CALBINDIN-D9K GENE-EXPRESSION IN THE UTERUS - STUDY OF THE 2 MESSENGER-RIBONUCLEIC-ACID SPECIES AND ANALYSIS OF AN IMPERFECT ESTROGEN-RESPONSIVE ELEMENT
F. Lhorset et al., CALBINDIN-D9K GENE-EXPRESSION IN THE UTERUS - STUDY OF THE 2 MESSENGER-RIBONUCLEIC-ACID SPECIES AND ANALYSIS OF AN IMPERFECT ESTROGEN-RESPONSIVE ELEMENT, Endocrinology, 134(1), 1994, pp. 11-18
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.