Hadi Sutedjo
Department of Chemistry, Faculty of Matematics and Natural Sciences Institut Teknologi Bandung

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Molecular Genetic Analysis of sup45 Paromomycin Hypersensitive Mutants In Saccharomyces cerevisiae Rukman Hertadi; Hadi Sutedjo; Akhmaloka Akhmaloka
Jurnal Matematika & Sains Vol 6, No 2 (2001)
Publisher : Institut Teknologi Bandung

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Translation termination in Saccharomyces cerevisiae is mediated by eRF-1 and eRF-3 protein encoded by SUP45 and SUP35 gene respectively. To probe more detail concerning the mechanism of translation termination in this organisms, the structure-function of the eRF-1 protein was studied by analyzing of sup45 mutants. In this study, we used three paramomycin hyper sensitive mutants, namely sup45-23, sup45-24 and sup45-38. Phenotypic characterization and quantitation of allosuppresor level showed that the mutants were allele specific mutants. Cloning and sequencing of the sup45 genes from these mutants showed that the sup45-23 exhibited Met48ATG → Ile48Ala and Gly431GGT → Gly431GGA mutation. While sup45-24 and sup45-38 exhibited single silent mutation, Gly431GGT → Gly431GGA . The structure-function analysis of sup45 gene of these mutant suggested that phenotypic mutants were not only due to the alteration of amino acid of eRF-1 protein in the cells.
Jurnal Kimia Terapan Indonesia Vol 6, No 1-2 (1996)
Publisher : Research Center for Chemistry - LIPI

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (4081.855 KB) | DOI: 10.14203/jkti.v6i1-2.236


Penicillin acylase plays an important role in the catalysis of benzylpenicillin hydrolytic reaction, prodeucing 6-aminopenicilanic acid (6- APA), a precursorfor the formation of penicillinderivatives. Cloning of the penicillin acylase gene of Escherichia coli B130 chromosomal DNA on pHC79 cosmid vector to increase the enzyme activity has been investigated. The cloning was cooducted with several steps, including isolation of the chromosomal DNA. digestion by restriction enzyme, ligation by T4-DNA ligase, transformation of the recombinant DNA, and selection of the transformans. Microbial assay utilizing Serratia marcescens was carried out for screening the penicillin acylase colony, whereas the determination of the enzyme activity was examined based on Kornfeld method. From 2070 colonies screened by S. marcescens, only 4 positive colonies were obtained. The enzyme activity of these colonies was 4-6 fold higher than the penicillin acylase activity from E. coli B 130.