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KMID : 0578319950050030230
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Molecules and Cells
1995 Volume.5 No. 3 p.230 ~ p.234
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Cloning and the Nucleotide Sequences of Rat c-Ki-ras Gene in Normal and Regenerating Liver
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Kim, Seung-Hyun
Park, Sun-Hee/Park, Jong-sang
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Abstract
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To examine the cause for high level expression of the c-Ki-ras gene during rat liver regeneration, we intended to compare the expression pattern of the rat c-Ki-ras gene in normal and regenerating liver. Above all, the unknown nueleotide sequence (exons 0, 3, 4A, or 4B) of the c-Ki-ras gene in normal liver should be identified, so we screened the rat liver cDNA library with the ^(32)P-labeled probe which was selectively amplified from a nanogram of rat liver λgt11 cDNA library using two oligonucleotides complementary to the known nucleotide sequence of the rat the c-Ki-ras exon 1. The whole cDNA of the c-Ki-ras gene was obtained from the positive plaque through the third screening, subcloned into the pTZ 19 vector, and sequenced by dideoxy chain termination method. A comparison of the rat gene with that of the homologous mouse Ki-ras gene revealed 98% nucleotide sequence homology within the coding regions, 82% homology for the untranslated region of exon 4B, and 88% homology for the untranslated exon 0. Between the rat and human Ki-ras genes, there are only 33 differences among the nucleotides (94%) comprising the four potential coding exons 1, 2, 3, 4B. In the untranslated ekon 0 revealed a homology of 88% (11 nucleotide fifferences/total 78 nucleotides). Next, to compare the nucleotide sequence of the rat c-Ki-ras gene in normal and regenerating liver, we have partially hepatectomized rat livers (i,e. the surgical removal of 70% of rat livers). Total RNA was isolated from the regenerating livers between 18-36 h after partial hepatectomy, reverse transcribed with AMV reverse transcriptase, and amplified by PCR (RT-PCR). The RT-PCR product was cloned to the pTZ 19 vector and sequenced. The sequencing data showed that there are quantitative differences, but no obvious qualitative differences between mRNA populations of normal and regenerating liver.
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