Inverse polymerase chain reaction mediated chromosome walking within the human glutamic acid decarboxylase gene.

Using inverse polymerase chain reaction (PCR), we have cloned partial intronic sequences from human glutamic acid decarboxylase (GAD) gene. A small ]53 bp core region was selected from the GAD cDNA sequence to design outward primers corresponding to its 3' and 5' ends. EcoRI digested human DNA which had been circularized by self-ligation and then linearized with SacIl was used as a substrate to can.y out PCR. This gave a 900 bp long product which was cloned into pUC]9. The sequence analysis of this fragment revealed the presence of introns in the region flanking the selected core DNA. In this work we used this technique to walk into the upsteam region of the GAD gene using sequence information from its cloned cDNA.

Analysis of a genetically unstable region in Streptomyces lividans 66–TK64.

Genetic and molecular analyses of an unstable region encompassing the gene loci cml arg and a 5.7 kb amplifiable unit of DNA were done. Spontaneous mutants from Cm1R→CmlS and the revertants from CmlS→CmlR were analysed for mutations at arg locus and amplification of amplifiable unit of DNA. Twenty-one revertants were analysed. Two of these had large-scale amplification and one of these was also Arg–. Nine of the revertants which were Arg had low-level or intermediate-level amplification of the 5.7 kb DNA sequence but no deletions of the flanking sequences were detected. Five of the CmIR' revertants, which were also Arg, had lost one of the two copies from the doublet of amplifiable unit of DNA. The remaining five revertants did not show any other change. The amplifiable unit of DNA, therefore, not only undergoes amplification but can also suffer specific deletion of one copy. Thus, this region as a whole is characterized by instability and the events appear to take place at more than one locus concomitantly with a high frequency.