• 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.

• Journal of Life Science
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• v.25 no.8
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• pp.903-909
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• 2015

Loop-mediated isothermal amplification (LAMP), a PCR-based diagnostic method, is based on autocycling strand displacement DNA synthesis in the presence of exonuclease-negative Bst DNA polymerase under isothermal conditions. With the help of four specific primers that recognize six different sequences of a target DNA, LAMP has high specificity in pathogenic identification in a short time. Hence, in the present study, LAMP is used as a diagnostic tool in the identification of the most dreadful aquatic pathogenic species, Vibrio alginolyticus, and to develop species-specific LAMP primers and optimization of LAMP reaction conditions such as annealing temperature, elongation time, and other PCR chemical concentrations, including MgSO₄, dNTPs, Betaine, and Bst polymerase. The optimized LAMP primers were also checked for specificity with other Vibrio species, which showed that the designed primers were very specific to V. alginolyticus After the first introduction of a species name like this one, the first part (“Vibrio” in this case) should be abbreviated to only the first letter.only. These are usually the most harmful pathogens of the Vibrio species that appear in shrimp and crabs. The results also revealed that the LAMP assay could be 10-fold more sensitive than conventional PCR in detecting V. alginolyticus. This could be the first report on using a rapid and highly sensitive technique, the LAMP assay, in the effective diagnosis of the pathogenic bacteria V. alginolyticus, which could help in the early detection of diseases, particularly in aquaculture.

• Korean Journal of Plant Tissue Culture
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• v.25 no.6
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• pp.453-475
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• 1998

During the 100 years since the initial discovery of meiotic phenomenon many brilliant aspects have been elucidated, but further researches based on light microscopy alone as an experimental tool have been found to have some limits and shortcomings. By the use of electron microscopy and armed with the advanced knowledges on modern genetics and biochemistry it has been possible to applu molecular technology in gaining information on the detailed aspects of meiosis. As synapsis takes place, a three-layered proteinous structure called the synatonemal complex starts to form in the space between the homologous chromosomes. To be more precise, it begins to form along the paired chromosomes early in the prophase I of meiotic division. The mechanism that leads to precise point-by-point pairing between homologous chromocomes division. The mechamism that leads to precise point-by-point pairing between homologous chromosomes remains to be ascertained. Several items of information, however, suggest that chromsome alignment leading to synapsis may be mediated somehow by the nuclear membrane. Pachytene bivalents in eukaryotes are firmly attached to the inner niclear membrane at both termini. This attached begins with unpaired leptotene chromosomes that already have developed a lateral element. Once attached, the loptotene chromosomes begin to synapse. A number of different models have been proposed to account for genetic recombination via exchange between DNA strands following their breakage and subsequent reunion in new arrangement. One of the models accounting for molecular recombination leading to chromatid exchange and chiasma formation was first proposed in 1964 by Holliday, and 30 years later still a modified version of his model is favored. Nicks are made by endomuclease at corresponding sites on one strant of each DNA duplex in nonsister chromatid of a bivalent during prophase 1 of meiosis. The nicked strands loop-out and two strands reassociate into an exchanged arrangement, which is sealed by ligase. The remaining intact strand of each duplex is nicked at a site opposite the cross-over, and the exposed ends are digested by exonuclease action. Considerable progress has been made in recent years in the effort to define the molecular and organization features of the centromere region in the yeast chromosome. Centromere core region of the DNA duplex is flanked by 15 densely packed nucleosomes on ons side and by 3 packed nucleosomes on the other side, that is, 2000 bp on one side and 400 400 bp in the other side. All the telomeres of a given species share a common DNA sequence. Two ends of each chromosome are virtually identical. At the end of each chromosome there exist two kinds of DNA sequence" simple telpmeric sequences and telpmere-associated sequencies. Various studies of telomere replication, function, and behabior are now in progress, all greatly aided by molecular methods. During nuclear division in mitosis as well as in meiosis, the nucleili disappear by the time of metaphase and reappear during nuclear reorganizations in telophase. When telophase begins, small nucleoli form at the NOR of each nucleolar-organizing chromosome, enlarge, and fuse to form one or more large nucleoli. Nucleolus is a special structure attached top a specific nucleolar-organizing region located at a specific site of a particular chromosome. The nucleolus is a vertical factory for the synthesis of rRNAs and the assenbly of ribosome subunit precursors.sors.

• Journal of fish pathology
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• v.6 no.2
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• pp.93-101
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• 1993

The RNAI mutation of Saccharomyces cerevisia is a recessive and temperature sensitive lethal mutation which interferes with the production of mRNA, rRNA, and tRNA. However, the precise role of RNAI gene have not been revealed until yet. We have cloned rna1-1 mutant gene from rna1-1 mutant yeast strain(R49 ; trpl, ura3-52, rna1-1). The 3.4kb BglII fragment of wild type RNAI clone(81-2-6) contains whole RNAI gene. The genomic southern blotting with BglII digested R49 genomic DNA as a probe shows the unique and identical band with wild type 3.4kb BglII fragment. Therefore, We prepared partial BglII genomic library(3~4kb BglII fragments) into BamH I site of pUC19. The rna 1-1 mutant clone was screened with Digoxigenin(DIG)-lableled probe by high density colony hybridization. The 5'-flanking region of rna1-1 gene was sequenced by dideoxy chain termination method. The 5'-flanking sequence of RNAI gene contains three TATA-like sequence ; TAATA, TATA and TTTTAA at position of -67, -45, and -36 from first ATG codon respectively. The 5'-flanking region of wild type RNA I gene from ATG codon to -103nt was deleted with Bal31 exonuclease digestion, generating $pUC{\Delta}$/RNA I. After constructing $pYEP{\Delta}RNA$ I (consists of -103nt deleting RNA I gene, URA3 gene, $2{\mu}m$ rep. origin), pYEPrna1-1(consists of Xba I fragment of pUCrna1-1. URA3 gene, $2{\mu}m$ rep. origin), and pYEPRNAI. each plasmid was transformed into host strain(trpl, ura3-52, rna1-1) by electroporation, respectively. Yeast transformant carrying $pYEP{\Delta}RNA$ I did not complement the thermal sensitivity of rna1-1 gene. It means that TATA-like sequences in 5'-flanking region is not TATA sequence for transcribing RNAI gene and there may be other essential sequence in upstream region for the transcription of RNAI gene.

• Tuberculosis and Respiratory Diseases
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• v.58 no.5
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• pp.490-497
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• 2005

Background : The paraoxonase enzyme plays a significant role in the detoxification of various organophosphorous compounds in mammals, and paraoxonase (PON) 1 is one of the endogenous free-radical scavenging systems in the human body. In this study, we investigated the interaction between cigarette smoking and the genetic polymorphism of PON1 with lung cancer in Korean males. Methods : Three hundred thirty five patients with lung cancer and an equal number of age-matched controls were enrolled in this study. Every subject was asked to complete a questionnaire concerning their smoking habits and alcohol drinking habits. A 5' exonuclease assay (TaqMan) was used to genotype the PON1 Q192R polymorphism. The effects of smoking habits and drinking habits, the PON1 Q192R polymorphism and their interactions were statistically analyzed. Results : Cigarette smoking and the Q/Q genotype of PON1 were significant risk factors for lung cancer. Individuals carrying the Q/Q genotype of PON1 were at a higher risk for lung cancer as compared with those individuals carrying the Q/R or R/R genotype (odds ratio, 2.84; 95% confidence interval, 1.69 - 4.79). When the groups were further stratified by the smoking status, the Q/Q PON1 was associated with lung cancer among the current or ex-smokers (odds ratio, 2.56; 95% confidence interval, 1.52 - 4.31). Current smokers or ex-smokers who had the Q/Q genotype showed an elevated risk for lung cancer (odds ratio: 15.50, 95% confidence interval: 6.76 - 35.54) as compared with the group of subjects who never smoked, and had the Q/R or R/R genotype. The odds ratios (95% confidence interval) of smokers with the PON1 Q/Q type compared to the nonsmokers with the PON1 Q/R or R/R type were 53.77 (6.55 - 441.14) for squamous cell carcinoma, 6.25 (1.38 - 28.32) for adenocarcinoma, and 59.94 (4.66 - 770.39) for small cell carcinoma, and these results were statistically significant. Conclusion : These results suggest that cigarette smoking and the PON1 Q/Q genotype are risk factors for lung cancer. The combination of cigarette smoking and the PON1 Q/Q genotype significantly increased the lung cancer risk irrespective of the histologic type of cancer.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 1995.06b
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• pp.77-96
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• 1995

To obtain basic informations for the improvement of seed potato production in Korea, some etiological properties of potato virus Y(PVY) distributed in the major seed potato production area(Daekwanryeong) were characterized, and the nucleotide and amino acid sequences of the coat protein gene of the PVY strains isolated were analyzed. PVY strains in Daekwonryeong, an alpine area, were identified to be two strains, PVYo and PVYN by symptoms of indicator plants, and their distribution in potato fields was similar. Major symptom on potato varieties by PVY was grouped as either mosaic alone or mosaic accompanied with veinal necrosis in the lower leaves. The symptom occurrence of the two symptoms was similar with Irish Cobbler, but Superior showed a higher rate of mosaic symptom than the other. The PVY strain which was isolated from potato cv. Superior showing typical mosaic symptoms produced symptoms of PVY-O on the indicator plants of Chenopodium amaranticolor, Nicotiana tabacum cv. Xanthi nc and Physalis floridana, but no symptom o Capsicum annum cv. Ace. Moreover, results from the enzyme-linked immunosorbent assay with monoclonal and polyclonal antibodies showed that the isolated PVY reacts strongly with PYV-O antibodies but does not react specifically with PVY-T antibodies. The purified virus particles were flexious with a size of 730$\times$11nm. On the basis of the above characteristics, the strain was identified to be a PVY-O and named as of PVY-K strain. The flight of vector aphids was observed in late May, however, the first occurrence of infected plants was in mid June with the bait plants surrounded with PVY-infected potato plants and early July with the bait plants surrounded with PVY-free potato plants. PVY infection rates by counting symptoms on bait plants (White Burley) were 1.1% with the field surrounded with PVY-free potato plants and 13.7% the fields surrounded with PVY-infected potato plants, showing the effect of infection pressure. The propagated PVY-K strain on tobacco(N. sylvestris) was purified, and the RNA of the virus was extracted by the method of phenol extraction. The size of PVY-K RNA was measured to be 9, 500 nucleotides on agarose gel electrophoresis. The double-stranded cDNAs of PVY-K coat protein(CP) gene derived by the method of polymerase chain reaction were transformed into the competent cells of E. coli JM 109, and 2 clones(pYK6 and pYK17) among 11 clones were confirmed to contain the full-length cDNA. Purified plasmids from pYK17 were cut with Sph I and Xba I were deleted with exonuclease III and were used for sequencing analysis. The PVY-K CP gene was comprised of 801 nucleotides when counted from the clevage site of CAG(Gln)-GCA(Ala) to the stop codon of TGA and encoded 267 amino acids. The molecular weight of the encoded polypeptides was calculated to be 34, 630 daltons. The base composition of the CP gene was 33.3% of adenine, 25.2% of guanine, 20.1% of cytosine and 21.4% of uracil. The polypeptide encoded by PVY-K CP gene was comprised of 22 alanines, 20 threonines, 19 glutamic acids and 18 glycines in order. The homology of nucleotide sequence of PVY-K CP gene with those of PVY-O(Japan), PVY-T(Japan), PVY-TH(Japan), PVYN(the Netherlands), and PVYN(France) was represented as 97.3%, 88.9%, 89.3%, 89.6% and 98.5%, respectively. The amino acid sequence homology of the polypeptide encoded by PVY-K CP gene with those encoded by viruses was represented as 97.4%, 92.5%, 92.9%, 92.9%, and 98.5%, respectively.

• Applied Biological Chemistry
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• v.39 no.6
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• pp.443-448
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• 1996

In order to investigate the function of xylA promoter(Pxyl) as regulatory region Pxyl-lacZ fusion gene was constructed by the insertion of xylA promoter to the multiple cloning site of upstream of lacZ gene in a multicopy numbered plasmid pMC1403 containing promoterless lac operon, which was designated pMCX191, and Pxyl-lacZ fragment from pMCX191 was inserted to low copy numbered plasmid pLG339, designated pLGX191. The expressions of ${\beta}-galactosidase$ in these recombinant plasmids containing Pxyl-lacZ fusion gene were induced strongly by the addition of xylose, repressed by the addition of 0.2% glucose in the presence of xylose. The catabolite repressions were derepressed by the addition of 1 mM cAMP as same as native xylA gene. The fragment of xylA promoter was partially deleted from the upstream of xylA promoter by exonuclease III to investigate the regulation site of xylA promoter and the degrees of deletion derivatives of xylA promoter were analyzed by the DNA base sequencing. By the investigations of the induction by xylose, repression by glucose and derepression by cAMP on xylose isomerase production, the regulation site of xylA promoter may be located in segment between -165 and -59 bp upstream from the initiation site of xylA translation.