• Microbiology and Biotechnology Letters
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• v.38 no.2
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• pp.158-162
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• 2010

The gene encoding N-terminally truncated Tod polymerase ($\Delta$Tod polymerase) from Thermus thermophilus HJ6 was expressed in Escherichia coli under the control of the lambda pR and pL tandem promoters on the expression vector pJLA503. The N-terminal domain (250 amino acids) of Tod polymerase was removed without significant effect on enzyme activity and stability, while no 5'$\rightarrow$3' exonuclease activity was detected. The $\Delta$Tod polymerase was verified to possess very efficient reverse transcriptase (RT) activity in the presence of $MgCl_2$. The cDNA can also be amplified in the polymerase chain reaction (PCR) with this mutant enzyme. The $\Delta$Tod polymerase was exhibited higher activity than the Taq polymerase in a one-step RT-PCR.

• Journal of Life Science
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• v.6 no.3
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• pp.185-192
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• 1996

Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, is required for T7 DNA replication, recombination, and repair. T7 gene 2.5 protein has two distinctive domains, DNA binding and C-terminal domain, directly involved in protein-protein interaction. Gene 2.5 protein participates in the DNA replication of Bacteriophage T7, which makes this protein essential for the T7 growth and DNA replication. What gene 2.5 protein makes important at T7 growth and DNA replication is its binding affinity to single-stranded DNA and the protein-protein important at T7 DNA replication proteins which are essential for the T7 DNA synthesis. We have constructed pGST2.5(WT) encoding the wild-type gene 2.5 protein and pGST2.5$\Delta $21C lacking C-terminal 21 amino acid residues. The purified GST-fusion proteins, GST2.5(WT) and GST2.5(WT)$\Delta$21C, were used for whether the carboxyl-terminal domain participates in the protein-protein interactions or not. GST2.5(WT) and GST2.5$\Delta$21C showed the difference in the protein-protein interaction. GST2.5(WT) interacted with T7 DNA polymerase and gene 4 protein, but GST2.5$\Delta$21C did not interact with either protein. Secondly, GST2.5(WT) interacts with gene 4 proteins (helicase/primase) but not GST2.5$\Delta$21C. these results proved the involvement of the carboxyl-terminal domain of gene 2.5 protein in the protein-protein interaction. We clearly conclude that carboxy-terminal domain of gene 2.5 protein is firmly involved in protein-protein interactions in T7 replication proteins.

• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.304-312
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• 2013

The thermotolerant methylotrophic yeast Hansenula polymorpha is attracting interest as a potential strain for the production of recombinant proteins and biofuels. However, only limited numbers of genome engineering tools are currently available for H. polymorpha. In the present study, we identified the HpPOL3 gene encoding the catalytic subunit of DNA polymerase ${\delta}$ of H. polymorpha and mutated the sequence encoding conserved amino acid residues that are important for its proofreading 3'${\rightarrow}$5' exonuclease activity. The resulting $HpPOL3^*$ gene encoding the error-prone proofreading-deficient DNA polymerase ${\delta}$ was cloned under a methanol oxidase promoter to construct the mutator plasmid pHIF8, which also contains additional elements for site-specific chromosomal integration, selection, and excision. In a H. polymorpha mutator strain chromosomally integrated with pHIF8, a $URA3^-$ mutant resistant to 5-fluoroorotic acid was generated at a 50-fold higher frequency than in the wild-type strain, due to the dominant negative expression of $HpPOL3^*$. Moreover, after obtaining the desired mutant, the mutator allele was readily removed from the chromosome by homologous recombination to avoid the uncontrolled accumulation of additional mutations. Our mutator system, which depends on the accumulation of random mutations that are incorporated during DNA replication, will be useful to generate strains with mutant phenotypes, especially those related to unknown or multiple genes on the chromosome.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.18
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• pp.8177-8186
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• 2016

The present study was aimed at determining the effects of alkylating and oxidative stress inducing agents on a newly identified variant of DNA polymerase beta ($pol{\beta}{\Delta}_{208-304}$) specific for ovarian cancer. $Pol{\beta}{\Delta}_{208-304}$ has a deletion of exons 11-13 which lie in the catalytic part of enzyme. We compared the effect of these chemicals on HeLa cells and HeLa cells stably transfected with this variant cloned into in pcDNAI/neo vector by MTT, colony forming and apoptosis assays. $Pol{\beta}{\Delta}_{208-304}$ cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards $H_2O_2$ and UV when compared with HeLa cells alone. It has been shown that cell death in $Pol{\beta}{\Delta}_{208-304}$ transfected HeLa cells is mediated by the caspase 9 cascade. Exon 11 has nucleotidyl selection activity, while exons 12 and 13 have dNTP selection activity. Hence deletion of this part may affect polymerizing activity although single strand binding and double strand binding activity may remain same. The lack of this part may adversely affect catalytic activity of DNA polymerase beta so that the variant may act as a dominant negative mutant. This would represent clinical significance if translated into a clinical setting because resistance to radiation or chemotherapy during the relapse of the disease could be potentially overcome by this approach.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.28 no.2
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• pp.209-218
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• 1995

Bacteriophage T7 gene 2.5 protein, a single-stranded DNA binding protein, has been implicated in T7 DNA replication, recombination, and repair. Purified gene 2.5 protein has been shown to interact with the phage encoded gene 5 protein (DNA polymerase) and gene 4 proteins (helicase and primase) and stimulates their activities. Genetic analysis of T7 phage defective in gene 2.5 shows that the gene 2.5 protein is essential for T7 DNA replication and growth. T7 phage that contain null mutants of gene 2.5 were constructed by homologous recombination. These mutant phage $(T7\Delta2.5)$ cannot grow in Escherichia coli. After infection of E. coli with $T7\Delta2.5$, host DNA synthesis is shut off, and $T7\Delta2.5$ DNA synthesis is reduced to less than $1\%$ of wild-type phage DNA synthesis (Kim and Richardson, 1993, Proc. Natl. Aca. Sci. USA, 90, 10173-10177). A truncated gene 2.5 protein $(GP2.5-\Delta21C)$ deleted the 21 carboxyl terminal amino acids was constructed by in vitro mutagenesis. $GP2.5-\Delta21C$ cannot substitute for wild-type gene 2.5 protein in vivo; the phage are not viable and exhibit less than $1\%$ of the DNA synthesis observed in wild-type phage-infected cells. $GP2.5-\Delta21C$ has been purified to apparent homogeneity from cells overexpressing its cloned gene. Purified $GP2.5-\Delta21C$ does not physically into「act with T1 gene 4 protein as measured by affinity chromatography and immunoblot analysis. The mutant protein cannot stimulate T7 gene 4 protein activity on RNA-primed DNA synthesis and primer synthesis. These results suggest that C-terminal domain of gene 2.5 protein is essential for protein-protein interactions.

• Journal of Life Science
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• v.19 no.3
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• pp.305-310
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• 2009

Plasmid pGKV21 contains a 229-nucleotide (nt) single-strand DNA initiation (ssi) signal. Using asymmetric PCR, we prepared a small single-stranded (ss) DNA fragment of the ssi signal and, using the 229-nt ssDNA fragment, determined the requirements of RNA polymerase for priming and DNA-protein interaction. The ssi fragment prepared was able to generate primer RNAs with almost the same efficiency as the $M13{\Delta}lac182/229$ phage DNA. However, the cssi (complementary strand of the ssi signal) fragment could not synthesize primer RNAs. This result suggests that the 229-nt ssi signal functions in a strand specific manner. Gel retardation and DNase I footprinting demonstrated that the synthesized ssi fragment could interact with both E. coli RNA polymerase and SSB protein to synthesize primer RNA. In Escherichia coli [pWVAp], an addition of rifampicin resulted in an accumulation of ssDNA, indicating that the host-encoded RNA polymerase is involved in the conversion of ssDNA to double-stranded plasmid DNA.

• Korean Journal of Plant Tissue Culture
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• v.24 no.1
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• pp.63-69
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• 1997

Erythropoietin (EPO) is a glycoprotein that mediates the growth and differentiation of erythroid progenitors. In order to produce recombinant human erythropoietin in tobacco plant, the EPO genomic DNA (5.4 kb) was cloned into plant expression vectors, pBI$\Delta$GUS121, pBD$\Delta$GUS121 and pPEV-1, and introduced in Nicotiana tabacum (var. Xanthi) via Agrobacterium tumefaciens-mediated transformation. After selection on MS media containing kanamycin (Km), 10 Km-resistant plants were obtained per each construct. The correct integration of EPO genomic DNA in the genome of transgenic plant was confirmed by polymerase chain reaction (PCR). Northern blot showed that transcripts of 1.8 kb length were produced in leaves of the plants, but there was no difference of mRNA amount according to promoter number and 5'-untranslated sequence (UTS). The proteins obtained from leaves of transgenic plants were immunologically detected by Western blot using rabbit anti-human EPO polyclonal antibody. The expressed protein appeared as smaller band of apparent mass of 30 kDa as compared to the EPO protein from human urine (37 kDa), suggesting that the modification (glycosylation) system in tobacco plant might be different from that of mammalian cells.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.1
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• pp.7-12
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• 2010

The full-length cDNA of the porcine peroxisomal ${\Delta}^3$,${\Delta}^2$-enoyl-CoA isomerase (PECI) gene encodes a monofunctional peroxisomal ${\Delta}^3$,${\Delta}^2$-enoyl-CoA isomerase. Cloning and sequencing of the porcine PECI cDNA revealed the presence of an 1185-base pair open reading frame predicted to encode a 394-amino acid protein by the 5'rapid amplification of cDNA ends (5'RACE) and EST sequences. The porcine PECI gene was expressed in seven tissues (heart, liver, spleen, lung, kidney, skeletal muscle, fat) which was revealed by reverse transcriptase-polymerase chain reaction (RT-PCR). The porcine PECI was mapped to SSC71/2 p11-13 using the somatic cell hybrid panel (SCHP) and the radiation hybrid panel (RH) (LOD score 12.84). The data showed that PECI was closely linked to marker S0383. A C/T single nucleotide polymorphism in PECI exon 10 (3'UTR) was detected as a PvuII PCR-RFLP. Association analysis in our experimental pig population showed that different genotypes of PECI gene were significantly associated with the Average Backfat thickness (ABF) (p<0.05) and Buttock backfat thickness (p<0.01).

• Journal of Life Science
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• v.29 no.8
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• pp.916-921
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• 2019

In this study, we constructed a biological system that exhibited thermotolerance, ethanol tolerance, and increased ethanol productivity using a random mutagenesis method. We attempted to isolate a thermotolerant mutant using proofreading-deficient DNA polymerase ${\delta}$ and ${\varepsilon}$ encoded by the pol3 and pol2 genes, respectively, in Saccharomyces cerevisiae. To obtain mutants that could grow at high temperatures ($38^{\circ}C$ and $40^{\circ}C$), random mutagenesis of AMY410 (pol2-4) and AMY126 (pol3-01) strains was induced. The parental strains (AMY410 and AMY126) grew poorly at temperatures higher than $38^{\circ}C$. By stepwise elevation of the incubation temperature, AMY410-Ht (heat tolerance) and AMY126-Ht strains that proliferated at $40^{\circ}C$ were obtained. These strains were further incubated in medium containing 6% and 8% ethanol and then AMY410-HEt (heat and ethanol tolerance) and AMY126-HEt strain with ethanol tolerance at an 8% ethanol concentration was obtained. The AMY126-HEt strain grew even at an ethanol concentration of 10%. Furthermore, following the addition of high concentrations of glucose (5% and 10%), an AMY126-HEt3 strain with increased ethanol productivity was isolated. This strain produced 24.7 g/l of ethanol (95% theoretical conversion yield) from 50 g/l of glucose. The findings demonstrate that a new biological system (yeast strain) showing various phenotypes can be easily and efficiently bred by random mutagenesis of a proofreading- deficient mutant.

• BMB Reports
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• v.33 no.2
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• pp.155-161
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• 2000

The saccharomyces cerevisiae Rad27, a structure-specific endonuclease for the okazaski fragment maturation has been known to interact genetically and biochemically with Dna2, an essential enzyme for DNA replication. In an attempt to define the significance of the interaction between the two enzymes, we expressed and purified both Dna2 and Rad27 proteins. In this report, Rad27 could not form a complex with Dna2 in the three different analyses. The analyses included glycerol gradient sedimentation, protein-column chromatography, and coinfection of baculoviruses followed by affinity purification. This is in striking contrast to the previous results that used crude extracts. These results suggest that the interaction between the two proteins is not sufficiently stable or indirect, and thus requires an additional protein(s) in order for Rad27 and Dna2 to form a stable physical complex. This result is consistent with our genetic findings that Schizosaccharomyces pombe Dna2 is capable of interacting with several proteins that include two subunits of polymerase $\delta$, DNA ligase I, as well as Fen-1. In addition, we found that the N-terminal modification of Rad27 abolished its enzymatic activity. Thus, as suspected, we found that on the basis of the structure determination, N-terminal methionine indeed plays an important role in the nucleolytic cleavage reaction.