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猪胃运钴胺素编码cDNA的分离与鉴定

Isolation and characterization of a cDNA encoding porcine gastric haptocorrin.

作者信息

Hewitt J E, Seetharam B, Leykam J, Alpers D H

机构信息

Department of Medicine, Washington University School of Medicine, St Louis, MO 63110.

出版信息

Eur J Biochem. 1990 Apr 20;189(1):125-30. doi: 10.1111/j.1432-1033.1990.tb15468.x.

Abstract

A cDNA encoding the gastric haptocorrin was isolated from a porcine gastric mucosal lambda gt11 cDNA library using oligonucleotide probes. The 1.4-kb cDNA contains a 1.25-kb open reading frame and 178 nucleotides of 3' noncoding region. Although no initiator methionine is present, primer extension analysis indicated that the transcription initiation site is only 100 bp upstream of the 5' end of this clone. Northern blot analysis showed that a single mRNA species of 1.6 kb exists in hog gastric mucosa. There was no cross-hybridization between this cDNA and the mRNA for haptocorrin in rat submaxillary gland or gastric RNA by Northern blot analysis. This lack of cross-reactivity was also seen on Southern blots, where cow, sheep and dog but neither rat nor mouse genomic DNAs showed cross-hybridizing bands. Comparison of the deduced amino acid sequence of this cDNA with that previously reported for rat intrinsic factor [Dieckgraefe, B.K. et al. (1988) Proc. Natl Acad. Sci. USA 85, 46-50] showed considerable similarity, suggesting that these cobalamin-binding proteins may have a common evolutionary origin.

摘要

利用寡核苷酸探针从猪胃黏膜λgt11 cDNA文库中分离出编码胃运钴胺蛋白的cDNA。该1.4kb的cDNA包含一个1.25kb的开放阅读框和178个核苷酸的3'非编码区。虽然不存在起始甲硫氨酸,但引物延伸分析表明转录起始位点仅位于该克隆5'端上游100bp处。Northern印迹分析显示,猪胃黏膜中存在一种1.6kb的单一mRNA。通过Northern印迹分析,该cDNA与大鼠颌下腺或胃RNA中的运钴胺蛋白mRNA之间没有交叉杂交。在Southern印迹上也观察到这种缺乏交叉反应性的情况,其中牛、羊和狗的基因组DNA显示出交叉杂交带,而大鼠和小鼠的基因组DNA则没有。将该cDNA推导的氨基酸序列与先前报道的大鼠内因子的氨基酸序列[Dieckgraefe, B.K.等人(1988年)《美国国家科学院院刊》85, 46 - 50]进行比较,显示出相当大的相似性,表明这些钴胺素结合蛋白可能有共同的进化起源。

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