Thesis (PH.D.)--Syracuse University, 2003. / "Publication number AAT 3113248."

The Smyth line chicken (SL), a model for autoimmune human vitiligo is characterized by spontaneous, posthatch, destruction of melanocytes. Morphological and immunological changes accompanying the SL vitiligo has been well characterized, but information on the mode of inheritance of the disease is limited. In this study, a comprehensive genetic analysis was conducted to get a better picture of the genetics of this animal model. The DNA fingerprint analysis has revealed moderate level of inbreeding within the SL and BL parental sublines. 5-AzaC treatment increased the incidence of vitiligo in BL controls, while no changes were noticed in the unrelated LBL controls, providing the evidence that the BL sublines are genetically susceptible controls. High Embryonic mortality and low incidence of vitiligo were observed when the SL was used as the female parent in SL/BL matings. The number of affected females were higher, when SL was used as the male parent, suggestive of sex-linked inheritance. However, a model involving polygenic inheritance and genomic imprinting, better explain these data. Based on the results of the above experiments, SL101 and BL101 were selected to produce an F$\sb2$ population for the mapping of vitiligo genes. Genome scan was conducted with 156 microsatellite (MS) markers and 75 polymorphic markers were selected for the gene mapping. The results of the linkage analysis showed that MS markers on chromosome 1, 2 and linkage group 36 are linked to vitiligo. Candidate gene analysis revealed linkage disequilibrium between vitiligo and endogenous virus (EV) genes. EV genes were found to be expressed in SL chicken and 5-Azacytidine treated vitiliginous BL chickens. In situ hybridization experiments revealed one EV locus in LBL chickens (1q14) and 3 in SL101 and BL101 birds (1p25, 2q26 and a microchromosome). The results from this study suggests the possible effect of EV genes on SL vitiligo. One of the loci mapped on chromosome 2 is most likely an EV gene or one that is linked to it. The genes mapped at the other two loci are not identified at this time and a detailed positional cloning strategy would be necessary to identify the genes from these regions.

We utilized the powerful molecular and genetic tools available for the analysis of neural development in Drosophila to characterize a mutation for its effects on imaginal sensory axons. Previous analysis demonstrated that loss of function alleles caused defects in axon anatomy. Molecular analysis revealed that these axon defects were due to a disruption in the 10.4 kDa cytoplasmic dynein light chain gene (Cdlc1). This molecular analysis involved the recovery of both genomic and cDNA clones, characterization of the transcription unit in both wild type and mutants, and sequence analysis. As verification that mutations in the cytoplasmic dynein light chain gene caused the axon defects, we generated transgenic flies in which expression of the gene was targeted to specific sensory neurons. When this targeting system was introduced into a Cdlc1$\rm\sp{null}$ genetic background the axon phenotype of the targeted neurons was rescued. Further, the rescuing effects of the targeted expression was restricted to the Cdlc1-positive neurons. Other sensory neurons that were not expressing the transgene in this system retained mutant axon phenotypes. The specificity of the rescue demonstrated that Cdlc1 function is cell autonomous, and that sensory neurons require Cdlc1 function for proper development of their axon projections. The dynein light chain has been shown to associate with the cytoplasmic dynein complex, myosin V and nitric oxide synthase. All of these molecules have neural function so it is important to determine whether any of these partners were involved in producing the axon defects we observed in Cdlc1 mutants. In Drosophila, mutant alleles are only available for genes encoding components of the cytoplasmic dynein complex. We used these alleles in a double mutant analysis to determine whether the axon phenotype was influenced by genetic interactions between mutations affecting the light chain and the dynein heavy chain and p150$\rm\sp{Glued}.$ Our data showed that loss of function mutations in the heavy chain gene acted as dominant suppressors of the Cdlc1 axon phenotype, while loss of function mutations in Glued acted as dominant enhancers of the phenotype. These results support a model in which the light chain functions with the cytoplasmic dynein complex during axon development.

Homosporous ferns such as Ceratopteris richardii produce only one type of spore; irrespective of the genotype, spores can develop into either ameristic males, or meristic, hermaphroditic gametophytes. Determination of sexual identity occurs after spore germination by the action of the hormone antheridiogen, which promotes male development and is secreted by the hermaphrodite. Meristem development antagonizes the antheridiogen response, whereas activation of maleness by antheridiogen involves the specific repression of female-associated traits, such as the meristem and the archegonia. To elucidate the possible relationship between the meristem and the antheridiogen sex determination pathway, I cloned a Ceratopteris gene (CKNOX-A or CK) homologous to the homeobox-containing, meristem-specific KNOTTED1 or KN1 gene. CK encodes a putative protein that contains an 88-aa region 76% identical to the KN1 homeodomain. Expression studies performed by RT-PCR and in situ hybridization showed that CK is most strongly expressed in tissues that contain a meristem, such as hermaphroditic gametophytes, and sporophyte apices and leaves. However, CK RNA was also detected in antheridial cells in males. Surprisingly, the cellular localization of the CK hybridization signal differed depending on the regions of the CK gene used as a probe. Using the CK 3$\sp\prime$ UTR probe, the hybridization signal localized to the nuclei, in contrast to the cytoplasmic localization with a homeobox-containing probe. Functional homology of the CK gene to KN1 was assessed by overexpressing CK in Arabidopsis plants (ecotype Columbia). Transformant phenotypes ranged between two classes: in one, leaf size was severely reduced and plants formed petite rosettes. In the other, plants displayed a loss of apical dominance, by overproducing rosette leaves that developed without a visible phyllotactic pattern, or by forming several inflorescences simultaneously. Contrary to the KN overexpressing phenotypes in Arabidopsis, leaf shape was not altered by lobe formation in CK transformants. In conclusion, CK can be recognized by the molecules that control the initiation and maintenance of the Arabidopsis meristem, which suggests that Ceratopteris CK may play a similar role to the KNOTTED gene of maize.

Many species of plethodontid salamanders exist as complexes of genetically fragmented, parapatrically and allopatrically distributed groups of populations. A complex describes a group of cryptic species with adjacent or slightly overlapping distributions. These complexes provide a unique opportunity to examine the mode and tempo of speciation, specifically illuminating the mechanisms that maintain genetic cohesion. My research focuses on the Desmognathus ochrophaeus complex, composed of a number of small plethodontid salamanders inhabiting streams and seeps in the Appalachian Mountains. I have examined the mode and tempo of speciation in this complex using multiple markers to characterize divergence, assess gene flow, and evaluate reproductive isolation. On a broad scale, markers are concordant in recognizing distinct, evolutionary lineages. On a finer scale, however, markers are less condordant, indicating differential gene flow of markers across regions of secondary contact. Data indicate species can show high degrees of genetic divergence and yet are still capable of interbreeding, unlike patterns seen in other organisms. Asymmetry in mating preferences, however, indicates divergence in mate recognition systems has occurred. Furthermore, levels of isolation generally correspond to gene flow inferred from markers, indicating that groups are behaviourally isolated. Phyloethological analysis indicates that loss of some courtship behavior patterns may contribute to reproductive isolation. Molecular and behavioural data indicate species in the Desmognathus ochrophaeus complex probably speciated in allopatry in the Southern Appalachians, sometime during the Pliocene, with some groups experiencing repeated periods of isolation followed by recontact.

Embryogenesis plays a central role in the plant life cycle. It starts after fertilization. A single zygote divides asymmetrically, giving rise a small apical cell and a larger basal cell. The small apical cell undergoes precise cell divisions, passes through 2, 4 and 8 cell stages, followed by the protoderm, globular, heart, torpedo and cotyledon stage, in the process of forming a mature embryo. In embryo development a large number of genes are estimated to be involved and interact with each other during embryogenesis. We demonstrated that the RSH gene was required for normal embryo development of Arabidopsis. Its essential role was determined by disrupting expression of the gene by the Ac/ DsE two-element transposon system, which caused the embryo mutation. The abnormal phenotype was traced to the first asymmetrical division of the zygote and the embryo development lost its precise programmed cell division pattern. The rsh mutant showed both apical-basal and radial pattern defects. The RSH gene mapped to chromosome I. The gene was cloned and sequenced. It encoded a HRGP-type protein of predicted size, 49 k Dalton. The pre-protein contained a signal peptide and 13 almost identical repeats. Each repeat had 28 amino acids. The rescue of homozygous rsh mutants by complementation with the wild-type RSH gene demonstrated that the rsh mutation is the consequence of the DsE insertion. The gene was found to be expressed through out the developing embryo. It was expressed in a tissue specific manner after embryo germination. The RSH gene expression pattern was first profiled using the GUS reporter gene assay. The Northern and RT-PCR confirmed the results. To localize the RSH protein at the cellular level, an EGFP gene was linked to the C-terminus of the RSH gene and expressed in wild-type Arabidopsis . Confocal microscopy showed that the fusion protein was localized to the cell wall. Wild type transformants expressing RSH-EGFP showed mutant phenotypes. All these results support the conclusion that the RSH protein plays an important role in cell division during embryogenesis.

To exploit DNA-mediated transformation as a tool for studying the mechanisms controlling differential gene expression in Dictyostelium discoideum, it is necessary to know the fate of exogenous sequences. I examined the ability of unlinked nonreplicating plasmid molecules to undergo homologous recombination during cotransformation of Dictyostelium amoebae. The transformation vector B10S confers resistance to the antibiotic G418 and was always presented to amoebae as a closed circle. Cotransforming DNA, containing a slime mold cDNA and sequences homologous to the primary vector, was presented either as a closed circle or as a linear molecule after digestion with restriction endonucleases which cut within one of three distinct regions of the plasmid. Remarkably, homologous recombination occurred in every clone examined. Moreover, the products of recombination were identical in all instances, irrespective of the presence or position of linearized ends. The ends of the linear templates were not recombinogenic. Accurate repair of the introduced double-strand break occurred frequently during recombination. The repair could occur inter-molecularly or, more likely, intra-molecularly, i.e. by recircularization. Recombination was predominantly nonreciprocal, and the recombinant products were present in separate head-to-tail tandem arrays. A single mechanism is proposed that links nonreciprocal recombination to the formation of homogenous head-to-tail tandem arrays.

A genetic approach using a colony color assay to monitor the mitotic segregation of a SUP11-marked chromosome III bearing a partially functional centromere was used to identify new genes involved in chromosome segregation. Two cold sensitive (Cs) alleles, cse1-1 and cse2-1, were identified which abolish the partial function of a mutant centromere but have a little effect on the function of wildtype centromeres and exhibit no increase mitotic recombination. The cse2-1 mutant is also temperature sensitive (Ts). Both cse1-1 and cse2-1 mutants are more sensitive to the microtubule depolymerizing drug nocodazole than wildtype cells and exhibit accumulation of large-budded cells with abnormal nuclear localization and deformed spindle structures at the nonpermissive temperature. Suppressor analysis suggests that the CSE1 and CSE2 gene products may functionally interact. CSE1 and CSE2 were cloned by complementing the Cs phenotypes of cse1-1 and cse2-1 strains, respectively. CSE1 is located adjacent to the HAP2 gene on the left arm of chromosome VII and encodes a putative 110-kD protein containing 960 amino acid residues. The CSE1 protein is highly negatively charged and contains two putative phosphorylation sites and two heptad repeats of hydrophobic amino acids. Gene disruption shows that CSE1 is essential for cell growth. The CSE2 gene is located 3 cM from CEN14 on the right arm of chromosome XIV and encodes a putative 17-kD protein containing 149 amino acid residues. The CSE2 protein contains two putative phosphorylation sites and a leucine zipper motif at the carboxy terminus. CSE2 is not essential for cell viability but disruption of CSE2 results in defective mitosis and meiosis, including slower growth at 30$\sp\circ$C, cold and temperature sensitivity, increased missegregation of the X69-chromosome, accumulation of large-budded cells with abnormal nuclear and spindle morphologies at the nonpermissive temperature, reduced sporulation efficiency, formation of abnormal asci, and lower spore viability. Analysis of double and triple mutants containing cse2::LEU2, a mutant centromere and cep1::URA3 (lacking a centromere-binding protein) indicates that the CSE2 protein could be directly involved in kinetochore function. Two high dosage suppressor genes, SCM1 and SCM2, were identified which rescue the Cs phenotypes of cse1-1 and cse2-1, respectively. SCM1 is located adjacent to KAR1 on the left arm of chromosome XIV and SCM2 is located on chromosome VII. The SCM1 gene encodes a putative 60-kD protein containing 542 amino acid residues. The SCM1 protein contains a putative phosphorylation site and two heptad repeats of hydrophobic amino acid residues. Disruption of SCM1 results in cell inviability indicating that SCM1 is essential for cell growth. SCM2 is also a novel gene indispensable for cell viability. In conclusion, I have discovered four novel genes, CSE1, CSE2, SCM1, and SCM2 which are involved in chromosome segregation and encode gene products that are functionally related and may physically interact.

Genetic and biochemical methods were used to study the structure and function of three nuclear encoded proteins that are believed to be located in the peptidyl transferase center of the mitochondrial ribosome in the yeast Saccharomyces cerevisiae. Mrp7p, the yeast mitochondrial homolog of bacterial ribosomal protein (r-protein) L27, contains an 84-amino-acid L27-like sequence followed by a nonconserved C-terminal extension of 260 amino acids. The structure-function relationships of the conserved and nonconserved sequence domains were studied by in vitro mutagenesis of MRP7. 56 mrp7 mutant alleles were examined for effects on respiratory growth and, in selected cases, on ribosome assembly. The results show that both sequence domains are essential for Mrp7p function. Deletions that truncate the coding sequence from the 3$\sp\prime$ end as well as single codon changes at each of three universally conserved positions in the L27-like domain caused defective assembly of the 54S large ribosomal subunit. When the two domains of Mrp7p were expressed as separate polypeptides, both associated independently with the large ribosomal subunit, but they could not provide Mrp7p function in trans. Cloning and sequencing of MRP7 from Kluyveromyces lactis revealed a high level of sequence conservation within the L27-1ike domains of ScMrp7p and KlMrp7p (88% identical amino acids) whereas sequence conservation in the C-terminal domains was significantly lower (48% identity), although five relatively short sequence blocks were highly conserved, implicating them as important functional elements. KlMRP7 was functional in S. cerevisiae. The yeast nuclear genes, RML16 and RML2, were shown to encode essential mitochondrial homologs of bacterial r-proteins L16 and L2, respectively. In gene expression studies, both genes were regulated transcriptionally in response to carbon source and the stable accumulation of both proteins was dependent on the presence of the 21S large rRNA. Site-directed mutagenesis showed that a seven-amino-acid sequence of Rml2p was essential for function, and the substitution of Gln for the universally conserved His-343 in Rm12p caused cold-sensitive respiratory growth but did not affect the assembly of 54S subunits.

Genetic and physical mapping of human and animal genomes has been greatly facilitated by using chromosome-specific DNA libraries. Although DNA libraries for most of the human chromosomes have been constructed, little has been documented for farm animals. The present study was undertaken to establish approaches for mapping single chromosomes. For this purpose, three experiments were carried out: microcell-mediated chromosome transfer; chromosome microdissection and cloning; and physical mapping of chicken chromosome 1 with chromosome 1-specific cosmid clones. In the first experiment, microcells prepared from pSV2-neo plasmid transfected chicken cells were fused to human HeLa cells. Three hybrid cell lines, each carrying a single chicken chromosome or a fragment of a chicken chromosome were established. In the second experiment, the chicken chromosome 1 was isolated by microdissection. The chromosomal DNA was amplified by PCR and DNA in microgram quantities was obtained. A chicken chromosome 1-specific DNA library was constructed from the PCR amplified DNA through conventional cloning. In the third experiment, a chicken genomic DNA cosmid library was constructed. Using chicken chromosome 1 specific DNA as a probe, aliquots of the cosmid library were screened and 6 cosmids located in different regions on chicken chromosome 1 were isolated and physically mapped to the chromosome by in situ hybridization. This study has demonstrated the applicability of the microcell-mediated chromosome transfer and chromosome microdissection for isolation of chromosome-specific DNA. The chicken chromosome 1-specific DNA library and chromosome assignment of chromosome specific cosmid clones will facilitate the development of a linkage map of the chicken genome.