Ph.D. - Biomedical Sciences (Genetics)

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    Tests of population genetic models of the segregation distorter system in wild populations of Drosophila melanogaster
    ( 1996) Anderson, John Bruce
    The Segregation Distorter system of meiotic drive in Drosophila melanogaster consists of a haplotype of second chromosome loci (SD) which together mediate the dysfunction of sperm carrying a variable number of 240 base pair satellite DNA repeats located in the centromere region of SD+ homologs. These alleles are found worldwide in D. melanogaster populations in an apparently stable polymorphism that is unexpected under the simplest model of meiotic drive dynamics. The present study investigates whether a high level of repeat number mutation from insensitive (Rspi) to sensitive (RspS) repeat number is the force that balances the drive effect of SD in removing Rspi alleles. Iterative computer simulations of six hypothetical models of mutational processes produced equilibria suggesting that mutation-drive balance can produce stable polymorphism, but at frequencies different from those found in nature. Rates of mutation required to maintain polymorphism are on the order of 10.3 to 10- 1 changes per generation. Southern blot analysis of native forms of SD and SD+ chromosomes from a natural Hawaii population indicated >100 and 18 copies of the Rsp repeat in canonical Rsps and Rspi forms respectively. This agrees with previous studies showing the correlation between copy number and sensitivity. No changes 10 repeat copy number in chromosomal isolines were observed through approximately 25 generations. The three native isolines were used to establish population cages, which were sampled at intervals for Sd, Rspi and Rsps frequencies. After 300 days, Rsps was lost from all cages, which therefore did not reproduce the polymorphism observed in nature. Minimum X2 analysis shows that none of the six mutational models was a good predictor of the observed frequencies. Therefore, the hypothesized models are not supported by these data. Cages containing only native Rspi and Rsps were established and changes in Rsp frequencies measured. Estimates of the relative fitness of the alleles the three cages was s = 0.087, 0.025 and 0.068 respectively. These selective coefficients between Rsp alleles from the same natural population are much less than those found in previous studies using lab chromosomes and are too low to explain the observed SD polymorphism in this population.
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    Genetic and molecular basis of heavy metal tolerance and the heat shock response in the Mediterranean fruit fly : Ceratitis capitata
    ( 1995) Sujinda Thanaphum
    The Mediterranean fruit fly (Medfly), Ceratitis capitata, is a major pest of agricultural commodities world wide. Genetic and molecular studies of heavy metal tolerance and the heat shock response in the Medfly can provide a better understanding of how eukaryotes respond to environmental stresses. Potential uses of heavy metal tolerance and a heat shock gene in biological control programs have also been evaluated. Three heavy metal tolerant strains were established. Genetic analyses of test crosses between these strains and a nonselected strain suggested different modes of inheritance underlying the heavy metal tolerance trait. An attempt to generate a Y-autosome translocation involving this trait appeared to not be feasible. PCR products were amplified from the Medfly genome based on conserved domains of a metallothionein gene. Genomic DNA sequences homologous to these PCR products demonstrated coding potential metallothionein proteins, although transcripts homologous to these sequences were not detected in poly(A) selected RNA. This coding potential included a long open reading frame with a potentially cysteine rich region and alignment to mammalian metallothionein proteins. A heat shock like gene (Cerhscl) was cloned from the Medfly using PCR. This PCR used heavy metal induced cDNA as a template and a PCR primer designed from a conserved domain of metallothionein proteins. The Cerhscl gene shows some structural characteristics of a heat inducible gene including the presence of several heat shock elements and the absence of an intron. The Cerhsc1 gene also exhibits a heat shock response resulting in a 10 to 20% increase in the level of transcription. Developmentally, expression of the Cerhsc1 gene is very abundant in the adult stage, less so in the pupal stage and not detectable in the embryo stages. Overall, the Cerhsc1 gene exhibits structural and functional similarities to both heat inducible and non-heat inducible genes from a wide range of organisms.
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    Molecular evolution, genetic diversity, and avian malaria in the Hawaiian honeycreepers
    ( 1994) Feldman, Robert A.
    This dissertation is an interdisciplinary study linking molecular and population genetics to basic problems in island ecology, evolution, and extinction. The Hawaiian honeycreepers (Aves: Fringillidae: Drepanidinae) are extremely morphologically diverse and have radiated into nearly all of the passerine behavioral niches. The group is currently threatened with extinction by anthropogenic disturbances including introduced diseases. Basic biological problems in the honeycreepers include resolving systematic issues, documenting population structure and identifying the role of disease in limiting remaining populations. A molecular systematic study tested representative honeycreeper species for monophyly. I sequenced 13 honeycreeper species and used 3 outgroup taxa, 2 cardueline finches and a titmouse, for a 790 bp fragment of the mitochondrial cytochrome b gene. Phylogenetic trees constructed using distance, parsimony, and maximum likelihood methods all grouped the honeycreepers monophyletically and placed P. montana and O. bairdi in a basal clade. Two Ore0mYstis species were polyphyletic, indicating that they have been misclassified. The basis for this misclassification was strong convergence of morphological and behavioral characters associated with insectivory. A population study of mitochondrial cytochrome b DNA sequence variability was conducted at three scales; single locations, single islands, and multiple islands. Mitochondrial variation was found for four species living in a center of distribution that is surrounded by highly disturbed regions. In the multiple location study, diversity was found for the Common Amakihi (Hemignathus virens). The multiple island study showed that a population of Kauai Amakihi is phylogenetically distinct from those on Maui and Hawaii and that the Maui and Hawaii populations share mitochondrial haplotypes. No variation was detected in the highly mobile Iiwi (Vestiaria coccinea) sampled from the same islands. A PCR-based test for avian malaria was developed that detects the Plasmodium 18S rDNA. Quantitative Competitive PCR experiments established the sensitivity limits of the test. The PCR test was used to estimate prevalence of avian malaria in Hawaiian birds living at high elevations above the normal range of mosquito vectors. Malaria was highest in the nomadic Apapane (Himatione sanguinea) followed by the sedentary Common Amakihi (Hemignathus virens). Malaria was not detected in the mobile Iiwi (Vestiaria coccinea) .
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    The history of the uhu transposable element in the Hawaiian Drosophila
    ( 1994) Wisotzkey, Robert Grier
    The uhu transposable element belongs to the class of elements that have short inverted repeats. It was originally isolated from Drosophila heteroneura, a Hawaiian picture-winged Drosophila endemic to the Island of Hawaii. Biogeographic and DNA sequence divergence data suggest an ancient origin for the uhu element in the Hawaiian Drusophila. Biogeographic data suggests that uhu arose more than 7 million years ago. Sequence divergence data and phylogenetic analysis suggests that uhu was present in a common ancestor of the species. The maximum distance between two isolates suggests that uhu has been in the Hawaiian Drosophila for 20 million years. Using in situ hybridization to polytene chromosomes, the copy number of uhu in the planitibia subgroup and the adiastola subgroups of the Hawaiian Drosophila is found to be higher in the species endemic to the younger islands than in the species endemic to the older islands. This trend is also seen for the loa transposable element in the planitibia subgroup. No complete loa elements are found in D. picticornis from the island of Kauai, while there are 10 to 20 potentially complete copies of loa in the other species. For the uhu element, the percentage of sites that are variable for the presence or absence of uhu is high in the species on the younger islands, while nearly all the sites in D. picticornis are fixed. This would indicate that uhu has more recently been active in the species on the younger islands. Since all of the species are single island endemics, and believed to have evolved on the island, the increase in copy number and evidence for transpositional activity is consistent with the idea that there has been increase in the activity of transposable element associated with a speciation event.