| In 1995, the first systematic analysis of the genome of the filamentous fungus, Neurospora crassa, was undertaken by the Neurospora Genome Project at the University of New Mexico. The purpose of the project was three-fold: (1) to analyze the expressed genes (transcriptome) of Neurospora; (2) to determine the nature of the encoded products or proteins (proteome); and (3) to identify genes expressed in a tissue-specific fashion, i.e., preferentially expressed genes. The studies reported in the following chapters build upon this successful project.;The high-throughput methods of partial cDNA sequencing and cDNA microarray technology provide molecular biologists the means to rapidly measure preferential gene expression in an organism. Both of these technologies have been employed (cDNA sequencing) and improved upon (cDNA microarray analysis) to analyze the expressed genes of Neurospora crassa.;In Chapter 1, an in-depth analysis of the expressed genes from an unfertilized sexual stage of Neurospora crassa is presented. This tissue was grown under mating conditions, and the cDNA library is referred to as the Westergaard library. The derived protein sequences corresponding to the partial cDNA sequences were analyzed with numerous computer algorithms to systematically characterize the nature of the encoded proteins and tissue-specific patterns of gene expression. Two major findings include: (1) The cDNA clones from unfertilized sexual tissue appear to express genes in a pattern very similar to a "vegetative" tissue of Neurospora; (2) Two genes, nmt1 and nmt2, both involved in thiamine biosynthesis, were found to be highly expressed in the Westergaard library relative to the other three libraries.;Chapter 2 presents an update of the first in silico functional classification of the known genes of a filamentous fungus, the Neurospora Proteome. A total of 3,397 cDNA clones were analyzed for this Proteome. Distinct patterns of tissue-specific gene expression and gene abundance were observed that distinguished all of the libraries (conidial, mycelial, perithecial, and Westergaard). Nearly half of the cDNAs sequenced to date appear to encode previously unidentified or novel genes.;Chapter 3 describes a proprietary chemical process for covalently linking DNA to a glass substrate in a manner that preserves the ability of the immobilized nucleic acid to hybridize to complementary sequences. This binding chemistry has the potential to further the new field of DNA microarray technology and analysis by increasing the reproducibility, consistency, and stability of results. |
