text: On Wed, 2 May 2007, Andrew Morton wrote:> > At some point I dream that SLUB could become the default but I thought > > this would take at least 6 month or so. If want to force this now then I > > will certainly have some busy weeks ahead.> > s/dream/promise/ ;)> > Six months sounds reasonable - I was kind of hoping for less. Make it> default-to-on in 2.6.23-rc1, see how it goes.Here is how I think the future could developCycle SLAB SLUB SLOB SLxB2.6.22 API fixes Stabilization API fixesMajor event: SLUB availability as experimental2.6.23 API upgrades Perf. Valid. EOLMajor events: SLUB performance validation. Switch off experimental (could even be the default) Slab allocators support targeted reclaim for at least one slab cache (dentry?) (vacate/move all objects in a slab)2.6.24 Earliest EOL Stable - ExperimentsMajor events: SLUB stable. Stable targeted reclaim for all major reclaimable slabs. Maybe experiments with another new allocator?2.6.25 EOL default - ?Death of SLAB. SLUB default. Hopefully new ideas on the horizon.-To unsubscribe from this list: send the line "unsubscribe linux-kernel" inthe body of a message to email@example.com majordomo info at http://vger.kernel.org/majordomo-info.htmlPlease read the FAQ at http://www.tux.org/lkml/


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Contribution to Book
Rules of Engagement Agreement [Book Chapter]ILT Faculty Bookshelf
Document TypeBook
Publication Date1-1-2007
AbstractAbout this book: "For those who think online learning can't be truly interactive, Patti Shank and her colleagues clearly demonstrate--in hundreds of examples--that it can. The real lesson in The Online Learning Idea Book is that technology doesn't build interactive learning; creative thinking and good, solid instructional design does. Using even a smidgen of the great ideas in this book will increase the learning effectiveness of any online program."--Marc J. Rosenberg, consultant, and author of Beyond E-Learning "Patti Shank has collected great ideas about online learning and teaching from all over the globe. If you are an online instructor or instructional designer looking for new ways to involve and engage your learners, you'll be inspired by this book!"--Terry Morris, associate professor, William Rainey Harper Colleges Filled with techniques, tools, tips, examples, resources, and dozens of "great ideas, this invaluable resource helps people who are looking to build online instructional materials -- or improve existing materials -- discover and implement what the best and brightest in industry and education are doing to make online learning more engaging and compelling. Increase your know-how in the following areas: * Look and Feel: how to increase ease-of-use* Graphics and Multimedia: how to make instructional graphics engaging and compelling* Activities: how to make instruction itself engaging and compelling* Tools: how to use a variety of online tools* Instructional Design: how to design better and faster.
This is a book chapter in the book: The Online Learning Idea Book, Volume 1: 95 Proven Ways to Enhance Technology-Based and Blended Learning
Citation InformationJoanna C. Dunlap. "Rules of Engagement Agreement [Book Chapter]" (2007)
Available at: http://works.bepress.com/joanna-dunlap/49/

subset: commoncrawl


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A reader of this blog wil surely notice that Carl Jung occupies a prominent place in my intellectual landscape. I discovered him in my youth and have returned to him again and again. It is a complicated relationship; every reading of him begins with pleasure and surprise but ends with frustration and disappointment. He was an astute observer, well-educated, a dedicated psychologist, a remarkably spiritual person. For all that, he is also deeply a man of his time, his writing caught between philology and scientism. Both of these are dessicative and, while that has its place in study, it leaves only traces of his subject matter's vitality available to the reader. For an individual so caught up in spiritual matters, his accounts of spiritual life are startlingly abstract.
It shouldn't be surprising, then, that so many who are actively engage in spiritual work disparage treating the spiritual world merely as a repository of archetypes. Psychologizing spirit is unsatisfying. However, in recoiling from it, we often overlook something quite important–spiritual matters are entangled with psychological matters. They aren't identical with each other, but they aren't disparate either. This is one reason why I keep coming back to Jung. While he didn't get it quite right, he did get it. While I can't simply rest intellectually within Jung's work, I can wrest from it the means for getting at that entanglement more clearly.


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So far in 2012, Mecklenburg County has issued more than $1 billion in construction permits, nearly double the value of permits issued by this time last year.
As of Tuesday, the county had issued 6,480 permits collectively worth $1.03 billion.
By the same time in 2011, 5,656 permits with a total value of $571.1 million had been issued.
This year, the permit with the largest value, at $123.7 million, is for a Charlotte Douglas International Airport hourly parking deck that will feature seven levels, 7,000 spaces and a rental car-return facility.
Archer Western Contractors is the general contractor for the project, which has an anticipated completion date of December 2014.


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Don't Miss!
May 29, 2008
Carved Fruit Centerpieces
Carved fruit centerpieces - some so beautiful they take your breath away. Recently I was watching "Fantasy Fruit Sculptures" on Food Network which was held on the island of Hawaii. It involved four fruit sculpting artists turning ordinary watermelons and other common fruits and vegetables (carrots, cucumbers, radishes, cabbage and cantaloupes) into magnificent carved centerpieces. The art of fruit and vegetable carving is called kae-sa-lak. It is a highly prized skill in China, Japan, Korea, and Thailand - originally used to decorate only the tables of the very rich.
The story of fruit and vegetable carving began over 700 years ago. Held at the full moon in December, many people released small, decorated rafts of flowers, carved fruit and candles onto the river to thank the soul of water for all their blessings.
Posted by Beautiful Bridal at 8:54 AM 0 comments
May 18, 2008
Sunflower Wedding Stamps
Sunflower wedding stamps - our choice to use on wedding invitations for our talented artist and youngest daughter, Danielle. Her quirky and colorful view of the world required a flower with sunny brightness that matched her eager-to-please personality. Plus, she chose April for her wedding date and here in Southeast Virginia that means rain, rain, and still more rain (makes for amazingly beautiful azaleas though). Sunflowers thrive in very hot weather and commonly bloom during summer and early fall. Not to worry - we loved the convenience and affordability of silk sunflowers anyway. When the tender breast of chicken glazed with lovely fresh peach preserves, ginger and honey was served, the garlic mashed potatoes eaten and the champagne toast finally made, no one seemed to care it wasn't the perfect time of year for a sunflower themed wedding.
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Pathogens 2013, 2(2), 422-435; doi:10.3390/pathogens2020422
Can We Prevent Antimicrobial Resistance by Using Antimicrobials Better?
Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, Garrod Building, Turner Street, Whitechapel, London, E1 2AD, UK
Medical Microbiology, Centre for Infection, Division of Clinical Studies, St George's, University of London, Cranmer Terrace, London SW17 0RE, UK
Author to whom correspondence should be addressed.
Received: 23 April 2013 / Revised: 24 May 2013 / Accepted: 1 June 2013 / Published: 10 June 2013
(This article belongs to the Special Issue Pathogen Infection Models)
View Full-Text | Download PDF [334 KB, uploaded 10 June 2013]
AbstractSince their development over 60 years ago, antimicrobials have become an integral part of healthcare practice worldwide. Recently, this has been put in jeopardy by the emergence of widespread antimicrobial resistance, which is one of the major problems facing modern medicine. In the past, the development of new antimicrobials kept us one step ahead of the problem of resistance, but only three new classes of antimicrobials have reached the market in the last thirty years. A time is therefore approaching when we may not have effective treatment against bacterial infections, particularly for those that are caused by Gram-negative organisms. An important strategy to reduce the development of antimicrobial resistance is to use antimicrobials more appropriately, in ways that will prevent resistance. This involves a consideration of the pharmacokinetic and pharmacodynamics properties of antimicrobials, the possible use of combinations, and more appropriate choice of antimicrobials, which may include rapid diagnostic testing and antimicrobial cycling. Examples given in this review include Mycobacterium tuberculosis, Gram-negative and Gram-positive organisms. We shall summarise the current evidence for these strategies and outline areas for future development. View Full-Text
Keywords: antimicrobial resistance; pharmacokinetics; pharmacodynamics; minimum inhibitory concentration (MIC); mutant prevention concentration (MPC); microbiota; colonisation resistance; antibiotic combination; bacteria-antimicrobial pairing; antimicrobial cycling
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).
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Soothill, G.; Hu, Y.; Coates, A. Can We Prevent Antimicrobial Resistance by Using Antimicrobials Better? Pathogens 2013, 2, 422-435.
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Soothill G, Hu Y, Coates A. Can We Prevent Antimicrobial Resistance by Using Antimicrobials Better? Pathogens. 2013; 2(2):422-435.Chicago/Turabian Style
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Tag: Hardwood
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Mark and Abbie Jury
Writer, gardener, TV presenter
My plant obsession
News and views from a Gloucestershire gardener.
Pottering around my Devon Cottage Garden
Because a community is more than just people
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Gardening in summer, painting in winter. Life is good.
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Plants seeds for everyone: wildflowers, medicinals and alpine species
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Patent application title: Method for Generating Hypermutable Plants
Nicholas C. Nicolaides (Boothwyn, PA, US)
Nicholas C. Nicolaides (Boothwyn, PA, US)
Luigi Grasso (Philadelphia, PA, US)
Phillip M. Sass (Audubon, PA, US)
Ken Kinzler (Bel Air, MD, US)
Bert Vogelstein (Baltimore, MD, US)
Morphotek, Inc.
IPC8 Class: AC12N1587FI
USPC Class:
Class name: Multicellular living organisms and unmodified parts thereof and related processes method of introducing a polynucleotide molecule into or rearrangement of genetic material within a plant or plant part
Publication date: 2011-03-03
Patent application number: 20110055964
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Patent application title: Method for Generating Hypermutable Plants
Bert Vogelstein
Nicholas C. Nicolaides
Luigi Grasso
Phillip M. Sass
Ken Kinzler
Origin: ,
IPC8 Class: AC12N1587FI
USPC Class:
Publication date: 03/03/2011
Patent application number: 20110055964
Blockade of mismatch repair in a plant can lead to hypermutation and a new
genotype and/or phenotype. One approach used to generate hypermutable
plants is through the expression of dominant negative alleles of mismatch
repair genes in transgenic plants or derived cells. By introducing these
genes into cells and transgenic plants, new cell lines and plant
varieties with novel and useful properties can be prepared more
efficiently than by relying on the natural rate of mutation. Moreover,
methods to inhibit the expression and activity of endogenous plant MMR
genes and their encoded products are also useful to generate hypermutable
1. A method for generating a mutation in a gene of interest in a plant
cell, comprising the steps of:growing a hypermutable plant cell
comprising the gene of interest and a dominant negative allele of a
mismatch repair gene;testing the cell to determine whether the gene of
interest harbors a mutation.
2. The method of claim 1 wherein the step of testing comprises analyzing a protein encoded by the gene of interest.
3. The method of claim 1 wherein the step of testing comprises analyzing a phenotype caused by the gene of interest.
4. The method of claim 1 wherein the plant cell is made by the process of introducing a polynucleotide comprising a dominant negative allele of a mismatch repair gene into a plant cell, whereby the cell becomes hypermutable.
5. The method of claim 4 wherein the step of testing comprises analyzing a protein encoded by the gene of interest.
6. The method of claim 4 wherein the step of testing comprises analyzing a phenotype caused by the gene of interest.
7. A method for generating a mutation in a gene of interest in a plant, comprising the steps of:growing a plant comprising the gene of interest and a polynucleotide encoding a dominant negative allele of a mismatch repair gene;testing the plant to determine whether the gene of interest harbors a mutation.
8. The method of claim 7 wherein the step of testing comprises analyzing a protein encoded by the gene of interest.
9. The method of claim 7 wherein the step of testing comprises analyzing a phenotype caused by the gene of interest.
10. The method of claim 7 wherein the plant is made by the process of introducing a polynucleotide comprising a dominant negative allele of a mismatch repair gene into a plant, whereby the plant becomes hypermutable.
11. The method of claim 10 wherein the step of testing comprises analyzing a protein encoded by the gene of interest.
12. The method of claim 10 wherein the step of testing comprises analyzing the phenotype of the gene of interest.
[0001]The invention is related to the area of mismatch repair genes. In particular it is related to the field of mutagenesis.
[0002]Within the past four years, the genetic cause of the Hereditary Nonpolyposis Colorectal Cancer Syndrome (HNPCC), also known as Lynch syndrome II, has been ascertained for the majority of kindreds affected with the disease (1). The molecular basis of HNPCC involves genetic instability resulting from defective mismatch repair (MMR). To date, six genes have been identified in humans that encode proteins which appear to participate in the MMR process, including the mutS homologs GTBP, hMSH2, and hMSH3 and the mutL homologs hMLH1, hPMS1, and hPMS2 (2-7). Germline mutations in four of these genes (hMSH2, hMLH1, hPMS1, and hPMS2) have been identified in HNPCC kindreds (2-7). Though the mutator defect that arises from the MMR deficiency can affect any DNA sequence, microsatellite sequences are particularly sensitive to MMR abnormalities (8,9). In addition to its occurrence in virtually all tumors arising in HNPCC patients, Microsatellite Instability (MI) is found in a small fraction of sporadic tumors with distinctive molecular and phenotypic properties (10).
[0003]HNPCC is inherited in an autosomal dominant fashion, so that the normal cells of affected family members contain one mutant allele of the relevant MMR gene (inherited from an affected parent) and one wild-type allele (inherited from the unaffected parent). During the early stages of tumor development, however, the wild-type allele is inactivated through a somatic mutation, leaving the cell with no functional MMR gene and resulting in a profound defect in MMR activity. Because a somatic mutation in addition to a germ-line mutation is required to generate defective MMR in the tumor cells, this mechanism is generally referred to as one involving two hits, analogous to the biallelic inactivation of tumor suppressor genes that initiate other hereditary cancers. In line with this two-hit mechanism, the non-neoplastic cells of HNPCC patients generally retain near normal levels of MMR activity due to the presence of the wild-type allele (11-12).
[0004]While MMR is a conserved process found in bacteria, yeast and mammalian cells (14-16), its activity has not been confirmed in plants. While sequences homologous to MMR genes have been identified in Arabidopsis thaliana, it is not known if they are functional in plants in the process of MMR (17-18). There is a need in the art for identification of the processes involved in genome stability in plants. There is a continuing need for methods and techniques for generating genetic diversity in agriculturally important crops.
[0005]It is an object of the invention to provide a method for making a hypermutable cell.
[0006]It is another object of the invention to provide a homogeneous composition of cultured, hypermutable, plant cells.
[0007]It is still another object of the invention to provide a hypermutable transgenic plant.
[0008]It is yet another object of the invention to provide a method for generating a mutation in a gene of interest in a plant cell.
[0009]It is still another object of the invention to provide a method for generating a mutation in a gene of interest in a plant.
[0010]It is an object of the invention to provide a method for generating a hypermutable plant.
[0011]It is another object of the invention to provide a vector for introducing a dominant negative MMR allele into a plant.
[0012]It is even another object of the invention to provide an isolated and purified polynucleotide encoding a plant MutL homolog.
[0013]It is another object of the invention to provide an isolated and purified protein which is a plant MutL homolog.
[0014]It is an object of the invention to provide a method for determining the presence of a mismatch repair (MMR) defect in a plant or a plant cell.
[0015]These and other objects of the invention are provided by one or more of the following embodiments. In one embodiment of the invention a method for making a hypermutable cell is provided. A polynucleotide comprising a dominant negative allele of a mismatch repair gene is introduced into a plant cell, whereby the cell becomes hypermutable.
[0016]In another aspect of the invention a homogeneous composition of cultured, hypermutable, plant cells is provided. The plant cells comprise a dominant negative allele of a mismatch repair gene.
[0017]Another aspect of the invention is a hypermutable transgenic plant. At least 50% of the cells of the plant comprise a dominant negative allele of a mismatch repair gene.
[0018]According to another aspect of the invention a method is provided for generating a mutation in a gene of interest in a plant cell. A hypermutable plant cell comprising the gene of interest and a dominant negative allele of a mismatch repair gene is grown. The cell is tested to determine whether the gene of interest harbors a newly acquired mutation.
[0019]Another embodiment of the invention is a method for generating a mutation in a gene of interest in a plant. A plant comprising the gene of interest and a polynucleotide encoding a dominant negative allele of a mismatch repair gene is grown. The plant is tested to determine whether the gene of interest harbors a newly acquired mutation.
[0020]According to another aspect of the invention a method is provided for generating a hypermutable plant. Endogenous mismatch repair (MMR) activity of a plant is inhibited. The plant becomes hypermutable as a result of the inhibition.
[0021]Another aspect of the invention is a vector for introducing a dominant negative MMR allele into a plant. The vector comprises a dominant negative MMR allele under the transcriptional control of a promoter which is functional in a plant.
[0022]Still another aspect of the invention provides an isolated and purified polynucleotide encoding Arabidopsis thaliana PMS2 as shown in SEQ ID NO: 12.
[0023]Another aspect of the invention provides an isolated and purified polynucleotide encoding Arabidopsis PMS134 as shown in SEQ ID NO: 14.
[0024]According to another embodiment of the invention an isolated and purified protein which is Arabidopsis PMS2 is provided. It has the amino acid sequence as shown in SEQ ID NO: 12.
[0025]Another embodiment of the invention is an isolated and purified protein which is Arabidopsis PMS134. It has the amino acid sequence as shown in SEQ ID NO: 14.
[0026]Still another aspect of the invention provides a method for determining the presence of a mismatch repair (MMR) defect in a plant or a plant cell. At least two microsatellite markers in test cells or a test plant are compared to the at least two microsatellite markers in cells of a normal plant. The test plant or plant cells are identified as having a mismatch repair defect if at least two microsatellite markers are found to be rearranged relative to the cells of the normal plant.
[0027]FIG. 1 A-C. Alignment of the Arabidopsis thaliana and human PMS2 cDNAs (SEQ ID NOS: 4 and 3, respectively). Similarity is 48.1%; identity is 48.1%. Black boxes show identical nucleotides.
[0028]FIG. 2. Alignment of the Arabidopsis thaliana and human PMS2 proteins (SEQ ID NOS: 11 and 12, respectively). Similarity is 41.5%; identity is 31.1%. Black boxes show identical residues.
[0029]FIG. 3. Alignment of the Arabidopsis thaliana MLH1 homolog and the human PMS2 proteins (SEQ ID NOS: 9 and 11, respectively). Similarity is 30%; identity is 18.4%. Black boxes show identical residues.
[0030]FIG. 4. Alignment of the Arabidopsis thaliana PMS1 homolog and the human PMS2 proteins (SEQ ID NOS: 10 and 11, respectively). Similarity is 24.4; identity is 15%. Black boxes show identical residues.
[0031]FIG. 5. Phylogenetic tree of Arabidopsis thaliana MutL homologs and the human PMS2 protein.
[0032]FIG. 6. Alignment of the Arabidopsis thaliana PMS134 and the human PMS134 cDNA (SEQ ID NOS: 6 and 5, respectively). Similarity is 24.4%; identity is 15%. Black boxes show identical residues.
[0033]FIG. 7. Alignment of the Arabidopsis thaliana PMS134 and the human PMS134 polypeptide (SEQ ID NOS: 14 and 13, respectively). Similarity is 65.1%; identity is 50.7%. Black boxes show identical residues.
[0034]FIGS. 8 A and B. Western blot analysis of bacteria expressing the hPMS134 (FIG. 8A) or the Arabidopsis thaliana PMS134 (FIG. 8B) polypeptides.
[0035]FIG. 9. Expression of plant dominant negative MMR genes produces hypermutability in bacteria, demonstrating the functionality of plant MMR proteins. Briefly, bacteria containing the empty vector of the TAC ATPMS134 expression vector were grown and plated on kanamycin-containing Lbagar plates. The host bacteria are susceptible to KAN bactericidal activity. Bacterial cultures expressing the hPMS134 gene resulted in genetic alteration of the bacterial host and the generation of clones that are KAN resistant.
[0036]FIG. 10. Schematic diagram of a plant dominant-negative MMR expression vector. Ag7 T. and NOS T.=gene 7 and Nopaline Synthase poly(A) signals, respectively. NOS Prom and CaMV Prom=Nopaline Synthase and Cauliflower Mosaic Virus promoters, respectively. L and R=left and right T=DNA border repeats, respectively. Arrows indicate direction of transcription.
[0037]FIG. 11. Transgenic plants containing the PMS134-KAN vector express the dominant negative hPMS134 gene. Message analysis for T1 plants shows steady state expression of dominant negative MMR genes in PMS134-Kan plants (A lanes) while non is observed in control plants (B lanes). Tubulin was used as an internal control to monitor sample loading and integrity.
[0038]FIG. 12. Microsatellite instability in plants expressing dominant negative MMR hPMS134 gene.
[0039]FIG. 13. MMR defective plants produce new phenotypes. Plants with decreased MMR produce offspring with two shoot apical meristems (SAM) in contrast to control plants exhibiting a single SAM. Seeds from the MMR defective plant have been obtained and offspring have the same "double-meristem" trait.
[0040]It is a discovery of the present inventors that plant cells have functional mismatch repair (MMR) systems which function similarly to mammalian MMR. Moreover, dominant negative alleles can be made and used to generate variability in plants and plant cells, as in mammalian cells. Other means of interfering with normal MMR activity can also be used as described in detail below. Dominant negative alleles of mismatch repair genes, when introduced into cells or plants, increase the rate of spontaneous or induced mutations by reducing the effectiveness of DNA repair and thereby render the cells or whole organism hypermutable. Hypermutable plant cells or plants can be utilized to develop new mutations in a gene of interest.
[0041]The process of mismatch repair, also called mismatch proofreading, is carried out by protein complexes in cells ranging from bacteria to mammalian cells (9, 14-16). A mismatch repair (MMR) gene is a gene that encodes one of the proteins of a mismatch repair complex. Although not wanting to be bound by any particular theory or mechanism of action, a mismatch repair complex is believed to detect distortions of a DNA helix resulting from non-complementary pairing of nucleotide bases. The non-complementary base on the newer DNA strand is excised, and the excised base is replaced with the appropriate base, which is complementary to the older DNA strand. In this way, cells eliminate many mutations which occur as a result of mistakes in DNA replication.
[0042]For purposes of example, this application discloses use of dominant negative alleles of MMR genes as a method for blocking or inhibiting MMR activity in plants. (Blocking or inhibiting are used synonymously herein, and denote any significant level of inhibition. They do not connote complete inhibition, although the terms include that possibility within their ambit.) However, any molecular method known by those skilled in the art to block MMR gene expression and/or function can be used, including but not limited to gene knockout (19), antisense technology (20), double stranded RNA interference (21), and polypeptide inhibitors (22).
[0043]Dominant negative alleles cause a mismatch repair defective phenotype even in the presence of a wild-type allele in the same cell. An example of a dominant negative allele of a mismatch repair gene is the human gene hPMS2-134, which carries a truncation mutation at codon 134 (13, U.S. Pat. No. 6,146,894). The mutation causes the product of this gene to prematurely terminate at the position of the 134th amino acid, resulting in a shortened polypeptide containing the N-terminal 133 amino acids. Such a mutation causes an increase in the rate of mutations which accumulate in cells after DNA replication. Expression of a dominant negative allele of a mismatch repair gene results in impairment of mismatch repair activity, even in the presence of the wild-type allele. Any allele which produces such effect can be used in this invention.
[0044]Dominant negative alleles of a mismatch repair gene can be obtained from the cells of humans, animals, yeast, bacteria, plants or other organisms as described by Nicolaides et. al. (23) and Hori et. al. (24). Alternatively such alleles can be made from wild-type alleles, typically by inserting a premature stop codon or other mutation which leads to a protein product which is able to complex with other members of the MMR complex but which is not functional. Such alleles can be identified by screening cells for defective mismatch repair activity. The cells may be mutagenized or not. Cells from plants exposed to chemical mutagens or radiation, e.g., can be screened for defective mismatch repair. Genomic DNA, a plasmid containing cDNA, or mRNA from any cell encoding a mismatch repair protein can be analyzed for variations from the wild type sequence. Dominant negative alleles of a mismatch repair gene can also be created artificially, for example, by producing variants of the hPMS2-134 allele or other mismatch repair genes (13, U.S. Pat. No. 6,146,894). Other truncated alleles of PMS2 or other MMR genes can be made. Such alleles are expected to behave similarly to hPMS2-134. An of various forms of site-directed mutagenesis can be used. The suitability of such alleles, whether natural or artificial, for use in generating hypermutable cells or plants can be evaluated by testing the mismatch repair activity caused by the allele in the presence of one or more wild-type alleles, to determine if the allele is dominant negative.
[0045]A cell or a plant into which a dominant negative allele of a mismatch repair gene has been introduced will become hypermutable. This means that the mutation rate (spontaneous or induced) of such cells or plants is elevated compared to cells or plants without such alleles. The degree of elevation of the mutation rate can be at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold that of the normal cell or plant.
[0046]According to one aspect of the invention, a polynucleotide encoding a dominant negative form of a mismatch repair protein is introduced into a cell or a transgenic plant. The gene can be any dominant negative allele encoding a protein which is part of a mismatch repair complex, for example, mutS or mutL homologs of the bacterial, yeast, fungal, insect, plant, or mammalian genes. The dominant negative allele can be naturally occurring or made in the laboratory. The polynucleotide can be in the form of genomic DNA, cDNA, RNA, or a chemically synthesized polynucleotide. The polynucleotide can be introduced into the cell by transfection.
[0047]Transfection is any process whereby a polynucleotide is introduced into a cell. The process of transfection can be carried out in a living plant, e.g., using a binary vector for gene transmission, or it can be carried out in vitro, e.g., using a suspension of one or more isolated cells in culture. The cell can be any type of plant cell.
[0048]In general, transfection can be carried out using a suspension of cells, or a single cell, but other methods can also be used as long as a sufficient fraction of the treated cells incorporates the polynucleotide to allow transfected cells to be readily isolated. The protein product of the polynucleotide may be transiently or stably expressed in the cell. Techniques for transfection are well known in the art of plant cell science. Available techniques for introducing polynucleotides include but are not limited to electroporation, transduction, Agrobacterium-mediated gene transfer, cell fusion, the use of calcium chloride, and packaging of the polynucleotide together with lipid for fusion with the cells of interest. Once a cell has been transfected with the mismatch repair gene, the cell can, e.g., be grown and reproduced in culture. If the transfection is stable, such that the gene is expressed at a consistent level for many cell generations, then a cell line results. Alternatively, a dominant negative MMR protein can be directly introduced by microinjection into a cell in order to inhibit MMR activity of the cell.
[0049]Root explants are incubated in 0.5 ug/ml of 2-4-dochlorophenoxy-ace-tic acid (2-4D) plus N6-Benzyl-Adenine in growth medium. After 4 weeks, suspension cells are isolated and digested with hemicellulase for protoplast preparation and transfection. Such isolated cells are typically cultured in the absence of other types of cells. Cells selected for the introduction of a dominant negative allele of a mismatch repair gene may be derived from a multicellular plant in the form of a primary cell culture or an immortalized cell line, or may be derived from suspensions of single-celled plants.
[0050]A polynucleotide encoding a dominant negative form of a mismatch repair protein can be introduced into the genome of a plant to form a transgenic plant. The plant can be any species for which suitable techniques are available to produce transgenic plants. For example, transgenic plants can be prepared from domestic agricultural crops, e.g. corn, wheat, soybean, rice, sorghum, barley, etc.; from plants used for the production of recombinant proteins, e.g., tobacco leaf; or experimental plants for research or product testing, e.g., Arabidopsis, pea, etc. The introduced polynucleotide may encode a protein native to the species or native to another species, whether plant, animal, bacterial, or fungal, for example.
[0051]Any method for making transgenic plants known in the art can be used. According to one process of producing a transgenic plant, the polynucleotide is transfected into the plant seedling The seed is germinated and develops into a mature plant in which the polynucleotide is incorporated and expressed. An alternative method for producing transgenic plants involves introducing the polynucleotide into the growing or mature plant by injection, electroporation, Agrobacterium-mediated transfer or transfection. With this method, if the polynucleotide is not incorporated into germline cells, the gene will not be passed on to the progeny. Therefore, a transgenic plant produced by this method will be useful to produce products from that individual plant.
[0052]To identify whether a gene was inserted into the germline, seedlings derived from such plants can be screened for the transgene. Genetic modification of a growing or mature plant is useful for evaluating the expression of hypermutable constructs and for evaluating effects on altering endogenous mismatch repair. Once transgenic plants are produced, they can be grown to produce and maintain a crop of transgenic plants.
[0053]Once a transfected cell line or a crop of transgenic plants has been produced, it can be used to generate new mutations in one or more gene(s) of interest. A gene of interest can be any gene naturally possessed by the cell line or transgenic plant or introduced into the cell line or transgenic plant. An advantage of using MMR-defective cells or plants to induce mutations is that the cell or plant need not be exposed to mutagenic chemicals or radiation, which may have secondary harmful effects, both on the object of the exposure and on the workers.
[0054]Mutations can be detected by analyzing the genotype of the cells or plants, for example by examining the sequence of genomic DNA, cDNA, messenger RNA, or amino acids associated with the gene of interest. Mutations can also be detected by testing a phenotype caused by the gene. A mutant phenotype can be detected, e.g., by identifying alterations in electrophoretic mobility, spectroscopic properties, or other physical or structural characteristics of a protein encoded by a mutant gene. One can also screen for altered function of the protein in situ, in isolated form, or in model systems. One can screen for alteration of any property of the cell or plant associated with the function of the gene of interest or its protein product. Finally, one can screen for macroscopic phenotypes such as but not limited to color, height, or the ability to grow in drought, high-salt, cold, hot, acidic, basic, pest-infested, or high ethylene environments.
[0055]The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples that will be provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.
Example 1
Isolation of Plant Mismatch Repair Genes
[0056]The ability to increase the hypermutability of host genomes has many commercial and medical applications. The generation of hypermutable plants such as those used in agriculture for livestock feed and human consumption are just one example of many types of applications that can be generated by creating hypermutable organisms. For instance, the creation of crops that are less susceptible to pests or soil pH would greatly increase yield of certain agricultural crops. In addition to greater production of goods, improved crops could increase the ability to grow many generations of crops on the same fields (25-27). Moreover, the ability to affect certain growth traits such as natural pest-resistance, drought-resistance, frost-resistance, increased production, or altered stalk size has many benefits for the production of agricultural products. Recently, it has been demonstrated that genes affecting the biologic activity of the plant growth hormone gibberellin results in crops with shorter stalk length that produce similar amounts of grain yields, however, the fact that the stalks are shorter makes these plants less susceptible to high winds and crop damage (28). The use of hypermutable crops could allow for the selection of shorter plants across many species such as corn, rice, etc, without having to first identify a gene to alter its activity. Another application of hypermutable agricultural products is the generation of crops with enhanced levels of vitamins and nutrients. One can select for enhanced vitamin production in seedlings of MMR defective plants. Recently, it has been demonstrated that altering a gene(s) within a vitamin biosynthetic pathway can result in the production of elevated levels of vitamin E (27,29).
[0057]Applications of hypermutable plants include use as crops for agricultural production, increased medicinal entities within plant extracts, chemicals and resins for industrial use, and their use as detoxifying organisms for environmental applications as described (25,26,29).
[0058]MutS and mutL homologs can be isolated from plant species libraries using degenerate RT-PCR, and standard Southern hybridization techniques as previously described (3,23,30). These may serve as reagents for producing MMR defective plant hosts. This process employs methods known by those skilled in the art of gene cloning.
[0059]One such approach is the use of degenerate PCR to clone MutS homologs following the methods used by Leach et. al. to clone the human MSH2 (3). Additional degenerate oligonucleotides can be generated and used against conserved domains of bacterial, yeast, and human MutS homologs. Various plant species cDNAs (obtainable from various commercial sources) can be amplified for MutS gene homologs by polymerase chain reaction (PCR). Products are cloned into T-tailed vectors (In Vitrogen) and analyzed by restriction endonuclease digestion. Clones with expected DNA fragment inserts are sequenced using M13 forward and reverse primers located on the vector backbone flanking the cloning site. Fragments containing MMR gene homologs are then used as probes to screen commercially available cDNA libraries from the appropriate species. cDNA contigs are generated to create a cDNA containing the sequence information for the full length MMR gene and its encoded polypeptide. One such example of cloning a plant MMR gene is provided below.
[0060]In order to clone mutl homologs, degenerate primers were synthesized to the conserved domains of the mutL gene family by aligning E. coli, yeast, mouse, and human mutL genes. These primers are directed to the polynucleotide sequences centered at nt 150 to 350 of the published human PMS2 cDNA (SEQ ID NO: 3). Degenerate PCR was carried out using RNA from Arabidopsis thaliana (AT) that was isolated using the RNeasy kit following the manufacturer's protocol (Qiagen). RNAs were reverse transcribed (RT) using Superscriptfl (Life Technologies) following the manufacturer's protocol. After RT, cDNAs were PCR amplified using degenerate primers in buffers described by Nicolaides et. al. 1995 (23, 30), and reactions were carried out at 95° C. for 30 sec for 1 cycle followed by 94° C. for 30 sec, 45° C. for 60 sec, and 72° C. for 60 sec for 20 cycles. PCR reactions were then diluted 1:10 in water and reamplified using the same primers and buffers. The secondary PCR reactions were carried out at 95° C. for 30 sec for 1 cycle followed by 94° C. for 30 sec, 52° C. for 90 sec, and 72° C. for 90 sec for 35 cycles. Reactions were analyzed by agarose gel electrophoresis. Products of the expected molecular weight were excised and cloned into T-tailed vectors (InVitrogen). Recombinant clones were sequenced and blasted against the public databases. The homolog was found to have homology to the mutL family of genes. Blast search analysis of GenBank found this gene to be part of a "putative" mismatch repair gene identified from the Arabidopsis genome project that has never been reported to be transcribed or capable of producing a message. In order to clone the full length, an Arabidopsis cDNA library was screened by PCR as well as cDNA from AT plants using 5' primers corresponding to the initiation codon (SEQ ID NO: 1:5'-atg caa gga gat tct tc-3') and the termination codon (SEQ ID NO: 2:5'-tca tgc caa tga gat ggt tgc-3') using buffers and conditions listed above. Amplifications were carried out at 95° C. for 30 sec for 1 cycle followed by 94° C. for 30 sec, 58° C. for 2 min, and 72° C. for 3 min for 35 cycles. Products were analyzed by gel electrophoresis. Products of the expected molecular weights were subcloned into T-tail vectors and sequenced using primers from the cloning vector or using internal primers. FIG. 1 shows the alignment of one Arabidopsis homolog, referred to as ATPMS2 (SEQ ID NO: 4), to the human PMS2 cDNA (SEQ ID NO:3) (FIG. 1) and the hPMS2 protein (FIG. 2; SEQ ID NO:11). This gene was found to be homologous (48% identity) to the human PMS2 (SEQ ID NO:3) cDNA and its encoded polypeptide (31% identity) (FIG. 2). Other homologs to the ATPMS2 were also identified from blast searching sequence databases. One mutL homolog is closely related to the MLH1 mammalian homolog and is referred to as ATMLH1 (shown in FIG. 3) and another is closely related to the mammalian PMS1 polypeptide referred to as ATPMS1 (shown in FIG. 4). A phylogenetic tree is shown in FIG. 5 showing the homology of the mutL homologs to the human PMS2 gene.
[0061]Degenerate primers can be used for isolating MMR genes from other plant species in a similar fashion.
Example 2
Generation of Dominant Negative Alleles of Plant Mismatch Repair Genes
[0062]To demonstrate that putative plant MMR proteins are truly involved in MMR biochemical process, cDNAs are cloned into constitutive (31,32) or inducible (33) bacterial expression vectors for functional studies. Various deletion mutants are generated to produce dominant negative MMR genes. Dominant negative alleles that are identified in the bacterial system are then useful for plant studies. Dominant negative MMR genes are prepared by over-expression of full-length MMR genes or by deletion analysis using standard protocols used by those skilled in the art of molecular biology. One such dominant MMR gene mutant was created by generating a construct with similar domains to that of the human dominant negative PMS2 gene (referred to as PMS134) (13, U.S. Pat. No. 6,146,894). To generate this vector, the ATPMS2 (SEQ ID NO: 4) and hPMS2 cDNA (SEQ ID NO: 3) sequences were aligned and the conserved domain was isolated. FIG. 6 shows a sequence alignment between the human and AT PMS134 cDNAs where a 52% identity is found between the two sequences. At the protein level these domains have a 51% identity (FIG. 7). Dominant negative hPMS134 and ATPMS134 genes were made by PCR and subcloned into bacterial expression vectors. The ATPMS134 was generated by PCR from the cloned cDNA using a sense primer (SEQ ID NO:1) corresponding to the N-terminus and an antisense primer (SEQ ID NO:15) 5' gtcgacttatcacttgtcatcgtcgtccttgtagtcgag-cgtagc-aactggctc-3' centered at nt 434 of the ATPMS2 cDNA (SEQ ID NO:4). This primer also contains a flag epitope that will allow protein detection followed by two termination codons. PCR products of the expected molecular weight were gel purified and cloned into T-tail vectors. Recombinant clones were sequenced to ensure authentic sequences. Inserts were then cloned into the inducible pTAC expression vector, which also contains the Ampicillin resistance gene as a selectable marker. The human PMS134 allele was also cloned into the pTAC expression vector as a positive control. Electrocompetent DH5alpha and DH10b bacterial cells (Life Technologies) were electroporated with empty vector, and the loaded vectors pTACATPMS134 and pTAChPMS134, using an electroporator (BioRAd) following the manufacturer's protocol. Bacterial cultures were then plated on to LB agar plates containing 100 μg/ml ampicillin and grown at 37° C. for 14 hours. Ten recombinant clones were then isolated and grown in 5 mls of LB broth containing 50 μg/ml ampicillin plus 50 μM IPTG for 18 hr at 37° C. One hundred microliters were then collected, spun down, and directly lysed in 2×SDS buffer for western blot analysis. For western analysis, equal number of cells were lysed directly in 2×SDS buffer (60 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.1 M 2-mercaptoethanol, 0.001% bromophenol blue) and boiled for 5 minutes. Lysate proteins are separated by electrophoresis on 4-12% NuPAGE gels (Novex). Gels are electroblotted onto Immobilon-P (Millipore) in 48 mM Tris base, 40 mM glycine, 0.0375% SDS, 20% methanol and blocked overnight at 4° C. in Tris-buffered saline plus 0.05% Tween-20 and 5% condensed milk. Filters are probed with a polyclonal antibody generated against MMR polypeptide sequence or a fused tag (e.g. FLAG, HIS, etc.) and a horseradish peroxidase conjugated secondary antibody, using chemiluminescence for detection (Pierce). FIG. 8 shows a western blot of a clone that expresses the human PMS134 protein (FIG. 8A, lane 2) using a human PMS2-specific antibody (directed to residues 2-20) of the hPMS134 sequence (see FIG. 1, and SEQ ID NO:6) or the Arabidopsis PMS134 protein (FIG. 8B, lane 2) using an anti-FLAG antibody directed to the fusion residues at the C-terminus of the protein. Cells expressing empty vector had no detectable expression.
[0063]Bacterial clones expressing the hPMS134, ATPMS134 or the empty vector were grown in liquid culture for 24 hr at 37° C. in the presence of 50 μg/ml ampicillin plus 50 μM IPTG. The next day, cultures were diluted 1:10 in medium containing 50 μM IPTG plus ampicillin or ampicillin plus 25 μg/ml kanamycin (AK) and cultures were grown for 18 hr at 37° C. The following day, a 0.1 μl aliquot (2 μl diluted in 1000 μl of LB medium and used 50 μl for plating) of cells grown in Amp medium were plated on LB-agar plates containing 40 μg/ml of 5-bromo-4-chloro-3-indolyl-B-D-galactoside (X-gal) plus 100 μg/ml ampicillin (AMP), while a 1 μl aliquot (1 μl diluted in 100 μl of LB medium and used 100 μl for plating) of cells grown in AK medium were plated on LB-agar plates containing X-gal and 50 μg/ml kanamycin (KAN). Plates were incubated for 18 hours at 37° C. The results from these studies show that cells expressing the hPMS134 or the ATPMS134 polypeptides displayed increased mutation rates in the genome of the DH10B bacterial strain which resulted in the production of KAN resistant clones (FIG. 9). Following the mutagenesis protocol described above, bacterial cells expressing the plant ATPMS134 were found to have an increase in the number of KAN resistant cells (12 clones) in contrast to cells expressing the empty vector, which yielded no KAN resistant clone. These data demonstrate that dominant negative ATPMS134 MMR genes are useful for creating hypermutable organisms that can generate phenotypically diverse offspring when put under selective conditions. Moreover, these data demonstrate that plants also use the conserved MMR process for genomic stability.
[0064]Dominant negative plant MMR gene mutants are also analyzed using mammalian cell systems. In this case, plant MMR gene cDNAs are cloned into eukaryotic expression vectors as described (13,34) and cells expressing dominant negative mutants are analyzed by measuring stability of endogenous microsatellite markers and biochemical activity of cell extracts from lines expressing dominant negative MMR gene alleles. Such methods are known by those skilled in the art and previously described (13).
Example 3
Inhibition of Plant MMR Activity by Dominant Negative MMR Alleles Produces Genetic Hypermutability and Microsatellite Instability
[0065]Dominant negative alleles of human and AT MMR genes identified using bacterial and or mammalian systems can be used for plants. To test the hypothesis that dominant negative MMR gene alleles produce global hypermutability in plants, the hPMS134 and ATPMS134 cDNAs were expressed in plants. These alleles have been found to work across species where the introduction of these genes into MMR proficient bacterial or mammalian cells renders the cells MMR deficient. Assays to carry out these studies are described below.
Engineering Plant Expression Vectors to Express the PMS134 Dominant Negative Alleles.
[0066]A BamH I fragment containing the hPMS134 cDNA was obtained from the pSG5PMS134 plasmid (ref 13) and cloned into the corresponding sites of the pEF1SP1-V5 vector (InVitrogen). The resulting vector (pEF-PMS134-sense) was then digested with Pme I to release a blunted DNA fragment containing the PMS134 cDNA. This fragment was then subcloned into the blunt Sma I and EcoICR I sites of the pGPTV-KAN binary plant expression vector (American Type Culture Collection). One clone, named pCMV-hPMS134-Kan (see FIG. 10), was sequenced to confirm that the vector contained authentic promoter and gene insert sequences. A schematic diagram of the pCMV-hPMS134-Kan vector is shown in FIG. 10.
Generation of hPMS134-Expressing Arabidopsis Thaliana Transgenic Plants.
[0067]Agrobacterium tumefaciens cells (agrobacteria) are used to shuttle genes into plants. To generate PMS134-expressing Arabidopsis thaliana (A. thaliana) plants, Agrobacterium tumefaciens cells (strain GV3101) were electroporated with pCMV-hPMS134-Kan or the pBI-121 (BRL) control binary vector. The pBI-121 control contains the CaMV promoter driving the expression of the β-glucuronidase cDNA (GUS) and serves as a control. Both vectors carry the neomycin phosphotransferase (NPTII) gene that allows selection of agrobacteria and plants that contain the expression vector. One-month old A. thaliana (ecotype Columbia) plants were infected by immersion in a solution containing 5% sucrose, 0.05% silwet, and binary vector-transformed agrobacteria cells for 10 seconds. These plants were then grown at 25° C. under a 16 hour day and 8 hour dark photoperiod. After 4 weeks, seeds (referred to as T1) were harvested and dried for 5 days at 4° C. Thirty thousands seeds from ten CMV-hPMS134-Kan-transformed plants and five thousand seeds from two pBI-121-transformed plants were sown in solid Murashige and Skoog (MS) media plates in the presence of 50 μg/ml of kanamycin (KAN). Three hundred plants were found to be KAN resistant and represented PMS134 expressing plants. These plants along with 300 control plants were grown in MS media for two weeks and then transferred to soil. Plants were grown for an additional four weeks under standard conditions at which time T2 seeds were harvested.
[0068]To confirm the integration and stability of the PMS134 gene in the plant genome, gene segregation and PCR analyses were conducted. Commonly, three out of four T1 plants transformed by agrobacteria technology are expected to carry the vector inserted within a single locus and are therefore considered heterozygous for the integrated gene. Approximately 75% of the seeds (T2) generated from most of the T1 plants were found KAN-resistant and this in accordance with the expected 1:2:1 ratio of null (no hPMS134 containing plants), heterozygous, and homozygous plants, respectively, in self-pollinating conditions. To confirm that these plants contained the hPMS134 expression vector, genomic DNA was isolated from leaves of T1 plants using the DNAzol-mediated technique following the manufacturer's suggestion (BRL-Life Technologies). One nanogram of genomic DNA was analyzed by polymerase chain reaction (PCR) to confirm the presence of the hPMS134 gene. PCR was carried out for 25 cycles with the following parameters: 95° C. for 30 seconds; 55° C. for 1 minute; and 72° C. for 2 minutes using hPMS134-specific sense (SEQ ID NO: 7: 5'-tct aga cat gga gcg agc tga gag ctc-3') and antisense (SEQ ID NO: 8:5'-tct aga agt tcc aac ctt cgc cga tgc-3') primers. Positive reactions were observed in DNA from pCMV-hPMS134-Kan-transformed plants and not from pBI-121-transformed plants, thus confirming the integration of this vector.
[0069]In order to assess the expression of hPMS134 in T1 plants, leaf tissue was collected from T1 plants, homogenized in liquid nitrogen using glass pestles, and suspended in RLT lysing buffer (Qiagen, RNeasy plant RNA extraction kit). Five micrograms of total RNA was purified according to the manufacturer's suggested protocol and then loaded onto a 1.2% agarose gel (1× MOPS buffer, 3% formaldehyde), size-fractionated by electrophoresis, and transferred onto N-Hybond+ membrane (Amersham). Each membrane was incubated at 55° C. in 10 ml of hybridization solution (North2South labeling kit, Pierce) containing 100 ng of PMS134, tubulin, or KAN cDNA probes, which were generated by PCR amplification, according to the manufacturer's directions. Membranes were washed three times in 2×SSC, 0.1% SDS at 55° C., and three times in 2×SSC at ambient temperature. Detection was carried out using enhanced chemiluminescence (ECL). Expression was also carried out by reverse trascriptase PCR as described above using polyA isolated mRNA that was isolated over a oligo dT column (Qiagen). A representative example of these studies are shown in FIG. 11. Here hPMS134 expression was detected in three out of ten analyzed pCMV-hPMS134-Kan transgenic lines, while no signal was found in the ten pBI-121 transformed plants analyzed. Immunoblot using whole lysates is used to confirm protein expression. Collectively these studies demonstrate the generation of hPMS134 expressing transgenic A. thaliana plants.
Molecular Characterization of PMS134-Expressing Plants
[0070]MMR is a process that is involved in correcting point mutations and "slippage" mutations within repetitive mono-, di-, and tri-nucleotide (microsatellite) repeats that occur throughout the genome of an organism after cellular replication. This process is very similar to a computer spell check function. The inactivation of MMR has been shown to result in global genomic hypermutation whereby cells with defective MMR experience over a one thousand-fold increase in point mutations and microsatellite instability (MI) (mutations within repetitive sequences) throughout their genomes per division. (35). MMR deficiency is the only known process capable of producing MI and has been used as a marker to detect cells with MMR dysfunction (36). Microsatellites serve as molecular tools to measure the inactivation of MMR that occurs by the defective MMR due to but not limited to expression of dominant negative MMR genes, double stranded RNA interference vectors, or inhibition by antisense nucleotides, or by gene knockout. In A. thaliana, a series of poly-A (A)n, (CA)n and (GA)n sequences were identified from genome searches using EMBL and GenBank databases. To demonstrate that hPMS134 expression could produce MI in A. thaliana, we analyzed microsatellites in T1 plants by PCR analyses. Initially we monitored three microsatellites, ATHACS, Nga280, and Nga128 with published primers that have been previously used to map the Arabidopsis genome (37). Briefly, DNA was extracted from A. thaliana leaves as described above. 10 ngs of plant genomic DNA was used as template for PCR amplification using the following amplification conditions: 94° C. for 15 sec, 55° C. for 15 sec and 72° C. for 30 seconds. PCR products were analyzed on 5% Metaphor agarose (BioWhittaker Molecular Applications) and ethidium bromide staining. In one transgenic pCMV-PMS134-Kan line, we detected a double product, likely representing a new allele of the polymorphic nga280 locus (FIG. 12). These data demonstrate the ability to produce MMR deficiency and MI in plants expressing the hPMS134 dominant negative allele and provide a molecular tool for screening MMR-defective plants.
[0071]Biochemical assays for mismatch repair. MMR activity in nuclear extracts is performed as described, using 24 finol of substrate as described (13). Complementation assays are done by adding ˜100 ng of purified MutL or MutS components to 100 μg of nuclear extract, adjusting the final KCl concentration to 100 mM. The substrates used in these experiments will contain a strand break 181 nucleotides 5' or 125 nucleotides 3' to the mismatch.
Example 4
Inactivation of MMR Leads to Plants with New Phenotypes
[0072]We demonstrated the ability of the defective MMR to produce molecular changes within plants. The objective of this section is to demonstrate the ability to generate MMR defective plants with macroscopic output traits. One way to measure for plants with new phenotypes is to grow plants under toxic conditions, such as but not limited to high levels of toxic ions, pest-infection, drought conditions, or extreme temperatures to identify a minority of plants with new output traits, i.e., resistance. Another way to score for plants with new phenotypes is through physical differences of MMR defective plants grown in standard conditions. An example of MMR-defective plants with new phenotypes include the generation of plants with double shoot apical meristems (FIG. 13) as well as plants with altered chlorophyll production rendering plants albino (data not shown). In FIG. 13, we show a typical wild type plant (left, labeled normal) and a plant produced from the MMR defective group (right, labeled MMR deficient). The double-meristem trait was not observed in greater than 500 normal plants. The double-meristem trait does not appear to be due to transgene integration since segregation analysis reveals the ability to generate double-meristem plants in the absence of transgene positive plants while MMR proficient control plants with other transgene vectors (pBI-121) did not produce this phenotype (data not shown). These data suggest that defective MMR produced a mutation or mutations within the plant genome that altered the normal biochemical function of the host to produce a new output trait.
[0073]These data demonstrate the ability to create plant subtypes with new genetic and phenotypic traits by blocking the endogenous MMR process of the plant cell or whole organism.
Example 5
Inhibition of Plant MMR Activity Using Molecular Approaches
[0074]This application teaches of the use of inhibiting MMR activity in a plant to produce genetically altered offspring with new phenotypes.
[0075]The inhibition of MMR activity in a host organism can be achieved by introducing a dominant negative allele as shown in FIGS. 11 and 12. Other ways to suppress MMR activity of a host is by: knocking out alleles of a MMR protein through homologous recombination (38); blocking MMR protein dimerization with other subunits (which is required for activity) by the introduction of polypeptides into the host via transfection methods; knocking out the expression of a MMR gene using anti-sense oligonucleotides (20), and/or the use of double stranded RNA interference genes (21).
MMR Gene Knockouts.
[0076]Data shown in EXAMPLE 1 demonstrate that plants contain MMR gene homologs that can be genetically engineered to produce altered biochemical activities. Data presented in EXAMPLES 3 and 4 demonstrate that defective MMR in plants can produce hypermutable parental plants and offspring. Together, these data demonstrate that inhibiting endogenous MMR genes by targeting vectors of the particular MMR gene can lead to hypermutability of a plant host that generate offspring with altered genetic loci and/or new phenotypes as described in EXAMPLES 3, 4, and 5. Hypermutable seedlings can also be produced with "knocked out" MMR genes using methods known by those skilled in the art. These can be used to produce genetically diverse offspring for commercial and medical applications (38). Cells will be confirmed to have lost the expression of the MMR gene using standard northern techniques and determined to be MMR defective using microsatellite analysis as described in EXAMPLE 3.
Blocking Polypeptides.
[0077]MMR subunits (MutS and MutL proteins) interact to form active MMR complexes. Peptides are able to specifically inhibit the binding of two proteins by competitive inhibition. Isolation of plant MMR genes allows for the elucidation of primary amino acid structures as described in EXAMPLE 1. Peptides containing some but not all of the domains can be synthesized from domains of the particular MMR gene and introduced into host plants using methods known by those skilled in the art (22). Like truncated PMS134, such peptides will compete with functional full length proteins for binding and form enzymatically inactive MMR complexes. The data indicate that the domains which are C-terminal to the 134 position in human PMS2 are dispensible for binding and necessary for enzymatic activity. As shown herein, a similar domain structure is also found in plant PMS2. Seedlings exhibiting hypermutability will be useful to produce genetically diverse offspring for commercial and medical applications.
RNA Blockade and Double Stranded Interference.
[0078]MMR subunits (MutS and MutL proteins) interact to form active MMR complexes. Peptides are able to specifically inhibit the binding of two proteins by competitive inhibition. Antisense oligonucleotides are synthesized against the cDNA sequence of plant MMR homologs identified in EXAMPLE 1 (20). Antisense molecules are then introduced into host plants using methods described in EXAMPLE 2 or through the bathing of seedlings or plantlets. Seedlings exhibiting hypermutability will be useful to produce genetically diverse offspring for commercial and medical applications.
[0079]Double stranded interference vectors are also useful for blocking expression/function of a plant MMR gene. The plant gene is expressed in both sense and antisense orientations from a transfection vector and the endogenous gene expression is suppressed by endogenous silencing processes (21).
[0080]Plants contain MMR genes that code for MMR functional proteins. Expression of dominant negative plant MMR proteins results in an increase in microsatellite instability and hypermutability in plants. This activity is due to the inhibition of MMR biochemical activity in these hosts. The data provided within this application demonstrates the blockade of MMR in a plant to produce genetic changes that lead to the production of offspring or cells with new output traits. This method is applicable to generate crop plants with new output traits as well as plant cells exhibiting new biochemicals for commercial use.
Each of which is Expressly Incorporated Herein for all Purposes
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Sequence CWU
15117DNAHuman sapiens 1atgcaaggag attcttc 17221DNAHuman sapiens 2tcatgccaat gagatggttg c 2132589DNAHuman sapiens 3atggagcgag ctgagagctc gagtacagaa cctgctaagg ccatcaaacc tattgatcgg 60aagtcagtcc atcagatttg ctctgggcag gtggtactga gtctaagcac tgcggtaaag 120gagttagtag aaaacagtct ggatgctggt gccactaata ttgatctaaa gcttaaggac 180tatggagtgg atcttattga agtttcagac aatggatgtg gggtagaaga agaaaacttc 240gaaggcttaa ctctgaaaca tcacacatct aagattcaag agtttgccga cctaactcag 300gttgaaactt ttggctttcg gggggaagct ctgagctcac tttgtgcact gagcgatgtc 360accatttcta cctgccacgc atcggcgaag gttggaactc gactgatgtt tgatcacaat 420gggaaaatta tccagaaaac cccctacccc cgccccagag ggaccacagt cagcgtgcag 480cagttatttt ccacactacc tgtgcgccat aaggaatttc aaaggaatat taagaaggag 540tatgccaaaa tggtccaggt cttacatgca tactgtatca tttcagcagg catccgtgta 600agttgcacca atcagcttgg acaaggaaaa cgacagcctg tggtatgcac aggtggaagc 660cccagcataa aggaaaatat cggctctgtg tttgggcaga agcagttgca aagcctcatt 720ccttttgttc agctgccccc tagtgactcc gtgtgtgaag agtacggttt gagctgttcg 780gatgctctgc ataatctttt ttacatctca ggtttcattt cacaatgcac gcatggagtt 840ggaaggagtt caacagacag acagtttttc tttatcaacc ggcggccttg tgacccagca 900aaggtctgca gactcgtgaa tgaggtctac cacatgtata atcgacacca gtatccattt 960gttgttctta acatttctgt tgattcagaa tgcgttgata tcaatgttac tccagataaa 1020aggcaaattt tgctacaaga ggaaaagctt ttgttggcag ttttaaagac ctctttgata 1080ggaatgtttg atagtgatgt caacaagcta aatgtcagtc agcagccact gctggatgtt 1140gaaggtaact taataaaaat gcatgcagcg gatttggaaa agcccatggt agaaaagcag 1200gatcaatccc cttcattaag gactggagaa gaaaaaaaag acgtgtccat ttccagactg 1260cgagaggcct tttctcttcg tcacacaaca gagaacaagc ctcacagccc aaagactcca 1320gaaccaagaa ggagccctct aggacagaaa aggggtatgc tgtcttctag cacttcaggt 1380gccatctctg acaaaggcgt cctgagacct cagaaagagg cagtgagttc cagtcacgga 1440cccagtgacc ctacggacag agcggaggtg gagaaggact cggggcacgg cagcacttcc 1500gtggattctg aggggttcag catcccagac acgggcagtc actgcagcag cgagtatgcg 1560gccagctccc caggggacag gggctcgcag gaacatgtgg actctcagga gaaagcgcct 1620gaaactgacg actctttttc agatgtggac tgccattcaa accaggaaga taccggatgt 1680aaatttcgag ttttgcctca gccaactaat ctcgcaaccc caaacacaaa gcgttttaaa 1740aaagaagaaa ttctttccag ttctgacatt tgtcaaaagt tagtaaatac tcaggacatg 1800tcagcctctc aggttgatgt agctgtgaaa attaataaga aagttgtgcc cctggacttt 1860tctatgagtt ctttagctaa acgaataaag cagttacatc atgaagcaca gcaaagtgaa 1920ggggaacaga attacaggaa gtttagggca aagatttgtc ctggagaaaa tcaagcagcc 1980gaagatgaac taagaaaaga gataagtaaa acgatgtttg cagaaatgga aatcattggt 2040cagtttaacc tgggatttat aataaccaaa ctgaatgagg atatcttcat agtggaccag 2100catgccacgg acgagaagta taacttcgag atgctgcagc agcacaccgt gctccagggg 2160cagaggctca tagcacctca gactctcaac ttaactgctg ttaatgaagc tgttctgata 2220gaaaatctgg aaatatttag aaagaatggc tttgattttg ttatcgatga aaatgctcca 2280gtcactgaaa gggctaaact gatttccttg ccaactagta aaaactggac cttcggaccc 2340caggacgtcg atgaactgat cttcatgctg agcgacagcc ctggggtcat gtgccggcct 2400tcccgagtca agcagatgtt tgcctccaga gcctgccgga agtcggtgat gattgggact 2460gctcttaaca caagcgagat gaagaaactg atcacccaca tgggggagat ggaccacccc 2520tggaactgtc cccatggaag gccaaccatg agacacatcg ccaacctggg tgtcatttct 2580cagaactag 258942340DNAArabidopsis thaliana 4atgcaaggag attcttctcc gtctccgacg actactagct ctcctttgat aagacctata 60aacagaaacg taattcacag aatctgttcc ggtcaagtca tcttagacct ctcttcggcc 120gtcaaggagc ttgtcgagaa tagtctcgac gccggcgcca ccagtataga gattaacctc 180cgagactacg gcgaagacta ttttcaggtc attgacaatg gttgtggcat ttccccaacc 240aatttcaagg tttgtgtcca aattctccga agaacttttg atgttcttgc acttaagcat 300catacttcta aattagagga tttcacagat cttttgaatt tgactactta tggttttaga 360ggagaagcct tgagctctct ctgtgcattg ggaaatctca ctgtggaaac aagaacaaag 420aatgagccag ttgctacgct cttgacgttt gatcattctg gtttgcttac tgctgaaaag 480aagactgctc gccaaattgg taccactgtc actgttagga agttgttctc taatttacct 540gtacgaagca aagagtttaa gcggaatata cgcaaagaat atgggaagct tgtatcttta 600ttgaacgcat atgcgcttat tgcgaaagga gtgcggtttg tctgctctaa cacgactggg 660aaaaacccaa agtctgttgt gctgaacaca caagggaggg gttcacttaa agataatatc 720ataacagttt tcggcattag tacctttaca agtctacagc ctggtactgg acgcaattta 780gcagatcgac agtatttctt tataaatggt cggcctgtag atatgccaaa agtcagcaag 840ttggtgaatg agttatataa agatacaagt tctcggaaat atccagttac cattctggat 900tttattgtgc ctggtggagc atgtgatttg aatgtcacgc ccgataaaag aaaggtgttc 960ttttctgacg agacttctgt tatcggttct ttgagggaag gtctgaacga gatatattcc 1020tccagtaatg cgtcttatat tgttaatagg ttcgaggaga attcggagca accagataag 1080gctggagttt cgtcgtttca gaagaaatca aatcttttgt cagaagggat agttctggat 1140gtcagttcta aaacaagact aggggaagct attgagaaag aaaatccatc cttaagggag 1200gttgaaattg ataatagttc gccaatggag aagtttaagt ttgagatcaa ggcatgtggg 1260acgaagaaag gggaaggttc tttatcagtc catgatgtaa ctcaccttga caagacacct 1320agcaaaggtt tgcctcagtt aaatgtgact gagaaagtta ctgatgcaag taaagacttg 1380agcagccgct ctagctttgc ccagtcaact ttgaatactt ttgttaccat gggaaaaaga 1440aaacatgaaa acataagcac catcctctct gaaacacctg tcctcagaaa ccaaacttct 1500agttatcgtg tggagaaaag caaatttgaa gttcgtgcct tagcttcaag gtgtctcgtg 1560gaaggcgatc aacttgatga tatggtcatc tcaaaggaag atatgacacc aagcgaaaga 1620gattctgaac taggcaatcg gatttctcct ggaacacaag ctgataatgt tgaaagacat 1680gagagagtac tcgggcaatt caatcttggg ttcatcattg caaaattgga gcgagatctg 1740ttcattgtgg atcagcatgc agctgatgag aaattcaact tcgaacattt agcaaggtca 1800actgtcctga accagcaacc cttactccag cctttgaact tggaactctc tccagaagaa 1860gaagtaactg tgttaatgca catggatatt atcagggaaa atggctttct tctagaggag 1920aatccaagtg ctcctcccgg aaaacacttt agactacgag ccattcctta tagcaagaat 1980atcacctttg gagtcgaaga tcttaaagac ctgatctcaa ctctaggaga taaccatggg 2040gaatgttcgg ttgctagtag ctacaaaacc agcaaaacag attcgatttg tccatcacga 2100gtccgtgcaa tgctagcatc ccgagcatgc agatcatctg tgatgatcgg agatccactc 2160agaaaaaacg aaatgcagaa gatagtagaa cacttggcag atctcgaatc tccttggaat 2220tgcccacacg gacgaccaac aatgcgtcat cttgtggact tgacaacttt actcacatta 2280cctgatgacg acaatgtcaa tgatgatgat gatgatgatg caaccatctc attggcatga 23405402DNAHuman sapiens 5atggagcgag ctgagagctc gagtacagaa cctgctaagg ccatcaaacc tattgatcgg 60aagtcagtcc atcagatttg ctctgggcag gtggtactga gtctaagcac tgcggtaaag 120gagttagtag aaaacagtct ggatgctggt gccactaata ttgatctaaa gcttaaggac 180tatggagtgg atcttattga agtttcagac aatggatgtg gggtagaaga agaaaacttc 240gaaggcttaa ctctgaaaca tcacacatct aagattcaag agtttgccga cctaactcag 300gttgaaactt ttggctttcg gggggaagct ctgagctcac tttgtgcact gagcgatgtc 360accatttcta cctgccacgc atcggcgaag gttggaactt ag 4026441DNAArabidopsis thaliana 6atgcaaggag attcttctcc gtctccgacg actactagct ctcctttgat aagacctata 60aacagaaacg taattcacag aatctgttcc ggtcaagtca tcttagacct ctcttcggcc 120gtcaaggagc ttgtcgagaa tagtctcgac gccggcgcca ccagtataga gattaacctc 180cgagactacg gcgaagacta ttttcaggtc attgacaatg gttgtggcat ttccccaacc 240aatttcaagg tttgtgtcca aattctccga agaacttttg atgttcttgc acttaagcat 300catacttcta aattagagga tttcacagat cttttgaatt tgactactta tggttttaga 360ggagaagcct tgagctctct ctgtgcattg ggaaatctca ctgtggaaac aagaacaaag 420aatgagccag ttgctacgct c 441727DNAHuman sapiens 7tctagacatg gagcgagctg agagctc 27827DNAHuman sapiens 8tctagaagtt ccaaccttcg ccgatgc 279737PRTArabidopsis thaliana 9Met Ile Asp Asp Ser Ser Leu Thr Ala Glu Met Glu Glu Glu Glu Ser1 5 10 15Pro Ala Thr Thr Ile Val Pro Arg Glu Pro Pro Lys Ile Gln Arg Leu 20 25 30Glu Glu Ser Val Val Asn Arg Ile Ala Ala Gly Glu Val Ile Gln Arg 35 40 45Pro Val Ser Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Asp 50 55 60Ser Ser Ser Ile Ser Val Val Val Lys Asp Gly Gly Leu Lys Leu Ile65 70 75 80Gln Val Ser Asp Asp Gly His Gly Ile Arg Arg Glu Asp Leu Pro Ile 85 90 95Leu Cys Glu Arg His Thr Thr Ser Lys Leu Thr Lys Phe Glu Asp Leu 100 105 110Phe Ser Leu Ser Ser Met Gly Phe Arg Gly Glu Ala Leu Ala Ser Met 115 120 125Thr Tyr Val Ala His Val Thr Val Thr Thr Ile Thr Lys Gly Gln Ile 130 135 140His Gly Tyr Arg Val Ser Tyr Arg Asp Gly Val Met Glu His Glu Pro145 150 155 160Lys Ala Cys Ala Ala Val Lys Gly Thr Gln Ile Met Val Glu Asn Leu 165 170 175Phe Tyr Asn Met Ile Ala Arg Arg Lys Thr Leu Gln Asn Ser Ala Asp 180 185 190Asp Tyr Gly Lys Ile Val Asp Leu Leu Ser Arg Met Ala Ile His Tyr 195 200 205Asn Asn Val Ser Phe Ser Cys Arg Lys His Gly Ala Val Lys Ala Asp 210 215 220Val His Ser Val Val Ser Pro Ser Arg Leu Asp Ser Ile Arg Ser Val225 230 235 240Tyr Gly Val Ser Val Ala Lys Asn Leu Met Lys Val Glu Val Ser Ser 245 250 255Cys Asp Ser Ser Gly Cys Thr Phe Asp Met Glu Gly Phe Ile Ser Asn 260 265 270Ser Asn Tyr Val Ala Lys Lys Thr Ile Leu Val Leu Phe Ile Asn Asp 275 280 285Arg Leu Val Glu Cys Ser Ala Leu Lys Arg Ala Ile Glu Ile Val Tyr 290 295 300Ala Ala Thr Leu Pro Lys Ala Ser Lys Pro Phe Val Tyr Met Ser Ile305 310 315 320Asn Leu Pro Arg Glu His Val Asp Ile Asn Ile His Pro Thr Lys Lys 325 330 335Glu Val Ser Leu Leu Asn Gln Glu Ile Ile Ile Glu Met Ile Gln Ser 340 345 350Glu Val Glu Val Lys Leu Arg Asn Ala Asn Asp Thr Arg Thr Phe Gln 355 360 365Glu Gln Lys Val Glu Tyr Ile Gln Ser Thr Leu Thr Ser Gln Lys Ser 370 375 380Asp Ser Pro Val Ser Gln Lys Pro Ser Gly Gln Lys Thr Gln Lys Val385 390 395 400Pro Val Asn Lys Met Val Arg Thr Asp Ser Ser Asp Pro Ala Gly Arg 405 410 415Leu His Ala Phe Leu Gln Pro Lys Pro Gln Ser Leu Pro Asp Lys Val 420 425 430Ser Ser Leu Ser Val Val Arg Ser Ser Val Arg Gln Arg Arg Asn Pro 435 440 445Lys Glu Thr Ala Asp Leu Ser Ser Val Gln Glu Leu Ile Ala Gly Val 450 455 460Asp Ser Cys Cys His Pro Gly Met Leu Glu Thr Val Arg Asn Cys Thr465 470 475 480Tyr Val Gly Met Ala Asp Asp Val Phe Ala Leu Val Gln Tyr Asn Thr 485 490 495His Leu Tyr Leu Ala Asn Val Val Asn Leu Ser Lys Glu Leu Met Tyr 500 505 510Gln Gln Thr Leu Arg Arg Phe Ala His Phe Asn Ala Ile Gln Leu Ser 515 520 525Asp Pro Ala Pro Leu Ser Glu Leu Ile Leu Leu Ala Leu Lys Glu Glu 530 535 540Asp Leu Asp Pro Gly Asn Asp Thr Lys Asp Asp Leu Lys Glu Arg Ile545 550 555 560Ala Glu Met Asn Thr Glu Leu Leu Lys Glu Lys Ala Glu Met Leu Glu 565 570 575Glu Tyr Phe Ser Val His Ile Asp Ser Ser Ala Asn Leu Ser Arg Leu 580 585 590Pro Val Ile Leu Asp Gln Tyr Thr Pro Asp Met Asp Arg Val Pro Glu 595 600 605Phe Leu Leu Cys Leu Gly Asn Asp Val Glu Trp Glu Asp Glu Lys Ser 610 615 620Cys Phe Gln Gly Val Ser Ala Ala Ile Gly Asn Phe Tyr Ala Met His625 630 635 640Pro Pro Leu Leu Pro Asn Pro Ser Gly Asp Gly Ile Gln Phe Tyr Ser 645 650 655Lys Arg Gly Glu Ser Ser Gln Glu Lys Ser Asp Leu Glu Gly Asn Val 660 665 670Asp Met Glu Asp Asn Leu Asp Gln Asp Leu Leu Ser Asp Ala Glu Asn 675 680 685Ala Trp Ala Gln Arg Glu Trp Ser Ile Gln His Val Leu Phe Pro Ser 690 695 700Met Arg Leu Phe Leu Lys Pro Pro Ala Ser Met Ala Ser Asn Gly Thr705 710 715 720Phe Val Lys Val Ala Ser Leu Glu Lys Leu Tyr Lys Ile Phe Glu Arg 725 730 735Cys101151PRTArabidopsis thaliana 10Met Lys Thr Ile Lys Pro Leu Pro Glu Gly Val Arg His Ser Met Arg1 5 10 15Ser Gly Ile Ile Met Phe Asp Met Ala Arg Val Val Glu Glu Leu Val 20 25 30Phe Asn Ser Leu Asp Ala Gly Ala Thr Lys Val Ser Ile Phe Val Gly 35 40 45Val Val Ser Cys Ser Val Lys Val Val Asp Asp Gly Ser Gly Val Ser 50 55 60Arg Asp Asp Leu Val Leu Leu Gly Glu Arg Tyr Ala Thr Ser Lys Phe65 70 75 80His Asp Phe Thr Asn Val Glu Thr Ala Ser Glu Thr Phe Gly Phe Arg 85 90 95Gly Glu Ala Leu Ala Ser Ile Ser Asp Ile Ser Leu Leu Glu Val Arg 100 105 110Thr Lys Ala Ile Gly Arg Pro Asn Gly Tyr Arg Lys Val Met Lys Gly 115 120 125Ser Lys Cys Leu His Leu Gly Ile Asp Asp Asp Arg Lys Asp Ser Gly 130 135 140Thr Thr Val Thr Val Arg Asp Leu Phe Tyr Ser Gln Pro Val Arg Arg145 150 155 160Lys Tyr Met Gln Ser Ser Pro Lys Lys Val Leu Glu Ser Ile Lys Lys 165 170 175Cys Val Phe Arg Ile Ala Leu Val His Ser Asn Val Ser Phe Ser Val 180 185 190Leu Asp Ile Glu Ser Asp Glu Glu Leu Phe Gln Thr Asn Pro Ser Ser 195 200 205Ser Ala Phe Ser Leu Leu Met Arg Asp Ala Gly Thr Glu Ala Val Asn 210 215 220Ser Leu Cys Lys Val Asn Val Thr Asp Gly Met Leu Asn Val Ser Gly225 230 235 240Phe Glu Cys Ala Asp Asp Trp Lys Pro Thr Asp Gly Gln Gln Thr Gly 245 250 255Arg Arg Asn Arg Leu Gln Ser Asn Pro Gly Tyr Ile Leu Cys Ile Ala 260 265 270Cys Pro Arg Arg Leu Tyr Glu Phe Ser Phe Glu Pro Ser Lys Thr His 275 280 285Val Glu Phe Lys Lys Trp Gly Pro Val Leu Ala Phe Ile Glu Arg Ile 290 295 300Thr Leu Ala Asn Trp Lys Lys Asp Arg Ile Leu Glu Leu Phe Asp Gly305 310 315 320Gly Ala Asp Ile Leu Ala Lys Gly Asp Arg Gln Asp Leu Ile Asp Asp 325 330 335Lys Ile Arg Leu Gln Asn Gly Ser Leu Phe Ser Ile Leu His Phe Leu 340 345 350Asp Ala Asp Trp Pro Glu Ala Met Glu Pro Ala Lys Lys Lys Leu Lys 355 360 365Arg Ser Asn Asp His Ala Pro Cys Ser Ser Leu Leu Phe Pro Ser Ala 370 375 380Asp Phe Lys Gln Asp Gly Asp Tyr Phe Ser Pro Arg Lys Asp Val Trp385 390 395 400Ser Pro Glu Cys Glu Val Glu Leu Lys Ile Gln Asn Pro Lys Glu Gln 405 410 415Gly Thr Val Ala Gly Phe Glu Ser Arg Thr Asp Ser Leu Leu Gln Ser 420 425 430Arg Asp Ile Glu Met Gln Thr Asn Glu Asp Phe Pro Gln Val Thr Asp 435 440 445Leu Leu Glu Thr Ser Leu Val Ala Asp Ser Lys Cys Arg Lys Gln Phe 450 455 460Leu Thr Arg Cys Gln Ile Thr Thr Pro Val Asn Ile Asn His Asp Phe465 470 475 480Met Lys Asp Ser Asp Val Leu Asn Phe Gln Phe Gln Gly Leu Lys Asp 485 490 495Glu Leu Asp Val Ser Asn Cys Ile Gly Lys His Leu Leu Arg Gly Cys 500 505 510Ser Ser Arg Val Ser Leu Thr Phe His Glu Pro Lys Leu Ser His Val 515 520 525Glu Gly Tyr Glu Ser Val Val Pro Met Ile Pro Asn Glu Lys Gln Ser 530 535 540Ser Pro Arg Val Leu Glu Thr Arg Glu Gly Gly Ser Tyr Cys Asp Val545 550 555 560Tyr Ser Asp Lys Thr Pro Asp Cys Ser Leu Gly Ser Ser Trp Gln Asp 565 570 575Thr Asp Trp Phe Thr Pro Gln Cys Ser Ser Asp Arg Gly Cys Val Gly 580 585 590Ile Gly Glu Asp Phe Asn Ile Thr Pro Ile Asp Thr Ala Glu Phe Asp 595 600 605Ser Tyr Asp Glu Lys Val Gly Ser Lys Lys Tyr Leu Ser Ser Val Asn 610 615 620Val Gly Ser Ser Val Thr Gly Ser Phe Cys Leu Ser Ser Glu Trp Ser625 630 635 640Pro Met Tyr Ser Thr Pro Ser Ala Thr Lys Trp Glu Ser Glu Tyr Gln 645 650 655Lys Gly Cys Arg Ile Leu Glu Gln Ser Leu Arg Leu Gly Arg Met Pro 660 665 670Asp Pro Glu Phe Cys Phe Ser Ala Ala Asn Asn Ile Lys Phe Asp His 675 680 685Glu Val Ile Pro Glu Met Asp Cys Cys Glu Thr Gly
Thr Asp Ser Phe 690 695 700Thr Ala Ile Gln Asn Cys Thr Gln Leu Ala Asp Lys Ile Cys Lys Ser705 710 715 720Ser Trp Gly His Ala Asp Asp Val Arg Ile Asp Gln Tyr Ser Ile Arg 725 730 735Lys Glu Lys Phe Ser Tyr Met Asp Gly Thr Gln Asn Asn Ala Gly Lys 740 745 750Gln Arg Ser Lys Arg Ser Arg Ser Ala Pro Pro Phe Tyr Arg Glu Lys 755 760 765Lys Arg Phe Ile Ser Leu Ser Cys Lys Ser Asp Thr Lys Pro Lys Asn 770 775 780Ser Asp Pro Ser Glu Pro Asp Asp Leu Glu Cys Leu Thr Gln Pro Cys785 790 795 800Asn Ala Ser Gln Met His Leu Lys Cys Ser Ile Leu Asp Asp Val Ser 805 810 815Tyr Asp His Ile Gln Glu Thr Glu Lys Arg Leu Ser Ser Ala Ser Asp 820 825 830Leu Lys Ala Ser Ala Gly Cys Arg Thr Val His Ser Glu Thr Gln Asp 835 840 845Glu Asp Val His Glu Asp Phe Ser Ser Glu Glu Phe Leu Asp Pro Ile 850 855 860Lys Ser Thr Thr Lys Trp Arg His Asn Cys Ala Val Ser Gln Val Pro865 870 875 880Lys Glu Ser His Glu Leu His Gly Gln Asp Gly Val Phe Asp Ile Ser 885 890 895Ser Gly Leu Leu His Leu Arg Ser Asp Glu Ser Leu Val Pro Glu Ser 900 905 910Ile Asn Arg His Ser Leu Glu Asp Ala Lys Val Leu Gln Gln Val Asp 915 920 925Lys Lys Tyr Ile Pro Ile Val Ala Cys Gly Thr Val Ala Ile Val Asp 930 935 940Gln His Ala Ala Asp Glu Arg Ile Arg Leu Glu Glu Leu Arg Thr Lys945 950 955 960Phe Ile Asn Asp Ala Leu Leu Ile Phe Val Leu Thr Leu Lys Val Leu 965 970 975Pro Glu Met Gly Tyr Gln Leu Leu Gln Ser Tyr Ser Glu Gln Ile Arg 980 985 990Asp Trp Gly Trp Ile Cys Asn Ile Thr Val Glu Gly Ser Thr Ser Phe 995 1000 1005Lys Lys Asn Met Ser Ile Ile Gln Arg Lys Pro Thr Pro Ile Thr Leu 1010 1015 1020Asn Ala Val Pro Cys Ile Leu Gly Val Asn Leu Ser Asp Val Asp Leu1025 1030 1035 1040Leu Glu Phe Leu Gln Gln Leu Ala Asp Thr Asp Gly Ser Ser Thr Ile 1045 1050 1055Pro Pro Ser Val Leu Arg Val Leu Asn Ser Lys Ala Cys Arg Gly Ala 1060 1065 1070Ile Met Phe Gly Asp Ser Leu Leu Pro Ser Glu Cys Ser Leu Ile Ile 1075 1080 1085Asp Gly Leu Lys Gln Thr Ser Leu Cys Phe Gln Cys Ala His Gly Arg 1090 1095 1100Pro Thr Thr Val Pro Leu Val Asp Leu Lys Ala Leu His Lys Gln Ile1105 1110 1115 1120Ala Lys Leu Ser Gly Arg Gln Val Trp His Gly Leu Gln Arg Arg Glu 1125 1130 1135Ile Thr Leu Asp Arg Ala Lys Ser Arg Leu Asp Asn Ala Lys Ser 1140 1145 115011862PRTHomo sapiens 11Met Glu Arg Ala Glu Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys1 5 10 15Pro Ile Asp Arg Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Val 20 25 30Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp 35 40 45Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp 50 55 60Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe65 70 75 80Glu Gly Leu Thr Leu Lys His His Thr Ser Lys Ile Gln Glu Phe Ala 85 90 95Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser 100 105 110Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys His Ala Ser 115 120 125Ala Lys Val Gly Thr Arg Leu Met Phe Asp His Asn Gly Lys Ile Ile 130 135 140Gln Lys Thr Pro Tyr Pro Arg Pro Arg Gly Thr Thr Val Ser Val Gln145 150 155 160Gln Leu Phe Ser Thr Leu Pro Val Arg His Lys Glu Phe Gln Arg Asn 165 170 175Ile Lys Lys Glu Tyr Ala Lys Met Val Gln Val Leu His Ala Tyr Cys 180 185 190Ile Ile Ser Ala Gly Ile Arg Val Ser Cys Thr Asn Gln Leu Gly Gln 195 200 205Gly Lys Arg Gln Pro Val Val Cys Thr Gly Gly Ser Pro Ser Ile Lys 210 215 220Glu Asn Ile Gly Ser Val Phe Gly Gln Lys Gln Leu Gln Ser Leu Ile225 230 235 240Pro Phe Val Gln Leu Pro Pro Ser Asp Ser Val Cys Glu Glu Tyr Gly 245 250 255Leu Ser Cys Ser Asp Ala Leu His Asn Leu Phe Tyr Ile Ser Gly Phe 260 265 270Ile Ser Gln Cys Thr His Gly Val Gly Arg Ser Ser Thr Asp Arg Gln 275 280 285Phe Phe Phe Ile Asn Arg Arg Pro Cys Asp Pro Ala Lys Val Cys Arg 290 295 300Leu Val Asn Glu Val Tyr His Met Tyr Asn Arg His Gln Tyr Pro Phe305 310 315 320Val Val Leu Asn Ile Ser Val Asp Ser Glu Cys Val Asp Ile Asn Val 325 330 335Thr Pro Asp Lys Arg Gln Ile Leu Leu Gln Glu Glu Lys Leu Leu Leu 340 345 350Ala Val Leu Lys Thr Ser Leu Ile Gly Met Phe Asp Ser Asp Val Asn 355 360 365Lys Leu Asn Val Ser Gln Gln Pro Leu Leu Asp Val Glu Gly Asn Leu 370 375 380Ile Lys Met His Ala Ala Asp Leu Glu Lys Pro Met Val Glu Lys Gln385 390 395 400Asp Gln Ser Pro Ser Leu Arg Thr Gly Glu Glu Lys Lys Asp Val Ser 405 410 415Ile Ser Arg Leu Arg Glu Ala Phe Ser Leu Arg His Thr Thr Glu Asn 420 425 430Lys Pro His Ser Pro Lys Thr Pro Glu Pro Arg Arg Ser Pro Leu Gly 435 440 445Gln Lys Arg Gly Met Leu Ser Ser Ser Thr Ser Gly Ala Ile Ser Asp 450 455 460Lys Gly Val Leu Arg Pro Gln Lys Glu Ala Val Ser Ser Ser His Gly465 470 475 480Pro Ser Asp Pro Thr Asp Arg Ala Glu Val Glu Lys Asp Ser Gly His 485 490 495Gly Ser Thr Ser Val Asp Ser Glu Gly Phe Ser Ile Pro Asp Thr Gly 500 505 510Ser His Cys Ser Ser Glu Tyr Ala Ala Ser Ser Pro Gly Asp Arg Gly 515 520 525Ser Gln Glu His Val Asp Ser Gln Glu Lys Ala Pro Glu Thr Asp Asp 530 535 540Ser Phe Ser Asp Val Asp Cys His Ser Asn Gln Glu Asp Thr Gly Cys545 550 555 560Lys Phe Arg Val Leu Pro Gln Pro Thr Asn Leu Ala Thr Pro Asn Thr 565 570 575Lys Arg Phe Lys Lys Glu Glu Ile Leu Ser Ser Ser Asp Ile Cys Gln 580 585 590Lys Leu Val Asn Thr Gln Asp Met Ser Ala Ser Gln Val Asp Val Ala 595 600 605Val Lys Ile Asn Lys Lys Val Val Pro Leu Asp Phe Ser Met Ser Ser 610 615 620Leu Ala Lys Arg Ile Lys Gln Leu His His Glu Ala Gln Gln Ser Glu625 630 635 640Gly Glu Gln Asn Tyr Arg Lys Phe Arg Ala Lys Ile Cys Pro Gly Glu 645 650 655Asn Gln Ala Ala Glu Asp Glu Leu Arg Lys Glu Ile Ser Lys Thr Met 660 665 670Phe Ala Glu Met Glu Ile Ile Gly Gln Phe Asn Leu Gly Phe Ile Ile 675 680 685Thr Lys Leu Asn Glu Asp Ile Phe Ile Val Asp Gln His Ala Thr Asp 690 695 700Glu Lys Tyr Asn Phe Glu Met Leu Gln Gln His Thr Val Leu Gln Gly705 710 715 720Gln Arg Leu Ile Ala Pro Gln Thr Leu Asn Leu Thr Ala Val Asn Glu 725 730 735Ala Val Leu Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp 740 745 750Phe Val Ile Asp Glu Asn Ala Pro Val Thr Glu Arg Ala Lys Leu Ile 755 760 765Ser Leu Pro Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp Val Asp 770 775 780Glu Leu Ile Phe Met Leu Ser Asp Ser Pro Gly Val Met Cys Arg Pro785 790 795 800Ser Arg Val Lys Gln Met Phe Ala Ser Arg Ala Cys Arg Lys Ser Val 805 810 815Met Ile Gly Thr Ala Leu Asn Thr Ser Glu Met Lys Lys Leu Ile Thr 820 825 830His Met Gly Glu Met Asp His Pro Trp Asn Cys Pro His Gly Arg Pro 835 840 845Thr Met Arg His Ile Ala Asn Leu Gly Val Ile Ser Gln Asn 850 855 86012779PRTArabidopsis thaliana 12Met Gln Gly Asp Ser Ser Pro Ser Pro Thr Thr Thr Ser Ser Pro Leu1 5 10 15Ile Arg Pro Ile Asn Arg Asn Val Ile His Arg Ile Cys Ser Gly Gln 20 25 30Val Ile Leu Asp Leu Ser Ser Ala Val Lys Glu Leu Val Glu Asn Ser 35 40 45Leu Asp Ala Gly Ala Thr Ser Ile Glu Ile Asn Leu Arg Asp Tyr Gly 50 55 60Glu Asp Tyr Phe Gln Val Ile Asp Asn Gly Cys Gly Ile Ser Pro Thr65 70 75 80Asn Phe Lys Val Cys Val Gln Ile Leu Arg Arg Thr Phe Asp Val Leu 85 90 95Ala Leu Lys His His Thr Ser Lys Leu Glu Asp Phe Thr Asp Leu Leu 100 105 110Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala Leu Ser Ser Leu Cys 115 120 125Ala Leu Gly Asn Leu Thr Val Glu Thr Arg Thr Lys Asn Glu Pro Val 130 135 140Ala Thr Leu Leu Thr Phe Asp His Ser Gly Leu Leu Thr Ala Glu Lys145 150 155 160Lys Thr Ala Arg Gln Ile Gly Thr Thr Val Thr Val Arg Lys Leu Phe 165 170 175Ser Asn Leu Pro Val Arg Ser Lys Glu Phe Lys Arg Asn Ile Arg Lys 180 185 190Glu Tyr Gly Lys Leu Val Ser Leu Leu Asn Ala Tyr Ala Leu Ile Ala 195 200 205Lys Gly Val Arg Phe Val Cys Ser Asn Thr Thr Gly Lys Asn Pro Lys 210 215 220Ser Val Val Leu Asn Thr Gln Gly Arg Gly Ser Leu Lys Asp Asn Ile225 230 235 240Ile Thr Val Phe Gly Ile Ser Thr Phe Thr Ser Leu Gln Pro Gly Thr 245 250 255Gly Arg Asn Leu Ala Asp Arg Gln Tyr Phe Phe Ile Asn Gly Arg Pro 260 265 270Val Asp Met Pro Lys Val Ser Lys Leu Val Asn Glu Leu Tyr Lys Asp 275 280 285Thr Ser Ser Arg Lys Tyr Pro Val Thr Ile Leu Asp Phe Ile Val Pro 290 295 300Gly Gly Ala Cys Asp Leu Asn Val Thr Pro Asp Lys Arg Lys Val Phe305 310 315 320Phe Ser Asp Glu Thr Ser Val Ile Gly Ser Leu Arg Glu Gly Leu Asn 325 330 335Glu Ile Tyr Ser Ser Ser Asn Ala Ser Tyr Ile Val Asn Arg Phe Glu 340 345 350Glu Asn Ser Glu Gln Pro Asp Lys Ala Gly Val Ser Ser Phe Gln Lys 355 360 365Lys Ser Asn Leu Leu Ser Glu Gly Ile Val Leu Asp Val Ser Ser Lys 370 375 380Thr Arg Leu Gly Glu Ala Ile Glu Lys Glu Asn Pro Ser Leu Arg Glu385 390 395 400Val Glu Ile Asp Asn Ser Ser Pro Met Glu Lys Phe Lys Phe Glu Ile 405 410 415Lys Ala Cys Gly Thr Lys Lys Gly Glu Gly Ser Leu Ser Val His Asp 420 425 430Val Thr His Leu Asp Lys Thr Pro Ser Lys Gly Leu Pro Gln Leu Asn 435 440 445Val Thr Glu Lys Val Thr Asp Ala Ser Lys Asp Leu Ser Ser Arg Ser 450 455 460Ser Phe Ala Gln Ser Thr Leu Asn Thr Phe Val Thr Met Gly Lys Arg465 470 475 480Lys His Glu Asn Ile Ser Thr Ile Leu Ser Glu Thr Pro Val Leu Arg 485 490 495Asn Gln Thr Ser Ser Tyr Arg Val Glu Lys Ser Lys Phe Glu Val Arg 500 505 510Ala Leu Ala Ser Arg Cys Leu Val Glu Gly Asp Gln Leu Asp Asp Met 515 520 525Val Ile Ser Lys Glu Asp Met Thr Pro Ser Glu Arg Asp Ser Glu Leu 530 535 540Gly Asn Arg Ile Ser Pro Gly Thr Gln Ala Asp Asn Val Glu Arg His545 550 555 560Glu Arg Val Leu Gly Gln Phe Asn Leu Gly Phe Ile Ile Ala Lys Leu 565 570 575Glu Arg Asp Leu Phe Ile Val Asp Gln His Ala Ala Asp Glu Lys Phe 580 585 590Asn Phe Glu His Leu Ala Arg Ser Thr Val Leu Asn Gln Gln Pro Leu 595 600 605Leu Gln Pro Leu Asn Leu Glu Leu Ser Pro Glu Glu Glu Val Thr Val 610 615 620Leu Met His Met Asp Ile Ile Arg Glu Asn Gly Phe Leu Leu Glu Glu625 630 635 640Asn Pro Ser Ala Pro Pro Gly Lys His Phe Arg Leu Arg Ala Ile Pro 645 650 655Tyr Ser Lys Asn Ile Thr Phe Gly Val Glu Asp Leu Lys Asp Leu Ile 660 665 670Ser Thr Leu Gly Asp Asn His Gly Glu Cys Ser Val Ala Ser Ser Tyr 675 680 685Lys Thr Ser Lys Thr Asp Ser Ile Cys Pro Ser Arg Val Arg Ala Met 690 695 700Leu Ala Ser Arg Ala Cys Arg Ser Ser Val Met Ile Gly Asp Pro Leu705 710 715 720Arg Lys Asn Glu Met Gln Lys Ile Val Glu His Leu Ala Asp Leu Glu 725 730 735Ser Pro Trp Asn Cys Pro His Gly Arg Pro Thr Met Arg His Leu Val 740 745 750Asp Leu Thr Thr Leu Leu Thr Leu Pro Asp Asp Asp Asn Val Asn Asp 755 760 765Asp Asp Asp Asp Asp Ala Thr Ile Ser Leu Ala 770 77513133PRTHomo sapiens 13Met Glu Arg Ala Glu Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys1 5 10 15Pro Ile Asp Arg Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Val 20 25 30Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp 35 40 45Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp 50 55 60Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe65 70 75 80Glu Gly Leu Thr Leu Lys His His Thr Ser Lys Ile Gln Glu Phe Ala 85 90 95Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser 100 105 110Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys His Ala Ser 115 120 125Ala Lys Val Gly Thr 13014147PRTArabidopsis thaliana 14Met Gln Gly Asp Ser Ser Pro Ser Pro Thr Thr Thr Ser Ser Pro Leu1 5 10 15Ile Arg Pro Ile Asn Arg Asn Val Ile His Arg Ile Cys Ser Gly Gln 20 25 30Val Ile Leu Asp Leu Ser Ser Ala Val Lys Glu Leu Val Glu Asn Ser 35 40 45Leu Asp Ala Gly Ala Thr Ser Ile Glu Ile Asn Leu Arg Asp Tyr Gly 50 55 60Glu Asp Tyr Phe Gln Val Ile Asp Asn Gly Cys Gly Ile Ser Pro Thr65 70 75 80Asn Phe Lys Val Cys Val Gln Ile Leu Arg Arg Thr Phe Asp Val Leu 85 90 95Ala Leu Lys His His Thr Ser Lys Leu Glu Asp Phe Thr Asp Leu Leu 100 105 110Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala Leu Ser Ser Leu Cys 115 120 125Ala Leu Gly Asn Leu Thr Val Glu Thr Arg Thr Lys Asn Glu Pro Val 130 135 140Ala Thr Leu1451554DNAArtificial SequencePCR primer 15gtcgacttat cacttgtcat cgtcgtcctt gtagtcgagc gtagcaactg gctc 54
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