Structure of the 5' region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites.
The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5' region of the transcription unit of these genes as well as localization of the 'cis' elements conferring their testis-specific expression gave contradictory results [Widlak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191-200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310-321]. In the present paper we solve these controversies and show that the 5' untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection 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 Hst70 gene transcripts with similar sizes but different 5' UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360 bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on 'cis' regulatory elements localized within exon 1 and the intron sequences.
Scieglinska
D
D
Department of Tumor Biology, Centre of Oncology, Maria Sklodowska-Curie Memorial Institute, Wybrzeze Armii Krajowej 15, 44-100 Gliwice, Poland.
Widłak
W
W
Konopka
W
W
Poutanen
M
M
Rahman
N
N
Huhtaniemi
I
I
Krawczyk
Z
Z
D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics*; D007438:Introns / Q000235:genetics*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D013090:Spermatocytes / Q000378:metabolism; D013737:Testis / Q000378:metabolism; D024363:Transcription Initiation Site
D020121:5' Untranslated Regions; D017931:DNA Primers; D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D012259:Ribonuclease, Pancreatic