Our long term goal of this proposal is to elucidate the insulin function at molecular level using recently developed biochemical techniques such as microsequencing and molecular cloning. Recent rapid advances in genetic engineering technology have had a dramatic impact on biomedical research in many areas related to diabetes. These advances have resulted in providing potentially unlimited supplies of insulin and also chemical probes for the analysis of DNA and RNA structure and of gene function and expression which are useful for a better understanding of the production of insulin in Beta cells. The insulin receptor is thought to play an important role in insulin action. Abnormality in the receptor number and/or structure have been observed in some forms of diabetes. The insulin receptor is a large glycoprotein with Mr=300,000-350,000 which is composed of two Mr=125,000(Alpha) and two Mr=90,000(Beta) subunits and is only available in very small quantities. Thus, it has not been possible to purify enough amount of receptor protein to further characterize it. Therefore, application of newly developed genetic engineering technology to the studies on insulin receptor would be highly desirable since it could not only reveal the complete DNA and amino acid sequence of the receptor but also provide us new strategies for more detailed understanding of the mechanism of insulln action and cause of diabetes. 1) Our approach for determining the complete amino acid sequence of the receptor is, therefore, i) to determine the partial amino acid sequence of the purified insulin receptor, ii) to clone cDNA coded for the receptor using synthetic oligonucleotide probes whose sequence will be predicted from the partial amino acid sequence of the receptor and iii) to deduce the total amino acid sequence of the receptor from the DNA sequence of the cloned cDNA. 2) Once we isolate the clones carrying insulin receptor cDNA sequence, we would be able to study a variety of projects including structure and organization of insulin receptor gene, genetic basis of diabetes, and mechanism of insulin receptor-gene expression. Furthermore, it will be possible to mutate the receptor gene and examine the function of the receptor. These studies would reveal new sights towards understanding diabetes and eventually curing the disease or preventing its complications.
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