Cloning of a DNA fragment displaying restriction fragment length polymorphisms between the genomes of spontaneously hypertensive and Wistar-Kyoto rats


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Abstract

A 3 kilobase (kb) EcoRl fragment cloned from the genome of the spontaneously hypertensive rat (SHR) displayed restriction fragment length polymorphism (RFLP) compared with the genome of the Wistar-Kyoto rat (WKY) when total genomic Southern blot analysis was performed for two restriction enzymes, Pstl and Pvull. Sequencing of the DNA fragment cloned from genomic SHR and WKY libraries revealed that this 3 kb EcoRI fragment harbours three point mutations. Two of them (C to T and A to T) are situated in the middle of the restriction sites for Pstl and Pvull, thus disrupting the recognition sites for these enzymes in the SHR genome. Southern blot analysis using total complementary (c) DNA obtained from cDNA libraries of aortic smooth muscle cells from SHR and a whole WKY kidney, with this 3 kb EcoRI fragment as a probe, showed polymorphic bands suggesting that these point mutations are reflected in the sequences of messenger (m) RNA transcribed from the gene encoded in this 3 kb fragment. Detection of two bands by a Northern blot analysis for RNA from various SHR tissues indicates that this 3 kb fragment is actively transcribed in vivo.

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