• Journal of Microbiology
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• v.34 no.4
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• pp.327-333
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• 1996

Yeast, like many other microbes, encounters large variations in ambient pH in their natural environments. Microorganisms capable of growing over a wide pH range require a versatile, efficient pH homeostatic mechanism protecting intracellular processes against extremes of pH. In several organisms, fusions to the bacterial lacZ gene have been extremely useful for the identification of genes expressed at different time during the life cycle or under different growth conditions. In this study, using the lacZ gene screening system, we surveyed a large number of yeast strains with lacZ insertion to identify genes regulated by pH. A yeast genomic library was constructed and inserted with lacZ by a shuttle mutagenesis procedure. The yeast transformants were individually picked up with a toothpick, replica-plated, and grown in alkaline pH medium. Among the 35,000 colonies screened, 10 candidate strains were identified initially by the $\beta$-gal assay. We finally confirmed two yeast strains carrying the genes whose expression are strictly dependent on pH of growth medium. One of the fusions showing a 10-fold induction in expression level in response to alkali pH was selected and further characterized. The pH-regulated gene was cloned by inverse PCR and a partial sequence of the gene was determined. Identification and characterization of the gene is currently under investigation.

• Genomics & Informatics
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• v.9 no.2
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• pp.64-68
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• 2011

Monomelic amyotrophy (MA), also known as Hirayama disease, occurs mainly in young men and manifests as weakness and wasting of the muscles of the distal upper limbs. Here, we sought to identify a genetic basis for MA. Given the predominance of MA in males, we focused on candidate neurological disease genes located on the X chromosome, selecting two X-linked candidate genes, androgen receptor (AR ) and ubiquitin-like modifier activating enzyme 1 (UBA1). Screening for genetic variants using patients' genomic DNA revealed three known genetic variants in the coding region of the AR gene: one nonsynonymous single-nucleotide polymorphism (SNP; rs78686797) encoding Leu57Gln, and two variants of polymorphic trinucleotide repeat segments that encode polyglutamine (CAG repeat; rs5902610) and polyglycine (GGC repeat; rs3138869) tracts. Notably, the Leu57Gln polymorphism was found in two patients with MA from 24 MA patients, whereas no variants were found in 142 healthy male controls. However, the numbers of CAG and GGC repeats in the AR gene were within the normal range. These data suggest that the Leu57Gln polymorphism encoded by the X-linked AR gene may contribute to the development of MA.

• Animal cells and systems
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• v.3 no.2
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• pp.229-236
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• 1999

The recA gene plays a central role in genetic recombination and SOS DNA repair in Escherichia coli (E. coli). We have previously identified a 42 kDa RecA-like protein inducible by a variety of DNA damages from a fluorescent Pseudomonas strain sp. and characterized its inducible kinetics. In the present study, we cloned and characterized the gene encoding the RecA-like protein by immunological screening of Pseudomonas genomic expression library using polyclonal E. coli anti-RecA antibodies as a probe. From 10$^{5}$ plaques screened, five putative clones were finally isolated. Southern blot analysis indicated that four clones had the same DNA inserts and the recA-like gene was located within the 3.2 kb EcoRI fragment of Pseudomonas chromosomal DNA. In addition, the cloned recA-like gene was transcribed into an RNA transcript approximately 1.1 kb in size, as judged by Northern blot analysis. The cellular level of RNA transcript of the cloned recA-like gene was increased to an average of 5.15- fold upon treatment with DNA damaging agents such as ultraviolet (UV)- light, nalidixic acid (NA), methyl methanesulfonate (MMS), and mitomycin-C (MMC). These results suggest that the cloned gene is inducible by DNA damage similarly to the recA gene in E. coli. However, the cloned gene did not restore the DNA damage sensitivity of the E. coli recA-mutant.

• Neonatal Medicine
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• v.17 no.2
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• pp.250-253
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• 2010

Citrullinemia type I is an urea cycle defect caused by mutations in the argininosuccinate synthetase (ASS1) gene. We report a novel argininosuccinate synthetase gene mutation in a Korean family with type I citrullinemia. Metabolic evaluation revealed significant hyperammonemia. Amino acid/acylcarnitine screening using tandem mass spectrometry showed high level of citrulline. Plasma amino acid analysis showed high level of citrulline and the urine organic acid analysis showed makedly increased level of orotic acid. To confirm diagnosis of citrullinemia we did mutation analysis of the ASS1 gene. The patient was found to have mutations of c.689G>C (p.G230A) and c.892G>A (p.E298K), which were new types of argininosuccinate synthetase gene mutation have never been reported in Korea. We report a novel case of argininosuccinate synthetase 1 gene mutation and suggest that the gene study to the family members is necessary to carry out when a patient is diagnosed as citrullinemia.

• Bulletin of the Korean Chemical Society
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• v.27 no.5
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• pp.657-662
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• 2006

Identification of accessible sites in targeted RNAs is a major limitation to the effectiveness of antisense oligonucleotides. A class of antisense oligodeoxynucleotides, known as the “10-23” DNA enzyme or DNAzyme, which is a small catalytic DNA, has been shown to efficiently cleave target RNA at purine-pyrimidine junctions in vitro. We have designed a strategy to identify accessible cleavage sites in the target RNA, which is hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool of random DNAzyme library. A pool of DNAzymes of 58 nucleotides-length that possess randomized annealing arms, catalytic core sequence, and fixed 5'/3'-end flanking sequences was designed and screened for their ability to cleave the target RNA. The screening procedure, which includes binding of DNAzyme pool to the target RNA under inactive condition, selection and amplification of active DNAzymes, incubation of the selected DNAzymes with the target RNA, and target site identification on sequencing gels, identified 16 potential cleavage sites in the target RNA. Corresponding DNAzymes were constructed for the selected target sites and were tested for RNA-cleavage in terms of kinetics and accessibility. These selected DNAzymes were effective in cleaving the target RNA in the presence of $Mg^{2+}$. This strategy can be applicable to identify accessible sites in any target RNA for antisense oligonucleotides-based gene inactivation methods.

• Korean Journal of Plant Resources
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• v.29 no.6
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• pp.658-669
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• 2016

Rice blast, caused by a fungus Magnaporthe oryzae, is one of the most devastating diseases of rice worldwide. Analyzing the valuable genetic resources is important in making progress towards blast resistance. Molecular screening of major rice blast resistance (R) genes was determined in 2,509 accessions of rice germplasm from different geographic regions of Asia and Europe using PCR based markers which showed linkage to twelve major blast R genes, Pik-p, Pi39, Pit, Pik-m, Pi-d(t)2, Pii, Pib, Pik, Pita, Pita/Pita-2, Pi5, and Piz-t. Out of 2,509 accessions, only two accessions had maximum nine blast resistance genes followed by eighteen accessions each with eight R genes. The polygenic combination of three genes was possessed by maximum number of accessions (824), while among others 48 accessions possessed seven genes, 119 accessions had six genes, 267 accessions had five genes, 487 accessions had four genes, 646 accessions had two genes, and 98 accessions had single R gene. The Pik-p gene appeared to be omnipresent and was detected in all germplasm. Furthermore, principal component analysis (PCA) indicated that Pita, Pita/Pita-2, Pi-d(t)2, Pib and Pit were the major genes responsible for resistance in the germplasm. The present investigation revealed that a set of 68 elite germplasm accessions would have a competitive edge over the current resistance donors being utilized in the breeding programs. Overall, these results might be useful to identify and incorporate the resistance genes from germplasm into elite cultivars through marker assisted selection in rice breeding.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.3
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• pp.1687-1689
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• 2013

We performed a case-control study to investigate whether SNPs of CHIP might affect the development of IA in Chinese Han nationality. We believe we are the first to have screened IA patients for mutations in the CHIP gene to determine the association with these variants. The study group comprised 224 Chinese Han nationality patients with at least one intracranial aneurysm and 238 unrelated healthy Han nationality controls. Genomic DNA was isolated from blood leukocytes. The entire coding regions of CHIP were genotyped by PCR amplification and DNA sequencing. Differences in genotype and allele frequencies between patients and controls were tested by the chi-square method. Genotype and allele frequencies of the SNP rs116166850 was demonstrated to be in Hardy-Weinberg equilibrium. No significant difference in genotype or allele frequencies between case and control groups was detected at the SNP. Our data do not support the hypothesis of a major role for the CHIP gene in IA development in the Chinese Han population.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.27 no.1
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• pp.17-27
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• 2001

A cell-based assay system for monitoring NF-$textsc{k}$B activity was developed to determine the influence of activated NF-$textsc{k}$B in human HaCaT cells. The pNF-$textsc{k}$B-SEAP-NPT plasmid that permits expression of the secreted alkaline phosphatase (SEAP) reported gene in response to the NF-$textsc{k}$B activity and contains neomycin phosphotransferase (NPT) gene for the geneticin resistance in host cells was constructed and transfected into human keratinocyte cell line HaCaT. Human HaCaT transfectant cells secreted the SEAP enzyme into the culture medium in a time-dependent manner until 72h. NF-$textsc{k}$B activities were measured in the SEAP reporter gene assay using a fluorescent detection method. The treatment of HaCaT cell transfectants with known antioxidants [e.g., N-acetyl-L-cysteine and vitamin C] showed inhibition of NF-$textsc{k}$B activity in a time-and concentration-dependent manner. The phorbol 12-myristate 13-acetate (PMA) known as a stimulator of NF-$textsc{k}$B expression demonstrated that it increased NF-$textsc{k}$B activity in a time- and concentration-dependent manner. This assay system could be used to determine the quantitative measurement of NF-$textsc{k}$B activity in the human skin and allow the screening of anti-inflammatory agents from various synthetic chemicals and natural products for dermatological purpose. Abbrevitions used: NF-$textsc{k}$B, nuclear factor kappa B; I-$textsc{k}$B, Inhibitory kappa B; SEAP, secreted alkaline phosphatase; NPT, neomycin phosphotransferease; PCR, polymerase chain reaction: dNTP, deoxynucleoside triphosphates; DMEM, dulbecco’s modified eagle medium; FBS, fetal bovine serum; PBs, phosphate-buffered saline; MUP, 4-methylumbellifery phosphate; NAC, N-acetyl-L-cysteine; DMSO, dimethyl sulfoxide; PMA, phorbol 12-myristate 13-acetate.

• Korean Journal of Microbiology
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• v.38 no.4
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• pp.230-230
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• 2002

Class Ⅲ chitin synthases in filamentous fungi are important for hyphal growth and differentiation of several filamentous fungi. A genomic clone containing the full gene encoding Chs4, a class Ⅲ chitin synthase in Penicillium chrysogenum, was cloned by PCR screening and colony hybridization from the genomic library. Nucleotide sequence analysis and transcript mapping of chs4 revealed an open reading frame (ORF) that consisted of 5 exons and 4 introns and encoded a putative protein of 915 amino acids. Nucleotide sequence analysis of the 5′flanking region of the ORF revealed a potential TATA box and several binding sites for transcription activators. The putative transcription initiation site at -716 position was identified by primer extension and the expression of the chs4 during the vegetative growth was confirmed by Northern blot analysis. Amino acid sequence analysis of the Chs4 revealed at least 5 transmembrane helices and several sites for past-transnational modifications. Comparison of the amino acid sequence of Chs4 with those of other fungi showed a close relationship between P chrysogenum and genus Aspergillus.

• Journal of Genetic Medicine
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• v.9 no.1
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• pp.42-46
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• 2012

Short-chain acyl-CoA dehydrogenase deficiency (SCADD; OMIM # 201470) is an autosomal recessive inborn error of mitochondrial fatty acid ${\beta}$-oxidation, presenting with a variety of clinical signs and symptoms. Developmental delay, hypertonia or hypotonia, ketotic hypoglycemia, and epilepsy are most frequently reported. In general, patients diagnosed through newborn screening have shown normal growth and development in contrast to those diagnosed as a result of clinically initiated evaluations. Here, the case of an asymptomatic Korean newborn with SCADD identified by tandem mass spectrometry is reported. The patient showed an elevated concentration of butyrylcarnitine detected on newborn screening. Urinary excretion of ethylmalonic acid was elevated by urine organic acid analysis. To confirm the diagnosis of SCADD, a direct sequencing analysis of 10 coding exons and the exon-intron boundaries of the ACADS gene were performed. Genetic analysis of ACADS showed the following novel compound heterozygous missense mutations: c.277C>A (p.Leu93Ile) on exon3 and c.682G>A (p.Glu288Lys) on exon6. These results will provide further evidence of mutational heterogeneity for SCADD.