Structure of the 5 ' region of the Hst70 gene transcription unit: presenceof an intron and multiple transcription initiation sites

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family o f Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5' region of the transcrip tion unit of these genes as well as localization of the 'cis' elements conf erring their testis-specific expression gave contradictory results [Widlak, Markkula, Krawczyk. Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1 264, 191-200; Dix. Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy ( 1996) Dev. Biol. 174, 310-321]. In the present paper we solve these controv ersies and show that the 5' untranslated region (UTR) of the Hst70 gene con tains an intron which is localized similar to that of the mouse Hsp70.2 gen e. Reverse transcriptase-mediated PCR. Northern blotting and RNase protecti on analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of hrst70 gene transcripts with similar sizes but different 5' UTR structures can be detected in total testicular RNA. Fu nctional analysis of the Hst70 gene promoter in transgenic mice and transie nt transfection assays proved that the DNA fragment of approx. 360 bp local ized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyl transferase transgene. These experiments also suggest that the expression o f the gene may depend on 'cis' regulatory elements localized within exon 1 and the intron sequences.