Developmentally regulated expression of Hsp70-2 and a Hsp70-2/lacZ transgene during spermatogenesis.
Germ cells synthesize large amounts of HSP70-2 protein during the meiotic phase of spermatogenesis. This developmentally regulated expression of HSP70-2 contrasts with the constitutive or inducible expression of other 70-kDa heat shock proteins (HSP70s). To better understand the genetic regulation of Hsp70-2, we used mRNA primer- extension, reverse transcriptase PCR (RT-PCR), and cDNA sequencing to determine that transcription began as far as 353 bp upstream of the start codon. We also identified a previously unrecognized 239-bp intron which is spliced out of the pre-mRNA transcript to leave a 114 nt 5'-untranslated region. Transgenic mice were then produced to delimit the upstream regulatory region required for developmental expression of Hsp70-2 during spermatogenesis. Results with multiple lines of transgenic mice containing promoter-reporter transgenes with varying lengths of Hsp7-2 sequence indicate that promoter sequences up to 640 bp upstream of the start codon and 287 bp upstream of the transcription start site are required for Hsp70-2/lacZ expression in spermatocytes. Histochemical detection of transgene beta- galactosidase activity was coincident with immunohistochemical detection of HSP70-2 protein, both in the first wave of spermatogenesis in juvenile mice and in ongoing spermatogenesis of adult mice. The distribution of Hsp7O-2 and Hsp7O-2/lacZ mRNAs was determined by Northern blot, in situ hybridization, and RT-PCR, and it was found that upregulation of expression of both Hsp7O-2 and Hsp7O-2/lacZ was specific to the meiotic phase of spermatogenesis.
Dix
D J
DJ
National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA. dix.david@epamail.gov
Rosario-Herrle
M
M
Gotoh
H
H
Mori
C
C
Goulding
E H
EH
Barrett
C V
CV
Eddy
E M
EM
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018507:Gene Expression Regulation, Developmental; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006720:Homozygote; D007438:Introns; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D013091:Spermatogenesis / Q000235:genetics*; D013737:Testis / Q000254:growth & development / Q000378:metabolism; D019076:Transgenes; D001616:beta-Galactosidase / Q000235:genetics*
D017931:DNA Primers; D018840:HSP70 Heat-Shock Proteins; D001616:beta-Galactosidase