Human prolactin gene expression. The use of an alternative noncoding exon in decidua and the IM-9-P3 lymphoblast cell line.

G E Dimattia,B Gellersen,M L Duckworth,H G Friesen

doi:10.1016/s0021-9258(17)46238-7

Abstract

The prolactin (PRL) gene is normally expressed in the anterior pituitary lactotrope and the decidualized stromal cell of the human endometrium. We have recently described the ectopic expression of the human PRL gene in a B-lymphoblastoid cell line, IM-9-P. The IM-9-P and decidual PRL mRNAs are approximately 150 nucleotides longer than the pituitary transcript. The protein coding sequence of the IM-9-P and decidual PRL cDNAs is identical to the pituitary PRL message, and the 3'-untranslated region (UTR) exhibited a 7-14-nucleotide elongation. The 5'-UTR of IM-9-P and decidual PRL cDNAs revealed an extension of 41 base pairs upstream of the normal transcription start site for the human pituitary PRL gene. This is preceded by an additional sequence that is not homologous to the DNA found 5' to the pituitary PRL cap site. Decidual and IM-9-P PRL mRNAs possess identical 5' ends having 5'-UTRs of 83-263 nucleotides longer than that of the pituitary mRNA. The decidual/IM-9-P-specific segment of the 5'-UTR is located as a new 5'-noncoding exon 5-7 kilobase pairs upstream of the human pituitary PRL gene, exon 1. The clonal IM-9-P3 cell line provides a unique and easily manageable resource with which to study decidual specific PRL gene transcription.

Highlights

From the Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, the §Znstitute for Hormone and Fertility Research, 2000 Hamburg 54, Federal Republic of Germany
The protein coding sequence of the IM-9-P and decidual PRL cDNAs is identical to the pituitary PRL message, and the 3’-untranslated region (UTR) exhibited a 7-14-nucleotide elongation
Isolation and Characterization of PRL cDNA Clones-Contrary to previous results in the literature, we have found recently that human decidual PRL mRNA, like the IM-9-P3 PRL message, is elongated relative to the pituitary transcript

Summary

PROCEDURES

Phage DNA from positive clones was isolated from confluent plates [28], digested with EcoRI, size fractionated on 0.8% agarose gels, and analvzed bv Southern blot hvbridization using random primer-labeled hPRL cDNA fragments-. 300,000 phage were screened from a human placental library (Clontech) using a- random primer-labeled hPRL cDNA [29] under stringent conditions as described. PRL cDNA fragments containing portions of the 5’untranslated sequence were subcloned into pGEM-3, and a-32P-labeled antisense or sense cRNA was synthesized using Sp6 or T7 RNA polymerase as outlined by the vendor of the in vitro transcription kit. Those RNA blots hybridized with random primer-labeled fragments of hPRL cDNA were done as described previously [18]. Blots were subjected to 24-h autoradiography to ensure removal of radioactive bands from previous hybridizations

RESULTS

E F G IM 9 P6 P3 P33 P33 P3 P6 IM-9 GFEDC 6 A

DISCUSSION