2013 — 2014 |
Larracuente, Amanda Marie |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
The Role of Satellite Rasirna in the Segregation Distorter System of Drosophila @ University of Rochester
DESCRIPTION (provided by applicant): Genomes are frequently in conflict with selfish genes that bias their transmission to the next generation in a process called meiotic drive, often at the cost of the organism. Segregation Distorter (SD) is a well-studied meiotic drive system in D. melanogaster: in SD/SD+ heterozygous males, the SD chromosome is transmitted to 95% of the progeny by killing sperm carrying sensitive alleles of its target locus, Responder (Rsp-a satellite repeat near the centromere of chromosome 2). The mechanism behind SD is unknown but it may involve RNA interference (RNAi), similar to other meiotic driver systems. Many features of spermatogenesis in eukaryotes cannot be explained without invoking a species history of genetic conflict (e.g. RNAi, rapid gene evolution). Understanding how selfish genes, like Sd, exploit RNAi to kill sperm will offer unique insight into the role of small RNAs in spermatogenesis. This proposal aims first to determine the cause of SD+ spermatid dysfunction in SD/SD+ heterozygotes, and second, to describe the distribution of Rsp repeat-associated short interfering RNAs (rasiRNAs) in the testis and test if they are disrupted in SD/SD+ heterozygotes. Third, this proposal will test the hypothesis that SD kills SD+-bearing spermatids by interfering with postmeiotic rasiRNA production in the testis. In the presence of SD, SD+ spermatids with a Rsp locus sensitive to distortion (Rsps), fail to condense their chromatin at a time when spermatids swap their histones for sperm-specific protamines to aid in condensing the nucleus as it is re- shaped into the sperm head. To determine which chromatin components show aberrant localization in SD/SD+ testes, this proposal will immunolocalize core histones and analyze the expression of GFP-tagged transition proteins, protamines and a protamine-associated chromatin component also involved in nuclear re-shaping (Aim 1). Preliminary results described in this proposal show Rsp rasiRNA expression in the testis after meiosis. This proposal will test the hypothesis that SD interferes with Rsp rasiRNA localization or expression by looking for a disruption of Rsp rasiRNAs in SD/SD+ testes using Fluorescence In Situ Hybridization (Aim 2). This proposal will also test the hypothesis that Rsp rasiRNAs originate from a genomic location outside of the satellite repeat itself, as preliminary results suggest. Thi will be accomplished by generating deletions of genomic regions containing Rsp repeats found outside of the satellite locus. The deletions will be used to test for a disruption of Rsp rasiRNAs and consequently, distortion against the Rsps-bearing chromosome (Aim 2). Finally, this proposal will test the hypothesis that SD interferes with postmeiotic rasiRNA production in the testis. To do this, I will sequence and compare small RNAs from dissections of SD/SD, SD/SD+ and SD+/SD+ testes enriched for mitotic and postmeiotic cells, separately, with Illumina short read technology (Aim 3). If SD affects Rsp rasiRNA production, then SD genotype will correlate with Rsp rasiRNA abundance in postmeiotic testis dissections.
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0.958 |
2016 — 2020 |
Larracuente, Amanda Marie |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
The Evolutionary and Functional Genomics of Satellite Dna @ University of Rochester
Project summary Eukaryotic genomes contain arrays of tandemly repeated non-coding sequences that we currently know little about?satellite DNAs. Typically found near centromeres, telomeres and on Y chromosomes, satellite DNAs can comprise over 50% of some eukaryotic genomes. They are known to change rapidly in sequence and genomic location, which can cause genetic incompatibilities between closely related species. The misregulation of satellite DNA can have serious consequences for genomic stability and cancer formation. Despite being a ubiquitous part of genomes and having important functional consequences, we know little about satellite DNA. The lack of genetic, genomic and molecular tools to study tandemly repeated sequences has stymied progress towards understanding satellite DNA evolution and function. For example, satellite DNAs are particularly challenging to sequence and assemble. Recent developments in next-generation sequencing technologies circumvent some of these problems. This proposal integrates genomic, molecular and cytological methods to study the evolutionary and functional genomics of satellite DNA in Drosophila genomes. The PI has developed new genomic and cytological methods to study the evolutionary dynamics, genomic structure and expression of satellite DNA with unprecedented resolution. The PI will use these methods to study changes in satellite DNA sequence, abundance and organization over evolutionary time and to determine the evolutionary forces responsible for these changes. This proposal aims to develop comprehensive models of satellite DNA evolution that take into consideration different types of natural selection based on the functional aspects of satellite DNAs. Little is currently known of satellite DNA function: the precise genetic manipulation of satellite DNAs with site-specific approaches had not been possible in the past due to a lack of unique target sites. The new genomic methods developed in this proposal provide an opportunity to discover unique sites flanking satellite DNAs that may serve as targets for genome editing techniques. The proposal will create precise genomic deletions of satellite DNA in Drosophila melanogaster to test specific hypotheses about the regulation, fitness effects and selfish genetic behavior of satellite DNA. This proposal will also use new molecular genetic techniques to manipulate the expression of satellite DNAs in the germline to ask questions about their functions in chromosome segregation and chromatin organization. These experiments will have broad implications not only for genome evolution, but also for understanding the regulation of satellite DNA in cancer and aging.
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0.958 |