Dix D J, Rosario-Herrle M, Gotoh H, Mori C, Goulding E H, Barrett C V, Eddy E M
National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA.
Dev Biol. 1996 Mar 15;174(2):310-21. doi: 10.1006/dbio.1996.0076.
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.
生殖细胞在精子发生的减数分裂阶段合成大量的HSP70-2蛋白。HSP70-2这种受发育调控的表达与其他70 kDa热休克蛋白（HSP70s）的组成型或诱导型表达形成对比。为了更好地理解Hsp70-2的基因调控，我们使用mRNA引物延伸、逆转录酶PCR（RT-PCR）和cDNA测序来确定转录起始于起始密码子上游353 bp处。我们还鉴定出一个先前未被识别的239 bp内含子，它从pre-mRNA转录本中剪接出来，留下一个114 nt的5'非翻译区。然后制备转基因小鼠以确定精子发生过程中Hsp70-2发育表达所需的上游调控区域。含有不同长度Hsp7-2序列的启动子-报告基因转基因的多系转基因小鼠的结果表明，起始密码子上游640 bp和转录起始位点上游287 bp的启动子序列是精子细胞中Hsp70-2/lacZ表达所必需的。在幼年小鼠的第一波精子发生和成年小鼠正在进行的精子发生中，转基因β-半乳糖苷酶活性的组织化学检测与HSP70-2蛋白的免疫组织化学检测一致。通过Northern印迹、原位杂交和RT-PCR确定了Hsp7O-2和Hsp7O-2/lacZ mRNA的分布，发现Hsp7O-2和Hsp7O-2/lacZ表达的上调都特异性地发生在精子发生的减数分裂阶段。
