CALBINDIN-D9K GENE-EXPRESSION IN THE UTERUS - STUDY OF THE 2 MESSENGER-RIBONUCLEIC-ACID SPECIES AND ANALYSIS OF AN IMPERFECT ESTROGEN-RESPONSIVE ELEMENT

The calbindin D9k (CaBP9k) gene is under strict estrogen control in th e rat uterus. This tissue contains two CaBP9k messenger RNA (mRNA) spe cies. We have used primer extension analysis, reverse transcriptase as sociated with polymerase chain reaction, and RNase H digestion to show that these two mRNA species have the same structural features, includ ing 5'- and 3'-ends, and poly(A) tail length. Our results suggest that the difference in electrophoretic mobilities of the two mRNA species might be due to interaction with another factor. We also analyzed the imperfect estrogen-responsive element (ERE) present on the first 5'-sp lice site of the rat CaBP9k gene. The oligonucleotide corresponding to the CaBP9k ERE was cloned in the plasmid pBLCAT2 (where the thymidine kinase promoter governs the expression of the chloramphenicol acetyl transferase gene) and transfected into MCF7 cells. This CaBP9k ERE was found to be a hormoneinducible enhancer that worked in an orientation -independent manner on a heterologous promoter and was functional at p hysiological hormone concentrations. One CaBP9k ERE conferred only wea k (about a-fold) estrogen induction, but two EREs cloned in tandem wer e strongly synergistic (14- to 16-fold). The CaBP9k ERE also bound to the partially purified estrogen receptor (ER) and to ER expressed in C OS cells by gel shift assay. Methylation interference showed that all the guanine residues in both half-sites of the CaBP9k ERE were protect ed by ER binding. Thus, ER binds to the CaBP9k ERE in a way similar to other EREs. The gel shift assay results indicate that the strong syne rgistic effect of two EREs cloned in tandem is not due to cooperative binding between the two elements. As the CaBP9k gene is under strong e strogenic control in the uterus in vivo, the imperfect CaBP9k ERE may cooperate with another trans-acting factor to become fully efficient.