Browsing by Author "Gregory C. Gibson, Committee Member"

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Development of Linkage and Association Methods to Map Disease Genes
(2002-10-28) Liu, Wenlei; Gregory C. Gibson, Committee Member; Bruce S. Weir, Committee Chair; Zhao-Bang Zeng, Committee Member; Dahlia M. Nielsen, Committee Member
Identification of disease susceptibility genes is one of the primary aims of contemporary genetic research. With the recent development in molecular biology techniques, large-scale gene mapping with a dense genome-spanning set of markers becomes a reality. The availability of markers throughout the genome has made linkage and association studies more feasible. In the first chapter, we review many linkage and association methods and point out the potential problems with current linkage and association analysis. In the second chapter, we modify two identity-by-state (IBS) test statistics of Lange (Lange K. 1986a, A test statistic for the affected-sib-set method. Annals of Human Genetics 50, 283--290; Lange K. 1986b, The affected sib-pair method using identity by descent relations. American Journal of Human Genetics 39, 148--150.) to allow for inbreeding in the population. We evaluate the power and false positive rates of the modified tests under three disease models using simulated data. When the population inbreeding coefficient is large, both the false positive rates and power are reduced when the modified test statistics were applied, although power remained high under a recessive disease model. Allowing for inbreeding is therefore appropriate at least for diseases known to be recessive. In the third chapter, we compute the proportions of affected sib pairs sharing 0, 1 and 2 marker alleles identity-by-decent (IBD) in an inbred population and express them in terms of higher order decent measures. We perform two consistency checks on the identity state probabilities and the two consistency checks verify our calculations. We did the same thing for affected sib pairs from first cousin marriage in an inbred population. In the fourth chapter, we study linkage and linkage disequilibrium (LD) simultaneously for single QTL using family data in an attempt to increase mapping resolution and reduce false positive rates. We estimate QTL allele frequencies, LD and recombination factions between the marker loci and the QTL locus and the QTL model parameters using an EM algorithm. After performing single analysis, we extend our model to study two marker loci simultaneously so that we can increase the accuracy of the estimations. Our simulation results show that our EM algorithm can give consistent estimates of all the parameters considered.
The Genetic Architecture of Complex Traits: Starvation Resistance in Drosophila melanogaster
(2004-09-02) Harbison, Susan Tracy; Trudy F. C. Mackay, Committee Chair; Michael D. Purugganan, Committee Member; Gregory C. Gibson, Committee Member; Bruce S. Weir, Committee Member
In nature, animals are often subjected to periods of sub-optimal food resources. Characteristic responses to starvation stress have been observed in bacteria, nematodes and yeast: they alter their morphology, become quiescent, and suspend reproduction until adequate food resources become available. Studies of laboratory and natural populations of Drosophila reveal a surprising amount of genetic variation for starvation tolerance. The presence of this genetic variation is an evolutionary puzzle, as variability would not be expected in a key trait related to individual survival. While starvation resistance has been positively correlated with lifespan and other stresses, it is often negatively correlated with fecundity, suggesting that a trade-off between reproduction and individual survival might be present. In order to evaluate this hypothesis, the suite of genes affecting starvation resistance and their properties must be known. Three complementary methods were used to identify genes affecting starvation resistance: a P-element insertional mutagenesis screen, which directly identifies candidate genes involved in the response to starvation stress; deficiency complementation mapping, which reveals small genomic regions contributing to variation in starvation resistance; and transcriptome analysis using microarrays, which has the potential to identify both types of genes. The starvation tolerance phenotype was assessed for 933 P-element insertion lines in two isogenic backgrounds: Canton-S and Samarkand. 383 insertions had a significant effect on starvation tolerance. The effect of the P-element inserts was generally negative and often sex-specific. Only 31 insertions significantly increased starvation tolerance. Significant insertions tag genes that are putatively involved in the starvation stress response. Deficiency complementation mapping was used to fine-map broad genomic regions (quantitative trait loci, or QTL) previously identified for starvation resistance. The five original QTL fractionated into thirteen smaller QTL, six of which had sex-specific effects. From these fine-mapped regions 26 genes were chosen for mutation complementation testing. Twelve of the 26 genes showed a significant effect on variation in starvation resistance between the two wild-type strains, Oregon-R and 2b. Transcriptome analysis was performed on a subset of the recombinant inbred mapping population used to identify broad QTL affecting starvation resistance: two lines resistant to starvation, two lines susceptible to starvation, and the two parental lines, Oregon-R and 2b. RNA samples were obtained in both the unstarved and starved states for these lines. Many genes were involved in the response to starvation stress: 3,528 unique probe sets exhibited significant differences in transcript abundance between the unstarved and starved states. 217 probe sets were identified as QTL that may affect variation in starvation resistance. 47 of these probe sets fell within the original QTL regions; nine probe sets fell within fine-mapped QTL found from the deficiency complementation tests. Further analysis revealed substantial epistasis at both the transcript level and the level of the phenotype, adding unexpected complexity to the attempt to map QTL using microarrays. Many of the genes implicated in this study have known phenotypes in cell fate specification/proliferation, feeding behavior, oogenesis, and metabolism, suggesting extensive pleiotropy. Mutational and transcriptional effects were often sex-specific. The large numbers of genes identified in this study suggest that a balance between mutation and selection may maintain variation in starvation resistance, rather than a trade-off among life history traits.
Insect response to Alphavirus infection
(2007-11-03) Mudiganti, Usharani; Dennis T. Brown, Committee Chair; Carla Mattos, Committee Member; Linda K. Hanley-Bowdoin, Committee Member; Gregory C. Gibson, Committee Member
Invertebrate cells survive Alphavirus infections to establish viral persistence, in contrast to cell death seen soon after infection in mammalian cells. Invertebrate response to prototype alphavirus, Sindbis, has been studied to a certain extent, using mosquitoes and cell lines derived from mosquitoes. Some of the observations made in studies using mosquito systems include formation of intracellular vesicles soon after infection with Sindbis, identification of antiviral activity in the media used to grow the mosquito cell lines and in Sindbis-infected mosquito cell lysates, controlled levels of virus production as persistence is established and superinfection exclusion by Sindbis-infected cells. The study presented here is designed to utilize array of genomic and genetic information available in Drosophila model to identify the candidate genes ⁄ gene products playing a role in establishment of alphavirus persistence. Observations described in Chapter I establish Drosophila S2 cells as a suitable invertebrate system to study alphavirus-insect interactions. Gene expression analysis identified increased expression of 18 transcripts coding for membrane trafficking and cytoskeletal components and 10 transcripts coding for Notch pathway components, at 5 days post-infection. Identification of upregulation of Notch pathway suggests similarities between mechanism of establishment of persistence of Alphaviruses and Herpesviruses. Transcript coding for TEP II, a wide-spectrum protease inhibitor is increased in expression at 5 days post-infection and upon superinfection at 5 days post-infection. We probed for inhibition of viral protease activity during early persistence and upon superinfection of Sindbis-infected cells with Sindbis. Inhibition of Sindbis viral protease nsP2 is identified to be involved in establishment of viral persistence and superinfection exclusion in cells derived from Mosquito and Drosophila
Quantitative Genetics and Genomics of Drosophila Life Span
(2006-11-10) Wilson, Rhonda Henderson; William R. Atchley, Committee Member; Gregory C. Gibson, Committee Member; Michael D. Purugganan, Committee Member; Trudy F. C. Mackay, Committee Chair
Limited life span and senescence are near-universal characteristics of eukaryotic organisms, controlled by many interacting quantitative trait loci (QTLs) with individually small effects, whose expression is sensitive to the environment. Understanding how genetic and environmental factors interact to limit life span and generate variation between individuals, populations and species, is important from both a human health and an evolutionary theory perspective. To begin to dissect the complex genetic architecture of longevity, it is necessary to identify the genes affecting life span and natural variation in life span. Here we have used quantitative complementation mapping to deficiencies, gene expression analysis, and functional tests to mutations at positional candidate genes to gain a better understanding of genes and categories of genes associated with the aging process. These complementary approaches have allowed us to identify several genomic regions as well as specific candidate genes affecting longevity and variation in longevity. Quantitative complementation tests to 69 overlapping deficiencies covering approximately 80% of the third chromosome yielded 11 QTLs affecting variation in life span between five old ("O") lines selected for postponed senescence and their five base ("B") control lines. Most QTLs were sex-specific, and all but one affected multiple O lines, suggesting that variation in life span for the B and selected O populations is most often attributable to the effects of common alleles. However, these 11 QTLs spanned over 4874 kb and contained approximately 598 genes. To identify and prioritize individual genetic loci affecting life span and variation in life span within our chromosomal regions as well as the remaining genome, we used whole genome expression analyses over multiple ages for one B and two O lines. Two separate analyses were used to compare changes in transcript abundance at the same chronological and physiological age between ages and lines. Over 26% of the genome was significantly altered between young and old flies and more than 5% of the genome showed significant changes between control and selected lines (indicating variation in aging effects) at multiple ages. Significant probe sets fell into a diverse group of biological processes and molecular functions, many associated with processes and pathways known to affect aging as well as many correlated traits in O lines. Examination of expression patterns for specific genes showed that O lines commonly exhibited a delayed response to aging, although different patterns of expression were observed as well. Transcriptional analyses were followed up with functional tests to mutants at positional candidate genes, which were selected, based on significant probe sets for either age or line effects and the availability of mutants. P-element insertion lines and their co-isogenic controls allowed us to test for age effects. Forty-four percent of 27 P-element mutants tested showed significant differences in mean life span from their co-isogenic control lines, with all but one decreasing life span. Quantitative complementation tests to these mutants provided an efficient method to test for variation in aging as 70% of the ten mutants tested yielded significant results. Candidate genes implicated in functional tests for both aging and variation in aging are involved in various categories of biological processes, including oogenesis, chromatin silencing, spermatogenesis, development, defense response, locomotor behavior, and cell death, suggesting that many of the processes that affect aging may affect variation in aging as well.