. AKHMALOKA
Institut Teknologi Bandung

Published : 4 Documents
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Molecular Dynamics Analysis of Thermostable DNA Pol I ITB-1 HERTADI, RUKMAN; NURBAITI, SANTI; AKHMALOKA, .
Microbiology Indonesia Vol 1, No 3 (2007): December 2007
Publisher : Indonesian Society for microbiology

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Abstract

One of the thermostable enzymes, which has been widely used in the biotechnological research, is DNA polymerase. The coding sequence of local DNA Pol I gene from a local thermophilic bacterium, namely DNA Pol I ITB-1, has been cloned, sequenced, and overexpressed. However, study on thermostability of this enzyme is very limited. In the present study, thermostability of the protein was evaluated by thermal unfolding simulation at 300, 400, and 500 K. Our simulation revealed that the secondary and tertiary structures of the protein was not significantly affected by thermal perturbation at 300 K, but they were affected and even gradually unfolded by that perturbation at 400 and 500 K. Evaluation of the root mean square fluctuation (RMSF) of individual residues from the simulation at 400 and 500 K revealed the distribution of the thermostability regions in the protein structure. From the RMSF analysis at 400 K, we found that thermostability of the 3’-5’ exonuclease domain was lower compared to that of the other domains. Where as from the RMSF analysis at 500 K, we found that in each domain of DNA pol I ITB-1 there was a single extraordinary thermostable a-helix which was likely to be the core of each corresponding domain. Thus our simulation provides a thermostability map of DNA Pol I ITB-1. Such information will be very valuable for the next genetic engineering work in determining a mutation target to modify thermostability of this enzyme.
The Effect of Mutation at Thr 295 of Saccharomyces cerevisiae eRF1 on Suppression of Nonsense Codons and eRF1 Structure SUSILOWATI, PRIMA ENDANG; ADITIAWATI, PINGKAN; MADAYANTI, FIDA; AKHMALOKA, .
Microbiology Indonesia Vol 2, No 1 (2008): April 2008
Publisher : Indonesian Society for microbiology

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Abstract

The termination of translation in Saccharomyces cerevisiae is controlled by two interacting polypeptide chain release factors, eRF1, and eRF3. Two regions in eRF1, at position 281-305 and 411-415, were proposed to be involved on the interaction to eRF3. In this study we have constructed and characterized eRF1 mutants at position 295 from threonine to alanine and serine residues resulting in eRF1(T295A) and eRF1(T295S) respectively. The mutations did not affect the viability or temperature sensitivity of the cells. The stop codons readthrough of the mutants were analyzed in vivo using PGK-stop codon-LACZ gene fusion and the results showed that thesuppression of the mutants was increased in all of the codon terminations. The suppression of the UAG codon was the high for both mutants, with a 7-fold increased for eRF1(T295A) and a 9 fold increase for eRF1(T295S). The suppressor activity of eRF1(T295S) was higher compared to that of eRF1(T295A), suggesting that the accuracy of translational termination in eRF1(T295S) was lower than that of eRF1(T295A). Computer modeling analysis using Swiss-Prot and Amber version 9.0 programs revealed that the overall structure of eRF1 mutants has no significant difference with the wild type. However, substitution of threonine to serine on eRF1(T295S) triggered a secondary structure change on the other motif of the C-terminal domain of eRF1. This observation did not occur for on eRF1(T295A). This suggests that the high stop codon suppression on eRF1(T295S) is probably due to the slight modification of the structure of the C terminal motif.
Cell Lysis Method Affects Assessment of Microbial Diversity Based on Ribotyping Analysis YOHANDINI, HENI; MADAYANTI, FIDA; ADITIAWATI, PINGKAN; AKHMALOKA, .
Microbiology Indonesia Vol 2, No 1 (2008): April 2008
Publisher : Indonesian Society for microbiology

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Abstract

The microbial community in Kawah Hujan, Kamojang, West Java, Indonesia, was analyzed using 16S-rRNA-gene-sequencing combining with denaturing-gradient-gel electrophoresis (DGGE) technique. Two different cell lysis methods, enzymatic-based, and physical treatment-based DNA extraction, were used to isolate chromosomal DNA for 16S rDNA gene-fragment amplification. The DGGE profiles showed some differences in banding pattern that were obtained from both cell lysis methods. The DNA sequence analysis of the individual DGGE bands revealed that most of the band sequences obtained by physical treatment were close to 16S rRNA gene fragments from the bacterial domain, while most of band sequences performed by enzymatic method had high homology with 16S rRNA gene fragments from archaeal domain. Further analysis of the sequences from both methods performed by comparisons with the Ribosomal Database Project showed that some of DGGE sequences from Kawah Hujan consisted unique 16S rDNA sequences.
16S Ribosomal RNA-Based Analysis of Thermophilic Bacteria in Gedongsongo Hot Spring AMININ1, AGUSTINA LULUSTYANINGATI NURUL; MADAYANTI, FIDA; ADITIAWATI, PINGKAN; AKHMALOKA, .
Microbiology Indonesia Vol 1, No 1 (2007): April 2007
Publisher : Indonesian Society for microbiology

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Abstract

Denaturing gradient gel electrophoresis was used to identify the bacterial community at the Gedongsongo (WGS-2) hot spring. The bacterial samples were obtained from both culture dependent and independent strategies. Partial 16S rRNA genes were amplified by a set of primers to produce at around 400 bp fragments, including the highly variable V9 region of the 16S rRNA genes. The DGGE profiles showed that there were a few distinct bands, namely G1-G3, and G8-G12, which represent the predominant bacteria in natural habitat and the medium.Further analysis of these bands showed that most of them, except for G7, have a high homology to the 16S rRNA gene sequences of Thermus sp. As for G7, the highest homology was shown to unculturable bacteria. In addition to the distinct bands in DGGE, there were other three thin bands, namely G4, G5, and G6, which possibly represent non dominant microorganisms in the natural habitat, but could grow on GS-A medium. Further analysis of these bands showed that G6 has 80% similarity to the 16S rRNA of Burkholderia sp., while G4 and G5 have a high homology to each other but only contained 10-15% homology to the sequences of 16S rRNA from unculturable microorganisms. The phylogenetic analyses of the last organisms showed that there was branching from Burkholderia. From all the data obtained it was suggested that the WGS-2 hot spring was predominantly occupied by the genus Thermus. In addition, there were a few novel microorganisms found in the hot spring.