A 252 bp upstream region of the rat spermatocyte-specific hst70 gene is sufficient to promote expression of the hst70-CAT hybrid gene in testis and brain of transgenic mice.
The rat hst70 gene belongs to a heat shock hsp70 multigene family and its expression has been detected so far solely in spermatocytes. To investigate the cis-elements responsible for testis-specific expression of the hst70 gene we produced several lines of transgenic mice carrying fragments of the 5'-flanking regions of the hst70 gene fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Hybrid genes of series B were constructed such that, besides the 780 bp, 343 bp and 163 bp 5'-flanking region these plasmids contained no other sequences of the hst70 gene. In hybrid genes of series D the CAT gene was ligated to 343 bp and 252 bp 5'-flanking regions together with the 57 bp of the 5'-end nontranslated (leader) sequences of the hst70 gene. We found that in 780/B, 343/B, 343/D and 252/D adult mice the transgene was specifically and highly expressed in testes. In developing testes the high CAT activity appeared in transgenic mice aged 3 weeks and older. None of the three 163/B transgenic lines exhibited CAT activity in any tissue analyzed. In all CAT expressing lines a weak but significant CAT activity (up to 5% of that in testis) was detected also in the brain. RNase protection assay confirmed that the endogenous hst70 gene transcripts are present in testis as well as in brain of nontransgenic rats and mice. Our data show that the cis-regulatory sequences responsible for testis-specific and developmentally regulated expression of the hst70 gene are localized within the 252 bp region 5' to the gene and neither the 5'-end nor 3'-end nontranslated sequences of the gene are important for this specificity.
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017931:DNA Primers; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D005810:Multigene Family; D016133:Polymerase Chain Reaction; D051381:Rats / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D013090:Spermatocytes / Q000378:metabolism*; D013737:Testis / Q000378:metabolism*
D017931:DNA Primers; D018840:HSP70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase