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ANALYSIS OF THE 5′ UTR OF THE HAMSTER RELAXIN GENE.
Renegar, Randall1, Owens, Charles1, Fu, Ping2, Tregear, Geoffrey2, 1 2
ABSTRACT- During pregnancy relaxin is expressed primarily in ovarian luteal cells of rats and mice and trophoblast giant cells of the hamster. The objective of this study was to obtain nucleotide sequence data for the regulatory region of the hamster relaxin gene and compare this information with that of the rat with the intent of gaining insight into regulatory elements that may direct tissue-specific expression of this hormone. A hamster genomic DNA library packaged in the Lambda Fix II vector was obtained from Stratagene. Plaques (106) were screened with the hamster relaxin cDNA, and the genomic insert (8 kb) of one positive clone was removed by restriction digestion (SpeI) and ligated into pcDNA2.1. DNA sequencing was performed by the dideoxy chain termination method with the initial primer (22 bp) made in accordance with the 5′ region of the hamster cDNA. Subsequent sequencing was accomplished by primer walking to yield 2081 bp of the 5′ UTR. Primer extension indicated a single transcription initiation start site (position 1) located 52 bp upstream of the ATG translation initiation codon. Comparison of the proximal 5′ UTR (642 bp) of the hamster with that previously reported for the rat revealed an overall sequence homology of 68%. Potential transcription factor binding sites were analyzed using TFSEARCH ver. 1.3. A consensus TATA-box element is present at position −31 and a GC-box was identified at position −83. Previous studies in the rat suggested that this latter region is an enhancer/positive regulatory element of relaxin expression in that species and thus may serve a similar function in the hamster. A potential binding site for members of the GATA family of transcription factors was present at position −295. Previous studies have demonstrated that GATA binding sites are involved in trophoblast giant cell-specific expression of mouse placental lactogen-I and proliferin and may therefore function in giant cell-specific relaxin expression in the hamster. A GATA-binding site is not present in the region of the rat promoter that has been reported. The information provided by this study will be useful in directing functional analysis of tissue-specific expression of the relaxin gene in the hamster.
KEY WORDS: relaxin, promoter, hamster
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