• BMB Reports
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• v.32 no.5
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• pp.502-505
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• 1999

cDNA fragments of ovine liver pyridoxal kinase were amplified by PCR using degenerate oligonucleotide primers derived from partial amino acids sequences of the enzyme. Using PCR products as probes, several overlapping cDNA clones were isolated independently from an ovine liver and a human brain cDNA library. The largest cDNA clone for each was selected for sequence analysis. The ovine liver cDNA encodes a polypeptide of 297 amino acid residues with Mr of 32,925, whereas the human clone is comprised of an open reading frame encoding 312 amino acid residues with Mr of 35,102. The deduced sequence of the human brain enzyme is completely identical to that of human testes cDNA recently reported (Hanna et al., 1997). The ovine enzymes have approximately 77% sequence identity with the human enzyme although the two sequences are completely different in the N-terminus comprising 32 residues. This result suggests that pyridoxal kinase is highly homologous in mammalian species.

• Journal of fish pathology
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• v.16 no.3
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• pp.215-217
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• 2003

This study examined whether the connexin (Cx) is an essential protein during oocyte maturation in the ovary of the goldfish (Carassius auratus). In mature female goldfish ovaries, at late vitellogenic stage, human insulin-like growth factor-I (IGF-I; 20 M) and human chorionic gonadotropin(hCG; 20 IU/㎖) were injected. Twelve hr after the injection, mature female goldfish ovaries were removed and stored at -80C until analysis by RT-PCR. From the goldfish Cx43 cDNA sequence (GenBank accession number AB078505), two degenerate primers were designated. In vivo, 12 hr after the treatment with hCG, goldfish Cx43 mRNA expression level was increased, while the levels of IGF-I was not changed. Goldfish Cx43 mRNA expressed after, but not before the hCG treatment. These results suggest that Cx43 mRNA was judged to be a gene, which was transcribed during oocyte maturation induced by hCG.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.732-736
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• 1999

The superoxide dismutase (SOD) in Lactococcus lactis was measured quantitatively and qualitatively under various culture conditions. The L. lactis SOD was induced by oxidative stress. As the concentration of paraquat to produce superoxide radicals increased, the growth of L. lactis decreased with concomitant increase of SOD activity. The SOD activity was found to be growth-phase dependent: when aerobically grown cells entered to the stationary phase, the activity increased gradually until the late stationary phase. From inhibition studies, L. lactis SOD was found to be insensitive to KCN and $H_2O_2$ which are known to inhibit Cu/ZnSOD and FeSOD, respectively. Moreover, as the concentration of manganese in the medium increased, the activity of SOD also increased. These data strongly suggested that L. lactis possessed a single manganese-containing SOD (MnSOD). Finally, a putative sod gene fragment of 510 bp was identified in L. lactis using a polymerase chain reaction (PCR) with degenerate primers designed from the deduced DNA sequences of known SOD genes.

• Korean Journal of Microbiology
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• v.35 no.1
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• pp.25-27
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• 1999

Degenerate primers corresponding to the sequences of the copper-binding regions in the fungal laccases were used to isolatc laccase gene specific fragment by PCR in Coprinus congregahts. A 144 bp DNA hagrnent was cloned and was identified to have 60-69 % homology with other fungal laccase genes. The predicted amino acid sequcnces showed 68-75% homology with other fungal laccase proteins.

• Journal of Plant Biology
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• v.38 no.4
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• pp.359-364
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• 1995

As the first step to elucidate the relationship between the structure and function of CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) in plants, the partial nucleotide sequence of soybean cytidylyltransferase cDNA was determined using a polymerase chain reaction (PCR). Degenerate oligonucleotide primers were synthesized from the conserved region revealed from the rat and yeast cytidylyltransferase DNA sequences. The catalytic domain region showed 78 and 76% homology with the rat and yeast amino acid sequences, respectivly. The hydropathy profile indicated that the C-terminal non-catalytic portion of the protein was very hydrophilic, and in the region between the catalytic domain and the C-terminal region, there was a large amphipathic $\alpha$-helical domain that was believed to bind the membrane surface in the active formation. There are 7 potential sites for phosphorylation by protein kinase C and 4 potential sites for phosphorylation by Ca2+/calmodulin kinase within the determined sequence.

• Journal of Microbiology
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• v.40 no.2
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• pp.151-155
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• 2002

A Superoxide Dismutase (SOD) gene from the obligate intracellular bacterium Orientia tsutsugamushi has been cloned by using the polymerase chain reaction with degenerate oligonucleotide primers corresponding to conserved regions of known SODs. Nucleotide sequencing revealed that the predicted amino acid sequence was significantly more homologous to known iron-containing SODs (FeSOD) than to manganese-containing SODs (MnSOD). Conserved regions in bacterial FeSOD could also be seen. Isolation of the oriential SOD gene may provide an opportunity to examine its role in the intracellular survival of this bacterium.

• Journal of Microbiology
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• v.35 no.3
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• pp.222-227
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• 1997

DNA fragments homologous to chitin synthase gene were amplified from the genomic DNA of Beauveria brongniartii by PCR using degenerate primers. Cloning and sequencing of the PCR-amplified fragments led to the identification of a gene, designated BbCHSl. Comparison of the deduced amino acid sequence of BbCHSl with those of other Euascomycetes revealed that BbCHSl is a gene for class II chitin synthase. The Blastp search of the deduced amino acid sequence of BbCHSl displayed the highest rate of similarity, 95.8%, with CHS2 of Metarhizium unisopliae. Phylogenetic analysis of the amino acid sequences confirmed the taxonomic and evolutionary position of B. brongniartii, which was previously derived by traditional fungal classification based on morphological features.

• KSBB Journal
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• v.19 no.4
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• pp.308-314
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• 2004

A degenerate set of PCR primers based on two conserved regions (heme binding region and oxygen ligand pocket) were designed and successfully applied to amplify DNA fragments of cytochrome P450 hydroxylase (CYP) genes from a rare actinomycetes, S. benihana. The PCR amplified products were employed as a DNA probe to clone the entire CYP genes from S. benihana genomic library. Five different CYP-positive cosmids were isolated by colony hybridization as well as PCR confirmation. The complete nucleotide sequencing of five different CYP genes revealed that each unique CYP showed a significant amino acid homology to previously-known CYP genes involved in streptomycetes secondary metabolism. In addition, four CYP genes (CYP502, CYP503, CYP504, CYP506) were found to be linked to ferredoxin genes in the chromosome, and the CYP503 gene showed the high degree of amino acid similarity to the previously well-characterized CYP105 family in streptomycetes.

• Applied Biological Chemistry
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• v.44 no.3
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• pp.153-161
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• 2001

In order to isolate genes involved in ecdysteroids biosynthesis in plants, total RNA was isolated from Achyranthes japonica Nakai, and RT-PCR was performed using degenerate primers selected based on the results of multi-alignment of four cytochrome P450 genes from plants and a putative ecdysone 20-hydroxylase gene from an insect. Fourteen partial cDNA clones showing unique base sequences were obtained, out of which six showed homologies at the levels of nucleotide and amino acid sequences to the other cytochrome P450 genes known to be involved in the ecdysteroid biosynthesis. Of the six clones, four showed relatively high homologies to a putative ecdysone 20-hydroxylase gene isolated from an insect.

• Horticultural Science & Technology
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• v.33 no.2
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• pp.242-250
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• 2015

Insertional mutagenesis induced by T-DNA or transposon tagging offers possibilities for analysis of gene function. However, its potential remains limited unless good methods for detecting the target locus are developed. We describe a PCR technique for efficient identification of DNA sequences adjacent to the inserted T-DNA in a higher plant, Chinese cabbage (Brassica rapa ssp. pekinensis). This strategy, which we named variable argument thermal asymmetric interlaced PCR (VA-TAIL PCR), was designed by modifying a single-step annealing-extension PCR by including a touch-up PCR protocol and using long gene-specific primers. Amplification efficiency of this PCR program was significantly increased by employing an autosegment extension method and linked sequence strategy in nested long gene-specific primers. For this technique, arbitrary degenerate (AD) primers specific to B. rapa were designed by analyzing the Integr8 proteome database. These primers showed higher accuracy and utility in the identification of flanking DNA sequences from individual transgenic Chinese cabbages in a large T-DNA inserted population. The VA-TAIL PCR method described in this study allows the identification of DNA regions flanking known DNA fragments. This method has potential biotechnological applications, being highly suitable for identification of target genomic loci in insertional mutagenesis screens.