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Genetic and molecular analysis of neurospora duplications and duplication-generating translocations

Shodhganga@INFLIBNET

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Title Genetic and molecular analysis of neurospora duplications and duplication-generating translocations
 
Contributor Kasbekar, D P
 
Subject Neurospora, Duplication, Duplication-generating translocations, Repeat-induced point mutation
 
Description In Neurospora, duplicated DNA sequences are substrates for repeat-induced point mutation (RIP), and my studies show that the RIP machinery is titrated out by > 300 kbp of duplicated DNA. RIP is a genome defense process which occurs during the premeiosis of a sexual cross and induces G: C to A: T mutations and cytosine methylation in duplicated DNA in an otherwise haploid genome. Both large (i.e., > 100 kbp) and small duplications (of the order of 0.5 - 5 kbp) are substrates for RIP. Strains bearing a large duplication (Dp strains) can be obtained from a cross of normal sequence strains (N) with some translocation strains. Dp segregants are identifiable by the barren phenotype of Dp x N crosses. That is, Dp x N crosses make normal-looking perithecia but produce very few ascospores. The barrenness is due to an RNAi-based process called meiotic silencing by unpaired DNA (MSUD). The semi-dominant Sad-1, Sad-2 and Sms-2 suppressors of MSUD suppress the barren phenotype of Dp-heterozygous crosses and can increase their productivity.Previous work from our laboratory revealed that five of six large Dp’s could dominantly suppress RIP in a small gene-sized probe duplication called Dp(erg-3). The suppressor Dp’s were all > 270 kbp, whereas the one non-suppressor Dp, Dp(B362i), was approximately 117 kbp. I screened another 35 Dp’s and found that 29 were suppressors and four non-suppressors. For 27 of the 34 suppressor Dp’s I estimated their minimum size to be > 270 kb, and for three non-suppressors Dp’s I estimated the maximum size to be < 200 kb. The size of the remaining Dp’s was not estimated because no covered markers were known for them. I also found that RIP is suppressed in a significant proportion of crosses that were multiply heterozygous for more than one non-suppressor Dp if the combined size of the duplicated DNA was close to or more than 300 kbp. These findings support our hypothesis that large Dp’s suppress RIP via titration of the RIP machinery and the “equivalence point” is ~ 300 kbp. I describe this work in Chapter 3.Breakpoint junction sequences can provide information regarding role of homologous and non-homologous end joining DNA repair pathways in the origin of rearrangements and thus give an idea of the mechanism of mutagenesis. Segment spanning breakpoints have been cloned from various rearrangements of Neurospora, but no nucleotide sequence information was available except for one reciprocal translocation T(IR;VIR)UK-T12. In Chapter 4, I describe further breakpoint localization of 24 Dp’s by analysis of RFLPs coverage and the determination of breakpoint junction sequences (i.e., deletion junctions on the donor and insertion junctions in the recipient LG) of 11translocations by performing inverse PCR with translocation DNA template and primers annealing just “outside” and “within” the translocated segments. Chromosomal rearrangements are known not only to mutate genes by disruption but also known to create gene fusions, which might generate novel chimeric proteins. I analyzed 30 breakpoint junction sequences of 11 Dp-generating translocations and found 13 gene disruptions, 10 T-specific novel fusion genes and about 3-6 bp micro-homology at some junctions. I also found that the breakpoint-disrupted eat-3gene is essential for ascospores formation in Neurospora although its homologue ami-1 is dispensable for this function in Podospora. I describe this work in Chapter 5.
Full text will be available after award of Degree
 
Date 2010-06-25T12:17:33Z
2010-06-25T12:17:33Z
2010-06-25
October 2006
21/5/2010
18/11/2010
 
Type Ph.D.
 
Identifier http://hdl.handle.net/10603/68
 
Language English
 
Rights Jawaharlal Nehru University
 
Format Acknowledgment, Synopsis, Appendix, Future plans, Reference and Publications
 
Publisher Delhi
Jawaharlal Nehru University
School of Life Sciences
 
Source Self Submission