M.S. / Molecular Biology / This thesis describes the cloning and analysis of PER6, a gene required for peroxisome biogenesis in Pichia pastoris. The gene was cloned by functional complementation of a per6 P. pastoris mutant strain that was one of a number of peroxisome-deficient mutants isolated in this laboratory. The complementing activity was localized to a small DNA fragment by subcloning and Northern filter hybridization analysis and the DNA sequence of the fragment was determined. The sequence revealed a 1296-bp open reading frame which potentially encodes a 432-amino acid protein of 49 kD. The gene was transcribed into a message of 1.4 kilobases that was constitutively expressed but induced several-fold in cells growing on methanol. A mutant strain with a deletion of a large portion of the open reading frame was constructed and used to genetically demonstrate that the cloned gene was identical to the defective gene in the originally isolated per6 mutant. The predicted amino acid sequence of the PER6 product revealed several interesting features, including a significant regional similarity to PAF-1, a gene known to be defective in some patients with Zellweger syndrome, a lethal human genetic disease caused by peroxisome deficiency. Finally, the PER6 product was produced in E. coli and purified to serve as antigen for antibody production.

Sex pre-selection in livestock offspring to produce dairy cows and specific male and females lines for heterosis in swine are important economic goals in animal husbandry. The long-term goal of the study is to separate porcine and bovine chromosome X- or Y-bearing sperms using sex-specific antibodies. H-Y antigen encoded by jumonji, AT-rich interactive domain 1d (Rbp2-like) (Jarid1d) is defined as a male histocompatibility antigen that causes rejection of male skin grafts by female recipients of the same inbred strain of rodents. Porcine and bovine JARID1D genes are estimated between 3.5 kb and 4.5 kb, however there was only 457 bp and 99 bp respectively found in the database. In this study, the porcine and bovine sex-specific genes, JARID1D were cloned using in silico cloning, PCR and cDNA library screening. We used human JARID1D gene as a probe or template to search the ESTs of porcine and bovine in NCBI and TIGR databases and combined the results from two databases and assembled the ESTs using CAP3 program. We also constructed porcine and bovine testis cDNA libraries, and performed porcine testis cDNA libraries screening. To study this model in the mouse, Jarid1d H-Y epitope TENSGKDI was synthesized and used to immune the rabbits. Dot blotting analysis revealed the specificity of the immuned antibodies to mouse Jarid1d H-Y epitope TENSGKDI, but western blotting analysis did not confirm the results. Immunofluorescence analysis of mouse sperms reacted with anti-mouse Jarid1d H-Y epitope TENSGKDI-specific antibody didn¡¦t show a 1:1 ratio of stained sperms to unstained sperms. The specificity of this antibody needed to be improved. The data shows partial JARID1D gene of pig was cloned, and the anti-mouse Jarid1d H-Y epitope TENSGKDI-specific antibodies were prepared, however the specificity of anti-mouse Jarid1d H-Y epitope TENSGKDI-specific antibodies to surface protein expression on mouse sperms needs more confirmations.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 165-188). Available also in a digital version from Dissertation Abstracts.

Title from PDF of title page (University of Missouri--Columbia, viewed on March 8, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Qisheng Song. Vita. Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 98-115).

Thesis (M. Phil.)--University of Hong Kong, 1987.

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