Ph.D. - Biomedical Sciences (Genetics)

Permanent URI for this collection

https://hdl.handle.net/10125/868

Browse

Recent Submissions

Now showing 1 - 10 of 13
  • Item
  • Item
    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.
  • Item
    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.
  • Item
    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) .
  • Item
    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.
  • Item
  • Item
    The thalassemias among Laotians, Chinese and Filipinos in Hawaii : prevalences, gene frequencies, geographic distributions, screening strategy
    ( 1991) Hall, Jana
    Both alpha and beta thalassemias and hemoglobin E structural variants are examined in three ethnic groups now residing in Hawaii: Chinese, Laotian and Filipino (N=2644). The purpose of this dissertation was; (I) to estimate prevalences and gene frequencies in the Hawaii sample, and to look at geographic distributions of the thalassemias within the original countries of these immigrants; and (II) to use red blood cell indices phenotypes to determine the possibility of developing new algorithms which would distinguish between normals and thalassemias, and between single, double and triple deletion alpha thalassemias with clinically acceptable rates of accuracy. High prevalences of singly and concomitantly inherited thalassemias (compound heterozygotes) were found in the Hawaii sample (Laotians 58.9%; Chinese 33.8%, Filipinos 40.5%) . Gene frequencies, corrected for bias, were estimated. Gene frequencies within Laos, the Philippines and China were estimated and mapped according to geographic region of family origin. In order to detect the thalassemias, red cell indices from the complete blood count generated by electronic particle counter were analyzed and yielded the algorithm l/(l+e^-L ) where L is the intercept constant -149.9 plus (RBCount constant 2.27 * RBCount measured) + (Hglobin constant -2.97 * Hglobin measured) + (Hcrit constant 0.72 * Hcrit measured) + (MCV constant 1.40 * MCV measured) + (MCH constant -5.10 * MCH measured) + (MCHConc constant 5.20 * MCHConc measured) . This algorithm identified normals and abnormals (thalassemias) with 92.3% accuracy. After thalassemia is indicated by the algorithm, the results of hemoglobin A2 measurement clearly delineate those with beta thalassemias and heterozygous and homozygous Hb E. A second discriminant algorithm was derived which distinguishes single, double and triple deletion alpha thalassemias with 94.6% accuracy. Inheritance of more than one form of thalassemia (concomitant inheritance or compound heterozygosity) may confound results. Both algorithms are relatively simple and can be readily applied by calculator; simple programming of microcomputers provides immediate results upon entry of the complete blood count indices. Inclusion of these two algorithms as steps in genetic screening strategies for the thalassemias may enhance effectiveness by more accurately targeting individuals at risk and providing presumptive diagnoses which will be of particular value where DNA analysis is not available.
  • Item
    Characterization of embryonic globin gene expansions in homo sapiens : mechanistic and evolutionary implications
    ( 1990) Titus, Elizabeth Anne Brumbaugh
    This research focused on the characterization of aberrant ζ-globin expansions as an important model system in the study of genetic recombination and gene rearrangements in Homo sapiens. There are normally two embryonic ζ-globin genes in the α-globin multigene family on chromosome 16p13.3. In this study, thirteen triple ζ-globin and two novel quadruple ζ-globin chromosomes from Laotian and Filipino populations in Hawaii were discovered and characterized by Southern blot gene mapping, PCR amplification and sequencing analyses in an effort to elucidate the structure and processes of gene duplication in the evolution of these amplified regions. Attempts to clone the reduplicated genes in genomic library constructs were unsuccessful, presumably due to sequence-specific idiosyncrasies and problems with methylation. Restriction patterns generated from a battery of enzymes in single and double digests indicate a similar homologous recombination event was responsible for the expansion haplotypes. The high frequencies (10-15%) of triplicated ζ-genes in the populations studied increases the likelihood of a homologous crossover event involving a triple ζ-globin chromosome in the generation of the quadruple arrangement. The identification of two distinct triple ζ-gene chromosomal arrangements, one in linkage with normal α-genes, and another associated with a - α3.7 deletion, indicates that the triple arrangement has undergone two recombination events in the Filipino population. Further, gene mapping and sequence analyses of PCR products of the ζ-gene expansions indicate that the recombinant genes are hybrids of 5'ψζ1 and 3'ζ2 genes, and that the ζ-globin sequences are highly conserved to date. The absence of single ζ-gene chromosomes in these populations suggests that the reciprocal chromosome in the recombinant expansion may be detrimental or lethal, similar to the α-thalassemia deletions. These analyses are suggestive of structural constraints within the molecule which lead to the production of similar zeta expansions in this region. As no correlating phenotypic advantage has been determined to date, the frequencies observed for the reduplicated ζ-globin genotypes are probably maintained by random genetic drift in these populations. The ζ-globin expansions may be an important event in the evolution of new genes or new gene functions in the α-globin complex, and their characterization is relevant to studies of molecular etiology of disease states and evolution in the human genome.
  • Item
    Molecular and evolutionary characterization of the transposable element Uhu from Hawaiian Drosophila
    ( 1990) Brezinsky, Laura
    I report the complete nucleotide sequence of three independent isolates of the transposable element Uhu from Drosophila heteroneura (an endemic Hawaiian Drosophila). The complete element is about 1650 base-pairs (bp) long, has 4650 bp inverse imperfect repeats at its ends, and contains a large open reading frame potentially encoding a 251 amino acid protein. The three randomly selected isolates of Uhu which I sequenced share 93.3% nucleotide sequence identity, indicating that Uhu is well conserved within the D. heteroneura genome. I demonstrate that Uhu belongs to a class of transposable elements which includes Tc1 from Caenorhabditis elegans, Barney from Caenorhabditis briggsae, and HB1 from Drosophila melanogaster. All of these elements share significant sequence similarity, are approximately 1600 base pairs long, have short inverse terminal repeats (ITRs), contain open reading frames (ORFs) with significant sequence identity, and appear to insert specifically at TA sequences generating target site duplications. Uhu has been found in five species of endemic Hawaiian Drosophila; D. heteroneura, D. silvestris, D. differens, D. planitibia, and D. picticornis. Because the phylogenetic relationship and approximate divergence times for these species are known, I was able to examine the evolutionary dynamics of Uhu. I have sequenced a 447 bp fragment from a total of 12 Uhu elements from these species. These data provides evidence for the degeneration of Uhu in D. picticornis indicating that it is being lost in this species. The nucleotide substitution rate of Uhu is estimated to be 1.02 x 10-8substitutions/position/year which is comparable to the estimated rate for structural genes in these flies. Phylogenetic analysis using distance matrix methods confirms previously proposed phylogenetic models of these Drosophila species indicating that Uhu has been distributed among these species by vertical transmission.
  • Item
    Sequence and structure of mtr, an amino acid transport gene, of Neurospora crassa
    ( 1989) Koo, Kenneth
    The gene product of the mtr locus of Neurospora crassa participates in the transport of neutral aliphatic and aromatic amino acids across the plasma membrane. Mutants of the mtr locus have been identified by other workers by selecting for resistance to the poisonous amino acid analog 4-methyltryptophan. Using a transformation and selection scheme. three cosmids were cloned which complement the mtr-6(r) mutant allele. Two of the cloned DNAs were found to be tightly linked to restriction fragment length polymorphisms (RFLPs) which flank the mtr locus on linkage group IV. The mtr- gene was subcloned as a 2.9 kbp fragment; nested deletion and restriction site subclones of the fragment were generated and used to determine the DNA sequence. DNA sequence analyses indicated the presence of a 845 bp open reading frame (ORF) with a 59 bp intron. The proposed mtr ORF contains sequences which are 100% homologous to N. crassa consensus sequences at the ATG start site, at the exon/intron splice boundaries. and internal intron sequences. The mtr ORF is followed downstream by a possible polyadenylation signal. The 2.9 kbp fragment hybridizes to a poly A+ mRNA transcript of 2,300 nt. 51 nuclease mapping analysis of the transcript confirms the size of the transcript and the presence of the intron. An upstream open reading frame (uORF), found in the same reading frame as the mtr ORF, is proposed to be present in the mRNA transcript. The proposed mtr ORF is predicted to translate into a 261 amino acid polypeptide with a molecular weight of 28,613 daltons. Codon usage of the mtr polypeptide is biased with 18 of the 61 sense codons being absent. The polypeptide is composed of 164 non-polar amino acid residues which represent 63% of the amino acids. Hydropathy analysis' of the mtr polypeptide indicates six possible transmembrane spanning domains with an average length of 23 amino acids which are proposed to span the membrane in alpha helical conformations. The mtr polypeptide does not have a signal sequence and the polypeptide is proposed to function as a dimeric protein. Homology to a RNP octamer motif supports the proposal that the mtr polypeptide functions as a ribonucleoprotein (RNP) particle.