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Abstract
Proacrosin, the zymogen form of the serine protease acrosin, is located within the acrosomal vesicle of mammalian spermatozoa and has been suggested 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 shown recently that 2.3 kilobase pairs of the 5'-flanking region of the rat proacrosin gene is sufficient to direct chloramphenicol acetyltransferase gene expression in a germ cell-specific and developmental stage-specific manner in the mouse. Additional transgenic lines have been generated which include two deletions in the 5'-flanking region and a tyrosinase minigene as marker for gene expression. Transgenic mice bearing 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 chloramphenicol acetyltransferase (CAT) activity in either testis or other tissues, but analysis via reverse transcription polymerase chain reaction confirmed low levels of reporter gene transcription in testis. Transgenic line TC bearing a longer fragment of 877 bp (from 45 to 922 bp upstream of ATG) showed a reporter gene expression and chloramphenicol acetyltransferase enzyme activity which was identical to that found in mice harboring the 2.3-kilobase pair 5'-flanking region. The analysis of the CAT gene expression during testicular development showed diploid transcription and haploid translation. It can be concluded that all sequences required for a basic level of testis-specific transcription of transgene are present within the 397-bp fragment, and other DNA sequences located outside of the 397-bp fragment but present within the 877-bp fragment can function as enhancer elements. Two fragments within the 877-bp region were identified by gel retardation assays as binding 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 and protamine 1, respectively.
Highlights
Bp showed a re- Earlier, we reported that 2.3 kb’ of the 5”flankingregion of porter geneexpression and chloramphenicol acetyl- the rat proacrosin gene wassufficient to direct transcriptionof transferase enzyme activity which wasidentical to that the bacterialchloramphenicol acetyltransferase (CAT)reporter found in mice harboring the 2.3-kilobase pair 5”flanking region
It can be concluded that all sequences required for a basic level of testis-specific transcription of transgene are present within the 397-bp fragment, and other DNA sequences located outside of the 397-bp fragment but present within the 877-bpfragment can function as enhancer elements
Construction of the Fusion Genes-The fusion gene constructs used for microinjectionare shown in Fig. 1.The constructprACRIII-CAT was cleaved from plasmid prACRII-pBLCAT3(Nayerniaet ul., 1992)
Summary
Cell suspension transgenic mice and as a control of the position effect on transfrom the testes of 7 animals was loaded onto a 1000-ml 2-496 bovine gene expression(Beermann et al.,1991).Three transgenicmice serum albumin gradient in a CELSEP chamber (Depart Inc., Washin( Ag-l l , A32, TC) for the prACRIII-CAT construct (877-bp 5' reton, D.C.) with a 10% bovine serum albumin cushion at the bottom. Of 24 animalsgenerated by coinjection ofprACRIV-CAT mouse p-actin cDNA using22-merprimers(upstreamprimer: 5'- (397-bp 5' region) and ptrTYR5, dot blot analysis of tail DNA. For Northern blot analysis 20-pg RNA samples were denatured in a assays were conducted on extracts of different organs of adult TC formaldehyde/formamide buffer and electrophoresed on 1.5% agarose transgenic mice and CAT activity was found only in testis extract gels.
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