Cahoon, Rebecca E. (Webster Groves, MO, US)
Scelonge, Christopher J. (Ankeny, IA, US)
Tao, Yumin (Fremont, CA, US)
Gordon-kamm, William J. (Urbandale, IA, US)
Bruce, Wesley B. (Raleigh, NC, US)
Newman, Lisa J. (Urbandale, IA, US)
Plaque It!
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E.I. DU PONT DE NEMOURS AND COMPANY (1007 Market Street, Wilmington, DE, US)
1. An isolated polynucleotide comprising a nucleotide sequence encoding a WUSCHEL polypeptide containing a 1+4 homeodomain, wherein the polypeptide has at least 80% sequence identity over its entire length to SEQ ID NO: 14 based on the CLUSTAL alignment method using default parameters of KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5, and wherein the polypeptide is capable of stimulating in vitro growth of plant tissue.
2. An isolated polynucleotide fully complementary to the polynucleotide of claim 1.
3. The isolated polynucleotide of claim 1, wherein the WUSCHEL polypeptide has at least 85% sequence identity over its entire length to SEQ ID NO: 14.
4. The isolated polynucleotide of claim 1, wherein the WUSCHEL polypeptide has at least 90% sequence identity over its entire length to SEQ ID NO: 14.
5. The isolated polynucleotide of claim 1, wherein the WUSCHEL polypeptide has at least 95% sequence identity over its entire length to SEQ ID NO: 14.
6. The isolated polynucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 13.
7. A chimeric gene comprising the polynucleotide of claim 1, operably linked to a regulatory sequence functional in a host cell.
8. A transgenic plant comprising the polynucleotide of claim 1.
9. A seed from the transgenic plant of claim 8, wherein the seed comprises the polynucleotide.
10. The seed of claim 9, wherein the seed is from a plant selected from the group consisting of corn, soybean, wheat, rice, alfalfa, sunflower, canola, or cotton.
11. A method for inducing meristem proliferation in a plant cell comprising: (a) transforming a plant cell with a polynucleotide of claim 1 operably linked to a regulatory sequence operable in the plant cell; and, (b) expressing the polynucleotide for a time sufficient to induce meristem proliferation.
12. The method of claim 11 wherein the polynucleotide is integrated into the plant cell genome to produce a transformed plant cell comprising the polynucleotide.
13. The method of claim 12 further comprising growing the transformed plant cell under plant growing conditions to produce a regenerated plant.
14. A plant produced by the method of claim 13.
15. A method for positive selection of a transformed cell, comprising: (a) transforming a plant cell with a polynucleotide of claim 1 operably linked to a regulatory sequence operable in the plant cell; and, (b) expressing the polynucleotide for a time sufficient to induce organogenesis and provide a positive selection means.
16. The method of claim 15 wherein the polynucleotide is integrated into the plant cell genome to produce a transformed plant cell comprising the polynucleotide.
17. The method of claim 16 further comprising growing the transformed plant cell under plant growing conditions to produce a regenerated plant.
18. A plant produced by the method of claim 17.
19. A method for transforming a plant cell comprising introducing the polynucleotide of claim 1 into the cell.
20. A transformed plant cell produced by the method of claim 19, wherein the cell comprises the polynucleotide.
21. A transformed plant regenerated from the plant cell of claim 20, wherein the plant comprises the polynucleotide.
22. A method to increase transformation frequency comprising: (a) introducing into a plant cell the isolated polynucleotide of claim 1 operably linked to a regulatory sequence operable in the plant cell; and, (b) expressing the polynucleotide in the plant cell; wherein plant cells expressing the polynucleotide have an increased transformation frequency as compared to control plant cells that do not have or express the polynucleotide.
23. A method to stimulate plant cell growth, wherein the method comprises: (a) introducing into a plant cell the isolated polynucleotide of claim 1 operably linked to a regulatory sequence operable in the plant cell; and, (b) expressing the polynucleotide in the plant cell;, wherein plant cells expressing the polynucleotide show stimulated plant cell growth as compared to control plant cells that do not have or express the polynucleotide.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation of co-pending U.S. application Ser. No. 10/744,572 filed Dec. 23, 2003, which is a Continuation-In-Part of co-pending U.S. application Ser. No. 09/807,946 filed Apr. 20, 2001, which is a national application that claims the benefit of PCT International Application No. PCT/US00/26648, filed Sep. 28, 2000, which in turn claims the benefit of U.S. Provisional Application No. 60/157,216 filed Oct. 1,1999. The entire contents of the above applications are herein incorporated by reference.
FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding Wuschel (WUS) proteins in plants and seeds.
BACKGROUND OF THE INVENTION
Organ formation in plants occurs via the activity of apical meristems. Plant meristems contain a pool of stem cells, which are able to self-maintain, and give rise to a variety of cell types including cells required for organ initiation. The initiation and maintenance of stem cells and their integration into organ-forming meristems are thus the basis for continuous plant development.
The Wuschel protein, designated hereafter as WUS, plays a key role in the initiation and maintenance of the apical meristem, which contains a pool of pluripotent stem cells (Endrizzi et al., 1996, Plant Journal 10:967-979; Laux et al., 1996, Development 122:87-96; and Mayer et al., 1998, Cell 95:805-815). Arabidopsis plants mutant for the WUS gene contain stem cells that are misspecified and that appear to undergo differentiation. WUS encodes a novel homeodomain protein, which presumably functions as a transcriptional regulator (Mayer et al., 1998, Cell 95:805-815). The stem cell population of Arabidopsis shoot meristems is believed to be maintained by a regulatory loop between the CLAVATA (CLV) genes which promote organ initiation and the WUS gene which is required for stem cell identity, with the CLV genes repressing WUS at the transcript level, and WUS expression being sufficient to induce meristem cell identity and the expression of the stem cell marker CLV3 (Brand et al. (2000) Science 289:617-619; Schoof et al. (2000) Cell 100:635-644). Constitutive expression of WUS in Arabidopsis has been recently shown to lead to adventitious shoot proliferation from leaves (in planta) (Laux, T., talk presented at the XVI International Botanical Congress Meeting, Aug. 1-7,1999, St. Louis, Mo.).
There is a great deal of interest in identifying the genes that encode proteins involved in development in plants, generally toward the objective of altering plant growth and architecture. WUS represents one such gene. However, the WUS gene can also be used for the novel application of stimulating in vitro growth of plant tissue and improving transformation. In this manner, this gene can expand the range of tissues types targeted for transformation. Specifically, the WUS gene may be used to improve meristem transformation frequencies and could result in genotype independent transformation of many important crops such as maize, soybean and sunflower. Furthermore, transformation into meristems would stimulate the formation of new apical initials reducing the chimeric nature of the transgenic events. Lastly, ectopic expression into non-meristematic cells would stimulate adventive meristem formation. This could lead to transformation of non-traditional tissues such as leaves, leaf bases, stem tissue, etc. Alternatively, transformation of a more traditional target such as callus or the scutellum of immature embryos could promote a “non-traditional” growth response, i.e. meristems in place of somatic embryos. In addition, WUS may also be used as a genetic marker for meristems.
Modulation of WUS is expected to modulate plant and/or plant tissue phenotype including cell growth stimulation, organogenesis, and somatic embryogenesis. WUS may also be used to improve transformation via somatic embryogenesis. Expression of Arabidopsis WUS can induce stem cells in vegetative tissues, which can differentiate into somatic embryos (Zuo, et al. (2002) Plant J 30:349-359). The ability to stimulate organogenesis and/or somatic embryogenesis may be used to generate an apomictic plant. Apomixis has economic potential because it can cause any genotype, regardless of how heterozygous, to breed true. It is a reproductive process that bypasses female meiosis and syngamy to produce embryos genetically identical to the maternal parent. With apomictic reproduction, progeny of specially adaptive or hybrid genotypes would maintain their genetic fidelity throughout repeated life cycles. In addition to fixing hybrid vigor, apomixis can make possible commercial hybrid production in crops where efficient male sterility or fertility restoration systems for producing hybrids are not available. Apomixis can make hybrid development more efficient. It also simplifies hybrid production and increases genetic diversity in plant species with good male sterility.
Accordingly, the availability of nucleic acid sequences encoding all or a portion of a WUS protein would facilitate studies to better understand programmed development in plants, provide genetic tools to enhance the efficiency of gene transfer into meristem tissue and help provide alternative transformation methods in several important crops.
SUMMARY OF THE INVENTION
This invention relates to isolated polynucleotides encoding WUS polypeptides. The invention further provides isolated WUS polypeptides. The invention also provides methods of using the polynucleotides to modulate the level of WUS, improve transformation efficiency, to stimulate plant cell growth, including stem cells, to stimulate organogenesis, to stimulate somatic embryogenesis, to induce apomixis, and to provide a positive selection for cells comprising the polynucleotide. The invention also relates to cells, plants and seeds comprising the polynucleotides of the invention or produced by the methods of the invention.
BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE LISTINGS
The invention can be more fully understood from the following detailed description and the accompanying drawing and Sequence Listing, which form a part of this application.
FIG. 1, parts A & B, shows an alignment of the amino acid sequences of WUS protein encoded by the nucleotide sequences derived from corn clone cpi1c.pk012.p19 (SEQ ID NO: 4), corn clone p0058.chpab57r (SEQ ID NO: 10), soybean clone ses4d.pk0033.c8 (SEQ ID NO: 20), soybean clone sgs5c.pk0002.f2 (SEQ ID NO: 22), and a contig assembled using soybean clone ssm.pk0060.h4 and NCBI GenBank Identifier (GI) No.4395781 (SEQ ID NO: 24), and the WUS protein from Arabidopsis thaliana (NCBI GI No. 4090200; SEQ ID NO: 25). Amino acids which are conserved among all and at least two sequences with an amino acid at that position are indicated with an asterisk (*). Dashes are used by the program to maximize alignment of the sequences.
FIG. 2, parts A & B, shows the stimulation of organogenesis by Wuschel. Epifluorescence microscopy shows the pattern of YFP expression and culture morphology in (A) maize embryo culture co-bombarded with Ubi:YFP and ubi:uidA control plasmids; and (B) maize embryo culture co-bombarded with Ubi:YFP and Ole:WUS2 plasmids. The control in panel (A) shows YFP expressing spots in tissue culture showing no developing projections, while panel (B) shows YFP expressing spots in the apices of outgrowths of tissue. Panel A—magnification=40×; Panel B—magnification=200×.
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns isolated polynucleotides comprising nucleotide sequences which encode polypeptides involved in the initiation and maintenance of stem cells in plants, i.e., polypeptides having Wuschel activity. The invention also provides isolated polypeptides having Wuschel activity.
The present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 70% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, and 12, (b) a second nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 70% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:14, 16, 18, and 20, (c) a third nucleotide sequence encoding a polypeptide of at least 180 amino acids having at least 70% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:24, (d) a fourth nucleotide sequence encoding a polypeptide of at least 230 amino acids having at least 70% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:22, (e) a fifth nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6, 8, and 10, and (f) a sixth nucleotide sequence comprising the complement of (a), (b), (c), (d), or (e).
In a second embodiment, it is preferred that the isolated polynucleotide of the claimed invention comprises a first nucleotide sequence which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 that codes for the polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24.
In a third embodiment, this invention concerns an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (or at least one of 40, or at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 and the complement of such nucleotide sequences.
In a fourth embodiment, this invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to at least one suitable regulatory sequence.
In a fifth embodiment, the present invention concerns an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, for example a baculovirus or a plant virus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
In a sixth embodiment, the invention also relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
In a seventh embodiment, the invention concerns an isolated WUS polypeptide selected from the group consisting of: (a) a polypeptide of at least 50 amino acids having at least 70% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, and 12, (b) a polypeptide of at least 100 amino acids having at least 70% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs: 14, 16, 18, and 20, (c) a polypeptide of at least 180 amino acids having at least 70% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO: 24, (d) a polypeptide of at least 230 amino acids having at least 70% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO: 22, and (e) a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6, 8, and 10.
In an eighth embodiment, the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a WUS polypeptide or enzyme activity in a host cell, for example a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level of the WUS polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level of the WUS polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of the WUS polypeptide or enzyme activity in the host cell that does not contain the isolated polynucleotide.
In a ninth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a WUS polypeptide, such as a plant WUS polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (or at least one of 40, or at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (for example, a cDNA inserted in a cloning vector) using the oligonucleotide primer. Optionally, the amplified nucleic acid fragment will encode a substantial portion of a WUS amino acid sequence.
In a tenth embodiment, this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a WUS polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
In an eleventh embodiment, this invention concerns a composition, such as a hybridization mixture, comprising an isolated polynucleotide of the present invention.
In a twelfth embodiment, this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, for example a plant cell, such as a monocot or a dicot, under conditions which allow expression of the WUS polynucleotide, and identifying transformed cells.
In a thirteenth embodiment, this invention relates to a method of altering the level of expression of a WUS protein in a host cell comprising: (a) transforming a host cell with a chimeric gene of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of the WUS protein in the transformed host cell.
In another embodiment, the invention relates to isolated polynucleotides, having at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or up to and including 100% identity over their entire length to at least one of the nucleic acid sequences selected from the group consisting of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42, 44, 46, 48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 80, which encode polypeptides having WUS activity. The invention also relates to isolated polynucleotides which are fully complementary to the nucleic acid sequences of this embodiment.
In another embodiment, the invention relates to isolated polynucleotides comprising at least a minimum whole integer number of contiguous nucleotides ranging from at least 30 contiguous nucleotides up to and including the full-length of the sequence. For example, the isolated polynucleotide comprises at least 30, 40, 50, 60, 75, 100, 150, 300, 500, 1000 contiguous nucleotides, up to and including the full-length of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42, 44, 46, 48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 80. Optionally, the isolated polynucleotide is a full-length polynucleotide encoding a polypeptide having WUS activity, which comprises at least 30, 40, 50, 60, 75, 100, 150, 300, 500, 1000 contiguous nucleotides, up to and including the full-length of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42, 44, 46, 48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 80. The invention also relates to isolated polynucleotides which are fully complementary to the nucleic acid sequences of this embodiment.
In another embodiment, the invention relates to isolated polynucleotides, or the complement thereof, which encode a polypeptide having WUS activity, wherein the encoded polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or up to and including 100% identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. The invention also relates to isolated polynucleotides which are fully complementary to the nucleic acid sequences of this embodiment.
In another embodiment, the invention relates to isolated polynucleotides which encode a polypeptide comprising a minimum whole integer number of contiguous amino acids from at least one polypeptide selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81, wherein the number of contiguous amino acids is selected from the range of 20 amino acids up to and including the full length of the polypeptide. For example, the isolated polynucleotide encodes a polypeptide comprising at least 20, 25, 30, 40, 50, 75,100, 200 contiguous amino acids, up to and including the full-length of at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. The invention also relates to isolated polynucleotides which are fully complementary to the nucleic acid sequences of this embodiment.
In another embodiment, the invention relates to isolated polynucleotides comprising primers capable of amplifying WUS polynucleotides from a nucleic acid library. In some embodiments, isolated polynucleotide amplification primers are selected from the group consisting of SEQ ID NOS: 82, 83, 84, 85, 86 and 87.
In another embodiment, the invention relates to DNA constructs, host cells, plants, and seeds comprising the isolated polynucleotides of the invention, or the complement thereof, particularly isolated polynucleotides having a certain percent identity to at least one nucleic acid sequence selected from the group consisting of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42, 44, 46, 48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 80, or encoding a polypeptide having a certain percent identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. Also included are isolated polynucleotides comprising at least a minimum whole integer number of contiguous nucleotides, or encoding a polypeptide comprising a minimum whole integer number of contiguous amino acids, as described above, of the sequences of the invention.
In another embodiment, the invention relates to isolated polynucleotides encoding polypeptides having WUS activity, which comprise conserved domains or consensus amino acid sequences. The isolated polynucleotides comprise nucleic acid sequences which encode a WUS polypeptide comprising at least one conserved polypeptide motif. The polypeptide motifs include the homeodomain motif, the (E/R)TLPLFP motif, and the A(A/S)LEL(ST)L motif. Amino acid variations within these motifs are known, and included in this embodiment. Also included is a 25 amino acid motif located between the (E/R)TLPLFP and the A(A/S)LEL(ST)L motifs. In some embodiments, the isolated polynucleotides have a certain percent sequence identity to the polynucleotides of the present invention, wherein the sequence identity in the region of the polynucleotide encoding a conserved motif may be less than 100%. In some embodiments, the isolated polynucleotides are selected from the group consisting of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42, 44, 46, 48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 80. Optionally, this embodiment includes polynucleotides which encode polypeptides in which a motif is absent, for example, polypeptides lacking the 25 amino acid motif (SEQ ID NO: 91 QPP(P/S)RPRHAVPVPAGE(T/P)IR(GN)GGG(S/G)S), the (E/R)TLPLFP motif or the A(A/S)LEL(ST)L motif. In some embodiments, the isolated polynucleotides have a certain percent sequence identity to the polynucleotides of the present invention, wherein at least one amino acid motif is absent. In some embodiments, the isolated polynucleotide is selected from the group consisting of SEQ ID NO: 30, 32, 39, 46, 49, 51, 53, 56, 58, 60, 63, 65, 67, 70, 72, 74, 77, and 80.
In another embodiment, the invention relates to isolated polynucleotides, or the complement thereof, which encode a polypeptide having WUS activity, wherein the encoded polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or up to and including 100% identity to at least one of the consensus amino acid sequences selected from the group consisting of SEQ ID NOS: 88, 89, and 90. The invention also relates to isolated polynucleotides which are fully complementary to the nucleic acid sequences of this embodiment.
In another embodiment, the invention relates to isolated polypeptides having WUS activity, which comprise conserved domains or consensus amino acid sequences. The isolated polypeptides comprise at least one conserved polypeptide motif. The polypeptide motifs include the homeodomain motif, the (E/R)TLPLFP motif, and the A(A/S)LEL(ST)L motif. Amino acid variations within these motifs are known, and included in this embodiment. Also included is a 25 amino acid motif located between the (E/R)TLPLFP and the A(A/S)LEL(ST)L motifs. In some embodiments, the isolated polypeptides have a certain percent sequence identity to the polypeptides of the present invention, wherein the sequence identity in the region of the polynucleotide encoding a conserved motif may be less than 100%. In some embodiments, the isolated polypeptides are selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. Optionally, this embodiment includes polypeptides in which at least one motif is absent. This embodiment includes, for example, polypeptides lacking the 25 amino acid motif (SEQ ID NO: 91), the (E/R)TLPLFP motif or the A(A/S)LEL(ST)L motif. In some embodiments, the isolated polypeptides have a certain percent sequence identity to the polypeptides of the present invention, wherein at least one motif is absent. In some embodiments, the isolated polypeptide is selected from the group consisting of SEQ ID NO: SEQ ID NO: 31, 33, 40, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81.
In another embodiment, the invention relates to isolated polypeptides having WUS activity, wherein the encoded polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or up to and including 100% identity to at least one of the consensus amino acid sequences selected from the group consisting of SEQ ID NOS: 88, 89, and 90.
In another embodiment, the invention relates to isolated polypeptides having WUS activity, wherein the polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or up to and including 100% identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81.
In another embodiment, the invention relates to isolated polypeptides having WUS activity, wherein the polypeptide comprises at least a minimum whole integer number of contiguous amino acids from at least one polypeptide selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81, wherein the number of contiguous amino acids is selected from the range of 20 amino acids up to and including the full length of the polypeptide. For example, the isolated polypeptide comprises at least 20, 25, 30, 40, 50, 75,100, 200 contiguous amino acids, up to and including the full-length of at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81.
In another embodiment, the invention relates to a method to modulate the level of WUS in a cell, plant cell, or plant. The method comprises introducing into the cell an isolated polynucleotide of the invention, and expressing the polynucleotide in the cell, wherein the expression of the polynucleotide modulates the level of WUS in the cell. The introduced polynucleotide optionally comprises a DNA construct operably linked to a promoter active in the cell, in either sense or antisense orientation. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. In some embodiments the isolated polynucleotide is selected from the group consisting of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42, 44, 46,48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67,69, 70, 72, 74, 76, 77, 79, and 80. The cell is optionally cultured under conditions in order to generate a plant wherein the level of WUS is modulated. In other embodiments, the introduced polynucleotide optionally comprises an RNA molecule, which is introduced into the cell, plant cell, or plant. In another embodiment, the method to modulate the level of WUS in a cell, plant cell, or plant comprises introducing an isolated WUS polypeptide of the invention into the cell, plant cell, or plant. In some embodiments the isolated polypeptide is selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments, the isolated polypeptide is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24.
In another embodiment, the invention relates to a method to stimulate plant cell growth. In some embodiments, stimulation of plant cell growth can provide a positive selection means, stimulate organogenesis, stimulate embryogenesis, including the production of asexually derived embryos, such as somatic embryos, and/or apomictic embryos. The method comprises introducing into the cell an isolated polynucleotide of the invention, and expressing the polynucleotide in the cell, wherein the expression of the polynucleotide modulates the level of WUS in the cell, thereby stimulating plant cell growth. The introduced polynucleotide optionally comprises a DNA construct operably linked to a promoter active in the cell, in either sense or antisense orientation. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. In some embodiments the isolated polynucleotide is selected from the group consisting of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42, 44, 46, 48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 80. In some embodiments the polynucleotide is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. The cell is optionally cultured under conditions in order to generate a plant. In other embodiments, the introduced polynucleotide optionally comprises an RNA molecule, which is introduced into the cell, plant cell, or plant, thereby stimulating cell growth. In another embodiment, the method to plant cell growth comprises introducing an isolated WUS polypeptide of the invention into the cell, plant cell, or plant. In some embodiments the isolated polypeptide is selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments the isolated polypeptide is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. In some embodiments, cells stimulated to proliferate by polynucleotides and/or polypeptides of the invention are subsequently transformed with another polynucleotide of interest. WUS stimulates growth in a non-cell autonomous manner, therefore in some embodiments proliferating cells stimulated by WUS, but not comprising stably incorporated WUS are used as the host cells for transformation with a polynucleotide of interest.
In another embodiment, the invention relates to a method of positive selection for transformed plant cells. The method comprises introducing into a plant cell an isolated polynucleotide which encodes a polypeptide having WUS activity, expressing the polynucleotide, and culturing the transformed cells under conditions which provide positive selection for cells comprising the polynucleotide. Optionally, the culture conditions comprise conditions in which neither exogenous plant hormones, nor chemical selection agents are provided. The conditions may also comprise the addition of exogenous plant hormones, in any range of lower concentration up to the normal plant cell culture concentration. If the plant cell comprises another selectable marker, the conditions may comprise the addition of the selecting agent or method. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments the isolated polynucleotide is selected from the group consisting of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42,44, 46, 48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 80. In some embodiments the polynucleotide is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23.
In another embodiment, the invention relates to a method to produce asexually derived embryos. The method comprises introducing into the cell an isolated polynucleotide of the invention, and expressing the polynucleotide in the cell, wherein the expression of the polynucleotide produces an asexually derived embryo. The introduced polynucleotide optionally comprises a DNA construct operably linked to a promoter active in the cell, in either sense or antisense orientation. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. In some embodiments the isolated polynucleotide is selected from the group consisting of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42, 44, 46, 48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 80. In some embodiments the polynucleotide is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. The produced embryo is optionally cultured under conditions in order to generate a plant. In other embodiments, the introduced polynucleotide optionally comprises an RNA molecule, which results in the production of an asexually derived embryo. In another embodiment, the method to produce asexually derived embryos comprises introducing an isolated WUS polypeptide of the invention into the cell, plant cell, or plant. In some embodiments the isolated polypeptide is selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments the isolated polypeptide is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. In some embodiments the asexually derived embryos are somatic embryos or apomictic embryos. In some embodiments, the plant regenerated from the embryo has an apomictic phenotype.
In another embodiment, the invention relates to a method to generate an apomictic plant. The method comprises introducing into a plant cell an isolated polynucleotide which encodes a polypeptide having WUS activity, regenerating a plant comprising the polynucleotide, wherein the plant has an apomictic phenotype. The introduced polynucleotide optionally comprises a DNA construct operably linked to a promoter active in the cell. Optionally, the promoter active in the cell is an inducible promoter, a tissue-preferred promoter, a developmentally regulated promoter, or a promoter, or combination of promoters, having more than one of these properties. In one embodiment, the promoter is a nucellus-preferred promoter, such as the barley nuc-1 promoter, or an inducible promoter, like In2. In some embodiments, WUS expression may be further regulated by having another gradient, temporal or spatial, super-imposed by separating the coding region or encoded polypeptide into at least two segments which are separably regulated. In some embodiments, the coding region is interrupted by a recombinase flanked spacer region, which is excised when the appropriate recombinase is provided in a controlled manner. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. In some embodiments the isolated polynucleotide is selected from the group consisting of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41,42, 44, 46, 48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 80. In some embodiments the polynucleotide is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In other embodiments a polypeptide having WUS activity is reconstituted via intein splicing. Only cells comprising both segments that have been expressed and combined will comprise a polypeptide having WUS activity.
In another embodiment, the invention relates to a method to increase transformation frequency. The method comprises introducing into a plant cell an isolated polynucleotide of the invention which encodes a polypeptide having WUS activity, and expressing the polynucleotide in the cell, wherein the expression of the polynucleotide increases transformation frequency. Transformation frequency is measured relative to a wild-type or control plant cell that does not contain and/or express the polynucleotide of the invention. The introduced polynucleotide optionally comprises a promoter active in the cell operably linked to the isolated polynucleotide. In some embodiments, the introduced polynucleotide further comprises a polynucleotide of interest, which may be operably linked to a promoter active in the cell, or a polynucleotide of interest may be separately introduced. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. In some embodiments the isolated polynucleotide is selected from the group consisting of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42, 44, 46, 48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 80. In some embodiments the polynucleotide is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. Optionally, a transformed cell produced by the method is further cultured under conditions in order to generate a plant. In other embodiments, the introduced polynucleotide optionally comprises an RNA molecule which is introduced into the cell, plant cell, or plant. In another embodiment, the method comprises introducing an isolated WUS polypeptide of the invention into the cell, plant cell, or plant. In some embodiments the isolated polypeptide is selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments the isolated polypeptide is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. In certain embodiments, the plant cell is a typically non-transformable and/or recalcitrant plant cell.
In another embodiment, the invention relates to a method to stimulate organogenesis in a plant. The method comprises introducing into a plant cell an isolated polynucleotide of the invention which encodes a polypeptide having WUS activity, and expressing the polynucleotide in the cell, wherein the expression of the polynucleotide stimulates organogenesis. The introduced polynucleotide optionally comprises a promoter active in the cell operably linked to the isolated polynucleotide. In some embodiments, the introduced polynucleotide further comprises a polynucleotide of interest, which may be operably linked to a promoter active in the cell, or a polynucleotide of interest may be separately introduced. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. In some embodiments the isolated polynucleotide is selected from the group consisting of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42, 44, 46, 48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 80. In some embodiments the polynucleotide is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. Optionally, a transformed cell produced by the method is further cultured under conditions in order to generate a plant. In other embodiments, the introduced polynucleotide optionally comprises an RNA molecule which is introduced into the cell, plant cell, or plant. In another embodiment, the method comprises introducing an isolated WUS polypeptide of the invention into the cell, plant cell, or plant. In some embodiments the isolated polypeptide is selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments the isolated polypeptide is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24.
In another embodiment, the invention relates to a method to stimulate somatic embryogenesis in a plant. The method comprises introducing into a plant cell an isolated polynucleotide of the invention which encodes a polypeptide having WUS activity, and expressing the polynucleotide in the cell, wherein the expression of the polynucleotide stimulates somatic embryogenesis. The introduced polynucleotide optionally comprises a promoter active in the cell operably linked to the isolated polynucleotide. In some embodiments, the introduced polynucleotide further comprises a polynucleotide of interest, which may be operably linked to a promoter active in the cell, or a polynucleotide of interest may be separately introduced. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments, the isolated polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. In some embodiments the isolated polynucleotide is selected from the group consisting of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42, 44, 46, 48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 80. In some embodiments the polynucleotide is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. Optionally, a transformed cell produced by the method is further cultured under conditions in order to generate a plant. In other embodiments, the introduced polynucleotide optionally comprises an RNA molecule which is introduced into the cell, plant cell, or plant. In another embodiment, the method comprises introducing an isolated WUS polypeptide of the invention into the cell, plant cell, or plant. In some embodiments the isolated polypeptide is selected from the group consisting of SEQ ID NOS: 27, 29, 31, 33, 36, 38, 40, 43, 45, 47, 50, 52, 54, 57, 59, 61, 64, 66, 68, 71, 73, 75, 78, and 81. In some embodiments the isolated polypeptide is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24.
In another embodiment, the invention relates to any transgenic host cell, plant cell, plant, and/or seed produced by the methods of the invention. This embodiment includes those instances wherein the host cell, plant cell, plant, and/or seed is transgenic and comprises a polynucleotide of the invention. This embodiment also includes those instances wherein a polynucleotide or polypeptide of the invention was used in a transient manner in order to generate host cells, plant cells, plants and/or seeds having the desired traits. In certain embodiments, the host cell, plant cell, plant, and/or seed produced by the methods of the invention may further comprise other polynucleotides of interest.
Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. Table 1 also identifies the cDNA clones as individual ESTs (“EST”), the sequences of the entire cDNA insert comprising the indicated cDNA clone (“full insert sequence” or “FIS”), contigs assembled from two or more EST, FIS, and/or PCR sequences (“Contig”), or sequences encoding the entire protein derived from an EST, an FIS, a contig, or an FIS and PCR fragment sequence (“complete gene sequence” or “CGS”). Nucleotide SEQ ID NOs: 1, 5,11, and 15 correspond to nucleotide SEQ ID NOs: 1, 3, 5, and 7, respectively, presented in U.S. Provisional Application No. 60/157216, filed Oct. 1, 1999. Amino acid SEQ ID NOs: 2, 6, 12, and 16 correspond to amino acid SEQ ID NOs: 2, 4, 6, and 8, respectively, presented in U.S. Provisional Application No. 60/157216, filed Oct. 1, 1999. Nucleotide SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, 21, and 23, and amino acid SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 25 retain the same sequence identifiers as presented in PCT International Application No. PCT/US00/26648 filed Sep. 28, 2000. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.
| TABLE 1 | ||||
| WUSCHEL Sequences | ||||
| Protein (Plant | SEQ ID NO: | |||
| Source) | Clone Designation | Status | (Nucleotide) | (Ami no Acid) |
| WUS (Corn) | Contig of | Contig | 1 | 2 |
| cpg1c.pk006.b16 | ||||
| cpi1c.pk012.p19 | ||||
| WUS (Corn) | cpi1c.pk012.p19 (FIS) | CGS | 3 | 4 |
| WUS (Corn) | p0016.ctsas50r | EST | 5 | 6 |
| WUS (Corn) | p0016.ctsas50r | FIS | 7 | 8 |
| WUS (Corn) | p0058.chpab57r (FIS) | CGS | 9 | 10 |
| WUS (Corn) | p0083.cldev71r | EST | 11 | 12 |
| WUS (Corn) | p0083.cldev71r | FIS | 13 | 14 |
| WUS (Soybean) | Contig of | Contig | 15 | 16 |
| ses4d.pk0033.c8 | < /tr>||||
| WUS (Soybean) | scr1c.pk001.d2 | FIS | 17 | 18 |
| WUS (Soybean) | ses4d.pk0033.c8 (FIS) | CGS | 19 | 20 |
| WUS (Soybean) | sgs5c.pk0002.f2 | CGS | 21 | 22 |
| WUS (Soybean) | Contig of | CGS | 23 | 24 |
| s sm.pk0060.h4 (FIS) | ||||
| NCBI GI No. 4395781 | ||||
| WUS (Corn) | Contig of | CGS | 26 | 27 |
| p 0016.ctsas50r (FIS) | ||||
| PCR fragments (1st intron | ||||
| spliced) | ||||
| WUS (Corn) | p0016.ctsas50r, 1st & | CGS | 28 | 29 |
| complete 2nd intron | ||||
| spliced | ||||
| WUS (Corn) | p0016.ctsas50r, 1st & | CGS | 30 | 31 |
| alternate 2nd intron | ||||
| spliced | ||||
| WUS (Corn) | Contig of | CGS | 32 | 33 |
| p 0083.cldev71r (FIS) | ||||
| PCR fragments | ||||
| WUS (Corn) | Genomic DNA from B73 | CGS | 34 | |
| correspondi ng to | ||||
| p0016.ctsas50r | ||||
| WUS (Corn) | Genomic DNA from B73 | CGS | 35 | 36 |
| corresponding to | ||||
| p0016.ctsas50r, 1st intron | ||||
| spliced | ||||
| WUS (Corn) | Genomic DNA from B73 | CGS | 37 | 38 |
| corresponding to | ||||
| p0016.ctsas50r, 1st & 2nd | ||||
| complete intron spliced | ||||
| WUS (Corn) | Genomic DNA from B73 | CGS | 39 | 40 |
| corresponding to | ||||
| p0016.ctsas50r, 1st & | ||||
| alternate 2nd intron | ||||
| spliced | ||||
| WUS (Corn) | Genomic DNA from | CGS | 41 | |
| Mo17 corresponding to | ||||
| p0016.ctsas50r | ||||
| WUS (Corn) | Genomic DNA from | CGS | 42 | 43 |
| Mo17 corresponding to | ||||
| p0016.ctsas50r, 1st intron | ||||
| spliced | ||||
| WUS (Corn) | Genomic DNA from | CGS | 44 | 45 |
| Mo17 corresponding to | ||||
| p0016.ctsas50r, 1st & 2nd | ||||
| complete intron spliced | ||||
| WUS (Corn) | Genomic DNA from | CGS | 46 | 47 |
| Mo17 corresponding to | ||||
| p0016.ctsas50r, 1st & | ||||
| alternate 2nd intron | ||||
| spliced | ||||
| WUS (Corn) | Genomic DNA from 07D | CGS | 48 | |
| correspondi ng to | ||||
| p0016.ctsas50r | ||||
| WUS (Corn) | Genomic DNA from 07D | CGS | 49 | 50 |
| corresponding to | ||||
| p0016.ctsas50r, 1st intron | ||||
| spliced | ||||
| WUS (Corn) | Genomic DNA from 07D | CGS | 51 | 52 |
| corresponding to | ||||
| p0016.ctsas50r, 1st & 2nd | ||||
| complete intron spliced | ||||
| WUS (Corn) | Genomic DNA from 07D | CGS | 53 | 54 |
| corresponding to | ||||
| p0016.ctsas50r, 1st & | ||||
| alternate 2nd intron | ||||
| spliced | ||||
| WUS (Corn) | Genomic DNA from KW3 | CGS | 55 | |
| correspondi ng to | ||||
| p0016.ctsas50r | ||||
| WUS (Corn) | Genomic DNA from KW3 | CGS | 56 | 57 |
| corresponding to | ||||
| p0016.ctsas50r, 1st intron | ||||
| spliced | ||||
| WUS (Corn) | Genomic DNA from KW3 | CGS | 58 | 59 |
| corresponding to | ||||
| p0016.ctsas50r, 1st & 2nd | ||||
| complete intron spliced | ||||
| WUS (Corn) | Genomic DNA from KW3 | CGS | 60 | 61 |
| corresponding to | ||||
| p0016.ctsas50r, 1st & | ||||
| alternate 2nd intron | ||||
| spliced | ||||
| WUS (Corn) | Genomic DNA from 3DT | CGS | 62 | |
| correspondi ng to | ||||
| p0016.ctsas50r | ||||
| WUS (Corn) | Genomic DNA from 3DT | CGS | 63 | 64 |
| corresponding to | ||||
| p0016.ctsas50r, 1st intron | ||||
| spliced | ||||
| WUS (Corn) | Genomic DNA from 3DT | CGS | 65 | 66 |
| corresponding to | ||||
| p0016.ctsas50r, 1st & 2nd | ||||
| complete intron spliced | ||||
| WUS (Corn) | Genomic DNA from 3DT | CGS | 67 | 68 |
| corresponding to | ||||
| p0016.ctsas50r, 1st & | ||||
| alternate 2nd intron | ||||
| spliced | ||||
| WUS (Corn) | Genomic DNA from 09B | CGS | 69 | |
| correspondi ng to | ||||
| p0016.ctsas50r | ||||
| WUS (Corn) | Genomic DNA from 09B | CGS | 70 | 71 |
| corresponding to | ||||
| p0016.ctsas50r, 1st intron | ||||
| spliced | ||||
| WUS (Corn) | Genomic DNA from 09B | CGS | 72 | 73 |
| corresponding to | ||||
| p0016.ctsas50r, 1st & 2nd | ||||
| complete intron spliced | ||||
| WUS (Corn) | Genomic DNA from 09B | CGS | 74 | 75 |
| corresponding to | ||||
| p0016.ctsas50r, 1st & | ||||
| alternate 2nd intron | ||||
| spliced | ||||
| WUS (Corn) | Genomic WUS5, gss | CGS | 76 | |
| WUS (Corn) | Genomic WUS5 gss, | CGS | 77 | 78 |
| single intron spliced | ||||
| WUS (Corn) | Genomic WUS6, gss | CGS | 79 | |
| WUS (Corn) | Genomic WUS6, gss, 1st | CGS | 80 | 81 | & 2nd intron spliced |
| WUS | Consensus WUS2, 1st | 88 | ||
| in tron spliced | ||||
| WUS | Consensus WUS2, 1st & | 89 | ||
| 2nd complete intron | ||||
| spliced | ||||
| WUS | Consensus WUS2, 1st & | 90 | ||
| alter nate 2nd intron | ||||
| spliced | ||||
| Motif | 25 amino acid motif | 91 | ||
The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
In the context of this disclosure, a number of terms shall be utilized. The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolated polynucleotide of the present invention may include a polynucleotide comprising at least 60 contiguous nucleotides, or at least 40 contiguous nucleotides, and optionally at least 30 contiguous nucleotides derived from SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23, or the complement of such sequences.
The length of the polynucleotide is given as an integer selected from the group consisting of from at least 30 to the length of the nucleic acid sequence from which the polynucleotide is a subsequence of. Therefore, an isolated polynucleotide of the present invention also includes polynucleotides comprising contiguous nucleotides of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 175, 200, 250, 300, 500 or up to and including the full-length of the polynucleotides of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 and the polynucleotides of SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42, 44, 46,48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, or 80. Optionally, the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5. The subsequences can be separated by any integer of nucleotides from 1 to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100, or 200 nucleotides. The subsequences of the present invention can comprise structural characteristics of the sequence from which it is derived, for example including but not limited to, signal sequences, translational start sites, polyadenylation sites, conserved motifs, introns, exons, UTR's, and the like.
As used herein “fully complementary” refers to a nucleic acid sequence which is 100% complementary to a reference nucleic acid sequence.
The term “apomixis” is used to describe asexual reproduction that replaces or substitutes for sexual methods of reproduction. When apomixis occurs, embryos are produced from maternal tissue and use only the maternal genome, and are referred to as “apomictic embryos”. A plant capable of producing embryos in the absence of fertilization is referred to as an “apomictic plant”, or a plant that has an “apomictic phenotype”.
As used herein, “Wuschel polynucleotide” or “WUS polynucleotide” means a polynucleotide encoding a polypeptide with Wuschel activity, or a polynucleotide capable of modulating the expression of mRNA or protein in a host cell. The term is also inclusive of fragments, variants, homologues, alleles or precursors with the any one of the above stated functions.
A “protein” or “polypeptide” is a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide. Each protein or polypeptide has a unique function. As used herein, “polypeptide” means proteins, protein fragments, modified proteins (e.g., glycosylated, phosphorylated, or other modifications), amino acid sequences and synthetic amino acid sequences. The polypeptide can be modified or not.
As used herein, “Wuschel polypolypeptide” or “WUS polypeptide” means a polypeptide having Wuschel activity, i.e., involved in the initiation and maintenance of stem cells in plants. Wuschel activity stimulates cell growth, including stem cells. Wuschel is a plant homeodomain protein, comprising an ‘atypical’ (compared to the animal homeodomain motif) helix-loop-helix-turn-helix homeodomain motif comprising extra amino acid residues in the loop and/or turn of the domain. Wuschel proteins may further comprise other conserved motifs, such as the two conserved Wuschel C-terminal domains, the (E/R)TLPLFP and A(A/S)LEL(S/T)L domains. The term is also inclusive of fragments, variants, homologues, with the any one of the above stated functions.
The term “isolated” refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in the cell other than the locus native to the material. The term “isolated” polynucleotide refers to a polynucleotide that is substantially free from other nucleic acid sequences, such as other chromosomal and extrachromosomal DNA and RNA, that normally accompany or interact with it as found in its naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
The term “recombinant” means, for example, that a nucleic acid sequence is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated nucleic acids by genetic engineering techniques.
As used herein, “contig” refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequences of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and thus their corresponding nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.
As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-à-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof. The terms “substantially similar” and “corresponding substantially” are used interchangeably herein.
Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications of the nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level of the polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell. For example, a substantially similar nucleic acid fragment representing at least one of 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell. The level of the polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level of the polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.
For example, it is well known in the art that silencing of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by using nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Consequently, an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (or at least one of 40, or at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of a WUS polypeptide in a host cell. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a virus or in a host cell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial) may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of a polypeptide or enzyme activity in a host cell that does not contain the isolated polynucleotide.
Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, or to screen for highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. More stringent conditions may use higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C.
Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, or at least about 80% identical to the amino acid sequences reported herein. Nucleic acid fragments that encode amino acid sequences that are about 85% identical to the amino acid sequences are reported herein. Nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences are also reported herein. Nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences are reported herein. Suitable nucleic acid fragments not only have the above identities but typically encode a polypeptide having at least 50 amino acids, at least 100 amino acids, at least 150 or 180 amino acids, at least 200 or 230 amino acids, or at least 250 amino acids.
Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers & Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman & Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson & Lipman (1988) PNAS 85:2444-2448; the algorithm of Karlin & Altschul (1990) PNAS 87:2264-2268, modified as in Karlin & Altschul (1993) PNAS 90:5873-5877.
Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, PILEUP, PRETTY, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucl. Acids Res. 16:10881-90; Huang et al. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.
Sequence alignments and percent identity calculations, particularly for sequences selected from the group consisting of SEQ ID NOS: 1-25, were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Percent sequence identity can also be calculated over the entire length of the sequences compared using the alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), for example as implemented in the GAP algorithm in the GCG™ software package (Accelrys, San Diego, Calif.). GAP Version 10 uses the following default parameters: % identity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3;% identity for an amino acid sequence using the BLOSUM62 scoring matrix (Henikoff & Henikoff, PNAS 89:10915-10919 (1992)), GAP Weight of 8 and Length Weight of 2.
Multiple alignment of the sequences, particularly for sequences selected from the group consisting of SEQ ID NOS: 1-25, was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Multiple alignments of polynucleotide and polypeptide sequences can also be generated using the PileUp program (Feng & Doolittle, J. Mol. Evol. 25:351-360 (1987)) in the GCG™ software package (Accelrys, San Diego, Calif.), which uses the following default parameters for amino acid sequences of the BLOSUM62 scoring matrix (Henikoff & Henikoff, PNAS 89:10915-10919 (1992)), GAP Weight=8 and Length Weight=2. For polynucleotide sequences, PILEUP uses the default parameters of GAP Weight=5, and Length Weight=1.
Optionally, one of skill can use the PRETTY program for alignment of polynucleotide and polypeptide sequences, as found in the GCG™ software package (Accelrys, San Diego, Calif.), which uses the following default parameters for amino acid sequences of the BLOSUM62 scoring matrix (Henikoff & Henikoff, PNAS 89:10915-10919 (1992)), GAP Weight=8 and Length Weight=2, and which also generates a consensus sequence for the alignment. For polynucleotide sequences, PRETTY uses the default parameters of GAP Weight=5, and Length Weight=1.
Substantially similar polynucleotides of the instant invention may also be characterized by the percent identity of the nucleic acid sequences to the polynucleotides disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Isolated polynucleotides of the present invention comprise nucleic acids sequences that have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the polynucleotides disclosed, e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 19, 21, or 23, or SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 42, 44, 46, 48, 49, 51, 53, 55, 56, 58, 60, 62, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, or 80. Sequence alignments and percent identity calculations may be performed using standard methods and algorithms, as described above.
As used herein, “substantially similar” in reference to polypeptides and amino acid sequences refers to polypeptides wherein changes (e.g., modification, substitution, deletion, insertion) in one or more amino acids do not affect the functional properties of the polypeptide, i.e., at least one activity of the polypeptide is retained, such as a protein-protein interaction, antibody binding, or enzymatic and/or biological activity. It is therefore understood that the invention encompasses more than the specific exemplary amino acid sequences and includes functional equivalents thereof. The terms “substantially similar” and “corresponding substantially” are used interchangeably herein. For these purposes, substantially similar polypeptides normally comprise amino acid sequences having a sequence identity of at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or greater.
A “substantial portion” of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also NCBI BLAST website). In general, a sequence of ten or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization). In addition, short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
“Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
“Synthetic nucleic acid fragments” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. “Chemically synthesized”, as related to a nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the nucleic acid fragments can be tailored for optimal gene expression based on optimization of the nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.
“Gene” refers to a polynucleotide that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native” refers to a polynucleotide as found in nature with its own regulatory sequences. “Chimeric” refers any polynucleotide that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign gene” refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.
As used herein, “heterologous” in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
“Coding sequence” refers to a nucleotide sequence that codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
“Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.
“Translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol. 3:225-236).
“3′ non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.
“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into polypeptides by the cell. “cDNA” refers to DNA that is complementary to and derived from an mRNA template. The cDNA can be single-stranded or converted to double stranded form using, for example, the Klenow fragment of DNA polymerase 1. “Sense-RNA” refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.
The term “operably linked” refers to the association of two or more nucleic acid fragments on a single polynucleotide so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. The term is also inclusive of protein trans-splicing events (e.g. inteins) which produce a single functional polypeptide.
The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein.
“Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.
“Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by reference).
“Altered levels” or “altered expression” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.
“Null mutant” refers here to a host cell which either lacks the expression of a certain polypeptide or expresses a polypeptide which is inactive or does not have any detectable expected enzymatic function.
“Mature protein” or the term “mature” when used in describing a protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed.
“Precursor protein” or the term “precursor” when used in describing a protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.
A “chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide. A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1627-1632).
“Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference). Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Flevin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
“Stable transformation” refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance. In contrast, “transient transformation” refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance. The term “transformation” as used herein refers to both stable transformation and transient transformation.
The terms “recombinant construct”, “expression construct” and “recombinant expression construct” are used interchangeably herein. These terms refer to a functional unit of genetic material that can be inserted into the genome of a cell using standard methodology well known to one skilled in the art. Such construct may be used by itself or may be used in conjunction with a vector. If a vector is used, the choice of vector is dependent upon the method that will be used to transform host plants as is well known to those skilled in the art.
In reference to transformation, particularly stable transformation, a “recalcitrant” cell or line is a cell or line wherein introduction of a polynucleotide of interest into the cell generally does not result in the recovery of stably transformed tissue that can be regenerated to produce a transformed plant. The term is inclusive of known recalcitrant genotypes, as well as older tissues, or tissue sources which generally do not respond and/or proliferate in standard culture conditions.
As used herein “transient transformation” refers to the transfer of a nucleic acid fragment or protein into the nucleus (or DNA-containing organelle) of a host organism resulting in gene expression without, necessarily, resulting in integration and stable inheritance.
As used herein, the term “positive selection” refers to any means by which a selectable phenotype or growth advantage is produced relative to control cells in the absence of chemical selection. The term is inclusive of positive selection.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”). “Motifs” refers to short regions of conserved sequences of nucleic acids or amino acids that comprise part of a longer sequence. These conserved motifs may be associated with a specific function. These conserved motifs could be used to identify new homologues in plants. It is expected that some or all of the motifs may be found in a homologue. Also, it is expected that one or two of the conserved amino acids in any given motif may differ in a true homologue.
“PCR” or “polymerase chain reaction” is well known by those skilled in the art as a technique used for the amplification of specific DNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).
As used herein, the term “plant” includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), plant tissue, seeds and plant cells, and progeny of the same. Plant cell, as used herein includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
As used herein, the term “organogenesis” refers to stem cell and/or meristematic activity that leads to the differentiation of organs.
As used herein, the term “asexually derived embryo” refers to any embryo generated in the absence of fertilization. The term is inclusive of apomictic and somatic embryos. As used herein, the term “somatic embryogenesis” refers to non-zygotic embryogenesis.
As used herein, the term “stem cells” refers to pleuripotent cells that give rise to both additional stem cells as well as cells that can differentiate into other cell types.
The present invention concerns isolated polynucleotides which encode polypeptides having WUS activity or that can modulate the level or activity of WUS in a cell, isolated polypeptides having WUS activity, and methods of using these sequences. Wuschel is involved in the initiation and maintenance of plant stem cells. Loss-of-function mutations in the WUS gene lead to shoot and floral meristems that fail to self-maintain. WUS mRNA expression is localized to a small group of cells in the central zone, below the L3 of the apical meristem, and affects stem cell fate in a non-cell autonomous manner (Mayer et al. (1998) Cell 95:805-815, herein incorporated by reference). Cells of the central zone divide relatively infrequently, while cells in the surrounding peripheral zone divide rapidly. Cell position in the meristem appears to regulate stem cell fate, via the interactions of many regulatory genes, including WUS, STM, CLV and the like (see, e.g., Byrne et al. (2003) Curr. Op. Gen. Dev. 13:551-557; Doerner (2000) Curr. Biol. 10:R826-R829; and Sharma, et al. (2003) PNAS 100:11823-11829, the contents of which are all herein incorporated by reference).
WUS encodes a homeodomain transcription factor polypeptide whose function is to bind a target DNA sequence and direct expression of the target gene. Binding of WUS to a DNA sequence was confirmed by Lohmann and co-workers (2001) Cell 105:793-803. The homeodomain region is approximately 61-64 amino acids with a substructure of alpha helix1-loop-alpha helix2-turn-alpha helix 3. Alpha helix3 makes the primary contact with the DNA sequence, although other substructures of the homeodomain polypeptide affect the level and specificity of interaction with the target DNA. The WUS homeodomain falls into a subcategory of homeodomain transcription factors depending on the presence and number of extra amino acids present in the loop and turn substructures relative to animal homeodomain transcription factors. One or two extra amino acids in the loop and four extra amino acids in the turn define the WUS family of transcription factors from other homeodomain transcription factors (“1+4” or “2+4'categories, Kamiya et al. (2003) Plant J 35:429-441). For example, WUS2 (p001 6.ctsas50r) is in the “2+4” category of homeodomain factors, like the Arabidopsis protein, whereas the other WUS sequences of the present invention belong to the “1+4” category (e.g., p0083.cldev71r, represented in SEQ ID NO: 33). Two secondary protein motifs have been identified (Stuurman et al. (2002) Genes Dev. 16:2213-2218) and include a highly conserved (E/R)TLPLFP and less conserved A(A/S)LEL(S/T)L amino acid sequences near the C-terminal end of the polypeptide. The role of these secondary motifs are not clear, but such leucine-rich motifs are generally involved in protein-protein interactions. The A(A/S)LEL(S/T)L shows significant similarity to C-terminal motifs identified in zn-finger proteins and ERFs, which have been implicated to act as a repressor domain via protein-protein interaction (Ohta, et al. (2001) Plant Cell 13:1959-1968; and Dinkins et al. (2003) Plant Science 165:33-41; herein incorporated by reference).
The present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 70% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, and 12, (b) a second nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 70% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs: 14, 16, 18, and 20, (c) a third nucleotide sequence encoding a polypeptide of at least 180 amino acids having at least 70% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO: 24, (d) a fourth nucleotide sequence encoding a polypeptide of at least 230 amino acids having at least 70% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO: 22, (e) a fifth nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs: 6, 8, and 10, and (f) a sixth nucleotide sequence comprising the complement of (a), (b), (c), (d), or (e).
The first nucleotide sequence may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, that codes for the polypeptide selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24.
Nucleic acid fragments encoding at least a portion of several WUS proteins have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).
For example, genes encoding other WUS proteins, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, an entire sequence can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.
In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220). Products generated by the 3′ and 5′ RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165). Consequently, a polynucleotide comprising a nucleotide sequence of at least one of 60 (or at least 40, or at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a WUS polypeptide, optionally a substantial portion of a plant WUS polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (or of at least 40, or at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (e.g., cDNA inserted in a cloning vector) using the oligonucleotide primer. Optionally, the amplified nucleic acid fragment will encode a portion of a WUS polypeptide.
Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).
In another embodiment, this invention concerns viruses and host cells comprising either the chimeric genes of the invention as described herein or an isolated polynucleotide of the invention as described herein. Examples of host cells which can be used to practice the invention include, but are not limited to, yeast, bacteria, and plants.
As was noted above, the nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed polypeptides are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering development (e.g., the initiation and maintenance of meristem apical initials) in those plants.
Overexpression of the proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. The chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3′ non-coding sequences encoding transcription termination signals may also be provided. The instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.
It is well known in the art that silencing of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by using nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed.
Reduction of the activity of specific genes (also known as gene silencing, or gene suppression) is desirable for several aspects of genetic engineering in plants. Many techniques for gene silencing are well known to one of skill in the art, including but not limited to antisense technology (see, e.g., Sheehy et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos. 5,107,065; 5,453, 566; and 5,759,829); cosuppression (e.g., Taylor (1997) Plant Cell 9:1245; Jorgensen (1990) Trends Biotech. 8(12):340-344; Flavell (1994) PNAS USA 91:3490-3496; Finnegan et al. (1994) Bio/Technology 12: 883-888; and Neuhuber et al. (1994) Mol. Gen. Genet. 244:230-241); RNA interference (Napoli et al. (1990) Plant Cell 2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13:139-141; Zamore et al. (2000) Cell 101:25-33; and Montgomery et al. (1998) PNAS USA 95:15502-15507), virus-induced gene silencing (Burton et al. (2000) Plant Cell 12:691-705; and Baulcombe (1999) Curr. Op. Plant Bio. 2:109-113); target-RNA-specific ribozymes (Haseloff et al. (1988) Nature 334: 585-591); hairpin structures (Smith et al. (2000) Nature 407:319-320; WO 99/53050; WO 02/00904; and WO 98/53083); ribozymes (Steinecke et al. ((1992) EMBO J. 11:1525; and Perriman et al. ((1993) Antisense Res. Dev. 3:253); oligonucleotide mediated targeted modification (e.g., WO 03/076574 and WO 99/25853); Zn-finger targeted molecules (e.g., WO 01/52620; WO 03/048345; and WO 00/42219); and other methods or combinations of the above methods known to those of skill in the art. The references cited above are herein incorporated by reference in their entirety.
Plasmid vectors comprising the instant isolated polynucleotide (or chimeric gene) may be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will likely result in different levels and patterns of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), and thus multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screen