of a foreign DNA into plant cell plastids.
Such 2 0 methods generally involve utilizing a plant cell source which contains an altered plant plastid morphology. By utilizing tissue derived from such plants in plastid transformation methods, efficiency of transformation of a foreign DNA into the plant cell plastid can be increased.
In one embodiment of the instant invention, plant tissue containing altered 25 plant plastid morphology is used for plastid transformation methods. Such alterations in plant plastid morphology include, but are not limited to. alterations in the plastid size, shape and number in respect to a wild-type plastid morphology from the target plant cell. In general, a wild-type plastid morphology consists of small, round organelles contained within the plant cell, depending on the species.
Furthermore, a 3 0 plant cell typically contains between about 50 and about i00 plastids.
The plant tissue source used in plastid transformation methods of the present invention contains an increase in the size of the plastids contained in the plant cells.
SUBSTITUTE SHEET (RULE 26) Such increases in the size of the plastids provides for a larger surface area for the foreign DNA to penetrate the plastid membrane during transformation.
The large plastids preferably contain approximately the same number of plastid genomes as would be contained in corresponding number of wild-type plastids. For example, in a wild-type plant cell containing 100 plastids per cell and 100 copies of the plastid genome in each plastid (a total of 10,000 copies of the plastid genome per cell), the corresponding mutant tissue source would preferably contain about the same number of plastid genomes, only contained in one, or several large plastid(s).
Alternatively, a plant tissue source with an increased number of plastids, with a corresponding reduced size, can also find use in the plastid transformation methods of the present invention.
As is understood in the art, additional methods for obtaining plants with alterations in the plastid size and number are known. The skilled artisan will recognize that a number of methods are available for providing for an alteration in plastid cell division. Such methods are described, for example, by Strepp, et al.
( 1998) Proc. Natl. Acad. Sci. USA, 95:4368-4.373.
Cell division, also referred to as cytokinesis, has been the focus of studies in many organisms such as bacterial, fungal, and animal cells. Division of bacterial cells 2 0 occurs through the formation of an FtsZ ring (also referred to as a Z
ring) at the site of division (Lutkenhaus, et al. ( 1997) Ann. Rev. Biochem. 66:93-116). The positioning and formation of the Z ring acts to further drive septation (cytokinesis). The ring is composed of a tubulin-like FtsZ protein which has GTPase activity. Mutations in the ftsZ gene in E. toll leads to the production of a temperature-sensitive filaments with 2 5 regularly spaced nucleoids at certain temperatures (Lutkenhaus ( 1992) In Prokaryotic Structure and Function: A New Perspective, ed. S Mohan, C Dow, pp 123-152.
Cambridge: Cambridge Univ. Press}. Such mutations in bacteria leads to the inability to divide correctly.
The plant cell plastid as well as the mitochondria are derived from prokaryotic 3 0 ancestors, and thus, the division apparatus of these organelles resembles that of bacteria. Recently, identification of ftsZ related sequences in Arabidopsis and Physcomitrella patens have been reported (Osteryoung, et al: ( 1995) Nature, 376:473-SUBSTITUTE S)EIEET (RULE 26) 474; and Strepp, et al. ( 1998), supra). The protein encoded by the Arabidopsis ftsZ
gene was found to be imported into the chloroplast amd was therefore specuiated to be a component of the plastid division machinery {Osteryoung, et al. ( 1995}, supra).
More recently, the involvement of FtsZ in plastid division was directly demonstrated.
The disruption of the ftsZ gene in a lower plant, Physcomitrella patens, impeded plastid division, thereby giving rise to mutant cell lines with one or a few large plastids {Strepp, et al. (1998), supra).
The use of plants with an altered number andlor size of plastids containing one or few large plastids could therefore be used as targets for plastid transformation of any plant species. Such plants containing an altered size and/or number can be obtained using various methods, including mutagenesis, antisense suppression, or co-suppression. Methods for the mutagenesis of plant genomes are well known in the art, and include chemical, such as ethylmethane sulfonate {EMS) and nitrosoguanidine (NTG), as well as physical mutagenesis methods such as fast neutron bombardment.
Other means for obtaining a plant source with an alteration in the size and/or number of plastids contained in the cell are also contemplated. For example, tissue for use in the transformation methods of the present invention can be obtained from plants grown in culture conditions which provide for such altered plastid content. For example, tissue obtained from plants grown in vitro under cuiture conditions in which inhibitors of bacterial cell division, such as S,5'-bis-(8-anilino-1-naphtaienesulfonate) (Yu, et al. ( 1998} J. Biol Chem. 273:10216-10222), are present, can be utilized as a cell source for the plastid transformation methods of the present invention.
In a preferred embodiment, such plants containing cells with an alteration in the size and/or number of plastids are generated by anti-sense expression of the FtsZ
2 5 gene. Once plastid transformation is achieved and homoplasmic plants are identified, the anti-sense transgene can be eliminated by out-crossing and the wild-type condition of 50 to 100 plastids per cell restored. Similarly, plants regenerated from plastid transformed tissue containing an altered number and/or size of plastids from mutations can also be reverted to the wild-type plastid conditions using such out-3 0 crossing methods.

SUBSTITUTE SHEET (RULE 26) In the case of the use of culture conditions for obtaining plant cells with an altered number and/or size of plastids, wild-type plastids can be obtained by releasing the tissue from such culture conditions.
In another embodiment of the present invention, novel nucleic acid sequences are provided which encode proteins related to proteins involved in bacterial cell and plastid division.
In particular, novel nucleic acid sequences fromArabidopsis, soybean, corn, Brassica are provided which encode FtsZ related proteins. Such nucleic acid sequences find use in the preparation of DNA constructs. Such constructs find use in the production of plants with an altered number and/or size of chloroplasts.
The skilled artisan will recognize that other DNA sequences useful for the production of plants with an altered number and/or size of chloroplasts are available in the art. The sequences include but are not limited to, ftsA, ftsL, ftsI, ftsQ, ftsN, ftsW, ftsK {Lutkenhaus, et al. (1997) supra), and the arc genes (Pyke, et al.
{1992) Plant Physiol. 99:1005-1008; Pyke et al. (1994) Plant Physiol. 104:201-207;
and Pyke ( 1997) Am. J. Botany 84:1017-1027).
In order to obtain additional ftsZ sequences, a genomic or other appropriate library prepared from the candidate plant source of interest can be probed with conserved sequences from one or more plant andlor bacterial ftsZ sequences) to 2 0 identify homoiogously related sequences. Positive clones can be analyzed by restriction enzyme digestion and/or sequencing. When a genomic library is used, one or more sequences can be identified providing both the coding region, as well as the transcriptional regulatory elements of the ftsZ gene from such plant source.
Probes can also be considerably shorter than the entire sequence. Oligonucleotides can be 2 5 used, for example, but should be at least about 10, preferably at least about 15, more preferably at least 20 nucleotides in length. When shorter length regions are used for comparison, a higher degree of sequence identity is required than for longer sequences. Shorter probes are often particularly useful for polymerase chain reactions (PCR), especially when highly conserved sequences can be identified. (See, Gould, et 3 0 al., PNAS USA ( 1989) 86:1934-1938.) When longer nucleic acid fragments (,100 bp) are employed as probes, especially when using complete or large cDNA sequences, one can still screen with SUBSTITUTE SHEET (RULE 26) moderately high stringencies (for example using 50% formamide at 37oC with minimal washing) in order to obtain signal from the target sample with 20-50%
deviation, i.e., homologous sequences. (For additional information regarding screening techniques see Beltz, et al., Meth. Enzymology ( 19$3) 100:266-285).
Another aspect of the present invention relates to isolated FtsZ
polynucleotides. The polynucleotide sequences of the present invention include isolated polynucleotides that encode the polypeptides of the invention having a deduced amino acid sequence selected from the group of sequences set forth in the Sequence Listing and to other polynucleotide sequences closely related to such sequences and variants thereof.
The invention provides a polynucleotide sequence identical over its entire length to each coding sequence as set forth in the Sequence Listing. The invention also provides the coding sequence for the mature polypeptide or a fragment thereof, as well as the coding sequence for the mature poiypeptide or a fragment thereof in a reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, pro-, or prepro- protein sequence. The polynucleotide can also include non-coding sequences, including for example, but not limited to, non-coding S' and 3' sequences, such as the transcribed, untranslated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, 2 0 polyadenylation signals, and additional coding sequence that encodes additional amino acids. Fox example, a marker sequence can be included to facilitate the purification of the fused polypeptide. Polynucleotides of the present invention also include polynucleotides comprising a structural gene and the naturally associated sequences that control gene expression.
2 5 The invention also includes polynucleotides of the formula:
X-(R 1 )n-(R2)-(R3)n-Y
wherein, at the 5' end, X is hydrogen, and at the 3' end, Y is hydrogen or a metal, R~
and R3 are any nucleic acid residue, n is an integer between 1 and 3000, preferably between 1 and 1000 and R~ is a nucleic acid sequence of the invention, particularly a 3 0 nucleic acid sequence selected from the group set forth in the Sequence Listing and preferably SEQ ID NOs: i,3,5,7,8,and 10-31. In the formula, R~ is oriented so that its 5' end residue is at the left, bound to R,, and its 3' end residue is at the right, bound to SUBSTITUTE SHEET (RULE 26) WO 00!32799 PCT/US99/28103 R~. Any stretch of nucleic acid residues denoted by either R group, where R is greater than l, may be either a heteropolymer or a homopolymer, preferably aheteropolymer.
The invention also relates to variants of the polynucleotides described herein that encode for variants of the polypeptides of the invention. Variants that are fragments of the poiynucleotides of the invention can be used to synthesize full-length polynucleotides of the invention. Preferred embodiments are polynucieotides encoding polypeptide variants wherein 5 to 10, 1 to 5, l to 3, 2, 1 or no amino acid residues of a polypeptide sequence of the invention are substituted, added or deleted, in any combination. Particularly preferred are substitutions, additions, and deletions that are silent such that they do not alter the properties or activities of the poiynucleotide or polypeptide.
Further preferred embodiments of the invention that are at least 50%, 60%, or 70% identical over their entire length to a polynucleotide encoding a polypeptide of the invention, and polynucleotides that are complementary to such polynucleotides.
More preferable are polynucleotides that comprise a region that is at least 80%
identical over its entire length to a palynucleotide encoding a polypeptide of the invention and polynucleotides that are complementary thereto. In this regard, polynucleotides at least 90% identical over their entire length are particularly preferred, those at least 95% identical are especially preferred. Further, those with at 2 0 least 97% identity are highly preferred and thaw with at least 98% and 99%
identity are particularly highly preferred, with those at least 99% being the most highly preferred.
Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptides 2 5 encoded by the polynucleotides set forth in the Sequence Listing.
The invention further relates to polynucleotides that hybridize to the above-described sequences. In particular, the invention relates to polynucleotides that hybridize under stringent conditions to the above-described polynucleotides.
As used herein, the terms "stringent conditions" and "stringent hybridization conditions" mean 3 0 that hybridization will generally occur if there is at Least 95% and preferably at least 97% identity between the sequences. An example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 50%
formamide, SUBSTITUTE SHEET (RULE 26) 5x SSC ( 150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH
7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 microgramslmilliiiter denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O.lx SSC at approximately 65°C. Other hybridization and wash conditions are well known and are exemplified in Sambrook, et al., Molecular Cloning: A
Laboratory Manual, Second Edition, cold Spring Harbor, NY ( 1989), particularly Chapter 11.
The invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set for in the Sequence Listing under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence or a fragment thereof; and isolating said polynucleotide sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers as described herein.
25 As discussed herein regarding polynucleotide assays of the invention, for example, polynucleotides of the invention can be used as a hybridization probe for RNA, cDNA, or genomic DNA to isolate full length cDNAs or genomic clones encoding a polypeptide and to isolate cDNA or genomic clones of other genes that have a high sequence similarity to a polynucleotide set forth in the Sequence Listing.
2 0 Such probes will generally comprise at least 15 bases. Preferably such probes will have at least 30 bases and can have at least 50 bases. Particularly preferred probes will have between 30 bases and 50 bases, inclusive.
The coding region of each gene that comprises or is comprised by a polynucleotide sequence set forth in the Sequence Listing may be isolated by 2 5 screening using a DNA sequence provided in the Sequence Listing to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to identify members of the library which hybridize to the probe.
For example, synthetic oligonucleotides are prepared which correspond to the FtsZ
EST
3 0 sequences. The oiigonucleotides are used as primers in polymerase chain reaction (PCR) techniques to obtain 5' and 3' terminal sequence of FtsZ genes.
Alternatively, where oligonucleotides of low degeneracy can be prepared from particularFtsZ

SUBSTITUTE SHEET (RULE 26) WO 00132799 PCTlUS99/2$103 peptides, such probes may be used directly to screen gene libraries forFtsZ
gene sequences. In particular, screening of cDNA libraries in phage vectors is useful in such methods due to lower levels of background hybridization.
Typically, a FtsZ sequence obtainable from the use of nucleic acid probes will show 60-70% sequence identity between the target FtsZ sequence and the encoding sequence used as a probe. However, lengthy sequences with as little as 50-60%
sequence identity may also be obtained. The nucleic acid probes may be a lengthy fragment of the nucleic acid sequence, or may also be a shorter, oligonucleotide probe.
When longer nucleic acid fragments are employed as probes (greater than about bp), one may screen at lower stringencies in order to obtain sequences from the target sample which have 20-50% deviation (i.e., 50-80% sequence homology) from the sequences used as probe. Oligonucieotide probes can be considerably shorter than the entire nucleic acid sequence encoding an FtsZ enzyme, but should be at least about I0, preferably at least about I5, and more preferably at least about 20 nucleotides. A
higher degree of sequence identity is desired when shorter regions are used as opposed to longer regions. It may thus be desirable to identify regions of highly conserved amino acid sequence to design oligonucleotide probes for detecting and recovering other related FtsZ genes. Shorter probes are often particularly useful for polymerase chain reactions (PCR), especially when highly conserved sequences can be identified.
2 0 (See, Gould, et al., PNAS USA ( 1989) 86:1934-1938.).
Another aspect of the present invention relates to FtsZ polypeptides. Such polypeptides include isolated polypeptides set forth in the Sequence Listing, as well as polypeptides and fragments thereof, particularly those polypeptides which exhibit FtsZ activity and also those polypeptides which have at least 50%, 60% or 70%
2 5 identity, preferably at least 80% identity, more preferably at least 90%
identity, and most preferably at least 95% identity to a polypeptide sequence selected from the group of sequences set forth in the Sequence Listing, and also include portions of such polypeptides, wherein such portion of the polypeptide preferably includes at least 30 amino acids and more preferably includes at least 50 amino acids.
3 0 "Identity", as is well understood in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence SUBSTITUTE SHEET (RULE 26) relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods including, but not limited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York ( 1988);
Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part l, Griffin, A.M. and Griffin, H.G., eds., Humana Press, New Jersey ( 1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press ( 1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York {1991);
and Carillo, H., and Lipman, D., SIAM J Applied Math, 48:1073 ( 1988}. Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available programs. Computer programs which can be used to determine identity between two sequences include, but are not limited to, GCG {Devereux, J., et al., Nucleic Acids Research I2{ 1 ):387 ( 1984); suite of five BLAST programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology, 12: 76-80 ( 1994); Birren, et al., Genome Analysis, 1: 543-559 ( 1997)).
The BLAST X program is publicly available from NCBI and other sources {BLAST
2 0 Manual, Altschul, S., et al., NCBI NLM NIH, Bethesda, MD 20894; Altschul, S., et al., J. Mal. Biol., 215:403-410 ( 1990)). The well known Smith Watenman algorithm can also be used to determine identity.
Parameters for polypeptide sequence comparison typically include the following:
2 5 Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 ( 1970}
Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.
Acad. Sci USA 89:10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4 3 0 A program which can be used with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison Wisconsin. The above SUBSTITUTE SHEET (RULE 26) parameters along with no penalty for end gap are the default parameters for peptide comparisons.
Parameters for polynucleotide sequence comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol. Biol. 4$:443-453 ( 1970) Comparison matrix: matches = +I0; mismatches = 0 Gap Penalty: 50 Gap Length Penalty: 3 A program which can be used with these parameters is publicly available as the ''gap" program from Genetics Computer Group, Madison Wisconsin. The above parameters are the default parameters for nucleic acid comparisons.
The invention also includes polypeptides of the formula:
X-(R~ )n-(R2)-(R3)n-Y
wherein, at the amino terminus, X is hydrogen, and at the carboxyl terminus, Y
is hydrogen or a metal, Rl and R3 are any amino acid residue, n is an integer between 1 and 1000, and RZ is an amino acid sequence of the invention, particularly an amino acid sequence selected from the group set forth in the Sequence Listing and preferably SEQ ID NOs: 2,4,6, and 9. In the formula, R~ is oriented so that its amino terminal residue is at the left, bound to R~, and its carboxy terminal residue is at the right, bound to R3. Any stretch of amino acid residues denoted by either R group, where R
2 0 is greater than I, may be either a heteropoiymer or a homopolymer, preferably a heteropolymer.
Polypeptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising a sequence selected from the group of a sequence contained in the Sequence Listing set forth herein .
The polypeptides of the present invention can be mature protein or can be part of a fusion protein.
Fragments and variants of the polypeptides are also considered to be a part of the invention. A fragment is a variant polypeptide which has an amino acid sequence that is entirely the same as part but not all of the amino acid sequence of the 3 0 previously described polypeptides. 'The fragments can be "free-standing"
or comprised within a larger polypeptide of which the fragment forms a part or a region, most preferably as a single continuous region. Preferred fragments are biologically SUBSTITUTE SHEET (RULE 26) WO 00/32799 PCTlUS99/28103 active fragments which are those fragments that mediate activities of the polypeptides of the invention, including those with similar activity or improved activity or with a decreased activity. Also included are those fragments that antigenic or immunogenic in an animal, particularly a human.
Variants of the polypeptide also include polypeptides that vary from the sequences set forth in the Sequence Listing by conservative amino acid substitutions, substitution of a residue by another with like characteristics. In general, such substitutions are among Ala, Val, Leu and lle; between Ser and Thr; between Asp and Glu; between Asn and Gln; between Lys and Arg; or between Phe and Tyr.
Particularly preferred are variants in which 5 to 10; i to 5; 1 to 3 or one amino acids) are substituted, deleted, or added, in any combination.
Variants that are fragments of the polypeptides of the invention can be used to produce the corresponding full length polypeptide by peptide synthesis.
Therefore, these variants can be used as intermediates for producing the full-length poiypeptides of the invention.
The polynucleotides and polypeptides of the invention can be used, for example, in the transformation of host cells, such as plant host cells, as further discussed herein.
The invention also provides polynucleotides that encode a polypeptide that is a 2 0 mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids within the mature polypeptide {for example, when the mature form of the protein has more than one polypeptide chain}. Such sequences can, for example, play a role in the processing of a protein from a precursor to a mature form, allow protein transport, shorten or lengthen protein half life, or facilitate manipulation of the protein 2 S in assays or production. It is contemplated that cellular enzymes can be used to remove any additional amino acids from the mature protein.
A precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. The inactive precursors generally are activated when the prosequences are removed. Some or all of 3 0 the prosequences may be removed prior to activation. Such precursor protein are generally called proproteins.

SUBSTITUTE SHEET (RULE 26) WO 00/32799 PCTIUS9912811f3 Homologous sequences are found when there is an identity of sequence and can be determined upon comparison of sequence information, nucleic acid or amino acid, or through hybridization reactions between a known FtsZ and a candidate source.
Conservative changes, such as Glu/Asp, VallIle, Ser/Thr, Arg/Lys and Gin/Asn can also be considered in determining sequence homology. Typically, a lengthy nucleic acid sequence can show as little as 50-60% sequence identity, and more preferably at least about 70% sequence identity, between the target sequence and the given FtsZ
sequence of interest excluding any deletions which can be present, and still be considered related. Amino acid sequences are considered homologous by as little as 25% sequence identity between the two complete mature proteins. (See generally, Doolittle, R.F., OF URFS and ORFS (University Science Books, CA, 1986.) In addition, not only can sequences provided herein be used to identify homologous FtsZ sequences, but the resulting sequences obtained therefrom can also provide a further method to obtain FtsZ sequences from other plant and/or bacterial sources. In particular, PCR can be a useful technique to obtain related FtsZ
sequences from sequence data provided herein. One skilled in the art will be able to design oligonucleotide probes based upon sequence comparisons or regions of typically highly conserved sequence.
Once the nucleic acid sequence is obtained, the transcription, or transcription 2 0 and translation (expression), of the FtsZ sequence in a host cell is desired to produce a ready source of the enzyme and/or modify the number and/or size of the plastids found therein. Other useful applications can be found when the host cell is a plant host cell, in vitro and in vivo.
Nucleic acids (genomic DNA, plasmid DNA, cDNA, synthetic DNA, mRNA, etc.) encoding FtsZ or amino acid sequences of the purified enzymes, which permit design of nucleic acid probes facilitating the isolation of DNA coding sequences therefor, are known in the art and are available for use in the methods of the present invention. It is generally recognized to an artisan skilled in the field to which the present invention pertains that the nucleic acid sequences provided herein and the 3 0 amino acid sequences derived therefrom can be used to isolate other potential FtsZ
genes from GenBank using DNA and peptide search techniques generally known in the art.

SUBSTITUTE SHEET (RULE 26) In addition to the sequences described in the present invention, DNA coding sequences useful in the present invention can be derived from algae, fungi, bacteria, plants, etc: Homology searches in existing databases using signature sequences corresponding to conserved nucleotide and amino acid sequences of FtsZ can be employed to isolate equivalent, related genes from other sources such as plants and microorganisms. Searches in EST databases can also be employed. Furthermore, the use of DNA sequences encoding enzymes functionally enzymatically equivalent to those disclosed herein, wherein such DNA sequences are degenerate equivalents of the nucleic acid sequences disclosed herein in accordance with the degeneracy of the genetic code, is also encompassed by the present invention. Demonstration of the functionality of coding sequences identified by any of these methods can be carried out by complementation of mutants of appropriate organisms, such as E. coli:
The sequences of the DNA coding regions can be optimized by gene resynthesis, based on cadon usage, for maximum expression in particular hosts.
The nucleic acid sequences which encode FtsZ can be used in various constructs, for example, as probes to obtain further sequences. Alternatively, these sequences can be used in conjunction with appropriate regulatory sequences to increase levels of the respective FtsZ sequence of interest in a host cell for recovery or study of 'the enzyme in vitro or in vivo or to decrease levels of the respective FtsZ
2 0 sequence of interest for some applications when the host cell is a plant entity, including plant cells, plant parts (including but not limited to seeds, cuttings or tissues) and plants.
Thus, depending upon the intended use, the constructs can contain the nucleic acid sequence which encodes the entire FtsZ protein, or a portion thereof. For 2 5 example, where antisense inhibition of a given FtsZ protein is desired, the entire FtsZ
sequence is not required. Furthermore, where FtsZ constructs are intended for use as probes, it can be advantageous to prepare constructs containing only a particular portion of a FtsZ encoding sequence, for example a sequence which is discovered to encode a highly conserved FtsZ region.
3 0 As discussed above, nucleic acid sequence encoding a plant or other FtsZ
proteins of this invention can include genomic, cDNA or rnRNA sequence. By "encoding" is meant that the sequence corresponds to a particular amino acid sequence SUBSTITUTE SHEET (RULE 26) either in a sense or anti-sense orientation. By "extrachromosomal" is meant that the sequence is outside of the plant genome of which it is naturally associated.
By "recombinant" is meant that the sequence contains a genetically engineered modification through manipulation via mutagenesis, restriction enzymes, and the like.
A cDNA sequence may or may not contain pre-processing sequences, such as transit peptide sequences or targeting sequences to facilitate delivery of the FtsZ
protein to a given organelle or membrane location. The use of any such precursor FtsZ DNA sequence is preferred for uses in plant cell expression. A genomic FtsZ
sequence can contain the transcription and translation initiation regions, introns, and/or transcript termination regions of the plant FtsZ, which sequences can be used in a variety of DNA constructs, with or without the FtsZ structural gene.
Thus, nucleic acid sequences corresponding to the FtsZ sequences of this invention can also provide signal sequences useful to direct grotein delivery into a particular organellar or membrane location, 5' upstream non-coding regulatory regions (promoters) having useful tissue and timing profiles, 3' downstream non-coding regulatory regions useful as transcriptionai and translational regulatory regions, and may lend insight into other features of the gene.
Once the desired plant or other FtsZ nucleic acid sequence is obtained, it can be manipulated in a variety of ways. Where the sequence involves non-coding 2 0 flanking regions, the flanking regions can be subjected to resection, mutagenesis, etc.
Thus, transitions, transversions, deletions, and insertions can be performed on the naturally occurring sequence. in addition, all or part of the sequence can be synthesized. In the structural gene, one or more codons can be modified to provide for a rnadified amino acid sequence, or one or more codon mutations can be 2 5 introduced to provide for a convenient restriction site or other purpose involved with construction or expression. The structural gene can be further modified by employing synthetic adapters, linkers to introduce one or more convenient restriction sites, or the like.
For the most part, the constructs will involve regulatory regions functianal in 3 0 plants which provide for altered size and number of plastids in a plant cell. The open reading frame, coding for the FtsZ protein, FtsZ-related protein or functional fragment thereof will be joined at its 5' end to a transcription initiation regulatory region such as SUBSTITUTE SHEET {RULE 26) WO 00/32799 PCTlUS99128103 the wild-type sequence naturally found 5' upstream to theFtsZ or FtsZ-related structural gene, or to a heterologous regulatory region from a gene naturally expressed in plant tissues. Examples of useful plant regulatory gene regions include those from T-DNA genes, such as nopaline or octopine synthase, plant virus genes, such as CaMV 35S, or from native plant genes.
The DNA sequence encoding a plant or other FtsZ protein of this invention can be employed in conjunction with all or part of the gene sequences normally associated with the FtsZ. In its component parts, a DNA sequence encoding FtsZ
is combined in a DNA construct having, in the 5' to 3' direction of transcription, a transcription initiation control region capable of promoting transcription and translation in a host cell, the DNA sequence encoding plant FtsZ and a transcription and translation termination region.
Potential host cells include both prokaryotic and eukaryotic cells. A host cell can be unicellular or found in a multicellar differentiated or undifferentiated organism depending upon the intended use. Cells of this invention can be distinguished by having a FtsZ sequence foreign to the wild-type cell present therein, for example, by having a recombinant nucleic acid construct encoding a FtsZ protein therein not native to the host species.
Depending upon the host, the regulatory regions will vary, including regions 2 0 from viral, plasmid or chromosomal genes, or the like. For expression in prokaryotic or eukaryotic microorganisms, particularly unicellular hosts, a wide variety of constitutive or regulatable promoters can be employed. Expression in a microorganism can provide a ready source of the plant enzyme. Among transcriptional initiation regions which have been described are regions from bacterial 2 5 and yeast hosts, such as E. colt, B. subtilis, Sacchromyces cerevisiae, including genes such as beta-galactosidase, T7 polymerase, tryptophan E and the like.
In a preferred embodiment, the constructs will involve regulatory regions functional in plants which provide for modified production of plant FtsZ, and.
possibly, modification of the plant cell plastid. The open reading frame coding for the 3 0 plant FtsZ or functional fragment thereof will be joined at its 5' end to a transcription initiation regulatory region. In embodiments wherein the expression of the FtsZ
protein is desired in a plant host, the use of all or part of the complete plant FtsZ gene SUBSTITUTE SHEET (RULE 26) is desired; namely all or part of the 5' upstream non-coding regions (promoter) together with the structural gene sequence and 3' downstream non-coding regionscan be employed.
If a different promoter is desired, such as a promoter native to the plant host of interest or a modified promoter, i.e., having transcription initiation regions derived from one gene source and translation initiation regions derived from a different gene source, numerous transcription initiation regions are available which provide for a wide variety of constitutive or regulatable, e.g., inducible, transcription of the structural gene functions. The transcription/translation initiation regions corresponding to such structural genes axe found immediately 5' upstream to the respective start codons. Among transcriptional initiation regions used for plants are such regions associated with the T-DNA structural genes such as for nopaline and mannopine synthases, the 19S and 35S promoters from CaMV, and the 5' upstream regions from other plant genes such as napin, ACP, SSU, PG, zero, phaseolin E, and the like. Enhanced promoters, such as double 35S, are also available for expression of FtsZ sequences. Fox such applications when 5' upstream non-coding regions are obtained from other genes regulated during seed maturation, those preferentially expressed in plant embryo tissue, such as ACP and napin-derived transcription initiation control regions, are desired. Such "seed-specific promoters" can be obtained and used in accordance with the teachings of issued U.S. Patent Numbers 5,608,152 and 5,530,194, which references are hereby incorporated by reference.
Transcription initiation regions which are preferentially expressed in seed tissue, i.e., which are undetectable in other plant parts, are considered desirable for TAG
modifications in order to minimize any disruptive or adverse effects of the gene product.
2 5 Regulatory transcript termination regions can be provided in DNA
constructs of this invention as well. Transcript termination regions can be provided by the DNA
sequence encoding the plant FtsZ or a convenient transcription termination region derived from a different gene source, for example, the transcript termination region which is naturally associated with the transcript initiation region. Where the transcript 3 0 termination region is from a different gene source, it will contain at least about 0.25 kb, preferably about 1-3 kb of sequence 3' to the structural gene from which the termination region is derived.

SUBSTITUTE SHEET (RULE 26) Plant expression or transcription constructs having a plant FtsZ as the DNA
sequence of interest for increased or decreased expression thereof can be employed with a wide variety of plant life, particularly, plant life involved in the production of vegetable oils for edible and industrial uses. Mast especially preferred are temperate oilseed crops. Plants of interest include, but are not limited to, rapeseed {Canola and High Erucic Acid varieties), sunflower, safflower, cotton, soybean, peanut, coconut and oil palms, and corn. Depending on the method for introducing the recombinant constructs into the host cell, other DNA sequences can be required.
Importantly, this invention is applicable to dicotyledenous and monocotyledenous species alike and will be readily applicable to new andlor improved transformation and regulation techniques.
The method of transformation is not critical to the instant invention; various methods of plant transformation are currently available. As newer methods are available to transform crops, they can be directly applied hereunder. For example, 25 many plant species naturally susceptible to Agrobacterium infection can be successfully transformed via tripartite or binary vector methods ofAgrobacterium-mediated transformation. In addition, techniques of microinjection, DNA
particle bombardment, and electroporation have been developed which allow for the transformation of various monocot and dicot plant species.
2 0 In developing the DNA construct, the various components of the construct or fragments thereof will normally be inserted into a convenient cloning vector which is capable of replication in a bacterial host, e.g., E. coli. Numerous vectors exist that have been described in the literature. After each cloning, the plasmid can be isolated and subjected to further manipulation, such as restriction, insertion of new fragments, 2 5 iigation, deletion, insertion, resection, etc., so as to tailor the components of the desired sequence. Once the construct has been completed, it can then be transferred to an appropriate vector for further manipulation in accordance with the manner of transformation of the host cell.
Normally, included with the DNA construct will be a structural gene having 3 0 the necessary regulatory regions for expression in a host and providing for selection of transforrnant cells. The gene can provide for resistance to a cytotoxic agent, e.g.
antibiotic, heavy metal, toxin, etc., complementation providing prototrophy to an SUBSTITUTE SHEET (RULE 26) auxotrophic host, viral immunity or the like. Depending upon the number of different host species in which the expression construct or corriponents thereof are introduced, one or more markers can be employed, where different conditioxis for selection are used for the different hosts. A number of markers have been developed for use for selection of transformed plant cells, such as those which provide resistance to various antibiotics, herbicides, or the like. The particular marker employed is not essential to this invention, one or another marker being preferred depending on the particular host and the manner of construction.
As mentioned above, the manner in which the DNA construct is introduced into the plant host is not critical to this invention. Any method which provides for efficient transformation can be employed. Various methods for plant cell transformation include the use of Ti- or Ri-plasmids, microinjection, eiectroporation, DNA particle bombardment, liposome fusion, or the like. In many instances, it will be desirable to have the construct bordered on one or both sides by T-DNA, particularly having the left and right borders, more particularly the right border. This is particularly useful when the construct uses A. tumefaciens or A. rhizogenes as a mode for transformation, although the T-DNA borders can find use with other modes of transformation.
Once a transgenic plant is obtained which contains cells with altered numbers 2 0 and/or sizes of chloroplasts, tissue containing such cells can then be used in plastid transformation experiments. For example, utilizing tissue containing cells with larger plastids provides for a larger target in plastid transformation methods, thus allowing for an increased probability of introduction of the foreign DNA into the plant cell plastid.
2 5 The DNA sequences, or polynucleotides, for use in plastid transformation of this invention will contain a plastid expression construct generally comprising a promoter functional in a plant cell plastid. and a DNA sequence of interest to be expressed in the transformed plastid cells.
Constructs and methods for use in transforming the plastids of higher plants 3 0 are described in Zoubenko et al. (Nuc Acid Res ( 1994) 22{ 19):3819-3824), Svab et al.
(Proc. Natl. Acad. Sci.(1990) 87:8526-8530 and Proc. Natl: Acad. Sci.(1993) 90:913-917) and Staub et al. (EMBO J. (i993) 12:601-606). Constructs and methods for use SUBSTITUTE SHEET (RULE 26) in transforming plastids of higher plants to express DNA sequences under the control of a nuclearly encoded, plastid targeted T7 polymerase are described in U.S.
Patent Number 5,576,198. The complete DNA sequences of the plastid genome of tobacco are reported by Shinozaki et al. (EMBO J. ( 1986) 5:2043-2049).
Stable transformation of tobacco plastid genomes by particle bombardment is reported (Svab et.al. ( 1990), supra) and Svab et al. ( 1993), supra). The methods described therein can be employed to obtain plants homoplasrnic for plastid expression constructs using the methods described herein. Briefly, such methods involve DNA bombardment of a target host explant, preferably from a tissue which is rich in metabolically active plastid organelles, such as green plant tissues including leaves, and cotyledons. The bombarded tissue is then cultured for --2 days on a cell division promoting media. The plant tissue is then transferred to a selective media containing an inhibitory amount of the particular selective agent, as well as the particular hormones and other substances necessary to obtain regeneration for that particular plant species. For example, in the above publications and the examples provided herein, the selective marker is the bacterial aadA gene and the selective agent is spectinomycin. The aadA gene product allows for continued growth and greening of cells whose chloroplasts comprise the marker gene product. Cells which do not contain the marker gene product are bleached. The bombarded explants will 2 0 form green shoots in approximately 3-8 weeks. Leaves from these shoots are then subcultured on the same selective media to ensure production and selection of homoplasmic shoots. As an alternative to a second round of shoot formation, the initial selected shoots can be grown to mature plants and segregation relied upon to provide transformed plants homopiastic for the inserted gene construct.
2 5 The transformed plants so selected can then be analyzed to determine whether the entire plastid content of the plant has been transformed (homoplastic transforrnants). Typically, following two rounds of shoot formation and spectinomycin selection, approximately 50% of the transgenic plantiets analyzed are homoplastic as determined by Southern blot analysis of plastid DNA. These plantlets 3 0 are selected for further cultivation, both for analysis of the transgenic plastid phenotype (where the nuclear viral polymerase expression construct is also present in SUBSTITUTE SKEET {RULE 26) the plastid transformant), or for use in methods to transform the viral polymerase construct into the nucleus of the transplastomic plants.
The methods of the present invention provide for a more efficient approach to obtaining homoplasmic plants. Wild-type plant cells typically contain 50 to i00 plastids per cell. However, once a transplastomic plant is obtained, the DNA
sequence contained in the plant cell nucleus can be crossed away from the transplastornic cells. The DNA sequence transformed into the nucleus encoding for the alteration can be crossed away from the plant containing the transformed plastids.
Once the DNA sequence has been crossed out, the plastids in the host plant cell can divide and revert back to normal (i.e. wild-type) plastid size and numbers. By applying the selective agent for which the plastid expression constructs provides resistance, cells containing a pure population of the plastids containing the foreign DNA can be obtained.
The vectors for use in plastid transformation preferably include means for providing a stable transfer of the plastid expression construct and selectable marker construct into the plastid genome. This is most conveniently provided by regions of homology to the target plastid genome. The regions of homology flank the construct to be transferred and provide for transfer to the plastid genome by homologous recombination, via a double crossover into the genome. The complete DNA
sequence 2 0 of the plastid genome of tobacco has been reported (Shinozaki et al., EMBO
J. ( 1986) 5:2043-2049). Complete DNA sequences of the plastid genomes from liverwort (Ohyama et al., Nature ( 1986) 322:572-574) and rice (Hiratsuka et al.. Mol.
Gen.
Genet. (1989) 217:185-194), have also been reported.
Where the regions of homology are present in the inverted repeat regions of the plastid genome {known as IRA and IRB), two copies of the transgene are expected per transformed plastid. Where the regions of homology are present outside the inverted repeat regions of the plastid genome, one copy of the transgene is expected per transformed plastid. The regions of homology within the plastid genome are approximately lkb in size. Smaller regions of homology can also be used, and as little 3 0 as 100 by can provide for homologous recombination into the plastid genome.
However, the frequency of recombination and thus the frequency of obtaining plants having transformed plastids decreases with decreasing size of the homology regions.

SUBSTITUTE SHEET (RULE 26) Examples of constructs comprising such regions of homology for tobacco plastid transformation are described in Svab et.al. (1990 supra) and Svab and Maliga (1993 supra). Regions useful for recombination into tobacco and Brassica plastid genomes are also described in the following examples. Similar homologous recombination and selection constructs can be prepared using plastid DNA from the target plant species.
Other means of transfer to the plastid gename are also considered herein, such as by methods involving the use of transposable elements. For example, the constructs to be transferred into the plastid genome can be flanked by the inverted repeat regions from a transposable marker which functions in plant plastids. A
DNA
construct which provides for transient expression of the transposase required to transfer the target DNA into the plastids is also introduced into the chloroplasts. In this manner, a variety of phenotypes can be obtained in plants transformed with the same expression construct depending on positional effects which can result from insertion of the expression constructs into various locations an the plastid genome.
1 S Appropriate transposons for use in such plastic transformation methods include bacterial TnlO, bacteriophage Mu and various other known bacterial transposons.
The DNA sequence of interest in the plastid promoter expression constructs can be an encoding sequence which is oriented for expression of a particular structural gene, such that the protein encoded by the structural gene sequence is produced in the 2 0 transformed plastid. In addition, the DNA sequence of interest can include a number of individual structural gene encoding regions such that an operon for expression of a number of genes from a single plastid promoter region is produced. Thus, it is possible to introduce and express multiple genes from an engineered or synthetic operon or from a pre-existing prokaryotic gene cluster. Such a method would allow 2 5 large scale and inexpensive production of valuable proteins and fine chemicals in a particular desired plant tissue or a particular stage of development, depending upon the promoter used to drive nuclear expression of the specific viral polymerase. Such an approach is not practical by standard nuclear transformation methods since each gene must be engineered into a monocistron including an encoded transit peptide for 3 0 plastid uptake and appropriate promoter and terminator signals. As a result, gene expression levels would be expected to vary widely between cistrons, and generation of a number of transgenic plant lines would be required. UItirnately crosses would be SUBSTITUTE SHEET (RULE 26) W4 00/32799 PCT/tJS99/28103 required to introduce all of these cistrons into one plant to get expression to the target biochemical pathway.
Alternatively, the DNA sequence of interest in the plastid construct can be a fragment of an endogenous plastid gene oriented such that an RNA complementary to the endogenous gene mRNA is produced in the transformed plastid. Such antisense constructs can be used to decrease the expression of the target plastid gene.
In order to provide a means of selecting the desired plant cells following plastid transformation, the polynucieotides for plastid transformation will also contain a construct which provides for expression of a marker gene. Expression of the marker gene product allows for selection of plant cells comprising plastid organelles which are expressing the marker protein. in the examples provided herein, a bacterial aadA
gene is expressed under the regulatory control of chloroplast 5' promoter and 3' transcription termination regions. The use of such an expression construct for plastid transformation of plant cells has been described by Svab and Maiiga { 1993, supra).
Expression of the aadA gene confers resistance to spectinomycin and streptomycin, and thus allows for the identification of plant cells expressing this marker gene.
Selection for the aadA marker gene is based on identification of plant cells which are not bleached by the presence of streptomycin, or more preferably spectinomycin, in the plant growth medium. Other genes which encode a product involved in 2 0 chioroplast metabolism can also be used as selectable markers. For example, genes which provide resistance to plant herbicides such as giyphosate, bromoxynil or imidazolinone can find particular use. Such genes have been reported by Stalker et al.
(J. Biol. Chem. (1985) 260:4724-4728; glyphosate resistant EPSP), Stalker et al. (J.
Biol. Chem. ( 1985) 263:6310-6314; bromoxynil resistant nitrilase gene), and 2 5 Sathasivan et al. (Nucl. Acids Res. { 1990) 18:2188; AHAS imidazolinone resistance gene).
The present invention also provides methods for obtaining a plastid transformed plant on medium containing glyphosate. At the initial event of transformation only a few plastids out of the many present in a plant cell are 3 0 transformed and therefore are able to express glyphosate resistant marker gene product. The rest of the untransformed plastids within the cell remains vulnerable to the effect of giyphosate. Therefore, although the cell contains transformed plastids, it SUBSTITUTE SHEET (RULE 26) is unable to divide and sort out the transformed plastid resulting in lack of recovery of transformed callus tissue which would give rise to the transformed regenerants. Thus, any method that reduces plastid number to one or few within the cell has the potential to survive the effect of glyphosate and be useful as selectable marker for plastid transformation.
The following examples are provided by way of illustration and not by way of limitation.
EXAMPLES
Example 1: Identification of Plant fts2 Sequences In order to obtain a plant tissue source with an altered number and/or size of plastids using antisense and/or sense expression of the bacterial FtsZ plant homologues, public as well as proprietary sequence databases are queried for homologous sequences in soybean, rice, Arabadsopsis, corn and Brassica. Two types of plant FtsZ proteins have been previously identified in GenBank, type IFtsZ
proteins exemplified by accession gi11079731 (SEQ ID N0:32), appear to be imported 2 0 into the plastid, while type II FtsZ proteins, exemplified by accession gi13608494 (SEQ ID N0:33) and gi1683524 (SEQ ID N0:34), appear to remain in the cytoplasm.
Homologs of both the type I FtsZ sequence as well as homologues of type IIFtsZ
genes are described below. The sequences used to search against the databases are:
type I FtsZ homologue search was (SEQ ID N0:32), and for type IIFtsZ searches, 2 5 (SEQ ID N0:33} is used.
Searches performed in proprietary databases containing sequences obtained from Arabidopsis identified DNA sequences which are related to the FtsZl sequence.
The sequence of SEQ ID NO:1 is identified as AtFtsZ 1. The deduced amino acid sequence encoded by SEQ ID NO: l is provided in SEQ ID N0:2. In addition, one 3 0 sequence (SEQ ID N0:3) was identified as related to the FtsZ2 sequence.
The deduced amino acid sequence encoded by SEQ ID N0:3 is provided in SEQ ID N0:4.
SUBSTITUTE SHEET (RULE 2b) Sequences were also identified in databases containing sequences obtained from Brassica. One sequence was identified as related to theArabidopsis FtsZl sequence. Based on sequence alignments between the two sequences, approximately 170 amino acids were predicted to be missing from the BrasSica sequence at the N-terminus. To obtain a full length coding sequence for the Brassica FtsZl (BnFtsZl) gene; RACE PCR using DNA obtained from Brassica leaves was performed using the primers SC258 (SEQ ID N0:35) and SC259 (SEQ ID N0:36). One reaction product was found to contain the most 5' sequence (SEQ ID NO:70) and was used to produce a full length sequence referred to as BnFtsZl (SEQ ID N0:5). The deduced amino acid sequence encoded by BnFtsZl is provided in SEQ ID N0:6) A FtsZI homoiog was also identified in tobacco with PCR using primers designed to the conserved amino acid domains of the Arabidopsis FtsZl sequence.
The PCR primers used are identified as SC252 (SEQ ID N0:37), SC253 (SEQ ID
N0:38), SC254 (SEQ ID N0:39) and SC255 {SEQ ID N0:40). The reaction products were cloned into TOPO TA (Invitrogen), and a single clone, referred to as xanthil-26-contig (SEQ ID N0:7), contained the most sequence. Additional primers were designed for use in RACE PCR to obtain full length coding sequence for the tobacco FtsZl homolog. For amplification of the 5' region, primers SC291 (SEQ ID
N0:41) and SC292 (SEQ ID N0:42) were used, and for amplification of the 3' sequence, primers SC293 (SEQ ID N0:43) and SC294 (SEQ ID N0:44) were used. The PCR
products were cloned in TOPO TA and sequenced. Clone xanftsZl-5'-15 (SEQ ID
N0:7 I ) was chosen to be the best for the 5' tobacco FtsZ I sequence since it contained the greatest amount of 5' sequence and overlap with xanthil-26-contig. This sequence was combined with the xanthil-26-contig to produce xanFtsZ1 (SEQ ID N0:8). The 2 5 deduced amino acid sequence is provided in SEQ ID N0:9.
FtsZ homolog sequences were identified in databases containing DNA
sequences obtained from corn by BLAST searches using the Arabidopsis FtsZ1 and FtsZ2 amino acid sequences. Ten sequence were identified as related to these FtsZ
sequences, provided in SEQ ID NOs:lO-19. The clones, when aligned, revealed six 3 0 contigs, and the best representative clone for each were chosen for further analysis.
Sequence analysis of SEQ ID NO:10 revealed a high homology to AtFtsZl, and was estimated to be missing 158 amino acids at the N-terminal end when compared to SUBSTITUTE SHEET {RULE 26) WO 00/32799 PCT/US99I2$103 Arabidopsis FtsZl. Clone SEQ ID N0:13 was found to overlap perfectly with SEQ
ID NO:10 for 153 nt at the 5'end and in addition had '167 nt additional nt at the 5' end that had amino acid-homology with theArabidopsis FtsZl. However, this clone was also not predicted to encode the full-length FtsZ, and was still missing 1 I3 amino acids at the N-terminal end when compared to Arabidopsis FtsZl. Interestingly, for clone SEQ ID N0:13, its homology with SEQ ID NO:10, ends at position 167nt and diverges. This could either be indicative of the presence of intronic sequence or a new class of FtsZ protein. Primer SC321 {SEQ ID N0:45) was designed to pull out the missing maize FtsZ 1 sequence by RACE PCR.
Sequence analysis of SEQ ID N0:18 revealed its high homology to FtsZ2, and was also predicted to not to be full-length and 'missing about 286 amino acids at the N-terminal end when compared to Arabidopsis FtsZ2. Primer SC322 {SEQ ID
N0:46) was designed to pull out the missing maize FtsZ2 sequence by RACE PCR.
Although SEQ ID N0;14 and SEQ ID N0:15 were identified with the highest BLAST
scores with FtsZ2.
Soybean FtsZ homolog sequences were identified in databases by BLAST
searches with Arabidopsis FtsZl and FtsZ2 amino acid sequences. Twelve sequences were obtained, and are provided in SEQ ID NOs:20-31. Sequence analysis of SEQ
ID
N0:20, SEQ ID N0:24 and SEQ ID N0:25 revealed high homology to FtsZl and 2 0 none to be full-length when compared to Arabidopsis FtsZ 1. SEQ ID N0:25 had the longest sequence at the N-terminal end and is predicted to be missing 64 amino acids at the N-terminal when compared to Arabidopsis FtsZl sequence. Sequences of SEQ
ID N0:20; SEQ iD N0:24 and SEQ ID NO:25 were used to correct the overlapping region. RACE PCR primers can now be designed to amplify the ends for obtaining a 2 S full length DNA sequence.
A sequence alignment between the Arabidopsis, Brassica, tobacco, soybean, and corn FtsZl protein sequences is provided in figure 1.
Example 2: Preparation of Plant Expression Constructs 2A. Nuclear Expression Constructs SUBSTITUTE SHEET (RULE 26) Constructs are prepared for transformation into a plant cell nucleus for alteration of the plastid size and/or number in the transformed plant cell.
Constructs can be prepared to alter the plastids constitutively, or in a tissue specific manner, for example, in leaf tissue, or seed tissue.
A plasmid containing the napin cassette derived from pCGN3223 (described in USPN 5,639,790, the entirety of which is incorporated herein by reference) was modified to make it more useful for cloning large DNA fragments containing multiple restriction sites, and to allow the cloning of multiple napin fusion genes into plant binary transformation vectors. An adapter comprised of the self annealed oligonucleotide of sequence CGCGATTTAAATGGCGCGCCCTGCAGGCGGCCGCCTGCAGGGCGCGCCAT
TTAAAT (SEQ ID N0:47} was Iigated into the cloning vector pBC SK+ (Stratagene) after digestion with the restriction endonuclease BssHII to construct vector pCGN7765. Plamids pCGN3223 and pCGN7765 were digested with NotI and Iigated together. The resultant vector, pCGN7770, contains the pCGN7765 backbone with the napin seed specific expression cassette from pCGN3223:
The cloning cassette, pCGN7787, essentially the same regulatory elements as pCGN7770, with the exception of the napin regulatory regions of pCGN7770 have been replaced with the double CAMV 35S promoter and the tml polyadenylation and 2 0 transcriptional termination region.
A binary vector for plant transformation, pCGN5139, was constructed from pCGN 1558 (McBride and Summerfelt, ( 1990) Plant Molecular Biology, 14:269-276).
The polylinker of pCGN 1558 was replaced as a HindIIT/Asp718 fragment with a polylinker containing unique restriction endonuclease sites, AscI, PacI, XbaI, SwaI, 2 5 BamHI,and NotI. The Asp718 and HindIII restriction endonuclease sites are retained in pCGN5139.
A series of turbo binary vectors are constructed to allow for the rapid cloning of DNA sequences into binary vectors containing transcriptional initiation regions (promoters) and transcriptional termination regions.
3 0 The plasmid pCGN8618 was constructed by ligating oligonucleotides 5'-TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID N0:48) and 5'-TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID N0:49) into SUBSTITUTE SHEET (RULE 26) SaII/XhoI-digested pCGN7770. A fragment containing the napin promoter, polylinker and napin 3' region was excised from pCGN8618 bydigestion with Asp718I; the fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment then ligated into pCGN5139 that had been digested with Asp718I and HindIII and blunt-s ended by filling in the 5' overhangs with Klenow fragment. A plasmid cantaining the insert oriented so that the napin promoter was closest to the blunted Asp718I
site of pCGN5139 and the napin 3' was closest to the blunted HindIII site was subjected to sequence analysis to confirm both the insert orientation and the integrity of cloning junctions. The resulting plasmid was designated pCGN8622.
The plasmid pCGN8619 was constructed by Iigating oligonucleotides 5'-TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC -3' (SEQ ID NO:50) and S'-TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID N0:51) into SaII/Xhol-digested pCGN7770. A fragment containing the napin promoter, polylinker and napin 3' region was removed frorri pCGN8619 by digestion with Asp718I; the fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment then Iigated into pCGN5139 that had been digested with Asp718I and HindIII and blunt-ended by filling in the 5' overhangs with HIenow fragment. A plasmid containing the insert oriented so that the napin promoter was closest to the blunted Asp718I
site of pCGN5139 and the napin 3' was closest to the blunted HindIII site was subjected to 2 0 sequence analysis to confirm both the insert orientation and the integrity of cloning junctions. The resulting plasmid was designated pCGN8623.
The plasmid pCGN8620 was constructed by ligating oligonucleotides 5'-TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGGAGCT -3' (SEQ ID N0:52}
and 5'-CCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID N0:53) into 2 5 SaII/SacI-digested pCGN7787. A fragment containing the d35S promoter, polylinker and tml 3' region was removed from pCGN8620 by complete digestion with Asp718I
and partial digestion with NotI. The fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment then ligated into pCGN5139 that had been digested with Asp718i and HindIII and blunt-ended by filling in the S' overhangs with Klenow 3 0 fragment. A plasmid containing the insert oriented so that the d35S
promoter was closest to the blunted Asp718I site of pCGN5139 and the tml 3' was closest to the blunted HindIII site was subjected to sequence analysis to confirm both the insert SUBSTITUTE SHEET (RULE 26}

orientation and the integrity of cloning junctions. The resulting plasmid was designated pCGN8624.
The plasmid pCGN8621 was constructed by ligating oligonucleotides 5'-TCGACCTGCAGGAAGCTTGCGGCCGCGGATCCAGCT -3' (SEQ ID N0:54) and 5'-GGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID N0:55) into SaII/SacI-digested pCGN7787. A fragment containing the d35S promoter, polylinker and tml 3' region was removed from pCGN862I by complete digestion with Asg718I
and partial digestion with NotI. The fragment was blunt-ended by filling in the 5' overhangs with Klenow fragment then ligated into pCGN5139 that had been digested with Asp718I and HindIII and blunt-ended by filling in the 5' overhangs withKlenow fragment. A plasmid containing the insert oriented so that the d35S promoter was closest to the blunted Asp718I site of pCGN5139 and the tml 3' was closest to the blunted HindIII site was subjected to sequence analysis to confirm both the insert orientation and the integrity of cloning junctions. The resulting plasmid was designated pCGN8625.
The plasmid construct pCGN8640 is a modification of pCGN8624 described above. A 938bp PstI fragment isolated from transposon Tn7 which encodes bacterial spectinomycin and streptomycin resistance (Fling et al. ( 1985}, Nucleic Acids Research 13(I9):7095-7106), a determinant for E. coli and Agrobacterium selection, 2 0 was blunt ended with Pfu polymerase. The blunt ended fragment was ligated into pCGN8624 that had been digested with SpeI and blunt ended with Pfu polymerase.
The region containing the PstI fragment was sequenced to confirm both the insert orientation and the integrity of cloning junctions.
The spectinomycin resistance marker was introduced into pCGN8622 and 2 5 pCGN8623 as follows. A 7.7 Kbp AvrII-SnaBI fragment from pCGN8640 was ligated to a 10.9 Kbp AvrII-SnaBI fragment from pCGN8623 or pCGN8622, described above. The resulting plasmids were pCGN8641 and pCGN8643, respectively.
The Arabidopsis FtsZl nucleotide sequence was used to construct the sense 3 0 expression vector pCGN649S for use in transformation of Arabidopsis.
Brassica and tobacco. For this construct, the Arabidopsis ftsZl sequence was PCR amplified.
To monitor protein expression of FtsZ1 in transformed lines, a c-myc tag (EQKLISEEDL
SUBSTITUTE SHEET (RULE 26) (SEQ ID N0:56)), was translationally fused to FtsZl at the C-terminal end. The PCR
amplification was done by first round of amplification with primers SC247 (SEQ
iD
N0:57) and SC260 (SEQ ID N0:58} followed by amplification with SC247 (SEQ ID
NO:S9) and SC261 (SEQ ID N0:60) using the product of the first amplification as the 5 template DNA, using standard amplification parameters. The final amplification product, FtsZl/c-myc fusion was cloned in the nuclear transfarmation vector pCGN8624 to create pCGN6495, which was used to nuclear transform Arabidopsis, canola and tobacco using standard protocols.
The turbo vector pCGN8624 was used for the antisense constructs such that 10 the antisense sequence is driven from d3SS promoter. For Arabidopsis the coding sequence (from ATG to TAG) was amplified with primers SC248 (SEQ ID N0:61) and SC2S0 (SEQ ID N0:62) using AtFtsZl as template. For Brassical primers SC276 (SEQ ID N0:63) and SC268 (SEQ ID N0:64) Were used with PCR fragment SC3-1-1 (SEQ ID N0:70} as template DNA to generate aHindIIIlPstI fragment and 15 cloned in pBSKS (Stratagene) to generate pCGN6528. Primer SC276 was designed to be located 140 bases downstream from ATG due to the presence of nonhomologous stretch of sequence compared to Arabidopsis FtsZl contained in the first 140 bases sequence fragment. The 3' half of the coding sequence was PCR amplified using primers SC269 (SEQ ID N0:65) and SC270 (SEQ iD N0:66) to produce a PstI/NotI
2 0 fragment, and subsequently cloned in pCGN6528 to generate pCGN6529. The HindIIIlPstI fragment containing BnFtsZl sequence (from 140b downstream of ATG
to TAG} was cloned in turbo vector pCGN8624 to generate final transformation vectors pCGN6S30 and pCGN6611. The HindIIIINotI fragment containing BnFtsZl sequence was also cloned into pCGN8643 vector for seed-specific antisense FtsZl 2 5 expression. For tobacco, primers SC30S and SC306 were designed to PCR
amplify FtsZ 1 sequence to produce a SseIINotI fragment using 5' RACE PCR library DNA
made from leaf RNA> and cloned into TOPO TA2.1 to produce pCGN6565. The SseIlNotI fragment from pCGN6565 was cloned in the turbo vector pCGN8624 to generate final transformation vector pCGN6566.
2A. Plastid Expression Constructs SUBSTITUTE SHEET (RULE 26) WO 00/32799 PCT/IfS99/2$103 Constructs and methods for use in transforming the plastids of higher plants are described in Zoubenko et al. (Nuc Acid Res {I994) 22(19):3819-3824), Svab et al.
(Proc. Natl. Acid. Sci. ( 1990) 87:8526-8530 and Proc. Natl. Acid. Sci. ( 1993) 90:913-917) and Staub et al. (EMBO J. (1993) 12:601-606): Constucts and methods for use in transforming plastids of higher plants to express DNA sequences under the control of a nuclearly encoded, plastid targeted T7 polymerise are described in U.S.
Patent Number 5,576,198. The complete DNA sequences of the plastid genome of tobacco are reported by Shinozaki et al. (EMBO J. ( 1986) 5:2043-2049).
A plastid expression construct, pMON49218, was constructed to express the synthetic CP4 sequence with the 14 amino acid GFP fusion from the promoter region of the l6SrDNA operon having the nuclear-encoded RNA polymerise region (PrrnPEP+NEP), and the terminator region from the plastid rps 16 gene. The DNA
sequence of the Prrn/NEP/G l OL:: I4aaGFP fusion SEQ ID N0:67.
Example 3: Plant Transformation And Analysis Constructs for the expression of sense or antisense sequences are transformed into tobacco cells using the methods described by Ursin et. al. (1991) Plant Cell 2 0 3:583-591.
Transgenic tobacco plants containing the nuclear FtsZ constructs were analyzed for alterations in plastid morphology. including size and number of plastids present in the plant cell.
Fifty-eight initial transformants {TI generation) obtained from transformation with FtsZl expression construct pCGN6495 were screened for the large plastid phenotype and divided into three categories. Thirty-four (34) lines contained less than 5 large plastids, 8 lines contained between 5-20 plastids and 16 lines more than 20 (wild-type# and more than wild-type#) plastids. One line, Nt6495-61, contained a single large plastid.
3 0 The screening method involved examining isolated mesophyll protoplasts at 100X magnification under light microscope. The large plastid containing transgenic SUBSTITUTE SHEET (RULE 26) plants appear to be phenotypically indistinguishable from wild-type under culture and greenhouse conditions.
Estimation of plastid DNA copy number from several large plastid Iines revealed no difference when compared to wild-type. Southern analysis was used to estimate transgene copy number in the large plastid lines and several lines with single integration events were identified. Western analysis of the large plastid lines with c-rnyc antibody confirmed expression of the introduced transgene (tagged by c-myc). T2 seeds were collected from selected plants from each of the three categories.
Example 4: Plastid Transformation and Analysis Leaf material from three transgenic lines, Nt6495-30 (with <5plastids/cell}, Nt6495-16 (with 5-20 plastids/cell) and Nt&495-69 {with 5-20 plastids/cell);
were obtained for evaluation of plastid transformation efficiency and direct glyphosate selection. Plastid transformation vector pMON49218 which contains aadA gene for spectinomycin selection and GFP as a marker was used to bombard 15 leaf explants of each of the three transgenic lines. For each series of bombardment of the transgenic line 15 wild type control leaves were used. The order of bombardment for the 2 0 transgenic line and the wild type leaves were randomized to eliminate any bias.
Transformation frequency of one event Nt6495-30 was approximately double that of the wild type control producing 7 versus 3 transformants respectively.
Nt6495-16 and Nt6495-69 had approximately the same transformation frequency (3 transformants) as the control. Thus, our preliminary analysis reveals that plastid transformation efficiency can have been enhanced by reducing the plastid number from wild type to less than 5 plastids per cell. Interestingly, alI of the plastid transformant regenerants from Nt6495 Lines were very much slower in growth and size compared to those from wild type. It appears that the presence of the selectable antibiotic spectinomycin dihydrochloride at a concentration of 500mg/ml can have 3 0 affected the regenerability of cells in the Nt6495 lines. Thus, it is possible that there could be more plastid transformed cells in the transgenic Nt6495 lines which were susceptible to the antibiotic and could not regenerate. To check if this was the case, SUBSTITUTE SHEET (RULE 26) kill curves with lower concentrations of spectinomycin dihydrochloride (S0, 100, 200, 300, 400 and 500mg/mI) can be used with each of the Nt6495-30, Nt6495-16 and Nt6495-69 lines to establish the concentration at which the regeneration of shoots are as good as in wild type. This concentration of spectinomycin dihydrochloride will then be used to repeat transformation frequency tests with the three Nt6495 lines.
To analyze for direct glyphosate selection, kill curves with varying levels of glyphosate will be established with the Nt6495 lines to find the best selection level.
Plastid transformation vector pMON49218 will be used to bombard the Nt6495 lines and tested for direct selection using the optimized glyphosate level.
In Arabidopsis, FtsZl nuclear expression construct pCGN6495 was used to transform Columbia ecotype. T1 seeds were collected and about i00 kanamycin resistant seedlings were analyzed for alteration in plastid size and number following the same protocols as outlined for the tobacco section of this report. The transgenic plants were divided into three groups based on plastid number-I) 20 independent lines containing few large plastid ( 1-5),{II) 23 lines lines containing 5-20 plastids and (III) 50 lines containing wild-type plastid number were obtained. Selected T2 plants from each category were analyzed for number of transgene integration loci and sent to the growth chamber for T3 seed collection to identify homozygous plants. Such 2 0 plants can be used in plastid transformations as described by Sikdar, et al. { 1998) Plant Cell Reports, 18:20-24.
Transformed plants selected for aadA marker gene expression or glyphosate resistance are analyzed to determine whether the entire plastid content of the plant has been transformed (homoplasmic transformants). Typically, following two rounds of 2 5 shoot formation and spectinomycin selection, approximately 50°l0 of the transgenic plantlets which are analyzed are homoplasmic, as determined by Southern blot analysis of plastid DNA. Homoplasmic plantlets are selected for further cultivation.
Southern blot analysis is used to confirm the integration of the chimeric expression cassettes in the plastid genome. Preparation, electrophoresis, and transfer 3 0 of DNA to filters is as described (Svab et al., ( 1993 supra)). Total plant cellular DNA
can be prepared as described by Dellaporta et al. ( 1983) Plant Mol. Biol.
Rep. 1:19-21).

SUBSTITUTE SHEET (RULE 26) To visually observe the expression of marker genes such as GFP from the chloroplasts of transformed plants, various tissues are visualized utilizing a dissecting microscope. Protoplasts and chloroplasts are isolated as described in Sidorov, et al.
( 1994) ~'heor. Appl. Genet. 88:525-529.
The above results demonstrate that the sequences of the present invention provide an efficient means for the production of plastid transformed plants.
Furthermore, such methods fmd use in plastid transformation methods involving the selection of transplastomic plants on herbicides, for example glyphosate.
All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. Ail publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications rnay be practiced within the scope of the appended claim.
SUBSTITUTE SKEET (RULE 26) WO 00!32799 PCTIUS99/28103 SEQUENCE LISTING
<110> Calgene LLC
<120> Methods for Transforming Plastids <130> 15595/00/WO
<150> 60/109,892 <151> 1998-11-25 <160> 73 <170> FastSEQ for Windows Version 4.0 <210>

<211>

<212>
DNA

<213>
Arabidopsis sp <400>

atggcgataattccgttagcacagcttaatgagctaacgatttcttcatcttcttcttcg60 tttcttaccaaatcgatatcttctcattcgttgcatagtagctgcatttgcgcaagttct120 agaatcagtcaattccgtggcggcttctctaaacgaagaagcgattcaacaaggtctaag180 tcgatgcgattgaggtgttccttctctccgatggaatctgcgagaattaaggtgattggt240 gtcggtggtggtggtaacaatgccgttaaccggatgatttcaagcggtttacagagtgtt300 gatttctatgcgataaacacggattcgcaagctctgttacagtcttctgctgagaaccca360 cttcaaattggagaacttttaactcgtgggcttggcactggtggaaacccgcttcttgga420 gaacaagctgcagaagaatcaaaagatgcaattgctaatgctcttaaaggatcagacctt480 gttttcataactgctggtatgggtggtggaacagggtctggtgctgcacctgtggtagct540 cagatttcgaaggatgctggttatttgactgttggtgttgttacctatccgtttagcttt600 gaaggacgtaaaagatctttgcaggcactggaagctattgaaaagctccaaaagaatgtt660 gatacccttatcgtgattccaaatgatcgtctgctagatattgctgatgaacagacgcca720 cttcaggacgcgtttcttcttgcagatgatgttttacgccaaggagtacaaggaatctca780 gatattattactatacctggactagtcaatgtagattttgcagatgtgaaggcagtcatg840 aaagattctggaactgcaatgctcggggtaggtgtttcttccagcaaaaaccgggcagaa900 gaagcagctgaacaagcaactttggctccattgatcggatcatccatacaatcagctact960 ggtgtcgtctacaacatcactggtggaaaagacataactttgcaggaagtgaaccgagta1020 tcacaggtcgtgacaagtttggcagacccatcggccaacatcatatttggagctgttgtg1080 gatgatcgctacaccggagagattcatgtaacgataatcgccacaggcttctctcagtca1140 ttccagaagacacttctgactgatccaagagcagctaaactccttgacaaaatgggatca1200 tcaggtcaacaagagaacaaaggaatgtctctgcctcaccagaagcagtctccatcaact1260 atctctaccaaatcgtcttctccccgtagacttttcttctagttttctttttttcctttt1320 cggtttcaagcatcaaaaatgtaacgatcttcaggctcaaatatcaattacatttgattt1380 tcaaaaaaaaaaaaaaaaggcggccgctctagaggatccaagcttacgtacgcgtgcatg1440 cgacgtcatagctcttctatagtgtcacctaaattcaattcactggccgtcgttt 1495 <210>

<211>

<212>
PRT

<213>
Arabidopsis sp <400> 2 Met Ala Ile Ile Pro Leu Ala Gln Leu Asn Glu Leu Thr I1e Ser Ser Ser Ser Ser Ser Phe Leu Thr Lys Ser Ile Ser Ser His Ser Leu His Ser Ser Cys Ile Cys Ala Ser Ser Arg Ile Ser Gln Phe Arg Gly Gly Phe Ser Lys Arg Arg Ser Asp Ser Thr Arg Ser Lys Ser Met Arg Leu Arg Cys Ser Phe Ser Pro Met Glu Ser Ala Arg Ile Lys Val Ile Gly Val Gly Gly Gly Gly Asn Asn A1a Val Asn Arg Met Ile Ser Ser Gly g5 90 95 Leu Gln Ser Val Asp Phe Tyr Ala Ile Asn Thr Asp Ser Gln Ala Leu Leu Gln Phe Ser Ala Glu Asn Pro Leu Gln Ile Gly Glu Leu Leu Thr Arg G1y Leu Gly Thr Gly Gly Asn Pro Leu Leu Gly Glu Gln Ala Ala Glu Glu Ser Lys Asp Ala Ile Ala Asn Ala Leu Lys Gly Ser Asp Leu Val Phe I1e Thr Ala Gly Met Gly Gly Gly Thr Gly Ser Gly Ala Ala Pro Val Val Ala Gln Ile Ser Lys Asp Ala Gly Tyr Leu Thr Val G1y Va1 Val Thr Tyr Pro Phe Ser Phe Glu Gly Arg Lys Arg Ser Leu Gln Ala Leu Glu Ala Ile Glu Lys Leu Gln Lys Asn Val Asp Thr Leu Ile Val Ile Pro Asn Asp Arg Leu Leu Asp Ile AIa Asp Glu Gln Thr Pro Leu Gln Asp Ala Phe Leu Leu Ala Asp Asp Val Leu Arg Gln Gly Val Gln Gly Ile Ser Asp Ile Ile Thr Ile Pro Gly Leu Val Asn Val Asp Phe Ala Asp Val Lys Ala Val Met Lys Asp Ser Gly Thr Ala Met Leu G1y Val Gly Val Ser Ser Ser Lys Asn Arg Ala Glu Glu Ala Ala Glu Gln Ala Thr Leu Ala Pro Leu Ile Gly Ser Ser Ile Gln Ser Ala Thr Gly Val Va1 Tyr Asn I1e Thr Gly Gly Lys Asp Ile Thr Leu Gln Glu Val Asn Arg Val Ser Gln Val Val Thr Ser Leu Ala Asp Pro Ser Ala Asn Ile Ile Phe Gly Ala Val Va1 Asp Asp Arg Tyr Thr Gly Glu Ile His Val Thr Ile Ile Ala Thr Gly Phe Ser Gln Ser Phe Gln Lys Thr Leu Leu Thr Asp Pro Arg Ala Ala Lys Leu Leu Asp Lys Met Gly Ser Ser Gly Gln Gln G1u Asn Lys Gly Met Ser Leu Pro His Gin Lys Gln Ser Pro Ser Thr Ile 5er Thr Lys Ser Ser Ser Pro Arg Arg Leu Phe Phe WO 00132799 PCT/US99/2$103 <210>

<211>

<212>
DNA

<213>
Arabidopsis sp <400>

tgttgttgccgctcagaaatctgaatcttctccaatcagaaactctccacggcattacca60 aagccaagctcaagatcctttcttgaaccttcacccggaaatatctatgcttagaggtga120 agggactagtacaatagtcaatccaagaaaggaaacgtcttctggacctgttgtcgagga180 ttttgaagagccatctgctccgagtaactacaatgaggcgaggattaaggttattggtgt240 gggaggtggtggatcaaatgctgtgaatcgtatgatagagagtgaaatgtcaggtgtgga300 gttctggattgtcaacactgatatccaggctatgagaatgtctcctgttttgcctgataa360 taggttacaaattggtaaggagttgactaggggtttaggtgctggaggaaatccagaaat420 cggtatgaatgctgctagagagagcaaagaagttattgaagaagctctttatggctcaga480 tatggtctttgtcacagctggaatgggcggtggaactggcactggtgcagcccctgtaat540 tgcaggaattgccaaggcgatgggtatattgacagttggtattgccacaacgcctttctc600 gtttgagggtcgaagaagaactgttcaggctcaagaagggcttgcatctctcagagacaa660 tgttgacactctcatcgtcattccaaatgacaagttgcttacagctgtctctcagtctac720 tccggtaacagaagcatttaatctagctgatgatatactccgtcagggggttcgtgggat780 atctgatatcattacgattcctggtttggtgaatgtggattttgctgatgtgagagctat840 aatggcaaatgcggggtcttcattgatgggaataggaactgcgacaggaaagagtcgggc900 aagagatgctgcgctaaatgcaatccaatcccctttgttagatattgggattgagagagc960 cactggaattgtttggaacattactggcggaagtgacttgacattgtttgaggtaaatgc1020 tgctgcggaagtaatatatgatcttgtcgatccaactgccaatcttatattcggtgctgt1080 tgtagatccagccctcagcggtcaagtaagcataaccctgatagctacgggtttcaaacg1140 acaagaagagggagaaggacgaacagttcagatggtacaagcagatgctgcgtcagttgg1200 agctacaagaagaccctcttcttcctttagagaaagcggttcagtggagatcccagagtt1260 cttgaagaagaaaggcagctctcgttatccccgagtctaaagcccaatctaatcactacc1320 ctgcacactgcagcaataacaaacgtgtgtgtactggtagtctggtactgccttctggga1380 tacagcaagatgtgttgatgtatgatcaagaatctgtgtgggtgtgtatatgttctgtca1440 ctgcctctggtcgtgttcttgaataggttgttttagaaatcggagtttctctctatgtca1500 cttccaaaacaaaaaaggagaagaagaatcacacttctcgaaccataaacatacttataa1560 gattatgagagttttagcagaaattattgtcaaaaaaaaaaaaaaaaaaaa 1611 <210>

<211>

<212>
PRT

<213>
Arabidopsis sp <400>

Met Leu Asn Pro Arg Arg Lys Gly G1u Glu Gly Thr Ser Thr Ile Val Thr Ser Glu Pro Ser Ser Ala Gly Pro Pro Val Val G1u Asp Phe Glu Ser Asn Lys Val Gly Val Tyr Ile Gly Gly Asn Gly Glu Ala Arg Ile Gly Ser Ile Glu Glu Met Asn Ser Ser Gly Ala Val Val Asn Arg Met Glu Phe Ile Gln Met Arg Trp Ala Met Ser Ile Pro Val Asn Thr Asp Val Leu Ile Gly Glu Leu Pro Lys Thr Arg Asp Gly Asn Arg Leu Gln Leu Gly a Gly Ile Gly Asn Ala Al Gly Asn Met Ala Arg Pro G1u Glu Ser Lys Glu Val Ile Glu Glu Ala Leu Tyr Gly Ser 'Asp Met Val Phe Val Thr Ala Gly Met Gly Gly Gly Thr Gly Thr Gly Ala Ala Pro Val Ile Ala Gly Ile Ala Lys Ala Met Gly Ile Leu Thr Val Gly Ile A1a Thr Thr Pro Phe Ser Phe Glu Gly Arg Arg Arg Thr Val Gln Ala Gln Glu Gly Leu A1a Ser Leu Arg Asp Asn Val Asp Thr Leu Ile Val Ile Pro Asn Asp Lys Leu Leu Thr Ala Val Ser Gln Ser Thr Pro Val Thr Glu Ala Phe Asn Leu Ala Asp Asp Ile Leu Arg Gln Gly Val Arg Gly Ile Ser Asp Ile ITe Thr Ile Pro Gly Leu Val Asn Val Asp Phe Ala Asp Val Arg Ala Ile Met Ala Asn Ala Gly Ser Ser Leu Met Gly Ile Gly Thr Aia Thr Gly Lys Ser Arg Ala Arg Asp Ala Ala Leu Asn A1a Ile Gln Ser Pro Leu Leu Asp Ile Gly Ile Glu Arg Ala Thr Gly Ile Val Trp Asn Ile Thr Gly Gly Ser Asp Leu fihr Leu Phe Glu Val Asn Ala Ala Ala Glu Val Ile Tyr Asp Leu Val Asp Pro Thr Ala Asn Leu Ile Phe Gly Ala Val Val Asp Pro Ala Leu Ser Gly Gln Val Ser Ile Thr Leu Ile Ala Thr Gly Phe Lys Arg Gln Glu Glu Gly Glu Gly Arg Thr Val Gln Met Val Gln Ala Asp Ala Ala Ser Val Gly Ala Thr Arg Arg Pro Ser Ser Ser Phe Arg Glu Ser Gly Ser Val Glu Ile Pro Glu Phe Leu Lys Lys Lys Gly Ser Ser Arg Tyr Pro Arg Val <210> 5 <211> 1450 <222> DNA
<213> Brassica sp <400>

atggcgattagtccgttggcacagcttaacgagctaccagtctcttcctcgtttcttgcg 60 acatcccactcgctgcacagtaccagaatcagtggcggcttctcaaaacaaaggtttaag 120 caaacacggttgagatgctccttctctccgatggagtctgcgaggattaaggtggttggt 180 gtcggcggtggtggtaacaatgccgtcaatcgcatgatttccagcggcttacagagtgtt 240 gatttctatgcgataaacacggactctcaagctctcttgcagtcttctgcgcagaaccct 300 cttcaaattggagagctcctaactcgtggccttgggactggtgggaacccgcttctagga 360 gaacaagctgctgaggaatctaaagacgcgattgctaatgctcttaaaggatctgacctt 420 gytttcattactgctggtatgggtggtggcactggctccggtgctgctcctgttgttgct 480 cagatctcgaaagacgctggttatttgaccgttggtgttgttacctatcccttcagcttc 540 gaaggtcgtaaaagatctttgcaggcacttgaagccattgaaaagctgcagaagaacgtg 600 gataccctcatcgtgataccaaatgatcgtctcctagatattgctgatgaacagacgcct 660 WO 00/32799 PCT/(1599/28103 cttcaagacgcttttcttctcgcggatgatgttttgcggcaaggagttcaaggaatctct720 gatattattactatacctggactggtcaatgtagattttgcggatgtgaagtcggttatg780 aaagattccggaactgcgatgctcggggtgggtgtttcttcaagcaagaaccgagcagaa840 gaagcagctgagcaagccactttggctccattgatcggatcatccattcaatcagctact900 ggtgtcgtctacaacatcaccggtggaaaagacattactttgcaggaagtgaaccgagta960 tctcaggtggtgacaagtttggcagacccatcggccaacatcatatttggagctgttgtg1020 gatgatcgatacactggagagattcatgtaacgataatagccacggggttctcacagtct1080 ttccagaagacacttctcagtgatccaagagcagctaaactactcgacaaaacgggatca1140 tcaggtcaacaacaagagaacaaaggcagtcaccagaggcagtctcctgcaactatcaac1200 accaaatcatcttctccccgtagattg.ttcttctagtatcttttgttttttaagcatatt1260 cctttatcaaaaatgtaacgatcttcaggctcaaatatcaattacttttctccagattat1320 ctcaaaagaagtaatttgttaaaccaaaaaaaaaaaaaaagggcggccgctctagaggat1380 ccaagcttacgtacgcgtgcatgcgacgtcatagctcttctatagtgtcacctaaattca1440 attcactggc <210> 6 <211> 411 <212> PRT
<213> Brassica sp <220>
<221> VARIANT
<222> (1)...(411 <223> Xaa = Any Amino Acid <400> 6 Met Ala Ile Ser Pro Leu Ala Gln Leu Asn Glu Leu Pro Val Ser Ser Ser Phe Leu Ala Thr Ser His Ser Leu His Ser Thr Arg Ile Ser Gly Gly Phe Ser Lys G1n Arg Phe Lys Gln Thr Arg Leu Arg Cys Ser Phe Ser Pro Met Glu Ser Ala Arg Ile Lys Val Val Gly Val Gly Gly Gly Gly Asn Asn Ala Val Asn Arg Met Ile Ser Ser Gly Leu Gln Ser Val Asp Phe Tyr Ala I1e Asn Thr Asp Ser Gln Ala Leu Leu Gln Ser Ser Ala Gln Asn Pro Leu Gln Ile Gly Glu Leu Leu Thr Arg Gly Leu Gly Thr Gly Gly Asn Pro Leu Leu G1y Glu Gln Ala Ala Glu Glu Ser Lys Asp Ala Ile Ala Asn Ala Leu Lys Gly Ser Asp Leu Xaa Phe Ile Thr Ala Gly Met G1y Gly Gly Thr Gly Ser Gly Ala Ala Pro Val Val Ala Gln Ile Ser Lys Asp Ala Gly Tyr Leu Thr Val Gly Val Val Thr Tyr Pro Phe Ser Phe Glu Gly Arg Lys Arg Ser Leu Gln Ala Leu Glu Ala Ile Glu Lys Leu Gln Lys Asn Val Asp Thr Leu Ile Val Ile Pro Asn Asp Arg Leu Leu Asp Ile Ala Asp Glu Gln Thr Pro Leu Gln Asp Ala Phe Leu Leu Ala Asp Asp Val Leu Arg Gln Gly Val Gln Gly Ile Ser WO 00/32799 PCTIUS99I2$i03 Asp Ile Thr 21e Pro Gly Leu Val Asp Phe Asp Val Ile Asn Val Ala Lys Ser Met Lys Asp Ser Gly Thr Leu Gly Gly Val Val Ala Met Val Ser Ser Lys Asn Arg Ala Glu Glu Glu Gln Thr Leu Ser Ala Ala Ala Ala Pro Ile Gly Ser Ser Ile Gln Thr Gly Val Tyr Leu Ser Ala Val Asn Ile Gly Gly Lys Asp I1e Thr Glu Val Arg Val Thr Leu Gln Asn Ser Gln Val Thr Ser Leu Ala Asp Ala Asn Ile Phe Val Pro Ser I7.e Gly Ala Val Asp Asp Arg Tyr Thr Ile His Thr Ile Val Gly Glu Val Ile Ala Gly Phe Ser Gln Ser Phe Thr Leu Ser Asp Thr Gln Lys Leu Pro Arg Ala Lys Leu Leu Asp Lys Ser Ser Gln Gln Ala Thr Gly Gly Gln Glu Lys Gly Ser His Gln Arg Pro Ala Ile Asn Asn Gln Ser Thr Thr Lys Ser Ser Pro Arg Arg Leu Ser Phe Phe <210> _ <211>

<212>
DNA

<213> iana sp Nicot <400>

tgccgttaaccggatgattt caagcggttt acagggtgttgacttctatgctataaacac 60 ggatgctcaagcactgctac agtctgctgc tgaaaacccgcttcaaattggagaacttct 120 gactcgtgggcttggtactg gtggtaatcc tcttttaggggaacaggcagtggaggagtc 180 gaaggaagccattgcaaatt ctctaaaagg ttcagatatggtgttcataacagcaggaat 240 gggtggaggtacaggatctg gtgctgctcc tgttgtggctcaaatagcaaaagaagcagg 300 ctatttgactgttggtgttg tcacataccc attcagctttgaaggacgtaaaagatccgt 360 gcaggctctggaagcaattg aaaaacttca gaaaaatgtagatacccttatagtaattcc 420 aatgaccgtctgctagatat tgctgatgag cagacaccacttcaagatgcttttcttctt 480 gctgatgatgtattacgcca aggtgtccaa ggaatttccgatataattactatacctggg 540 cttgtaaatgtggattttgc cgatgtaaag gtagtgatgaaagattctggaactgctatg 600 cttggagttggggtttcatc aagcaagaac cgtgctgaagaagcagccgaacaagcaact 660 cttgcccctcttaattggat cgtccattca atcgccactggggtagtatccaccattcca 720 ggaggaaaagaccataactt tgcagaaagt gaatagggtgtctcaggttgttacagtctg 780 gctgatccctcccgctaaca tcatatttgg tgctgttgtggatgagcgctacaatggcga 840 aatacacgtgaccataattg caactggttt tacccagtcttttcagaagactcttctctc 900 tgacccacgaggtgcaaagc ttgttgataa aggcccagtaatccaagaaagcatggcatc 960 acctgttaccctgaggtcat caacctcacc ttcgacaacatcacgaacacctactcggag 1020 gctgttcttttagctccttt atatagtttg ttacggcttcatttttctcttttcttactt 1080 ttttcttttttactttcttt gtatttacat,gttttgctgattggtgtttgcatttggctg 1140 tagacatagtgatgattctt atcaagtgca tcacattcatactcgaaaaaaaaaaaaaaa 1200 aaaaaaagtactctgcgttg ttacccactg ttaagggcgaattctgcagatatcccatca 1260 cactggcggccgctcgagca tgcatctaga gggcc 2295 <210>

<211>

WO 00/32799 PCT/US99/2$103 <212> DNA

<213> Nicotiana sp <400> 8 atggccacca tctcaaaccc agcagagatagcagcttcttctccttcctttgctttttac60 cactcttcct ttattcctaa acaatgctgcttcaccaaagctcgccggaaaagcttatgt120 aaacctcaac gtttcagcat ttcaagttcatttactccttttgattctgctaagattaag180 gttatcggcg tcggtggcgg tggtaacaatgccgttaaccggatgatttcaagcggttta240 cagggtgttg acttctatgc tataaacacggatgctcaagcactgctgcagtctgctgct300 gaaaacccgc ttcaaattgg agaacttctgactcgtgggcttggtactggtggtaatcct360 cttttagggg aacaggcagc ggaggagtcgaaggaagccattgcaaattctctaaaaggt420 tcagatatgg tgttcataac agcaggaatgggtggaggtacaggatctggtgctgctcct480 gttgtggctc aaatagcaaa agaagcaggctatttgactgttggtgttgtcacataccca540 ttcagctttg aaggacgtaa aagatccgtgcaggctctggaagcaattgaaaaacttcag600 aaaaatgtag atacccttat agtaattcccaatgaccgtctgctagatattgctgatgag660 cagacaccac ttcaagatgc ttttcttcttgctgatgatgtattacgccaaggtgtccaa720 ggaatttccg atataattac tatacctgggcttgtaaatgtggattttgccgatgtaaag780 gtagtgatga aagattctgg aactgctatgcttggagttggggtttcatcaagcaagaac840 cgtgctgaag aagcagccga acaagcaactcttgcccctcttattggatcgtccattcaa900 tcagccactg gggtagtatc caccattccaggaggaaaagacataactttgcagaaagtg960 aatagggtgt ctcaggttgt tacagtctggctgatccctcccgctaacatcatatttggt1020 gctgttgtgg atgagcgcta caatggcgaaatacacgtgaccataattgcaactggtttt1080 acccagtctt ttcagaagac tcttctctctgacccacgaggtgcaaagcttgttgataaa1140 ggcccagtaa tccaagaaag catggcatcacctgttaccctgaggtcatcaacctcacct1200 tcgacaacat cacgaacacc tactcggaggctgttcttttagctcctttatatag 1255 <210> 9 <211> 413 <212> PRT

<213> Nicotiana sp <400> 9 Met Ala Thr Ile Ser Asn Glu Ile Ala Ser Pro Ser Pro Ala Ala Ser Phe Ala Phe Tyr His Ser Ile Pro Gln Cys Phe Thr Ser Phe Lys Cys Lys Ala Arg Arg Lys Ser Lys Pro Arg Phe Ile Sex Leu Cys Gln Ser Ser Ser Phe Thr Pro Phe Lys Val Gly Val Asp Ser Ala Lys Ile Ile Gly Gly Gly Gly Asn Asn Ile Ser G1y Leu Ala Va1 Asn Arg Met Ser Gln Gly Val Asp Phe Tyr Ala Gln Leu Leu Ala Ile Asn Thr Asp Ala Gln Ser Ala Ala Glu Asn Gln Ile Glu Leu Thr Arg Pro Leu Gly Leu Gly Leu Gly Thr Gly Gly Leu Leu Glu Gln Ala Glu Asn Pro Gly Ala Glu Sex Lys Glu Ala Ile Ser Leu Gly Sex Met Val Ala Asn Lys Asp Phe Tle Thr Ala Gly Met Gly Thr Ser Gly Ala Pro Gly Gly Gly Ala Val Va1 Ala Gln Ile Ala Ala Gly Leu Thr Gly Val Lys Glu Tyr Val Val Thr Tyr Pro Phe Ser Gly Arg Arg Ser Gln Ala Phe Glu Lys Val _ 7 _ WO 00/32799 ~ PCT/US99/28103 Leu Glu Ile Glu Lys Leu Gln Lys Asp Thr Ile Val Ala Asn Val Leu Ile Pro Asp Arg Leu Leu Asp Ile Glu Gln Pro Leu Asn Ala Asp Thr Gln Asp Phe Leu Leu Ala Asp Asp Arg Gln Val Gln Ala Val Leu Gly Gly Ile Asp Ile Ile Thr Ile Pro Val Asn Asp Phe Ser Gly Leu Val A1a Asp Lys Val Val Met Lys Asp Thr Ala Leu Gly Val Ser Gly Met Val Gly Ser Ser Sex Lys Asn Arg Glu Ala Glu Gln Val Ala G1u Ala Ala Thr Ala Pro Leu Ile Gly Ser Gln Ser Thr Gly Leu Ser Ile Ala Val Val Thr Ile Pro Gly Gly Lys Thr Leu Lys Val Sex Asp Ile Gln Asn Arg Ser Gln Val Va1 Thr Val Ile Pro Ala Asn Val Trp Leu Pro Ile Ile Gly Ala Val Val Asp Glu Asn Gly Ile His Phe Arg Tyr Glu Val Thr Ile A1a Thr Gly Phe Thr Phe Gln Thr Leu Ile Gln Ser Lys Leu Ser Pro Arg Gly Ala Lys Leu Lys Gly Val Ile Asp Val Asp Pro Gln Glu Met Ala Ser Pro Val Thr Ser Ser Ser Pro Ser Leu Arg Thr Ser Thr Ser Arg Thr Pro Thr Arg Phe Phe Thr Arg Leu <210> 10 <211> 12?8 <212> DNA

<213> Zea mays <220>

<221> misc_feature <222> (1)...(1278) <223> n ,T,C or G
= A

<400> 10 gatcttgtcttcataacagc tgggatggga gggggtactggatctggtgctgctccagtt60 gttgcccagatatcaaagga agctggttat cttactgttggtgttgtcacctatccattc120 agtttcgagggccgtaagcg ctctgtacag gcattggaagcactagagaagctggaaaag180 agtgtagacacacttattgt gattccaaat gataagttattagatgttgcggatgaaaac240 atgcccttgcaagatgcatt tctccttgca gatgatgtccttcgtcagggtgttcaagga300 atatcagacatcatcacaat accgggactt gtcaatgttgattttgctgatgtaaaagct360 gtcatgaaaaactctggaac tgccatgctc ggtgttggtgtttcttccagcaaaaatcgg420 gcccaagaagctgctgaaca ggcaacactt gctcctttgattggatcatccatcgaggca480 gctactggcgttgtgtataa tattactggt gggaaggacatcactttgcaagaagtgaac540 aaggtgtcccagattgtgac aagcctagct gacccatctgcgaacataattttcggtgct600 gtcgttgatgaccgttacac tggtgagata catgtgacaatcattgcgacaggatttcca660 cagtccttccagaaatccct tttggcggat ccaaagggagcacgtatagtggaatccaaa720 gagaaagcagcaaccctcgc ccataaagca gcagcagctgcagttcaaccggtccctgct780 tctgcttggtctcgaagact cttctcctga gaagctcatttggtgaaccgtgactcgtag840 _ g _ tgcattagatttgcatttagcgtgttgagggcagtccctaaggtgatcttcggatatctg 900 gagatttatagcttgggctagtgttcggtagtggtagaataagtttcagtgtatgtatcg 960 ttgctttgctttatgtttttgaggatcaggcggtgaggctgagagaagtgctcagcaact 1020 caacattgaactgttgtagaagatctttgattgcttttattgctgcaacatgccaacaac 1080 cctctgttggattcamcmnaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1140 aaaaaaaaaaaaaaaaaaaaaaaanncaaaaaaaaaaaaaaaaaaaaagggcggccgccg 1200 actagtgagctcgtcgacccgggaattaattccggaccggtacctgcaggcgtaccagct 1260 ttccctatagtgagtcgt 1278 <210>

<211>

<212>
DNA

<213>
Zea mays <400>

gctccagttgttgcccagatatcaaaggaagctggttatcttactgttggtgttgtcacc 60 tatccattcagtttcgagggccgtaagcgctctgtacaggcattggaagcactagagaag 120 ctggaaaagagtgtagacacacttattgtgattccaaatgataagttattagatgttgcg 180 gatgaaaacatgcccttgcaagatgcatttctccttgcagatgatgtccttcgtcagggt 240 gttcaaggaatatcagacatcatcacaataccgggacttgtca 283 <210>

<211>

<212>
DNA

<213>
Zea mays <220>

<221> feature misc _ <222>
(1).
.(287) <223>
n = A,T,C
or G

<400>

gggccgtaagcgctctgtacaggcattggaagcactagagaagctggaaaagagtgtaga 60 cacacttattgtgattccanatnatnngttattagatgttgcggatgaaaacatgccctt 120 gcaagatgcatttctccttgcagatgatgtccttcgtcagggtgttcaaggaatatcaga 180 catcatcacaataccgggacttgtcaatgttgattttgctgatgtaaaagctgtcatgaa 240 aaactctggaactgccatgctcggtgttggtgtttcttccagcaaaa 287 <210>

<211>

<212>
DNA

<213>
Zea mays <400>

gctataaacaccgattcccaagcccttattaattcacaagcgcaatatcctctgcaaatt 60 ggagagcagttgacccgcggcttaggtgccggtggaaatccgaatttgggagagcaggct 120 gctgaggaatcaagagaaaccatagccactgccctgagggattcagatcttgtcttcata 180 acagctgggatgggagggggtactggatctggtgctgctccagttgttgcccagatatca 240 aaggaagctggttatcttactgttggtgttgtcacctatccattcagtttcgagggccgt 300 aagcgctctgtacaggcaaagtatctgagccccccttcactcctgaattttaattcaaac 360 tgtcatatctcgttctgcgactttcttttgctcgatggaagcattagtttgtagtcataa 420 caatgacatccagccacatttattgctgatgatgtatacaatggtaggtcaaagaaatgt 480 agcatcatgccatcacctgtagttcatctcatcattttgttcctacttttctgcgtggtt 540 gatgcccaaaacaatatacaactatgtggttgtactgttgcattgccttgtggagggatg 600 tttatgttgtgaaatatttcaaaacacatgtcattatgaatattccctcctgtggttgtg 660 _ g _ gggacttgtttcaaatgctatgaattaagaacaaggcaacataaagtgttaaatgttaac720 cgtctttcgtccatgaaacattattcccttgaggataatgggccttggacaaaggctgat780 gagagtataattaccaagcttaaatcttcgtaataaaatttcaatagatattgtaagata840 acataaaataaagggtataaaaaggggtaaataaatcatagacgaattatattatattta900 cttaatatattgaatcattgaatacaataatacctctgccttggcaaaggttggattccg960 aaaaatgtgattgcaagttaccagaatgcgtgaacagtaaaggaatactgttcactattt1020 ataggcacaggacacagcctgtggaggaattcaattatacccgtcataagagtttacaca1080 ttgacttagacctttatggactaaaagatcattgctatcttt 1122 <210>

<211>

<212>
DNA

<213> mays Zea <220>

<221> feature misc <222> _ (1). .(291) <223> A,T,C or n = G

<400>

aaaatagtgtggacaccctaatcgtcatcccaaatgataagttgctgtctgctgtttctc60 caaatacacctgtaactgaagcatttaatctggctgatgatattcttcgtcaaggcattc120 gtggcatatctgatataattacggttcctgggnaggttaatgttgattttgctgacgtac180 gtgctatcatgcaaaatgcagggtcatccttgatgggtatagggactgctacaggaaagt240 caagagcaagggatgctgctcttaacgccatccagtcgccgctgcttgata 291 <210>

<211>

<212>
DNA

<213> mat's Zea <220>

<221> feature misc <222> _ (1). .(415) <223> A,T,C or n = G

<400>

gagcaagggatgctgctcttaacgccatccagtcgccgctgcttgatattggaattgaaa60 gagccacaggcattgtgtggaatatcactgggggaactgacctgactttgtttgaggtga120 atgctgcggccgaaattatctacgaccttgtcgatccaaacgctaatctgatatttggcg180 ccgtcatagacccgtcactgagtgggcaggtgagcataaccttgatagctactggcttca240 .

aacggcaggatgaaccagaaggccgcgtgtcgaagggtgggcaacaaggtgagaatggcc300 gacgcccatccccagcanagggcaacaacacggtggaaattccaaaattcccgccaacaa360 aaagggcccttccnncttcccacnattttgactggtcctgtctgcacctgtatga 415 <210>

<211>

<212>
DNA

<213> mat's Zea <220>

<221> feature misc <222> _ (1). .(744) <223> A,T,C or n = G

<400>

aattcccgggtcgacccacgcgtcccgcggacgcgtgggtggaatatcactggagggaac 60 gatctaaccttgacagaggtgaatgctgcagctgaagtaatctatgatcttgttgaccct 120 ggtgcaaatctgatttttggctctgttatagatccgtcatacactggtcaagtgagcata 180 actctaattgcaactggtttcaaacgccaggaggaaagtgagagccggtcttcacaggct 240 ggaggagacaagcaaccgcggtcgctcggctggttttctcccacttcccaggaggaaggt 300 catgcattgcaaatcccanagttcctacagaggaaagggcgtccagggtttcacgagtct 360 gaacacactttggatcaatgtttttcttgtcatagtttggtacgatgcaggtttggtttc 420 tgggtctcttaggtagcaaggtagaacagatgttcctgaacccgcacatactaatctgtg 480 tgcaaacttcngccgctgagtaccattggcttgggctgctttgcttctcangaacctgca 540 gtgaggtctcaatttgctagttagtatgattaaaagtnaagcgctgagaccaaattatac 600 gttccgtgtgaatgattacttgctcnctgccattttcttttcaaaaaaaaaaaaaaaaaa 660 aaaaaggcggcgctntanaggatccaagcttacttcccctgcatncgacncanagctntt 720 ntatagngtnacctaaattcaatc 744 <210>

<211>

<212>
DNA

<213>
Zea mays <220>

<221> feature misc -<222>
(lj..
.(230) <223>
n = A,T,C
or G

<400>

ggctgctgaggaatcaagagaaaccatagccactgccctgagggattcagatcttgtttt 60 cataacagctgggatgnnagggggtgctgctccaattgttgcccagatatcaaaggaagc 120 tggttatcttactgttggtgttgtcacctatccattcaatttcgagggccgtaagcgctc 180 tttacaggcaagtatctgagccccccttcactcctgaattagaattcaaa 230 <210>

<211>

<212>
DNA

<213>
Zea mays <220>

<221> feature misc <222> _ (1j. .(318) <223>
n = A,T,C
or G

<400>

caggcattgtgtggaatatcactgggggaactgacctaactttgtttgaggtgaatgctg 60 cggccgaaattatctacgaccttgtcgatccaaatgctaatctgatatttggtgccgtca 220 tagacccgtcactgagtgggcaggtgagcataacctgatagctactggcttcaaacggca 180 ggatgaaccagaaggccgcgtgtcgaagggtgggcaacaaagtgagaatggccgacgccc 240 gtcccccgcagagggcagcagcacggtggagttccagagtcctgcgacgtagagganctt 300 ctcgcttcccagagttga 318 <210>

<211>

<212>
DNA

<213>
Zea mays <220>

<221> misc feature <222> {1)...(472) <223> n = A,T,C or G
<400> 19 cgacgcccaaggtgacgaatgctgtcagccacgctgtgctacacgggggaaacaatgcaa60 anacattacctgcctcactcntgcttgctcctgtaaatataatgatngtcgctgctacat120 natatttactcctgctgctgcttgaggccattattctgtacgtaaatgaagccactacta180 ctctcacacagcatgcgccggccgacgacgtacgtacgtgtattatatacgctctacccc240 gtgagcttttgttcgagtgatacgtgatccatccatgcatggatgcttatgtatgtatat300 gtgttagtcgtctcagggaaccgggcancanaagggggtgttgtattanatttacgtctt360 ctggtgattaaataanaaaggggtatgttggatgtgtgcaaaaaaaaaaaaaaaanaaaa420 aaaaaaaaaaaaaaaaaaagggcggccgccgactagtgagctcgtcgaccc 471 <210>

<211>

<212>
DNA

<213>
Glycine sp <220>

<221> feature misc _ <222>
(1).
.{1085) <223>
n =
A,T,C
or G

<400>

cggctcgaggaaccctattaaaattggagaagttctgactcgtggattaggtacgggcgg60 gaatccacttttgggggaacaagctgcagaggaatcaagagatgctattgctgatgctct120 taaaggatcagatttggtgtttataacggctgggatgggtgggggaaccgggtctggtgc180 tgccccagttgtagcccaaatatcaaaagaggcaggttacttgactgtaggtgttgttac240 ctatcccttcagttttgaaggacgtaagagatccttgcaggcctttgaagccatcgaaag300 gctgcagaaaaatgttgacacmmttatagtgawtccmaatgmccgtctgcttgacawagy360 tratragcaratgcctcttcaaggatgctttccgytttgcagatgacgttytmsggcaag420 gagtmcagggaatatcagacattatamctgtacctggacttkkcaaatgtggattttgca480 agatgtaaaagctgtgatgaaagactctgggactgcaatgcttggagtaggtgtttccty540 cggtaaaaaaccgagcagaagaagcagccgaacaggctactttggctcctttaattggat600 cctctattcagtcaagctactggggtagtgtataatattactggagggaaaggacataac660 cctgcaggaagtgracagggtttytmaggtkgkgacyarkttggctgatccttctgctaa720 .

tattatatttggggctgtcgttgatgatcgctacacgggggagattcacgtgactatcat780 tgcaactggcttctcacagtcttttcagaagaagttgctaacagatccaagggcttgcaa840 agctgcttgacaaggtggctgagggccaagaaagcaaggcagtccctcctcccctcaagt900 cctcaaacaaggttgaatctagaccatccccgcgaaagctctttttttagttgcatggtt960 .

ctttttaccctttttcatttttccaattattattattatattatatnggccgatcaaaaa1020 aaaaaaaaaaggcggccgccgactagtgagctcgtcgacccgggaattaattccggaccg1080 gtacc <210>

<211>

<212>
DNA

<213>
G3.ycine sp <400>

ccagctggcgaaaggggatgtgctgcaaggcgattaagttgggtacgcagggttttccca60 gtcacgacgttgtaaaacgacggcagtgaattgaatttaggtgacactatagaagagcta120 tgacgtcgcatgcacgcgtacgtaagctcggaattcggctcgagaggctactttggctcc180 tttaattggatcctctattcagtcagctactggggtagtgtataatattactggaggaaa240 ggacataaccctgcaggaagtgaacagggtttctcaggttgtgactagtttggctgatcc 300 ttctgctaatattatatttggggctgtcgttgatgatcgctacactggggagattcacgt 360 gactatcattgcaactggcttctcacagtcttttcagaagaagttgctaacagatccaag 420 ggctgcaaagctgcttgacaaggtggctgagggccaagaaagcaaggtagtccctcctcc 480 cctcaagtcctcaaacaaggttgaatctagaccatccccgcgaaagctcttttttttagt 540 tgcatggttctttttaccctttttcatttttccaattattattattatattatattggcc 600 gatcaaaaaaaaaattattatattatattgtaggacacaatgatcttgatgcttaattaa 660 gtgagatatcattctcttgatgttaaaaaaaaaaaaaaagggcggccgccgactagtgag 720 ctcgtcgacccgggaattaattccggaccggtacctgcaggcgtaccagctttccctata 780 gtgagtcgtattagagg 797 <210>

<211>

<212>
DNA

<213>
Glycine sp <220>

<221> feature misc _ .(714) <222>
(1).

<223>
n = A,T,C.or G

<400>

aattcggctcgagacggctgcgagaagacgacagaagggggttaccgttatcatgcaagc 60 tgataatggggcctctgaagttcttgttccgttattataaaactgagtccttcactctct 120 ctcgaaccagctcacagaaacaatgatctcctacgccgacatgctcaagggatcacatgg 180 atgtcaacaacttcaactatcctccattgtcagagatgtaaactacagctgtggctcgtg 240 tggttatgagctgaacttgaactccagcaaccgcaacacttgttctctcattgactcaaa 300 gtccataaagagaggcatcatctccttcttctccgtggatgagagcaggttcactcagat 360 ccagcaacttcactggccttcttggatgccctttttcaactccaagcgccaaagaaccaa 420 gcttttttgccgcagctgtgggaaccaccttggctatgcttacactttgcctctcaatct 480 caatcccgggatggcatctctgatgattcagaatctatgatatcaaactaaccgctttgt 540 taccttctttctgcgaggaaccaagtcaaaagttagangatatgggcaaggtttgagact 600 gcatcttcctccactcttggtggtctaattcttgaaagggacagaaacatattcatcagt 660 tcttggttggttggaatgngaattaatgnattctaccttttgacattatgaagg 714 <210>

<211>

<222>
DNA

<213>
Glycine sp <400>

cgggctcgagattactggaggaaaggacataaccctgcaggaagtgaacagggtttctca 60 ggttgtgactagtttggctgatccttctgctaatattatatttggggctgtcgttgatga 120 tcgctacactggggagattcacgtgactatcattgcaactggcttctcacagtcttttca 180 gaagaagttgctaacagatccaagggctgcaaagctgcttgacaaggtggctgagggcca 240 agaaagcaaggtagtccctcctcccctcaagtcctcaaacaaggttgaatctagaccatc 300 cccgcgaaagctctttttttagttgcatggttctttttaccctttttcatttttccaatt 360 attattattatattatattggccgatcaaaaaaaaaattattatattatattgtaggaca 420 caatgatcttgatgcttaattaagtgagatatcattctcttgatgttctttcccctccaa 480 aaaaaaaaaaaaagggcggccgccgactagtgagctcgtcgaccc 525 <210>

<211>

<212>
DNA

<213>
Glycine sp <400>

cggctcgaggaaccctattaaaattggagaagttctgactcgtggattaggtacgggcgg 60 gaatccacttttgggggaacaagctgcagaggaatcaagagatgctattgctgatgctct 120 taaaggatcagatttggtgtttataacggctgggatgggtgggggaaccgggtctggtgc 180 tgccccagttgtagcccaaatatcaaaagaggcaggttacttgactgtaggtgttgttac 240 ctatcccttcagttttgaaggacgtaagagatccttgcaggcctttgaagccatcgaaag 300 gctgcagaaaaatgttgacacacttatagtgattccaaatgaccgtctgcttgacatagc 360 tgatgagcagatgcctcttcaggatgcttttccgtcttgcagatgacgttctacggcaag 420 gagtacagggaatatcagacattatamctgwcctggacttgtcaatgtggatttttgcag 480 atgtaaaagctgtgatgaaagactctgggactgcaatgcttggagtaggtgtttcctccg 540 gtaaaaaccgagcagaagaagcagccsaacaggctactttggctyctttaattggatcct 600 ctatttcagtcagctactgggggtagtgtataatattactggaggaaaggacataaccct 660 scaggaagtgaacagggkttctcaggttgtgactaagtttggctgatccttctgctaata 720 ttatatttggggctgtcgttgatgatcgctacacgggggagattcacgtgactatcattg 780 caactggcttctcacagtcttttcagaagaagttgctaacagatccaagggctgcaaagc 840 tgcttgacaaggtggctgagggccaagaaagcaaggcagtccctcctcccctcaagtcct 900 caaacaaggttgaatctagaccatccccgcgaaagctctttttttagttgcatggttctt 960 tttaccctttttcatttttccaattattattattatattatattggccgatcaaaaaaaa 1020 aaaaaaagggcggccgccgactagtgagctcgtcgacccgggaattaattccggaccggt 1080 acc 1083 <210>

<211>

<212>
DNA

<213>
Glycine sp <220>

<221> feature misc _ <222>
(1?.
.(2335) <223>
n = A,T,C
or G

<400>

cggctcgaggcccagaacaacaaaaattgctcctcaacgcctaagtcgtcgtttcggttc 60 ggtgagatgctcctacgcttacgtagataacgccaaaattaaggttgtcggcatcggcgg 120 tggcggcaacaatgccgttaatcgcatgatcggaagtggtttgcagggtgtagacttcta 180 tgcgataaataccgatgctcaggcactattaaattctgctgctgagaaccctattaaaat 240 tggagaagttctgactcgtggattaggtacaggtgggaatccacttttgggggaacaagc 300 tgcggaggaatccagagatgctattgctgatgctcttaaaggatcagatttggtatttat 360 aacggctgggatgggtgggggaaccgggtcttggtgctgccccagttgtagnccaaatat 420 caaaagaggcaggntactttgactgtaggtgttggtacctatcccttcagttttgaagga 480 cgtaagagatgcttgcaggcctttgaagccatcgaaaggctgcagaaaaatgttgcacac 540 ttatagttattccaaatgatcgtctgcttgacatancttgatgaaccagatgcctattca 600 aggatgctttycgytytkcarawkatgttytamcgsaargsgkacagggaatatcaagac 660 attwtaacaggtacctggacttgtmaatgtagattttgctgatgtaaaamctgkgataaa 720 gacttctgggactgcaatgcttggtgtaggtgtttcatccggtaaaaccgaccagaagaa 780 gcagcagaacagggctactttggctcctttaattggatcatctattcagtcagctactgg 840 ggtagtgtataatattactggaggaaaggacataaccctgcaggaagtgaacagggtttc 900 tcaggtggtgactagtttggctgatccttctgctaatattatatttggagcttgttgttg 960 atgatcgcttacactggggagattcacgtgactataattgcaactggcttctcacagtct 1020 tttcagaagaagttgctaacagatccaagggctgcaaagctgcttgacaaagtggctgag 1080 ggccaagaaagcaaggcagtccctcctccccccaagtcctcaatcaaggttgaatctaga 1140 ccatccccgcgaaagctctttttgtagttgcatggttcttttacccttttcttttttcca 1200 attattatattgtaagtcattctgtagtacaatgatcttgatgcttaatttagtgagata 1260 tcattctcttgatgttaaaaaaaaaaaaaaaaaaaaaaaaaaagggcggccgccgactag 1320 tgagctcgtc gaccc 1335 <210>

<211>

<212>
DNA

<213>
Glycine sp <220>

<221> feature misc _ <222>
(1).
.(902) <223>
n = A,T,C
or G

<400>

aattcggctcgagtaccagggttggtgaatgtagattttgctgatgttcgggctataatg 60 gccaatgcaggttcttcactaatggggataggaactgcaactggaaaatcaagggcaaga 120 gatgctgcattaaatgccatccagtcacctttactggatattggtataragagggctact 180 kgaattgtttggaacawaactggtgggactgatctgrccttgtttgaggtaaacacggca 240 gcagaggttatttatgacctcgtggaccctactgctaatttaatatttggagcagtaata 300 gatccatcactcagtggtcaagtgagcataacattaattgcttactgrattcaaagcgyc 360 aagaggagagtgaagggaggcctctgcaggccagtcaactcactcaagcagacacaacct 420 tcggcaccaattggcggtcttcctctttcactgatggtggtttgtttgagataccagaat 480 tcctaaagaagaraggaggttcacgctatccgagggcgtaatctttttcatcctaatttc 540 ttttgatcccttgcatttcttcacccttggatatacatagcaattggtctagttcttarg 600 tccctgtcttgscctttttcggatttwrkcaaragttgkgkatacagttkgttcatgaaa 660 gtttattacttyccactgkccagacttatgggkctaaaccgganggtattksarcatgga 720 tgcttttcttggcatatttgaattagtttattagcttgtacagagatttcagtaatgctg 780 agagcttgttatagttctttggcatgttatagaaaattcattattattaaaaaaaaaaaa 840 aaaggcggccgccgactagtgagctcgtcgacccgggaattnattccggaccggtacctg 900 ca 902 <210>

<211>

<212>
DNA

<213>
Glycine sp <220>

<221> feature misc _ <222>
(1).
.(856) <223>
n = A,T,C
or G

<400>

aattcggctcgagattggtgaaccgtagactttgctgatgttcgagctataatggccaat 60 gcagggtcttcacttatggggataggaactgcaactgggaaaacaagggcmarggawgct 120 gcattaaatgctatccagtcmccctttactggatatttggtataraaagggctactggaa 180 ttgtatggaacataacyggkggaagtgatttgaccttgtttgaaggtaaatgttgcasca 240 raagttatatatgmccttgtggmccccactgstaatttaatatttgggscagwaatagat 300 ccatcactccagtgggcaagtaagcatammwtaatcgcaactggattcaagcgtcaagag 360 gaaaagtgaagggagaccctatgcaggccagtcaactcacacaaggagatnccgttggta 420 tcaatcggcgatyttctactttcactgatggtagcttttgttggagatccctggaattct 480 taaagaagaaggggcgctcacgttatccaagagtttaatactcttttccccaactcctta 540 atccctccttgcatctctttmccaascaatttttagggatacaaatctcatcagtctaag 600 gtattagatcacggtttttgccccttttttcatttttaggttcgcattgtgcantamagt 660 tgttcattgaaagcgaagttactttccaaaaccgttgttttctgarttgaaggcttggtt 720 ggcatgttttwataagtttattagcttgtatttttgtncagagaataatatatcagtaat 780 ggtcagtgcttgttataaanccncnaaaaaaaaaaaaaaaaaaagggcggccgccgacta 840 gtgagctcgt cgaccc 856 <210>

<221>

<212>
DNA

<213>
Glycine sp <220>

<221> feature misc _ .(1060) <222>
(1).

<223>
n = A,T,C
or G

<400>

aattcggctcgaggtcacaacccctttttcatttgaagggcgaagaagggcagttcaagc60 acaagaaggaattgctgcattaagagataatgttgatacactgatagttattccaaatga120 caaactgctgactgcagtttctcaatctacccctgtaactgaagcattcaatctggctga180 tgatattcttagacaaggtgttcgtggtatatctgatattattacgataccaggattggt240 gaatgtagactttgcagatgttcgagctataatggccaatgcaggttcttcacttatggg300 gataggaactgcaactggcaaaacaagggcaagagacgctgcattaaatgctatccagtc360 acctttactagatattggtatagaaagggctaccggaattgtatggaacataactggtgg420 aaagwgatttgaccttgtttgaggtaaatgctgcagcagaagttatatatgaccttgtgg480 accccactgytaatttaatatttggagcagtaatagatccatcactcagtggtcaagtaa540 gcatcacattaattgcaactggattcaagccgtcaagaggaaaagtgaagggagacctat600 acaggccarccaatttacacaaggagatacggttggtatcaaccsgcgatcttcctcttt660 cactgatggkagctttgttgagayccctgaattcttaaagaaraaggggcgctcaygtta720 tccgagagcttaatactcttctccccaatttcttaatcccttgatttctttacaaagtaa780 tttttagggatacaaatctcatcagtctaggtattagatcccgttttgcccctttttttt840 ttcatttttaggttcgcattgggcatactgttgttcaagaaagcaaagtactttcaaaac900 cgttgtttactgagtcgaggcttgttggcaggttttaataagtttattagcttgtatttt960 ttgtacagagaatatatcagtaatggtcagggcttgttatnnnanncccnnnnannaaan1020 aaaaaaaaaggcggccgccgactagtgagctcgtcgaccc 1060 <210>

<211>

<212>
DNA

<223>
Glycine sp <220>

<221> feature misc -<222>
(1).
..(727) <223>
n = A,T,C
or G

<400>

atctcncaaaatgcatgncnctgtgtgtggcatatattcaaaatgacttggcccagggtg60 gggttttantttgctcttagaaaatgtgttgagcctgcacatanaagattggagttgttg120 attctcagtggattgttcaccaaggtattccctcactagggaatcagggtgantctcaaa180 caggaaagcnccatggcaggggntgagggancggtgtanaaaggagtggccatgttccag240 agtcggtggcaaatgctgaatacgcgtatcacaactccattggaattgatacatctaatt300 ccactgctcattaggtgacttcggcctaagttgacttgtaaacatattgttactaccctt360 agccttacgcgtagaattttcccttaaaaaaaaaaatatattcctatgtaacgttacgta420 catgcaatgcaatcacaatatagagtcctagctagggaccaaacatcatttcgatgtaga480 aattgctgtacttaacagtgagtaaatctagtgaagagaattattattgctgctaacgaa540 ggtgcttataggaaatggaaatgctagtgaatccttaaattggaggctgacaacgaagtt600 ctttagggtttttgggattaaagaaaacgaaatgtcataattatcatacccttgggatga660 ggagacaggactattactataaaaaaaaaaaaaaagggcggccgccgactagtgagctcg720 tcgaccc 727 <210>

<211>

<212>
DNA

<213>
Glycine sp <220>

<221> feature misc _ <222>
(1).
.(1185) <223>
n = A,T,C
or G

<400>

cggctcgagctggaatgggtgggggaactggcacaggtggagctccaattattgctagta 60 ttgcaaagtcaatgggtatattgacggttggtattgtcaccacccctttctcgtttgaag 120 ggagaaagagatctattcaagcccaagaaggaattacagccttaagagataatgttgaca 180 cgcttatagttattccaaatgacaagctactaacggcagtttctcaatctacccctgtaa 240 ctgaagcattcaatctggctgatgatattcttcgacagggtgttcgtggcatatctgata 300 ttattacaataccagggttggtgaatgtagattttgctgatgttcgggctataatggcca 360 atgcaggttcttcactaatggggataggaactgcaactggaaaatcaagggcaagagatg 420 ctgcattaaatgccatccagtcaccwttmctggatattggtatagararggctactggaa 480 ttgtttggaacawaactggkgggactgatcttgaccttgtttgaggtaaacacggcarca 540 rraggttatttatgacctcgtggaccctactgctaatttaatatttggagcagtaataga 600 tccatcactcagtggkcaagtgagcataacattaattgctactggattcaagcgtcaaga 660 ggarartgaarggaggcctntgcaggccagtcaactcactcaagcagacacaaccttcgg 720 caccaattggcggtcttcctctttcactgatggtggtttgtttgagataccagaattcct 780 aaagaagagaggaggttcacgctatccgagggcgtaatctttttcatcctaatttctttg 840 atcccttgcatttcttcacccttggatatacatagcattggtctagttcttaggtccctg 900 tcttgccctttttcggattttagtcagagttgtgtatacagtttgttcatgaaagtttat 960 tacttcccactgtccagacttatgggtctaaccggaggtattgcagcatggatgcttttc 1020 ttggcatatttgaattagtttattagcttgtacagagatttcagtaatgctgagagcttg 1080 ttatagttctttggcatgttatagaaaattcattattattattcatcccnccaaaaaaaa 1140 aaaaaaaaaaaaagggcggccgccgactagtgagctcgtcgaccc 1185 <210>

<211>

<212>
DNA

<213>
Glycine sp <220>

<221> feature misc -<222>
(1)..
.(700) <223>
n = A,T,C
or G

<400>

aattcggctcgagattgtcaccacccctttctcgtttgaagggagaaagagatctattca 60 agcccaagaaggaattacagccttaagagataatgttgacacgcttatagttattccaaa 120 tgacaagctactaacggcagtttctcaatctacccctgtaactgaagcattcaatctggc 180 tgatgatattcttcgacagggtggtccgtggcatatctgatattattacaataccagggt 240 tggtgaatgtagattttgctgatgttcgggctataatggccaatgcaggttcttcactaa 300 tggggataggaactgcaactggaaaatcaagggcaagagatgctgcattaaatgccatcc 360 agtcacctttactggatattggtatagagagggctactggaattgtttggaacataactg 420 gtgggactgatctgccttgtttgaggtaaacacngcagcaganggtatttatgacctcgn 480 ggccctactgctaattaatatttggagcagaatagatccatcctcatggcaagtgacata 540 cattnantgctctggattcaagcgtcaagangagaagtgaagggangcctttgcaggcca 600 gcactcactcagcagacacaaccttngnaccaattggcggcttcctctttcactgatggg 660 nggttggttgagatncnanaattcctaaagaaaaanagag 700 <210>

<211>

<212>
DNA

<213>
Arabidopsis sp <400>

ccacgcgtccggaggaagtaaacaatggcgataattccgttagcacagcttaatgagcta 60 acgatttcttcatcttcttcttcgtttcttaccaaatcgatatcttctcattcgttgcat 120 agtagctgcatttgcgcaagttctagaatcagtcaattccgtggcggcttctctaaacga 180 agaagcgattcaacaaggtctaagtcgatgcgattgaggtgttccttctctccgatggaa 240 tctgcgagaattaaggtgattggtgtcggtggtggtggtaacaatgccgttaaccggatg 300 atttcaagcggtttacagagtgttgatttctatgcgataaacacggattcgcaagctctg 360 ttacagttttctgctgagaacccacttcaaattggagaacttttaactcgtgggcttggc 420 actggtggaaacccgcttcttggagaacaagctgcagaagaatcaaaagatgcaattgct 480 aatgctcttaaaggatcagaccttgttttcataactgctggtatgggtggtggaacaggg 540 tctggtgctgcacctgtggtagctcagatttcgaaggatgctggttatttgactgttggt 600 gttgttacctatccgtttagctttgaaggacgtaaaagatctttgcaggcactggaagct 660 attgaaaagctccaaaagaatgttgatacccttatcgtgattccaaatgatcgtctgcta 720 gatattgctgatgaacagacgccacttcaggacgcgtttcttcttgcagatgatgtttta 780 cgccaaggagtacaaggaatctcagatattattactatacctggactagtcaatgtggat 840 tttgcagatgtgaaggcagtcatgaaagattctggaactgcaatgctcggggtaggtgtt 900 tcttccagcaaaaaccgggcagaagaagcagctgaacaagcaactttggctccattgatc 960 ggatcatccatacaatcagctactggtgtcgtctacaacatcactggtggaaaagacata 1020 actttgcaggaagtgaaccgagtatcacaggtcgtgacaagtttggcagacccatcggcc 1080 aacatcatatttggagctgttgtggatgatcgctacaccggagagattcatgtaacgata 1140 atcgccacaggcttctctcagtcattccagaagacacttctgactgatccaagagcagct 1200 aaactccttgacaaaatgggatcatcaggtcaacaagagaacaaaggaatgtctctgcct 1260 caccagaagcagtctccatcaactatctctaccaaatcgtcttctccccgtagacttttc 1320 ttctagttttctttttttccttttcggtttcaagcatcaaaaatgtaacgatcttcaggc 1380 tcaaatatcaattacatttgattttcctccaaaaaaaaaaaaaaa 1425 <210>

<211>

<212>
DNA

<213>
Arabidopsis sp <400>

tgttgttgccgctcagaaatctgaatcttctccaatcagaaactctccacggcattacca.60 aagccaagctcaagatcctttcttgaaccttcacccggaaatatctatgcttagaggtga 120 agggactagtacaatagtcaatccaagaaaggaaacgtcttctggacctgttgtcgagga 180 ttttgaagagccatctgctccgagtaactacaatgaggcgaggattaaggttattggtgt 240 gggaggtggtggatcaaatgctgtgaatcgtatgatagagagtgaaatgtcaggtgtgga 300 gttctggattgtcaacactgatatccaggctatgagaatgtctcctgttttgcctgataa 360 taggttacaaattggtaaggagttgactaggggtttaggtgctggaggaaatccagaaat 420 cggtatgaatgctgctagagagagcaaagaagttattgaagaagctctttatggctcaga 480 tatggtctttgtcacagctggaatgggcggtggaactggcactggtgcagcccctgtaat 540 tgcaggaattgccaaggcgatgggtatattgacagttggtattgccacaacgcctttctc 600 gtttgagggtcgaagaagaactgttcaggctcaagaagggcttgcatctctcagagacaa 660 tgttgacactctcatcgtcattccaaatgacaagttgcttacagctgtctctcagtctac 720 tccggtaacagaagcatttaatctagctgatgatatactccgtcagggggttcgtgggat 780 atctgatatcattacgattcctggtttggtgaatgtggattttgctgatgtgagagctat 840 aatggcaaatgcggggtcttcattgatgggaataggaactgcgacaggaaagagtcgggc 900 WO 00132799 PCTlUS99/28103 aagagatgctgcgctaaatgcaatccaatcccctttgttagatattgggattgagagagc 960 cactggaattgtttggaacattactggcggaagtgacttgacattgtttgaggtaaatgc 1020 tgctgcggaagtaatatatgatcttgtcgatccaactgccaatcttatattcgtgctgtt 1080 gtagatccagccctcagcggtcaagtaagcataaccctgatagctacgggtttcaaacga 1140 caagaagagggagaaggacgaacagttcagatggtacaagcagatgctgcgtcagttgga 1200 gctacaagaagaccctcttcttcctttagagaaagcggttcagtggagatcccagagttc 1260 ttgaagaagaaaggcagctctcgttatccccgagtctaaagcccaatctaatcactaccc 1320 tgcacactgcagcaataacaaacgtgtgtgtactggtagtctggtactgccttctgggat 1380 acagcaagatgtgttgatgtatgatcaagaatctgtgtgggtgtgtatatgttctgtcac 1440 tgcctctggtcgtgttcttgaataggttgttttagaaatcggagtttctctctatgtcac 1500 ttccaaaacaaaaaaggagaagaagaatcacacttctcgaaccataaacatacttataag 1560 attatgagagttttagcagaaattattgtcaaaaaaaaaaaaaaaaaaaaa 1611 <210> 34 <211> 299 <212> DNA

<213> Arabidopsis sp <220>

<221> miscfeature _ <222> (1).
.(299) <223> n = A,T,C
or G

<400> 34 agtaattgaaaaatgacacatacctcttttnctacaatcaaagtttataactaagaagaa 60 caaatgcaatagtaataataaagatttgaggacatactgtgacaaagaccatatctgagc 120 cataaagagcttctncaataactgctttgctctctctagcagcattcataccgatttcng 180 gatttcctccagcacctaaacccctagtcaactccttaccaatttgtaacctattatcag 240 gcaaaacaggagacattctcatagcctggatatcagtgttgacaatccagaactccaca 299 <210> 35 <211> 25 <212> DNA

<213> Artificial Sequence <220>
<223> Synthetic Oligonucleotide <400> 35 cttgccgcaa aacatcatcc gcgag 25 <210> 36 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic Oligonucleotide <400> 36 cacccacccc gagcatcgca gttccgga 28 <210> 37 <211> 41 <212> DNA

<213> Artificial Sequence <220>
<223> Synthetic Oligonucleotide <400> 37 ttggtgtcgg tggtggtggt aacaatgccg ttaaccggat g 42 <210> 38 <211> 41 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic Oligonucleotide <400> 38 ggtaacaatg ccgttaaccg gatgatttca agcggtttac a 41 <210> 39 <211> 41 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic Oligonucleotide <400> 39 gctgctcttg gatcagtcag aagtgtcttc tggaatgact g 41 <210> 40 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic Oligonucleotide <400> 40 tttgtcaagg agtttagctg ctcttggatc agtcagaag 39 <210> 41 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic Oligonucleotide <400> 41 cagcaccaga tcctgtacct ccacccat 28 <210> 42 <211> 30 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 42 ctgaatgggt atgtgacaac accaacagtc 30 <210> 43 <211> 28 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 43 gctgatgatg tattacgcca aggtgtcc 28 <210> 44 <211> 28 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 44 ctggaactgc tatgcttgga gttggggt 28 <210> 45 <211> 34 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 45 tcgaaactga atggataggt gacaacacca acag 34 <210> 46 <211> 36 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 46 tggatcgaca aggtcgtaga taatttcggc cgcagc 36 <210> 47 <211> 56 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 47 cgcgatttaa atggcgcgcc ctgcaggcggccgcctgcag ggcgcgccat ttaaat56 <210> 48 <211> 32 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 48 tcgaggatcc gcggccgcaa gcttcctgcagg 32 <210> 49 <211> 32 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 49 tcgacctgca ggaagcttgc ggccgcggatcc 32 <210> 50 <211> 32 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic 0ligonucleotide <400> 50 tcgacctgca ggaagcttgc ggccgcggatcc 32 <210> 51 <211> 32 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 51 tcgaggatcc gcggccgcaa gcttcctgcagg 32 <210> 52 <211> 36 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 52 tcgaggatcc gcggccgcaa gcttcctgca ggagct 36 <210> 53 <211> 28 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 53 cctgcaggaa gcttgcggcc gcggatcc 28 <210> 54 <211> 36 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 54 tcgacctgca ggaagcttgc ggccgcggat ccagct 36 <210> 55 <211> 28 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 55 ggatccgcgg ccgcaagctt cctgcagg 28 <210> 56 <211> 10 <222> PRT

<213> Artificial Sequence <220>

<223> c-myc Tag <400> 56 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu <210> 57 <221> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic Oligonucleotide <400> 57 acgtgcggcc gcatggcgat aattccgtta gcacagc 37 <210> 58 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic Oligonucleotide <400> 58 actgataagc ttttgctcga agaaaagtct acgggga 37 <210> 59 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic Oligonucleotide <400> 59 acgtgcggcc gcatggcgat aattccgtta gcacagc 37 <210> 60 <211> 44 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic Oligonucleotide <400> 60 cgtcctgcag gctacaagtc ttcctcactg ataagctttt gctc 44 <210> 61 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Synthetic Oligonucleotide <400> 61 acgtcctgca ggatggcgat aattccgtta gcacagc 37 <210> 62 <211> 38 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic 0ligonucleotide <400> 62 acgtgcggcc gcctagaaga aaagtctacggggagaag 38 <210> 63 <211> 32 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligoilucleotide <400> 63 acgtaagctt tccttctctc cgatggagtctg 32 <210> 64 <211> 33 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 64 acgtctgcag cttttcaatg gcttcaagtgcct 33 <210> 65 <211> 34 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 65 acgtctgcag aagaacgtgg ataccctcatcgtg 34 <210> 66 <211> 38 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Oligonucleotide <400> 66 acgtgcggcc gcctagaaga acaatctacggggagaag 38 <210> 67 <211> 244 <212> DNA
<213> Artificial Sequence <220>
<223> Prrn/NEP/Gene101eader/14 amino acid from GFP
fusion <400>

gaattcggtacccccgtcgttcaatgagaatggataagaggctcgtggga ttgacgtgag60 ggggcagggatggctatatttctgggagcgaactccgggcgaatactgaa gcgcttggat120 acaagttatccttggaaggaaagacaattccggatcctctagaaataatt ttgtttaact180 ttaagaaggagatatacccatgggtaaaggagaagaacttttcactggag ttgtcccaag240 catg 244 <210>

<211>

<212>
DNA

<213>
Artificial Sequence <220>

<223>
Synthetic Oligonucleotide <400>

gtcctgcaggatggccaccatctcaaacccag 32 <210>

<211>

<212>
DNA

<213>
Artificial Sequence <220>

<223>
Synthetic Oligonucleotide <400>

tagcggccgcctatataaaggagctaaaagaacagcc 37 <z1o>
7a <211>

<212>
DNA

<213>
Arabidopsis sp <400>

atggcgattagtccgttggcacagcttaacgagctaccagtctcttcctc gtttsttgcg60 acatcccactcgctgcacagtaccagaatcagtggcgggcttctcaaaac aaaggtttaa120 gcaaacacggttgagatgctccttctctccgatggagtctgcgaggatta aggtggttgg180 tgtcggcggtggtggtaacaatgccgtcaatcgcatgatttccagcggct tacagagtgt240 tgatttctatgcgataaacacggactctcaagctctcttgcagtcttctg cgcagaaccc300 tcttcaaattggagagctcctaactcgtggccttgggactggtgggaacc cgcttctagg360 agaacaagctgctgaggaatctaaagacgcgattgctaatgctcttaaag gatctgacct420 tgytttcattactgctggtatgggtggtggcactggctccggtgctgctc ctgttgttgc480 tcagatctccaaagacgctggttatttgaccgttggtgttgttacctatc ccttcagctt540 cgaaggtcgtaaaagatctttgcaggcacttgaagccattgaaaagctgc agaakaacgt600 ggataccctcatcgtgataccaaatgatcgtctcctagatattgctgatg aacagacgcc660 tcttcaaga 669 WO U0/32799 PCT/US99/2$103 <210> 71 <211> 646 <222> DNA
<213> Nicotiana sp <400>

ggccctctagatgcatgctcgagcggccgccagtgtgatggatatctgcagaattcgccc 60 ttaagcagtggtaacaacgcagagtacgcgggggtaaaccaaacagacagagagagcaga 120 aacagcaatggccaccatctcaaacccagcagagatagcagcttcttctccttcctttgc 180 tttttaccactcttcctttattcctaaacaatgctgcttcaccaaagctcgccggaaaag 240 cttatgtaaacctcaacgtttcagcatttcaagttcatttactccttttgattctgctaa 300 gattaaggttatcggcgtcggtggcggtggtaacaatgccgttaaccgtatgattggcag 360 tggcttacagggtgttgacttctatgctataaacacggatgctcaagcactgctgcagtc 420 tgctgctgaaaatccacttcaaattggagagcttctgactcgtgggcttggtactggtgg 480 caatcctcttttaggggaacaggcagcagaggagtcgaaggaagccattgcaaattctct 540 aaaaggttcagatatggtgttcataacagcaggaatgggtggaggtacaggatctggtgc 600 tgaagggcgaattccagcacactggcggccgttactagtggatccg 646 <210> 72 <211> 325 <212> PRT
<213> Glycine sp <400> 72 Gly Ser Arg Pro Arg Thr Thr Lys Tle Ala Pro Gln Arg Leu Ser Arg Arg Phe Gly Ser Val Arg Cys Ser Tyr Ala Tyr Val Asp Asn Ala Lys Ile Lys Val Val Gly Ile Gly Gly Gly Gly Asn Asn Ala Val Asn Arg Met Ile Gly Ser Gly Leu G1n Gly Val Asp Phe Tyr Ala Ile Asn Thr Asp Ala Gln Ala Leu Leu Asn Ser Ala Ala Glu Asn Pro Ile Lys Ile Gly Glu Val Leu Thr Arg Gly Leu Gly Thr Gly Gly Asn Pro Leu Leu Gly Glu Gln Ala Ala Glu Glu Ser Arg Asp Ala Ile Ala Asp Ala Leu Lys Gly Ser Asp Leu Val Phe Ile Thr Ala Gly Met Gly Gly Gly Thr Gly Ser Gly Ala Ala Pro Va1 Va1 Ala Gln Ile Ser Lys Glu Ala Gly Tyr Leu Thr Val Gly Val Val Th,r Tyr Pro Phe Ser Phe Glu Gly Arg Lys Arg Ser Leu Gln Ala Phe Glu Ala Ile Glu Arg Leu Gln Lys Asn Val Asp Thr Leu Ile Val Ile P'ro Asn Asp Arg Leu Leu Asp Ile Ala Asp Glu Gln Met Pro Leu Gln Asp Ala Phe Pro Phe Ala Asp Asp Val Leu Arg Gln Gly Val G1n Giy Ile Ser Asp Ile Ile Thr Val Pro Gly Leu Val Asn Val Asp Phe Ala Asp Val Lys Ala Val Met Lys Asp Ser Gly Thr Ala Met Leu Gly Val Gly Val Ser Ser Gly Lys Asn Arg Ala Glu Glu Ala Ala Glu Gln Ala Thr Leu Ala Pro Leu Ile Gly Ser Ser I1e Gln Ser Ala Thr Gly Val Val Tyr Asn Ile Thr Gly Gly Lys Asp Ile Thr Leu Gln Glu Val Asn Arg Val Ser G1n Val Val Thr Ser Leu Ala Asp Pro Ser Ala Asn Ile Ile Phe Gly Ala Val Val Asp Asp Arg Tyr Thr Gly Glu Ile <210> 73 <211> 357 <212> PRT
<213> Zea mays <220>
<221> VARIANT
<222> (1)..-.(357}
<223> Xaa = Any Amino Acid <400> 73 Asp Leu Val Phe Ile Thr Ala Gly Met Gly Gly Gly Thr Gly Ser Gly Ala Ala Pro Val Val Ala Gln Ile Ser Lys Glu Ala Gly Tyr Leu Thr Val Gly Val Val Thr Tyr Pro Phe Ser Phe Glu Gly Arg Lys Arg Ser Val Gln Ala Leu Glu Ala Leu Glu Lys Leu Glu Lys Ser Val Asp Thr Leu Ile Va1 I1e Pro Asn Asp Lys Leu Leu Asp Val Ala Asp Glu Asn Met Pro Leu Gln Asp Ala Phe Leu Leu Ala Asp Asp Val Leu Arg Gln Gly Val Gln Gly Ile Ser Asp I1e Ile Thr Ile Pro Gly Leu Val Asn Val Asp Phe Ala Asp Val Lys Ala Va1 Met Lys Asn Ser Gly Thr Ala Met Leu Gly Val Gly Val Ser Ser Ser Lys Asn Arg Ala Gln Glu Ala Ala Glu Gln Ala Thr Leu Ala Pro Leu Ile Gly Ser Ser Ile Glu Ala A1a Thr Gly Val Val Tyr Asn Ile Thr Gly Gly Lys Asp Ile Thr Leu Gln Glu Val Asn Lys Val Ser Gln Ile Val Thr Ser Leu Ala Asp Pro Ser Ala Asn Ile Ile Phe Gly Ala Va1 Val Asp Asp Arg Tyr Thr Gly Glu Ile His Val Thr Ile Ile Ala Thr Gly Phe Pro Gln Ser Phe Gln Lys Ser Leu Leu Ala Asp Pro Lys Gly Ala Arg Ile Val Glu Ser Lys Glu Lys Ala Ala Thr Leu Ala His Lys Ala Ala Ala Ala Ala Val GIn _ 28 _ Pro Val Pro Ala Ser A1a Trp Ser Arg Arg Leu Phe Ser Xaa Glu Ala His Leu Val Asn Arg Asp Ser Xaa Cys Ile Arg Phe Ala Phe Ser Val Leu Arg Ala Val Pro Lys Val TIe Phe Gly Tyr Leu Glu Ile Tyr Ser Leu Gly Xaa Cys Ser Val Val Val G1u Xaa Val Ser Val Tyr Val Ser Leu Leu Cys Phe Met Phe Leu Arg Ile Xaa Arg Xaa Gly Xaa Glu Lys Cys Ser Ala Thr Gln His Xaa Thr Val Xaa Lys Ile Phe Asp Cys Phe Ile A1a Ala Thr Cys

Claims (31)

What is claimed is:

1. In a method for transforming a plant cell plastid comprising the steps of introducing into cells of a plant a construct comprising a promoter functional in a plant cell plastid operably associated with a DNA sequence of interest and transforming said plant cell plastid with said construct, wherein the improvement comprises introducing said construct into a plant cell having an altered plant plastid morphology selected from the group consisting of altered plastid size and altered plastid number in said plant cell.

2. The method according to Claim 1, wherein said plastid size is increased from a wild-type plant plastid morphology.

3. The method according to Claim 2, further wherein said plastid number is decreased from a wild-type plant plastid morphology.

4. The method according to Claim 1, wherein said plastid size is decreased from a wild-type plant plastid morphology.

5. The method according to Claim 4, further wherein said plastid number is increased from a wild-type plant plastid morphology.

6. The method according to Claim 2, wherein said plant cell is obtained from a plant tissue source in which plastid division is inhibited.

7. The method according to Claim 6, wherein said plastid division is inhibited by introduction into cells of the plant tissue source a second DNA
construct comprising in the 5' to 3' direction of transcription a promoter functional in a plant cell, a DNA sequence coding for a gene involved in plastid cell division and a transcriptional termination sequence functional in a plant cell.

8. The method according to Claim 7, wherein said DNA sequence is in an antisense orientation.

9. The method according to Claim 8, wherein said construct contains a DNA
sequence coding for an FtsZ protein.

10. The method according to Claim 7, wherein said DNA sequence is in a sense orientation.

11. The method according to Claim 10, wherein said DNA sequence provides for sense suppression.

12. The method according to Claim 6, wherein said plastid division is inhibited by growing a plant under culture conditions which inhibit the division of plant cell plastids.

13. The method according to Claim 12, wherein said culture conditions comprise growing the plant tissue source under exposure to an inhibitor of bacterial cell division.

14. The method according to Claim 13, wherein said inhibitor is 5,5'-Bis-(8-anilino-1-naphthalenesulfonate).

15. The method according to Claim 6, wherein said plastid division is inhibited by genetic mutagenesis.

16. An isolated DNA sequence encoding a plant FtsZ protein from Arabidopsis thaliana.

17. The DNA sequence of Claim 16, wherein said FtsZ protein is encoded by a sequence which includes a sequence selected from the group consisting of SEQ
ID
Nos: 1 and 3.

18. An isolated DNA sequence encoding a plant FtsZ protein from Brassica.

19. The DNA sequence of Claim 18, wherein said FtsZ protein is encoded by a sequence of SEQ ID NO:5.

20. An isolated DNA sequence encoding a plant FtsZ protein from soybean.

21. The DNA sequence of Claim 20, wherein said FtsZ protein is encoded by a sequence which includes a sequence selected from the group consisting of SEQ
ID
NOs:20-31.

22. An isolated DNA sequence encoding a plant FtsZ protein from corn.

23. The DNA encoding sequence of Claim 22, wherein said FtsZ protein is encoded by a sequence which includes a sequence selected from the group consisting of SEQ ID Nos:10-19.

24. A recombinant DNA construct comprising any of the DNA encoding sequences of Claims 16-23.

25. A plant cell comprising the DNA construct of 24.

26. A plant comprising a cell of Claim 25.

27. A method for improving the selectability of plant comprising, transforming a plant cell source having an altered plastid morphology with a construct comprising a promoter functional in a plant cell plastid operably associated with a nucleic acid sequence encoding a selectable marker.

28. The method according to Claim 27, wherein said nucleic acid sequence encodes an herbicide tolerance gene.

29. The method according to Claim 27, wherein said nucleic acid sequence encodes a glyphosate tolerance gene.

30. A method for preparing a plant cell source with increased plastid transformation efficiency comprising, transforming a plant cell with a construct comprising a promoter functional in plant cell operably associated with a nucleic acid sequence encoding a FtsZ
protein.

31. A method for transforming a plant cell plastid comprising, introducing into a plant cell having altered plastid morphology a first nucleic acid construct comprising a promoter functional in a plant cell plastid operably associated with a nucleic acid sequence of interest.