A study of the guinea pig relaxin gene(s)

Abstract

This dissertation study was designed to elucidate the nucleotide sequence of the guinea pig relaxin gene and hence to derive the amino acid sequence of guinea pig preprorelaxin, to show how many relaxin gene(s) are present in the guinea pig genome, and to study the transcription of a relaxin gene in the guinea pig endometrium during the reproductive cycle. A lactating guinea pig mammary gland cDNA library was screened with a radioactive full-length rat preprorelaxin cDNA probe. Seven positive clones were identified. The characterization of the inserts with the PCR (Polymerase Chain Reaction) technique and Southern hybridization suggested that they are truncated molecules. In order to obtain general information of the guinea pig relaxin gene, the clone containing the longest insert (approximately 600 bps) was submitted to sequence analysis. Computer-assisted sequencing data analysis show that this insert was similar to the mRNA sequence of the pig preprorelaxin. The conclusion from this study was that the guinea pig relaxin gene sequence is similar to that of the pig relaxin gene. Based on the preliminary information from these studies, several sets of oligonucleotide primers were selected from different regions of the mRNA sequence of pig preprorelaxin. These synthetic primers were used to screen a cDNA "pool" prepared from the guinea pig endometrium in late pregnancy by a PCR technique. One set of these PCR primers successfully amplified a relaxin related cDNA fragment (286 bps). Sequence analysis of this PCR product confirmed that it encodes an intact B chain of the guinea pig preprorelaxin plus part of the signal peptide and C peptide of this molecule. The rest part of the guinea pig endometrial relaxin gene was elucidated by a RACE-PCR and subcloning strategy. This is the first report of any part of the guinea pig relaxin gene sequence. Availability of this sequence allowed a study of the physiology of guinea pig relaxin. The second question of this dissertation was studied by a Southern analysis of guinea pig genomic DNA digests with a guinea pig relaxin specific cDNA probe. It was shown that there are two different relaxin genes in the guinea pig genome, a situation similar to the human genome, but different to the genomes of other mammals studied thus far. One relaxin gene has been shown to be expressed in the guinea pig endometrium in this dissertation using PCR and direct sequencing. Whether ,or where, the second relaxin gene is expressed needs further investigation. The availability of the guinea pig relaxin gene sequence allowed a more convincing study of the expression of this gene in the guinea pig endometrium. Guinea pig endometrial mRNAs from different stages of the reproductive cycle were hybridized with the radioactive guinea pig relaxin cDNA probe. Northern analyses obtained from this study showed that the transcription pattern of the relaxin gene in this organ is maximal in the late pregnancy, minimal during mid pregnancy, appears in the estrous cycle and disappears rapidly after parturition. This is the first study of the transcription of the relaxin gene in the guinea pig endometrium during the reproductive cycle with a species-specific cDNA probe. It provides proof that relaxin is indeed synthesized in the guinea pig endometrium, not sequestered from other sources. The loss of transcription activity of the relaxin gene, in the endometrium, during lactation indicates that there are other sites of relaxin synthesis as sources of the plasma relaxin found in lactation.