FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF THE TRANSCRIPTIONAL REGULATORY REGION OF THE PROACROSIN GENE

Proacrosin, the zymogen form of the serine protease acrosin, is locate d within the acrosomal vesicle of mammalian spermatozoa and has been s uggested to be involved in the fertilization process, In mouse and rat , expression of the proacrosin gene starts in pachytene spermatocytes and continues through the early stages of spermiogenesis, We have show n recently that 2.3 kilobase pairs of the 5'-flanking region of the ra t proacrosin gene is sufficient to direct chloramphenicol acetyltransf erase gene expression in a germ cell-specific and developmental stage- specific manner in the mouse, Additional transgenic lines have been ge nerated which include two deletions in the 5'-flanking region and a ty rosinase minigene as marker for gene expression, Transgenic mice beari ng these two truncated fragments showed different patterns of reporter gene expression, Transgenic lines (BM, B3, B2) harboring the 397-base pair (bp) fragment (from 45 to 442 bp upstream of ATG) showed no chlo ramphenicol acetyltransferase (CAT) activity in either testis or other tissues, but analysis via reverse transcription polymerase chain reac tion confirmed low levels of reporter gene transcription in testis, Tr ansgenic line TC bearing a longer fragment of 877 bp (from 45 to 922 b p upstream of ATG) showed a reporter gene expression and chloramphenic ol acetyl-transferase enzyme activity which was identical to that foun d in mice harboring the 2.3-kilobase pair 5'-flanking region, The anal ysis of the CAT gene expression during testicular development showed d iploid transcription and haploid translation, It can be concluded that all sequences required for a basic level of testis-specific transcrip tion of transgene are present within the 397-bp fragment, and other DN A sequences located outside of the 397-bp fragment but present within the 877-bp fragment can function as enhancer elements. Two fragments w ithin the 877-bp region were identified by gel retardation assays as b inding exclusively to nuclear factor(s) from testis protein extracts. In both fragments we identified sequence elements which are present in the promoter region of the germ cell-specific genes for histone H2B a nd protamine 1, respectively.