PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus

Date

2000-10-01

Author

Ozturk, ZN
Ögel, Zümrüt Begüm

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

198
views

0
downloads

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resulted in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.

Subject Keywords

Biotechnology, Applied Microbiology and Biotechnology, Biochemistry, Bioengineering, Molecular Biology

URI

https://hdl.handle.net/11511/57260

Journal

MOLECULAR BIOTECHNOLOGY

DOI

https://doi.org/10.1385/mb:16:2:109

Collections

Graduate School of Natural and Applied Sciences, Article

Suggestions

OpenMETU
Core

Homologous expression of aspartokinase (ask) gene in Streptomyces clavuligerus and its hom-deleted mutant: Effects on cephamycin C production Özcengiz, Gülay; Ünsaldi, Eser; Taşkin, Bilgin; Liras, Paloma; Piret, Jacqueline (Informa UK Limited, 2010-05-01) Abstract In this study, the effect of homologous multiple copies of the ask gene, which encodes aspartokinase catalyzing the first step of the aspartate pathway, on cephamycin C biosynthesis in S. clavuligerus NRRL 3585 and its hom mutant was investigated. The intracellular pool levels of aspartate pathway amino acids accorded well with the Ask activity levels in TB3585 and AK39. When compared with the control strain carrying vector alone without any gene insert, amplification of the ask gene in the w...
RT-PCR amplification of a Rhizopus oryzae lactate dehydrogenase gene fragment Hakki, EE; Akkaya, Mahinur (Elsevier BV, 2001-02-01) No amino acid or DNA sequence information in sequence databases was found for a fungal lactate dehydrogenase (LDH) isozyme. Highly conserved regions in the lactate dehydrogenase enzymes of all taxonomies are found to be beta alpha beta nucleotide binding and substrate binding sites, also catalysis/active site. The conserved regions were selected as PCR primer target regions. The degenerate primers were designed according to the codon usage, determined by analyzing a number of different genes of Rhizopus spe...
Kinetics of amino acid production by over-producer mutant microorganisms Özilgen, Mustafa (Elsevier BV, 1988-2) Experimental data were modeled based on the literature for the production of nine different amino acids by ten different mutant over-producer microorganisms. Mathematical modeling helped to better understand common aspects of these fermentations, which were not stressed previously by plotting the data only. A Luedeking-Piret type of a model was found to be valid for representing all the amino acid production data. This result indicated the similarity among the amino acid production patterns of all the mutan...
Recombinant therapeutic protease production by Bacillus sp. Korkmaz, Nuriye; Çalık, Pınar; Department of Chemical Engineering (2007) The first aim of this study is the development of extracellular recombinant therapeutic protease streptokinase producing Bacillus sp., and the second aim is to determine fermentation characteristics for streptokinase production. In this context, the signal (pre-) DNA sequence of B.licheniformis (DSM1969) extracellular serine alkaline protease enzyme gene (subC: Acc. No. X03341) was ligated to 5’ end of the streptokinase gene (skc: Acc. No. S46536) by SOE (Gene Splicing by Overlap Extension) method through P...
Overexpression of a serine alkaline protease gene in Bacillus licheniformis and its impact on the metabolic reaction network Çalık, Pınar; Oliver, SG; Ozdamar, TH (Elsevier BV, 2003-05-20) This work reports on cloning of serine alkaline protease (SAP) encoding gene subC to a multi-copy plasmid and its expression in Bacillus licheniformis with the quantitative impact of overexpression of the subC gene on metabolic flux distributions. Bioprocess characteristics of the wild-type and the recombinant B. licheniformis were investigated in a defined simple synthetic medium with glucose as the sole carbon source under well-defined bioreactor-operation conditions. Significant physiological changes wer...

Citation Formats

Z. Ozturk and Z. B. Ögel, “PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus,” MOLECULAR BIOTECHNOLOGY, pp. 109–115, 2000, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/57260.