This research proposal will extend our recent studies elucidating the role of a new, testis-specific, homeo box-containing gene in the process of spermatogenesis and ongoing investigations examining DNA modifications. A cDNA containing sequences homologous to the homeo box region of genes important in the development of organisms as diverse as Drosophila and humans has been identified in a cDNA library prepared from mature mouse testis. This new homeo box-containing gene will be characterized at the molecular level by (a) obtaining a full-length cDNA for the homeo box-containing transcript and characterizing the clone by DNA sequence analysis; (b) isolating the gene; and (c) mapping to the appropriate chromosome. Examination of its tissue-, cellular-, and developmental stage-specific expression will be undertaken by Northern blot hybridization, nuclear run-off transcription, and in situ hybridization. Determination of function will ask if homeo-box containing transcripts are translated, and if so, when during the process of differentiation of the germ cells. Polysomes will be prepared from germ cells and tissues and assayed for the presence of homeo box-containing transcripts. Characterization of the size and cellular distribution of the protein products by cell-free translation of the homeo box-containing transcripts and by production of an antibody to amino acid sequences of the homeo box-containing polypeptide will be carried out. A study of the effect on germ cell development of interfering with the expression of the homeo box-containing gene, specifically within the germ line, will be accomplished by producing transgenic mice containing exogenous DNA composed of a construct of a testis cell specific promoter(s) and a portion of the homeo box-containing cDNA clone in an anti-sense orientation. Current studies determining the role of the pattern of methylation and DNase I hypersensitivity of single or low-copy genes during spermatogenesis and the regulation of their expression will be continued by examining specific sites of methylation, especially those at the 5' end of the genes, in premeiotic, meiotic, and post-meiotic cells.
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