Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. We have studied a patient with leprechaunism (leprechaun/Minn-1), a genetic syndrome associated with intrauterine growth retardation and extreme insulin resistance. Genomic DNA from the patient was amplified by the polymerase chain reaction catalyzed by Thermus aquaticus (Taq) DNA polymerase, and the amplified DNA was directly sequenced. A nonsense mutation was identified at codon 897 in exon 14 in the paternal allele of the patient's insulin receptor gene. Levels of insulin receptor mRNA are decreased to less than 10% of normal in Epstein-Barr virus-transformed lymphoblasts and cultured skin fibroblasts from this patient. Thus, this nonsense mutation appears to cause a decrease in the levels of insulin receptor mRNA. In addition, we have obtained indirect evidence that the patient's maternal allele of the insulin receptor gene contains a cis-acting dominant mutation that also decreases the level of mRNA, but by a different mechanism. The nucleotide sequence of the entire protein-coding domain and the sequences of the intron-exon boundaries for all 22 exons of the maternal allele were normal. Presumably, the mutation in the maternal allele maps elsewhere in the insulin receptor gene. Thus, we conclude that the patient is a compound heterozygote for two cis-acting dominant mutations in the insulin receptor gene: (i) a nonsense mutation in the paternal allele that reduces the level of insulin receptor mRNA and (ii) an as yet unidentified mutation in the maternal allele that either decreases the rate of transcription or decreases the stability of the mRNA.
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PMID:A nonsense mutation causing decreased levels of insulin receptor mRNA: detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction. 230 May 53

Insulin resistance is an early predictor of development of noninsulin-dependent diabetes mellitus (NIDDM) in Pima Indians, a population with the highest reported prevalence of NIDDM. The insulin receptor plays a central role in mediating insulin action, and previous studies have demonstrated that mutations in the insulin receptor gene may cause insulin resistance. Therefore, we have cloned the insulin receptor cDNA from an insulin-resistant Pima Indian to determine if there is a mutation in the patient's insulin receptor gene. We obtained nine cDNA clones spanning exons 4-10 and 12-22 of the patient's insulin receptor gene. Polymorphisms in the nucleotide sequences for codons 523 (Ala), 1058 (His), and 1062 (Leu) provided useful markers to differentiate the patient's two alleles of the insulin receptor gene. These substitutions were silent, in that they did not alter the predicted amino acid sequence. The sequence of exons 1-3 and 11 was determined directly from genomic DNA that had been amplified using the polymerase chain reaction catalyzed by Taq DNA polymerase. Other investigators have reported defects in insulin binding and insulin receptor tyrosine kinase activity in diabetic Pima Indians. However, we did not detect any mutations in this patient's insulin receptor gene. Thus, these observations are consistent with the interpretation that the defects in insulin receptor function are acquired rather than derived from defects in the primary structure of the receptor.
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PMID:The amino acid sequence of the insulin receptor is normal in an insulin-resistant Pima Indian. 231 37

Two sisters presented with severe insulin resistance and markedly decreased insulin binding to erythrocytes, cultured fibroblasts and transformed lymphocytes. The dose-response curve of insulin-stimulated amino acid uptake in the fibroblasts was shifted to the right. The molecular weight of the insulin receptor on the transformed lymphocytes from the patients was 210,000 and could not be dissociated to alpha- and beta-subunits by dithiothreitol treatment. However, the proreceptor was cleaved by trypsin and this led to the production of alpha-subunit with normal insulin binding. We performed cDNA sequence analysis of the cleavage site of the insulin proreceptor from the patients. The polymerase chain reaction was used to obtain a large amount of cDNA coding for the region including the interconnecting site. A thermostable DNA polymerase, Taq polymerase, successfully produced enough cDNA for the region to be sequenced. The results showed an AGG (Arg) to AGT (Ser) point mutation, resulting in the change of the interconnecting sequence of the two subunits from -Arg-Lys-Arg-Arg- to -Arg-Lys-Arg-Ser-. These results suggest that the tertiary structure change of the cleavage site leads to production of unprocessed insulin proreceptors.
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PMID:Unprocessed insulin proreceptors due to point mutation at the cleavage site. 268 Mar 65

The polymerase chain reaction catalyzed by Taq DNA polymerase has been used for site-specific mutagenesis. The amplification was primed by two oligodeoxyribonucleotides complementary to insulin receptor cDNA. To direct the synthesis of mutant DNA, mismatches were introduced into one of the primers. Six different mutations were constructed by this technique. Of twelve clones whose sequences were determined, ten (83%) had the correct sequence. This technique, which does not require the use of single-stranded DNA templates, provides a simple and efficient approach to site-specific mutagenesis.
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PMID:Use of polymerase chain reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis. 274 78