Spermatogenesis involves sequential changes of precursor cells to haploid spermatozoa through regulated mitotic, meiotic, and spermiogenic cell differentiation events. During meiotic process, somatic histones are replaced by spermatogenic cell-specific histones. The major objective of this application is to investigate regulation of somatic and testis-specific histone genes and also the role of testis-specific histones in assembly of new chromatin structure and meiosis during spermatogenesis. The opposing mechanisms of spermatogenic cell-specific transcription and repression of S-phase specific transcription elements in a testis-specific histone TH2B gene will be investigated by determining the DNA sequence elements required for the cell- specific transcription and the binding sites of nuclear factors on the regulatory elements. The DNA elements will be identified by sequential deletions of the 5' or 3' flanking sequence of the TH2B gene fused with a reporter gene, chloramphenicol acetyl transferase (CAT). Expression of CAT under the control of TH2B regulatory sequences will be examined by introduction of the fused genes into rat spermatogenic cells. The TH2B gene has the DNA element(s) which allows the gene expressed in a cell-cycle specific manner but the elements are repressed in the cells. The mechanism of repression will be investigated. The role of testis-specific histones in assembly of new chromatin structure and meiosis will be investigated by introducing rat H2B and TH2B genes into the yeast which do not produce H2B histones. The amino acid sequences of TH2B which alter the binding of H2B histones to DNA will be determined. Rat H1 and TH1 histone genes and also combination of H1 + H2B and TH1 + TH2B genes will be introduced into yeast cells to investigate effects of the histones on chromatin structure and meiosis. We will sequence the H2A gene on the 3' side of TH2B gene to determine if the histone gene encodes the testis-specific H2A histones.
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