During late pregnancy, placental and decidual relaxins are autocrine/paracrine hormones partly responsible for the controlled loss of collagen in the fetal membranes allowing them to strteh and accommodate to the rapidly growing fetus. Overexpression of relaxins could lead to the overproduction of the enzymes that degrade collagen, thereby overly weakening the membranes and resulting in their premature rupture. This leads to preterm birth, which is a major health problem in the minority population of this country. The goal of this proposal is to study the differential expression of the human relaxin genes in the decidua and placenta as a function of gestation, by analysis of the promoter regions and identification of the regulatory elements at their 5'-flanking sequences. The first specific aim is to isolate the genomic clones of relaxins H1 and H2 containing at least 2.0 kb length of the 5'-flanking region and includes the single intron using inverse PCR and plaque hybridiution screening of a human genomic library. The second specific aim will identify and characterize the promoter regions by performing chloramphenicol acetyltransferase (CAT) assays using cell lysates of a human cell line known to express both relaxin genes, which were previously transfected with various 5'-end deletion plasmids fused with the bacterial CAT gene. The third specific aim will focus on the specific DNA sequences with putative regulatory functions and which may affect the tissue specific expression of the relaxin genes in the decidua and placenta. Binding of specific transcription factors from crude nuclear extracts prepared from decidua and placental trophoblast cells as well as selected cell lines to these specific DNA sequences will be analyzed by gel electrophoretic mobility shift assays, in vitro transcription and DNase protection assays.
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