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T. Usui, Y. Nakai, T. Tsukada, H. Takahashi, J. Fukata, Y. Naito, S. Nakaishi, T. Tominaga, N. Murakami and H. Imura

ABSTRACT

A fragment of human genomic DNA containing the entire pro-opiomelanocortin (POMC) gene was introduced by transfection into the rat glial cell line C6. Blot analysis using poly(A)-rich RNA from the transformed C6 cells showed several hybridization bands. One band was similar in size (1·2 kb) to the POMC mRNA of human pituitary, while two were larger (2·6 and 2·2 kb) and the fourth smaller (800 bp). S1 nuclease mapping revealed that the POMC transcripts in transformed C6 cells were similar to those in non-pituitary tissues. Immunoreactive ACTH (ir-ACTH) was measurable in both the culture medium and cells. Gel chromatography showed that ir-ACTH in the medium eluted at a position identical to that of so-called big ACTH (approximately 40 kDa) which is found in the plasma of patients with ectopic ACTH syndrome. The human POMC gene could thus be expressed in the non-pituitary rat glial cell line C6, although the transcripts and translation products in C6 cells differ from those in the human pituitary. These results suggest that the transformed C6 cell may be a useful tool for studying the regulation of human POMC gene expression in non-pituitary cells.

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M Tanaka, M Suzuki, T Kawana, M Segawa, M Yoshikawa, M Mori, M Kobayashi, N Nakai and T R Saito

In addition to the known four alternative first exons E11, E12, E13 and E14 of the rat prolactin receptor (PRL-R) gene, a novel first exon, E15, was identified by cDNA cloning of the 5′-end region of PRL-R mRNA in the rat liver. Genomic fragments containing E15 and its 5′- or 3′-flanking regions were also cloned from rat kidney genomic DNA. A sequence search for E15 revealed that E15 is located 49 kb upstream of exon 2 of the PRL-R gene in rat chromosome 2q16. RT-PCR analysis revealed that E15 was preferentially expressed in the liver, brain and kidney. Expression profiles of E12-, E13- and E15-PRL-R mRNAs in the liver of male and female rats at 5 days of age and those at 8 weeks of age were examined by RT-PCR. The levels of E12-PRL-R mRNA in the female rat increased remarkably in rats at 8 weeks of age compared with those at 5 days of age, and the levels of E15-PRL-R mRNA in the male rat decreased markedly at 8 weeks of age compared with those at 5 days of age. In the female rat, the levels of E12-PRL-R mRNA at 8 weeks of age decreased with ovariectomy performed at 4 weeks of age and recovered with the administration of β-oestradiol. On the contrary, the levels of E15-PRL-R mRNA increased with ovariectomy and decreased with the oestrogen treatment. In the male rat liver, the levels of E12-PRL-R mRNA at 8 weeks of age increased strikingly with castration performed at 4 weeks of age and became undetectable with the administration of testosterone. The levels of E15-PRL-R mRNA increased slightly with castration and were restored by testosterone treatment. Removal of gonadal tissues and sex steroid hormone treatment had no effect on the expression levels of E13-PRL-R mRNA in both female and male rat livers. These results indicated that the expression of the PRL-R gene in the liver is regulated by the differential effects of sex steroid hormones on the transcription of the multiple first exons including the novel one.