• 한국신재생에너지학회:학술대회논문집
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• 2005.11a
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• pp.631-633
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• 2005

For biohydrogen production, hydrogenase is a key enzyme. In the present work we performed search of [NiFe]-hydrogenases from hydrogen producing microorganisms using degenerate polymerase chain reaction (PCR) strategy. Degenerate primers were designed from the conserved region of [NiFe]-hydrogenase group I especially on structural genes encoding for catalytic subunit of [NiFe]-hydrogenase from bacteria producing hydrogen. Most of [NiFe]-hydrogenase (group I) are expressed via complex mechanism with aid of auxiliary protein and localized through twin-arginine translocation pathway. [NiFe]-hydrogenase is composed of large and small subunits for catalytic activity. It is known that only small subunit has signal peptide for periplasmic localization and large & small subunitscome together before localization. During this process, large subunit is treated by endopeptidase for maturation. Based on these information we used signal peptide sequence and C-terminal of large subunit by recognized by endopeptidase as templates for degenerate primers. About 2,900 bp of PCR products were successfully amplified using the designed degenerate primers from genomic DNAs of several microorganisms. The amplified PCR products were inserted into T-vector and then sequenced to confirm.

• Journal of Microbiology
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• v.33 no.3
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• pp.252-259
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• 1995

The PCR was employed to detect and characterize the bovine immunodeficiency-like virus (BIV), which is a newly recognized member of the I entivirinae of the retroviruses. Degenerate primers representing the conserved regions in the pol genes of the Lentivirinae, were used to detect proviral DNA obtained from the bovine embryonic spleen cell cultures infected with BIV. The PCR amplified DNA fragment was molecularly cloned and sequenced. The BIV DNA fragment contained a sequence identical to that reported by Garvey et al. (Garvey et al., 1990. Virology, 175, 391-409). With the degenerate primers, peripheral blood mononuclear cells (PBMCs) of sick cattle and cells cultured with BIV were tested to determine genetic variation of BIV pol conserved sequence. We found the sequence heterogeneity within cultures and most variations occurred at the third base of codons that would not lead to amino acid substitutions. Another change was GAG (Glu) to AAG (Lys) within the BIV isolates. Interestingly, the altered sequence is also found in other lentiviruses such as HIV-2, SIV mac, CAEV and EIAV.

• Journal of Microbiology and Biotechnology
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• v.17 no.3
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• pp.481-489
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• 2007

[ $\alpha$ ]-L-Arabinofuranosidases (AFases; EC 3.2.1.55) are exo-type enzymes, which hydrolyze terminal nonreducing arabinose residues from various polysaccharides such as arabinan and arabinoxylan. Genome-wide BLAST search showed that various bacterial strains possess the putative AFase genes with well-conserved motif sequences at the nucleotide and amino acid sequence levels. In this study, two sets of degenerate PCR primers were designed and tested to detect putative AFase genes, based on their three highly conserved amino acid blocks (PGGNFV, GNEMDG; and DEWNVW). Among 20 Bacillus-associated species, 13 species were revealed to have putative AFase genes in their genome and they share over 67% of amino acid identities with each other. Based on the partial sequence obtained from an isolate, an AFase from Geobacillus sp. was cloned and expressed in E. coli. Enzymatic characterization has verified that the resulting enzyme corresponds to a typical AFase. Accordingly, degenerate PCR primers developed in this work can be used for fast, easy, and specific detection and isolation of putative AFase genes from bacterial cells.

• Korean Journal of Microbiology
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• v.42 no.1
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• pp.77-81
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• 2006

Degenerate primers corresponding to the sequences of the N-terminal regions of Mn-peroxidase isozymes were used to isolate the genomic fragments encoding the isozymes of Mn-peroxidase, CVMP1, CVMP2, CVMP3 and CVMP5 from the white-rot fungus Trametes versicolor KN9522. Three isozymes except one gave the expected PCR products (cmp1, cmp2 and cmp5) of about 900 base pairs, respectively. DNA sequence data obtained from each PCR products were used to analyze the BLAST program search on the National Center for Biotechnology Information. cmp1, cmp2 and cmp5 were similar to MPG-I (GenBank accession number Z30668) and PGV-II (GenBank accession number, Z54279) gene T. versicolor PRL572. PCR products of cmp1 and cmp2 showed 77%, 95% base sequence similarities to MPG-I gene and cmp5 showed about 88% similarity to PGV-II gene from T. versicolor PRL572. From this experiment, we could isolate genomic DNA fragments with degenerate primers designed from the N-terminal amino acid sequences of Mn-peroxidase isozyme family.

• Journal of Veterinary Science
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• v.24 no.3
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• pp.40.1-40.6
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• 2023

Analysis of the VP1 gene sequence of the foot and mouth disease virus (FMDV) is critical to understanding viral evolution and disease epidemiology. A standard set of primers have been used for the detection and sequence analysis of the VP1 gene of FMDV directly from suspected clinical samples with limited success. The study validated VP1-specific degenerate primer-based reverse transcription polymerase chain reaction (RT-PCR) for the qualitative detection and sequencing of serotype O FMDV lineages circulating in India. The novel degenerate primer-based RT-PCR amplifying the VP1 gene can circumvent the genetic heterogeneity observed in viruses after cell culture adaptation and facilitate precise viral gene sequence analysis from clinical samples.

• Journal of Microbiology and Biotechnology
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• v.22 no.6
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• pp.729-735
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• 2012

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.

• Microbiology and Biotechnology Letters
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• v.32 no.3
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• pp.282-285
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• 2004

The polyene antibiotics including nystatin, pimaricin, amphotericin and candicidin are a family of most promising antifungal polyketide compounds, typically produced by rare actinomycetes species. The biosynthetic gene clusters for these polyenes have been previously investigated, revealing the presence of highly homologous biosynthetic genes among polyene-producers such as polyketide synthase (PKS) and cytochrome P450 hydroxylase (CYP) genes. Based on amino acid sequence alignment among actinomycetes CYP genes, the highly-conserved regions specific for only polyene CYP genes were identified and chosen for degenerate PCR primers, followed by the PCR-screening with various actinomycetes genomic DNAs. Among tested several polyene non-producing actinomycetes strains, Pseudonorcardia autotrophica strain was selected based on the presence of PCR product with polyene-specific CYP gene primers, and then confirmed to contain a cryptic novel polyene hydroxylase gene in the chromosome. These results suggest that the polyene-specific hydroxylase gene PCR should be an efficient way of screening and isolating potentially-valuable cryptic polyene antibiotic biosynthetic genes from various microorganisms including rare actinomycetes.

• Food Science and Biotechnology
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• v.15 no.6
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• pp.967-974
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• 2006

Cyclomaltodextrinases (CDases), maitogenic amylases, and neopullulanases share highly conserved primary structures and similar characteristics, and are thus classified into the same family. BLAST search has showed that a variety of bacterial strains harbor putative CDase family genes with several well-conserved motif amino acid sequences. In this study, four degenerate polymerase chain reaction (PCR) primer sets were designed for the detection of CDase genes, on the basis of their highly conserved amino acid blocks (WYQIFP, DGWRLD, LGSHDT, and KCMVW). The PCR detection conditions were optimized and the detection specificity of each for the primer sets was tested against the genomic DNAs isolated from 23 different Bacillus-associated species. Consequently, all tested primer sets evidenced successful amplification of specific PCR products in length, which share 55-98% amino acid sequence identity with known and putative CDases. The primers developed herein, therefore, can be applied for the easy and efficient detection and isolation of CDase family genes for the modification of functional food carbohydrates.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 1999.04a
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• pp.50.1-50
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• 1999

• Journal of Plant Biology
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• v.39 no.2
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• pp.85-92
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• 1996

Small GTP-binding proteins are divided into three major group: Ras, Rho and Ypt/Rab. They have the conserved regions designed G1 to G5 that are critical in GDP/GTP exchange, GTP-induced conformational change and GTP hydrolysis. We isolated and characterized genomic DNA or cDNAfragments encoding G1 to G3 domains of small GTP-binding protein Rab and Rho from several plant species using two different PCR-based cloning strategies. Seven rab DNA fragments were isolated from 4 different plants, mung-bean, tobacco, rice and pepper using two degenerate primers corresponding to the GTP-binding domain G1 and G3 in small GTP-binding proteins. The amino acid sequences among these rab DNA fragments and other known small GTP-binding proteins shows that they belong to the Ypt/Rab family. Six rho DNA fragments were isolated from 5 different plants, mung-bean, rice, Arabidopsis, Allium and Gonyaulax using the nested PCR method that involves four degenerate primers corresponding to the GTP-binding domain G1, G3 and G4. The rho DNA fragments cloned show more than 90% homology to each other. Sequence comparison between plant and other known Rho family genes suggests that they are closely related (67 to 82% amino acid identity). Sequence analysis and southern blot analysis of rab and rho in mung-bean suggest than thses genes are encoded by multigene family in mung-bean.