Determination of male rat (rattus norvegicus) kidney prolactin receptor mRNA diversity

Prolactin receptor (PRLR) and its cognate hormone (prolactin, PRL) involve in over 300 different functions, including osmoregulation, immunoregulation, fat store regulation, proliferation, tumorigenesis and behaviour as well as its well known role in lactation and reproduction. Rat prolactin gene (r-prlr) is about 190 kb and codes at least two different isoforms, named Long and Short forms (LF and SF). So far, 11 variants in human, 4 variants in mouse and 2 variants in pig and rat were identified. Some of these variants are formed either by substitution of exons in 3' ends of mRNAs (such as exons 10 and 11, in the LF and SF, respectively) or exclusion of one or few internal exons of 'the core exons', namely between exons 3 to 9. The aim of this project was to investigate possibility of new rat PRLR variants and also to identify the 3' UTR of PRLR mRNAs. In order to this, first the 'internal exon' and the 3' end variants were searched and then, the possibility of expression of mouse SF exon variants (exons 11 and 12), their expression in rats are not known, in rat organs were investigated. Finally, the 3' UTR sequences of rat L- and SF PRLRs were tried to be revealed. PCR primer sets, were used to investigate the 'internal exon' and the 3' end variants, were designed using rat prlr genome and its mRNA sequences. It is known that mouse prlr gene contains extra two more exons (exons 11 and 12) and their expression is not known in rats. A homology search were performed in rat and other available rodent prlr genes and it is found that these mouse exons (11 and 12) are highly conserved in the rat and some rodents. Therefore, primer sets, targeting the potential exons, were designed using rat prlr sequence. For determination of 3' UTR regions of rat PRLR mRNAs, primers were designed, targeting 3' UTR regions and poly A ends of the mRNAs. Organs (kidney, liver and testis) were collected from 22-24 weeks-old male and female rats and they were used for total RNA and mRNA isolations. Single strand cDNAs were synthesised by a reverse transcription reaction (RT-PCR) using either poly T or poly T Adaptor primers and then target PRLR regions were amplified using specific primers by either Standard PCR and/or other PCR applications (3' RACE, Nested PCR and Stepdown PCR). PCR products were run on 1-1.5% agarose gels and bands corresponding to expected amplicon sizes were sent for sequencing and provided sequences were analysed using appropriate programs. Amplification products of agarose gels showed that all organs express the core region of PRLR gene (exons 3 to 9) as well as known rat PRLR variants (e.i L- and SFs). On the other hand, no specific amplification products were obtained for 3' (exons 10 and 11) and internal exon variants. In order to find out whether mouse exons (11 and 12, expressed in mouse but not known in rat) were also expressed in rats, PCR reactions were performed using specific primers but no rat prlr gene specific products were observed in both sexes and all organs. Using gene specific primers or gene specific and adaptor primer sets, it was found that SF PRLR mRNA has a 3' UTR region, about 500 bp, and LF PRLR mRNA contains a 3' UTR region, longer than 2 kbp as its about 2000 bp were successfully amplified and sequenced. Although the thesis project is on male rat kidney, in this study both sexes (male and female) and their organs (kidneys, livers and testis) were studied for identification of new PRLR variants. In both sexes and all organs, rat PRLR L- and SFs were successfully amplified but no new PRLR variants (3' end or internal exon) were observed. It also seems that highly conserved mouse exons were not expressed in rat kidney, liver or testis.