Tissue-specific expression of squirrel monkey chorionic gonadotropin

Pituitary gonadotropins LH and FSH play central roles in reproductive function. In Old World primates, LH stimulates ovulation in females and testosterone production in males. Recent studies have found that squirrel monkeys and other New World primates lack expression of LH in the pituitary. Instead, chorionic gonadotropin (CG), which is normally only expressed in the placenta of Old World primates, is the active luteotropic pituitary hormone in these animals. The goal of this study was to investigate the tissue-specific regulation of squirrel monkey CG. We isolated the squirrel monkey CGβ gene and promoter from genomic DNA from squirrel monkey B-lymphoblasts and compared the promoter sequence to that of the common marmoset, another New World primate, and human and rhesus macaque CGβ and LHβ. Using reporter gene assays, we found that a squirrel monkey CGβ promoter fragment (−1898/+9) is active in both mouse pituitary LβT2 and human placenta JEG3 cells, but not in rat adrenal PC12 cells. Furthermore, within this construct separate cis-elements are responsible for pituitary- and placenta-specific expression. Pituitary-specific expression is governed by Egr-1 binding sites in the proximal 250 bp of the promoter, whereas placenta-specific expression is controlled by AP-2 sites further upstream. Thus, selective expression of the squirrel monkey CGβ promoter in pituitary and placental cells is governed by distinct cis-elements that exhibit homology with human LHβ and marmoset CGβ promoters, respectively.     

Multihormonal regulation of the human prolactin gene expression from 5000 bp of its upstream sequence

We have cloned DNA sequences extending up to 6000 bp upstream from the first exon of the human prolactin (hPRL) gene. 5000 bp of these upstream sequences were fused to a CAT reporter gene and shown to provide tissue-specific transient expression in rat pituitary GH3 cells.

Multihormonal response was found in this transient expression assay, leading to significant 2- to 5-fold induction by addition of 8-chlorophenylthio-cyclic AMP, thyrotropin-releasing hormone, epidermal growth factor, basic fibroblast growth factor, phorbol myristate acetate, a calcium channel agonist (Bay K-8644) and triiodothyronine. A 3-fold inhibition was observed in the presence of the glucocorticoid agonist dexamethasone.

The sequence of the hPRL promoter was determined up to coordinate — 3470. Computer similarity search between the rat and human sequences showed two highly conserved regions corresponding to the proximal and distal tissue specific enhancers described in both PRL promoters.     

Transient induction of a variant hepatic lipase messenger RNA by corticotropic hormone in rat adrenals

Hepatic lipase (HL) is present not only in liver, but also in steroidogenic organs, where it is thought to mediate cellular uptake of plasma cholesterol. In rat adrenals and ovaries, the HL gene is transcribed into a variant messenger RNA (mRNA) that lacks exons 1 and 2. Treatment of male Wistar rats with corticotropin resulted in a transient 9-fold increase in the variant HL mRNA in the adrenals, which was paralleled by synthesis of 47- to 49-kilodalton HL-related proteins. In contrast, a delayed, but sustained, 6-fold increase in adrenal HL activity was observed. This difference in time course suggests that the HL activity does not reflect HL-like proteins expressed from the variant mRNA. By Northern blotting, the variant HL mRNA was 2.6 kilobase. By screening a rat genomic library, the 5' end of the variant HL mRNA was located in intron 2 immediately upstream of exon 3. Primer extension analysis mapped the 5' end at nucleotide 465 upstream of exon 3. In promoter-reporter assays, the intron 2 region (-233/+350 with respect to the putative start site) showed no apparent basal activity in HepG2 hepatoma and NCI-H295R adrenocortical cells. The putative promoter in intron 2 was up-regulated in NCI-H295R human adrenocortical cells by treatment with 8-bromo-cyclic adenosine monophosphate. We conclude that intron 2 of the rat HL gene has an alternative promoter with low activity in adrenals, ovaries, and liver. In rat adrenals, this promoter is transiently activated by corticotropin.     

Gene encoding human growth hormone-releasing factor precursor: structure, sequence, and chromosomal assignment.

We have isolated and characterized overlapping clones from phage lambda and cosmid human genomic libraries that predict the entire structure of the gene encoding the precursor to human growth hormone-releasing factor. The gene includes five exons spanning 10 kilobase pairs of human genomic DNA. There appears to be a segregation of distinct functional regions of the GRF precursor and its mRNA into the five exons of the gene. The DNA sequences of all exons, intron/exon boundaries, and 5' and 3' flanking regions are presented. Dot-blot analysis of DNA from high resolution dual-laser-sorted human chromosomes indicates that the single-copy growth hormone-releasing factor gene is located on human chromosome 20.