Semester

Spring

Date of Graduation

2003

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Biochemistry

Committee Chair

Joginder Nath

Committee Co-Chair

Andrew J. Wyrobek

Abstract

Variations in gene expression are the basis of differences in cell and tissue function, response to DNA damaging agents, susceptibility to genetic disease, and cellular differentiation. The purpose of this dissertation research was to characterize variation in basal gene expression among adult mouse tissues for selected stress response, DNA repair and damage control genes and to utilize variation in temporal gene expression patterns to identify candidate genes associated with germ cell differentiation from mitosis through meiosis in the prepubertal mouse testis. To accomplish these goals, high throughput analyses of gene expression were performed using custom cDNA and random oligonucleotide microarrays. CDNA microarray technology was optimized by evaluating the effects of multiple hybridization and image analysis methodologies on the magnitude of background-subtracted hybridization signal intensities. The results showed that hybridizing lower probe quantities in a buffer developed at Lawrence Livermore National Laboratory to tryptone-blocked microarrays improved signal intensities. In addition, the error in expression ratio measurements was significantly reduced when microarray images were preprocessed. A custom cDNA microarray comprised of 417 genes and enriched for stress response, DNA repair, and damage control genes was used to investigate basal gene expression differences among adult mouse testis, brain, liver, spleen, and heart. Genes with functions related to stress response exhibited the most variation in expression among tissues whereas DNA repair-associated gene expression varied the least. Random oligonucleotide microarrays comprised of ∼10,000 genes were used to profile changes in gene expression during the first wave of spermatogenesis in the prepubertal mouse testis. Approximately 550 genes were differentially expressed as male germ cells differentiated from spermatogonia to primary spermatocytes. These findings suggest that the 313 unannotated sequences and 178 genes with known functions in other biological pathways have spermatogenesis-associated roles. This dissertation research showed that microarrays are a useful tool for quantitating the expression of large numbers of genes in parallel under normal physiological conditions and during differentiation. It has also provided candidate genes for future investigations of the molecular mechanisms underlying (1) tissue-specific DNA damage response and genetic disease susceptibility and (2) cellular differentiation during the onset and progression of spermatogenesis.

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