猪Pit-1基因第三内含子序列的克隆测序

根据不同物种间同一基因核苷酸序列的保守性及相似性的特点 ,在人和鼠的Pit 1基因第三外显子上设计上游引物 ,而将下游引物设计在猪Pit 1基因第四外显子上。利用聚合酶链式反应 (PCR)技术 ,扩增出猪Pit 1基因第三内含子序列 ,并经由酶切及序列同源性比对确认该序列即为猪Pit 1基因第三内含子序列。此序列的确定为下一步进行遗传变异分析的研究奠定了基础     

Phylogenetic specificity of prolactin gene expression with conservation of Pit-1 function.

In mammals, the pituitary POU homeodomain protein, Pit-1, binds to proximal and distal 5'-flanking sequences of the PRL gene that dictate tissue-specific expression. These DNA sequences are highly conserved among mammals but are dramatically different from PRL 5' sequences in the teleost species, Oncorhynchus tschawytscha (chinook salmon). To analyze the molecular basis for pituitary-specific gene expression in a distantly related vertebrate, we transfected CAT reporter gene constructs containing 2.4 kilobases (kb) 5'-flanking sequence from the salmon PRL (sPRL) gene into various cell types. Expression of the sPRL gene was restricted to pituitary cells, but in rat pituitary GH4 cells levels of expression were at least 90-fold lower than those obtained with a -3 kb rat PRL (rPRL) construct. Conversely, in primary teleost pituitary cells, -2.4 kb sPRL/CAT was expressed at levels about 10-fold higher than -3 kb rPRL/CAT. To determine whether species-specific transactivation by Pit-1 was sufficient to explain these species differences in PRL gene expression, we isolated a cDNA clone encoding the salmon Pit-1 POU domain and constructed a rat Pit-1 expression vector that contained salmon Pit-1 POU domain sequences substituted in frame. The chimeric Pit-1 encoded 14 amino acids unique to salmon. Coexpression of rat Pit-1 with salmon or rat PRL/CAT in transfected HeLa cells resulted in specific and strikingly comparable levels of promoter activation. Moreover, the specificity and efficacy of the chimeric salmon/rat Pit-1 was similar to wild type rat Pit-1 in activating salmon and rat PRL/CAT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transcriptional induction of the human prolactin gene by cAMP requires two cis-acting elements and at least the pituitary-specific factor Pit-1.

To identify the cis-acting elements responsible for cAMP stimulation of human prolactin (hPRL) promoter activity, pituitary GC cells were transfected with 5'-deleted hPRL promoters fused to the chloramphenicol acetyltransferase reporter gene. The proximal regulatory region (coordinates -250 to -42) was sufficient to confer strong cAMP stimulation (+/- 25 fold). Further 5' and 3' deletions performed within this proximal region demonstrated that two types of cis-acting elements are involved in the cAMP regulation: (i) the binding sites of the pituitary-specific factor Pit-1, and (ii) the sequence between coordinates -115 and -85 (named fragment A), which contains a TGACG motif. We show by gel-shift and Southwestern experiments that fragment A binds Pit-1 monomer and also a ubiquitous factor that is neither cAMP-responsive element-binding protein nor activator protein-1. Strong cAMP induction was observed when fragment A was juxtaposed to a Pit-1 binding site. That Pit-1 plays an important role was supported further by the finding that the hPRL proximal region conferred cAMP regulation when linked to the herpes simplex virus thymidine kinase promoter only in pituitary GC cells and not in other heterologous cells, which do not express Pit-1. Furthermore, we observed that concatenated Pit-1 binding sites were able to confer cAMP responsiveness to the thymidine kinase promoter in GC cells.     

Estrogen-modulated estrogen receptor x Pit-1 protein complex formation and prolactin gene activation require novel protein synthesis

Both estrogen receptor (ER) and Pit-1 proteins are essential for the estrogen-activated expression of the rat prolactin gene. Our results show that ER.Pit-1 protein complex formation is reduced by estrogen in GH3 and PR1 rat pituitary tumor cells. In the latter, this decrease was blocked by cycloheximide, a protein synthesis inhibitor. On the other hand, the direct addition of estrogen to PR1 cell lysates had no effect on the formation of ER.Pit-1 complexes. Estrogen-activated prolactin gene expression was also inhibited by cycloheximide, suggesting that some form of protein synthesis is involved in ER.Pit-1 complex formation and subsequent prolactin gene activation. In support of this notion, we showed that estrogen-induced regulation of ER.Pit-1 complex formation could be transferred from cell lysates prepared from estrogen-treated PR1 cells to control cell lysates. This is not true for GH3 cells; instead, direct administration of estrogen to GH3 cell lysates readily abolished ER.Pit-1 protein complex formation in a dose-dependent manner, and such estrogen-induced regulation was blocked by the antiestrogen ICI 182,780. These findings thus indicate that 1) interaction between ER and Pit-1 proteins is estrogen-regulated in ways specific to different cell types, and 2) auxiliary protein factor synthesis may be involved in this process.