Gene Discovery Using Computational and Microarray Analysis of Transcription in the Drosophila melanogaster Testis

Abstract

Identification and annotation of all the genes in the sequenced Drosophila genome is a work in progress. Wild-type testis function requires many genes and is thus of potentially high value for the identification of transcription units. We therefore undertook a survey of the repertoire of genes expressed in the Drosophila testis by computational and microarray analysis. We generated 3141 high-quality testis expressed sequence tags (ESTs). Testis ESTs computationally collapsed into 1560 cDNA set used for further analysis. Of those, 11% correspond to named genes, and 33% provide biological evidence for a predicted gene. A surprising 47% fail to align with existing ESTs and 16% with predicted genes in the current genome release. EST frequency and microarray expression profiles indicate that the testis mRNA population is highly complex and shows an extended range of transcript abundance. Furthermore, >80% of the genes expressed in the testis showed onefold overexpression relative to ovaries, or gonadectomized flies. Additionally, >3% showed more than threefold overexpression at p <0.05. Surprisingly, 22% of the genes most highly overexpressed in testis match Drosophila genomic sequence, but not predicted genes. These data strongly support the idea that sequencing additional cDNA libraries from defined tissues, such as testis, will be important tools for refined annotation of the Drosophila genome. Additionally, these data suggest that the number of genes in Drosophila will significantly exceed the conservative estimate of 13,601.

Paucity of Genes on the Drosophila X Chromosome Showing Male-Biased Expression

“…1A). Recent work has shown that sex-biased gene expression is significant in Drosophila (6)(7)(8). This first report of sex-biased expression of the full (predicted) genome also strongly indicates that there is significant sex-biased expression-especially in gonads (Fig.…”

“…Testis and male-specific accessory gland ESTs are biased toward autosomal locations (8,9), which suggests that the X chromosome is not a favored location for genes differentially expressed in the male soma. In contrast, the chromosomal distribution of ovary ESTs from the Berkeley Drosophila Genome Project (10) was random.…”

Evolution in the Fast Lane: Rapidly Evolving Sex-Related Genes in Drosophila

“…These genes, annotated according to their presence in various EST libraries (see materials and methods), can be used to test the extent and details of molecular sexual dimorphism in expression (Andrews et al 2000;Parisi et al 2004) as well as the evolution of reproductive (testis and ovary) genes relative to presumably nonreproductive (head) genes (Coulthart and Singh 1988;Civetta and Singh 1995;Jagadeeshan and Singh 2005). Previous studies, which have used a limited number of genes, have suggested that the testis transcriptome may have a greater proportion of rapidly evolving genes relative to ovary or head (Civetta and Singh 1995;Jagadeeshan and Singh 2005) and that rates of evolution of testis-expressed genes are far higher than rapidly evolving genes expressed in ovary and head.…”

The libraries that made SUCEST

“…Increasing importance has also been attributed to ESTs as a tool for the annotation of complete genome sequences of mammalians and plants. Unique ESTs provided biological evidence of hundreds of predicted genes, newly discovered genes, or transcript isoforms leading to considerable advance in gene identification mission in multicellular organisms (Andrews et al, 2000). Today, more than ten million ESTs are currently available through the dbEST entry of GenBank (http://www.ncbi.nlm.nih.gov/dbEST/dbEST_sumary.htm l); however, only 14% of dbEST release 022301 of February 23, 2001 corresponds to plant sequences.…”