The long term objective of this application is the better understanding of the biochemical basis of mammalian spermatogenesis.
The specific aims concern the roles of testis-specific chromosomal proteins that occur during restricted stages of germ cell development in spermatocytes and spermatids. These studies may provide information useful for the diagnosis or treatment of male infertility, chromosome abnormalities generated during spermatogenesis, and malignant growths of male germ cells (seminomas). Two groups of testis-specific chromosomal proteins are under study. They are of interest because they presumably play critical roles in specific nuclear events during spermatogenesis and because they are specific gene products whose regulated synthesis reflects the precise program of hormonally influenced gene expression that underlies spermatogenesis. One group of proteins are the testis-specific histone variants (H1t, TH2A, TH2B & TH3) that are synthesized during meiosis, thereby replacing to varying degrees the somatic type histones. Only H1t among these proteins has been characterized in detail, and little is known of the factors regulating their synthesis or of the details of their genetic organization. A second group of testis-specific chromosomal proteins are the TP family (TP1-4) found in elongating spermatids. They seem to serve in the transition from necleosomal to protamine associated DNA. Since they are synthesized well after the completion of meiosis, they either result from haploid gene expression, or they may possibly be translated from messenger RNA synthesized earlier in spermatogenesis and stored in an inactive form. TP1 is the only member of this group to have been well characterized as a protein. We propose to use recombinant DNA technology to characterize the rat genes for H1t and TP1. We anticipate in one or both cases that isolation of the gene for one member of the family will lead to isolation of genes for other members due to gene clustering. Initially DNA copies of mRNA (cDNA) will be cloned in bacterial plasmids. This will depend in part on use of synthetic oligonucleotides. The cDNA clones will then permit selection of recombinant lambda bacteriophage containing the genes for each protein and perhaps genes for other members of the respective families. DNA sequence analysis of isolated genes will provide the complete amino acid sequence for a number of these proteins. The clones will also serve as specific hybridization probes to allow assay of the mRNA for each protein and thus detection of hypothetical stored message particles.
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