Progress 07/01/05 to 06/30/09
Outputs
OUTPUTS: Genetic experiments were conducted to test the hypothesis that in a RAD51-minus genetic background the nature of the homolog determines whether HR with the homolog or NHEJ is the favored repair pathway. All four genetic experiments (1.1-1.4) designed to test the hypothesis have been completed. The results of these experiments consistently support the view that the homolog is NOT used to any appreciable degree as the repair template in RAD51 minus plants. Efforts were made to obtain a dmc1 defective mutant to facilitate the functional analysis of this gene. Homozygous mutants of four previously isolated Mu insertion of alleles of dmc1 do not have any obvious phenotypes. Because these four alleles carry Mu insertions either in the 5' UTR or in the intron, dmc1 function may not be completely lost in these alleles. In an effort to obtain a null dmc1 mutant, a screen was conducted for derivative alleles in which DNA sequences flanking the Mu insertions in dmc1 had been lost. This screen was conducted for all four Mu insertion alleles of dmc1 that had been introduced into a RAD51 minus genetic background, in which NHEJ is the dominant pathway to repair Mu-derived DSBs. Progeny from RAD51 minus mutants that carry Mu insertion alleles of dmc1 were screened for Mu adjacent deletions. However, no adjacent deletions were identified from more than ~400 progeny. Project outputs have been disseminated to the scientific community via seminars and poster presentations. PARTICIPANTS: Patrick Schnable (PI), Sanzhen Liu (graduate student), Jin Li (graduate student), Mitzi Wilkening (research associate), Megan Brown (lab asst), Chor-Han Tan (lab asst), Ho Man Tang (lab asst), Rana Kasper (lab asst), Wan Chin Lim (lab asst), Barbara Pawlikowski (lab asst), Jia Ling Pik (lab asst), Jennifer Trump (lab asst). Graduate student attended a scientific conference and summer training workshop. TARGET AUDIENCES: Maize genetics research community and seed companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results of all four experiments indicate that NHEJ is a dominant pathway to repair Mu-derived double-stranded breaks (DSBs) in RAD51 minus plants. Based on this finding, derivative alleles would be expected to occur at high rates in a RAD51 minus background. The finding that no deletion derivatives of dmc1a Mu insertion alleles were recovered from approximately ~400 progeny from RAD51 minus plants indicates that the rate at which deletion derivatives arise is influenced by factors that remain to be elucidated, but may include Mu activity and the type of inserted Mu transposon. All of this points to a need for further research on the mechanisms responsible for repair of Mu-induced DSBs.
Publications
- No publications reported this period
|
Progress 07/01/07 to 06/30/08
Outputs
OUTPUTS: Genetic experiments were conducted to test the hypothesis that in a RAD51-minus genetic background the nature of the homolog determines whether HR with the homolog or NHEJ is the favored repair pathway. A genetic screen was conducted to isolate mutants in the dmc1 gene. Project outputs have been disseminated to the scientific community via seminars and poster presentations. PARTICIPANTS: Patrick Schnable (PI), Sanzhen Liu (graduate student), Rana Kasper (lab asst), Wan Chin Lim (lab asst), Barbara Pawlikowski (lab asst), Jia Ling Pik (lab asst), Jennifer Trump (lab asst). ). Graduate student attended a scientific conference and summer training workshop. TARGET AUDIENCES: Maize genetics research community and seed companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
All four genetic experiments designed to test the hypothesis that in a RAD51-minus genetic background the nature of the homolog determines whether HR with the homolog or NHEJ is the favored repair pathway have been completed. The results of these experiments consistently support the view that the homolog is NOT used to any appreciable degree as the repair template in RAD51 minus plants. For functional analysis of dmc1 it would be desirable to use mutant alleles that contain deletions of dmc1 coding sequences. Using the three previously isolated Mu insertion alleles of dmc1, a screen is being to identify derivative alleles in which DNA sequences flanking Mu insertions have been lost. To enhance the recovery of such alleles, this screen is being conducted in a RAD51 minus genetic background, in which NHEJ (rather than HR) is the dominant pathway to repair Mu-derived DSBs. Progeny from RAD51 minus mutants that carry the dmc1a-mtm alleles were generated during the summer of 2008. During the remainder of this funding period, these progeny will be screened for adjacent deletions, which would be expected to disrupt dmc1a gene function.
Publications
- Li, J., Wen, T.-J., and Schnable, P.S. 2008. The role of RAD51 in the repair of MuDR-induced DSBs in Zea mays L. Genetics 178:57-66.
- Jackson, B.N., Aluru, S., and Schnable, P.S. 2008. Consensus genetic maps as median orders from inconsistent sources. ACM/IEEE Transactions Computational Biology and Bioinformatics 5(2):161-71.
|
Progress 07/01/06 to 06/30/07
Outputs
OUTPUTS: Crosses have been performed to test whether the locus (Experiment 1.1) or MuDR homozygosity (Experiment 1.2) affects the outcomes of DSB repair. Similarly, crosses to test whether the homolog is used as a template for gene conversion (Experiment 1.4) have been conducted. The progeny of these crosses will be subjected to genetic and molecular analyses during the coming year. The previously isolated dmc1a-93F11 allele does not condition any obvious phenotype. This is not surprising given that the Mu insertion associated with this allele is located in intron 2. To obtain a knock-out allele, a screen was conducted to isolate a deletion derivative of this allele in which dmc1a sequences (including portions of exon 3) adjacent to the Mu insertion have been lost. Based on observations that such deletions arise at elevated frequencies in RAD51 minus plant this screen was conducted in both RAD51 plus and RAD51 minus genetic backgrounds. No adjacent deletions were recovered from
either genetic background. In a parallel effort to recovery a knock-out allele of dmc1a, three additional Mu transposon insertion alleles of dmc1a (dmc1a-mtm18778, dmc1a-mtm18780, dmc1a-mtm21046) were isolated using the reverse genetics (MTM) resource at Cold Spring Harbor Laboratories. All three alleles contain Mu insertions in the 5' UTR of dmc1a. So far, no obvious phenotypes have been to be associated with any of these mutants. Project outputs have been disseminated to the scientific community via publications and poster presentations. These genetic experiments must be conducted in plants with Mu activity. It was observed that in RAD51 minus plants Mu activity causes plants to be much weaker than RAD51 minus plants that lack Mu activity. This feature complicates these experiments because it is difficult to obtain sufficient seeds for analysis.
PARTICIPANTS: Individuals: Patrick S. Schnable, PI; Jin Li, Graduate student; Sanzhen Liu, Graduate Student; Mitzi Wilkening, Research Associate. Partner Organizations: UC Berkeley; Pioneer Hi-Bred, International.
Impacts
Although progeny from the genetic crosses are still under analysis, it can already be concluded that the rate of gene conversion in a1-m5216/A1-5216 heterozygotes is less than 2% (Experiment 1.4). The finding that no deletion derivatives of dmc1a-93F11 were recovered from approximately 200 progeny from RAD51 minus plants indicates that the rate at which deletion derivatives arise is influenced by factors that remain to be elucidated, but may include the type of inserted Mu transposon. The observation that RAD51 minus plants that carry an active Mu transposon system are weak indicates both that Mu transposons are creating double-strand breaks (DSBs) during somatic development and that RAD51 is required to repair these DBSs.
Publications
- Yandeau-Nelson, M.D., Xia, Y., Li, J., Neuffer, M.G., and Schnable, P.S. 2006. Unequal sister chromatid and homolog recombination at a tandem duplication of the a1 locus in maize. Genetics 173:2211-2226.
- Yandeau-Nelson, M.D., Nikolau, B.J., and Schnable, P.S. 2006. Effects of trans-acting genetic modifiers on the rates and distribution of meiotic recombination across the a1-sh2 interval of maize. Genetics 174:101-112.
- Li, J., Hsia, A.P., and Schnable, P.S. 2007. Recent advances in plant recombination. Current Opinion in Plant Science 10:131-135.
- Li, J., Harper, L.C., Golubovskaya, I., Wang, C.R., Weber, D.F., Meeley, R.B., McElverd, J., Bowen, B., Cande, W.Z., and Schnable, P.S. 2007. Functional analysis of maize RAD51 in meiosis and DSBs repair. Genetics 176:1469-1482.
|
Progress 07/01/05 to 06/30/06
Outputs
Genotypes have been constructed to test whether the locus (Experiment 1.1) or MuDR homozygosity (Experiment 1.2) affects the outcomes of DSB repair. Similarly, genotypes required to test whether the homolog is used as a template for homologous recombination (Experiment 1.4) have been constructed. Genotypes are being constructed to test whether presence of an rdt transposon insertion affects the outcomes of DSB repair (Experiment 1.3). A screen for deletion derivatives of the dmc1 genes is underway (Experiment 2).
Impacts
By enhancing understanding of DSB repair and recombination in plants these studies will suggest methods to regulate recombination rates and perhaps to develop a gene knock-out system and other tools needed to understand the functions of plant genes and to engineer plant genomes. Because maize is an agronomically important crop species, the findings and tools generated by the proposed project will be directly applicable to crop improvement.
Publications
- No publications reported this period
|
|