Hamil K G, Hall S H
Laboratories for Reproductive Biology, University of North Carolina, Chapel Hill 27599.
Endocrinology. 1994 Mar;134(3):1205-12. doi: 10.1210/endo.134.3.8161377.
The molecular mechanisms underlying the pleiotropic effects of FSH were investigated by screening a plasmid cDNA library for clones hybridizing to FSH-regulated RNAs. Recombinant colonies were selected at random, and plasmids were purified, radiolabeled, and hybridized to Northern blots containing RNA extracted from control and FSH-treated Sertoli cells. Of 210 clones screened by this method, 10 hybridized to transcripts that were regulated either positively or negatively by FSH. DNA sequence comparisons with the GenBank database revealed that 3 clones that hybridized to positively regulated RNAs have sequences similar to known or putative transcription regulating factors. Clone 99 encodes the rat homolog of transforming growth factor-beta 1-stimulated clone 22 (TSC-22), which contains a putative leucine zipper region. Clone 18 has 93% sequence identity in the coding region with the cDNA for mouse nuclear factor-kappa B p50 subunit. Clone 325 encodes rat TIS11b, which contains zinc finger-like motifs thought to confer DNA-binding capacity. Among the other cDNAs, 2 have strong sequence similarity to RNA-binding proteins, including splicing factors, 1 corresponds to the rat mitochondrial transcript that encompasses the abundant 12S and 16S ribosomal RNAs, and another has 93% homology with the human B-cell translocation gene-1 (BTG-1), which encodes a putative antiproliferative factor. The remaining 3 clones had no identity with sequences in the GenBank database. Regulation of rat TSC-22 mRNA was analyzed in primary Sertoli cell cultures. TSC-22 mRNA transiently increased 4-fold in the presence of FSH, reached maximal levels at 3 h, and returned to prestimulation levels by 12 h. The FSH-stimulated increase was independent of protein synthesis because it occurred in the presence of cycloheximide and FSH. TSC-22 mRNA was detected in all tissues examined in male and female rats, and the highest levels in the 16-day animal were observed in the testis, ovary, uterus, and lung. Testicular 1.8-kilobase (kb) TSC-22 mRNA decreased by 50% from 14 to 60 days of age. A 5-kb transcript became detectable by 30 days and decreased after 50 days of age. Ovarian 1.8-kb TSC-22 transcript levels increased about 2-fold during the same maturation period.
通过筛选质粒cDNA文库以寻找与FSH调节的RNA杂交的克隆，研究了FSH多效性作用的分子机制。随机选择重组菌落，纯化质粒，进行放射性标记，并与包含从对照和FSH处理的支持细胞中提取的RNA的Northern印迹杂交。用这种方法筛选的210个克隆中，有10个与受FSH正向或负向调节的转录本杂交。与GenBank数据库进行DNA序列比较显示，3个与正向调节的RNA杂交的克隆具有与已知或推定的转录调节因子相似的序列。克隆99编码转化生长因子-β1刺激克隆22（TSC-22）的大鼠同源物，其包含一个推定的亮氨酸拉链区域。克隆18在编码区与小鼠核因子-κB p50亚基的cDNA具有93%的序列同一性。克隆325编码大鼠TIS11b，其包含被认为赋予DNA结合能力的锌指样基序。在其他cDNA中，2个与RNA结合蛋白有很强的序列相似性，包括剪接因子，1个对应于包含丰富的12S和16S核糖体RNA的大鼠线粒体转录本，另一个与人类B细胞易位基因-1（BTG-1）有93%的同源性，BTG-1编码一种推定的抗增殖因子。其余3个克隆与GenBank数据库中的序列无同一性。在原代支持细胞培养物中分析了大鼠TSC-22 mRNA的调节。在FSH存在下，TSC-22 mRNA瞬时增加4倍，在3小时达到最高水平，并在12小时回到刺激前水平。FSH刺激的增加与蛋白质合成无关，因为它在放线菌酮和FSH存在下发生。在雄性和雌性大鼠的所有检测组织中都检测到了TSC-22 mRNA，在16日龄动物中，睾丸、卵巢、子宫和肺中的水平最高。睾丸1.8千碱基（kb）的TSC-22 mRNA在14至60日龄时下降了50%。一个
