• Journal of Microbiology and Biotechnology
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• 제8권1호
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• pp.21-27
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• 1998

The xylA gene of Bacillus stearothermophilus encoding the major ${\beta}$-xylosidase was previously cloned and sequenced. In the present study we examined the regulation of the cloned xylA gene expression in Bauillus subtilis MW15 carrying the xylA::aprA fusion plasmids. The induction of the fused xylA gene expression remained uninfluenced by any of the carbon sources tested but the gene expression was repressed about 2-3 fold in the presence of glucose. Two CRE-like sequences (CRE-1: nucleotides ＋ 124 to ＋136 and CRE-2: ＋247 to ＋259) were recognized within the reading frame region of the xylA gene. The deletion experiments showed that the CRE-2 sequence had a role in catabolite repression (CR) as a true CRE of the xylA gene, but the CRE-1 had no effect on CR of the xylA gene expression. Surprisingly, the deletion of the CRE- 1 sequence reduced about 2~3 fold of the expression of the xylA fused gene. The repression ratios of the xylA gene expression were estimated to be about 0.4 from the assay of subtilisin activity, and about 0.3 at the level of transcription by determining the amounts of xylA transcripts in B. subtilis. While, the level of CR of the xylA gene was assessed to be about l0-fold in previous work when the relative amounts of the xylA transcripts were measured in B. stearothermophilus.

• 한국환경성돌연변이발암원학회지
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• 제19권1호
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• pp.7-13
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• 1999

The mouse lymphoma thymidine kinase (tk+/-) gene assay (MOLY) using L5178Y tk+/- mouse lymphoma cell line is one of the mammalian forward mutation assays. It is well known that MOLY has many advantages and more sensitive than the other mammalian forward mutation assays such as x-linked hyposanthine phosphoribosyltransferase (hprt) gene assay. The target gene of MOLY is a heterozygous tk+/- gene located in 11 chromosome of L5178Y tk+/- cell, so it is able to detect the wide range of genetic changes like point mutation, deletion, rearrangement, and mitotic recombination within tk gene or deletion of entire chromosome 11. MOLY has relatively short expression time (2-3 days) compared to 1 week of hprt gene assay. MOLY can also induce relatively high mutant frequency so a large number of events can be recorded. The bimodal distribution of colony size which may indicate gene mutation and chromosome breakage potential of chemicals according to mutation scale such as large normal-growing mutants and small slow-growing mutants can be observed in this assay. The statistical analysis of data can be performed using the MUTANT program developed by York Electronic Research in association with Hazelton as recommended by the UKEMS (United Kingdom Environmental Mutagen Society) guidelines. This report reviewed MOLY using the microtiter cloning technique (microwell assay).

• 생명과학회지
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• 제20권5호
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• pp.703-709
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• 2010

Glucuronidation은 NNAL [4-(methylnitrosamno)-1-(3-pyridyl)-1-butanol]의 주요 pathway이며, UGT2B의 family인 UGT2B17 (UGT, uridine diphospho-glucuronosyltransferase) 유전자는 발암원의 glucuronidation에 관여 한다. UGT2B17 결손은 NNAL의 감소 수준과 특정 암에 있어 위험도를 증가시킨다. UGT2B17 유전자의 copy 수는 사람에서 개인별로 0~2로 다양하다. 본 연구에서는 UGT2B17 결손이 폐암의 위험도와 연관성을 가지는 가를 알아보기 위해 한국인인 271명의 대조군과 176명의 폐암환자의 샘플로 PCR 방법으로 CNV를 조사하였다. 그 결과, 현재까지 보고된 백인과 흑인에 비해 한국인에서 결실 대립형질이 현저히 높게 나타났다. 백인에서 유전자 두 개 모두가 결실된 0 copy 수가 약 10%를 나타낸 것에 비해, 본 연구의 한국인에서는 0 copy 수가 약 74%를 나타내었다. 더욱이 양 쪽 결실이 여성그룹에서 전반적으로 남성그룹에 비해 높게 나타났다. 그러나 UGT2B17 유전자가 CNV와 폐암과의 연관성은 찾을 수 없었다. 이러한 결과는 UGT2B17 유전자의 결실이 폐암의 감수성과는 연관되어 있지 않으나, UGT2B17 CNV 다형성이 인종간의 진화적 분석의 유용한 마커로 사용이 가능할 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제20권1호
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• pp.146-152
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• 2010

Streptomyces clavuligerus NRRL3585 produces a clinically important $\beta$-lactamase inhibitor, clavulanic acid (CA). In order to increase the production of CA, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (gap) was deleted in S. clavuligerus NRRL3585 to overcome the limited glyceraldehyde-3-phosphate pool; the replicative and integrative expressions of ccaR (specific regulator of the CA biosynthetic operon) and claR (Lys-type transcriptional activator) genes were transformed together into a deletion mutant to improve clavulanic acid production. We constructed two recombinant plasmids to enhance the production of CA in the gap1 deletion mutant of S. clavuligerus NRRL3585: pHN11 was constructed for overexpression of ccaR-claR, whereas pHN12 was constructed for their chromosomal integration. Both pHN11 and pHN12 transformants enhanced the production of CA by 2.59-fold and 5.85-fold, respectively, compared with the gap1 deletion mutant. For further enhancement of CA, we fed the pHN11 and pHN12 transformants ornithine and glycerol. Compared with the gap1 deletion mutant, ornithine increased CA production by 3.24- and 6.51-fold in the pHN11 and pHN12 transformants, respectively, glycerol increased CA by 2.96- and 6.21-fold, respectively, and ornithine and glycerol together increased CA by 3.72- and 7.02-fold, respectively.

• Journal of Microbiology and Biotechnology
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• 제33권11호
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• pp.1420-1427
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• 2023

The forkhead domain genes are important for development and morphogenesis in fungi. Six forkhead genes fkhA-fkhF have been found in the genome of the model filamentous Ascomycete Aspergillus nidulans. To identify the fkh gene(s) associated with fungal development, we examined mRNA levels of these six genes and found that the level of fkhB and fkhD mRNA was significantly elevated during asexual development and in conidia. To investigate the roles of FkhB and FkhD, we generated fkhB and fkhD deletion mutants and complemented strains and investigated their phenotypes. The deletion of fkhB, but not fkhD, affected fungal growth and both sexual and asexual development. The fkhB deletion mutant exhibited decreased colony size with distinctly pigmented (reddish) asexual spores and a significantly lower number of conidia compared with these features in the wild type (WT), although the level of sterigmatocystin was unaffected by the absence of fkhB. Furthermore, the fkhB deletion mutant produced sexual fruiting bodies (cleistothecia) smaller than those of WT, implying that the fkhB gene is involved in both asexual and sexual development. In addition, fkhB deletion reduced fungal tolerance to heat stress and decreased trehalose accumulation in conidia. Overall, these results suggest that fkhB plays a key role in proper fungal growth, development, and conidial stress tolerance in A. nidulans.

• Journal of Microbiology
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• 제41권2호
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• pp.129-136
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• 2003

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMPI) exhibits considerable sequence heterogeneity among EBV isolates. Seven distinct EBV strains have been defined based on sequence polymorphisms in the LMPI gene, which are designated China 1, China 2, China 3, Alaskan, Mediterranean, NC, and the B95-8 strains. In this study, we analyzed a 30-bp deletion and sequence variations in the carboxy-terminal region of the LMPl gene in 12 EBV isolates from spontaneous lym-phoblastoid cell lines derived from individuals with non-EBV associated cancers in Korea. Eleven of the 12 isolates showed a 30-bp deletion spanning LMPI amino acids 342 to 353, suggesting a high prevalence of the LMPI 30-bp deletion variant among EBV isolates in Korea. In addition, all 12 isolates had a 15-bp common deletion in the 33-bp repeat region and multiple base-pair changes relative to the prototype B95-8 EBV strain along with variations in the number of the 33-bp repeats. The bp changes at positions 168746, 168694, 168687, 168395, 168357, 168355, 168631, 168320, 168308, 168295, and 168225 were highly conserved among the isolates. Comparative analysis of sequence change patterns in the LMPI carboxy-terminal coding region identified nine 30-bp deletion variants as China 1, two deletion variants as a possible interstrain between the Alaskan and China 1 strains, and a single undeleted variant as a possible variant of the Alaskan strain. These results suggest the predominance of the China 1 EBV strain in the Korean population.

• 대한미생물학회지
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• 제35권3호
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• pp.215-224
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• 2000

In this study, the distribution of the mec regulator genes and the presence of the mutation in mecI gene and mec promoter region among 50 MRSA clinical isolates derived from a single university hospital in Korea were analyzed. Among 50 MRSA strains, 13 strains had a deletion of mecI gene, and 37 strains were found to have mutations in mecI gene or mecA promoter region corresponding to a presumptive operator of mecA, i.e., the binding site of the repressor protein. Furthermore, in order to track the evolution of methicillin-resistant Staphylococcus aureus (MRSA) distributed in Korea, we determined the MRSA clonotype by combined use of genetic organization patterns of mec regulator genes, ribotype, and coagulase type. As the result, 48 of 50 MRSA strains could be classified into four distinct clones. Clonotype I is characterized by the coagulase type 3, deletion of mecI gene, and ribotype 1 shared by NCTC10442, the first reported MRSA isolate in England (9 strains). Clonotype II is characterized by the coagulase type 4, C to T substitution at position 202 of mecI gene, and ribotypes 2, 3 and 4 shared by 85/3619 strain isolated in Austria (10 strains). Clonotype III is characterized by the coagulase type 2, mutations of mecA promoter region and/or mecI, and ribotypes 4, 5, and 6 shared by N315 strain isolated in Japan (25 strains). Clonotype IV is characterized by the coagulase type 4, deletion of mecI gene, and ribotype 7 (4 strains). The clonality of two strains could not be determined due to their undefined ribotype.

• Journal of Microbiology and Biotechnology
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• 제30권5호
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• pp.662-667
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• 2020

Epidermal growth factor receptor (EGFR) mutations are not only genetic markers for diagnosis but also biomarkers of clinical-response against tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). Among the EGFR mutations, the in-frame deletion mutation in EGFR exon 19 kinase domain (EGFR exon 19-del) is the most frequent mutation, accounting for about 45% of EGFR mutations in NSCLCs. Development of sensitive method for detecting the EGFR mutation is highly required to make a better screening for drug-response in the treatment of NSCLC patients. Here, we developed a fluorometric tandem gene amplification assay for sensitive detection of low-abundance EGFR exon 19-del mutant genomic DNA. The method consists of pre-amplification with PCR, thermal cycling of ligation by Taq ligase, and subsequent rolling circle amplification (RCA). PCR-amplified DNA from genomic DNA samples was used as splint DNA to conjugate both ends of linear padlock DNA, generating circular padlock DNA template for RCA. Long stretches of ssDNA harboring multiple copies of G-quadruplex structure was generated in RCA and detected by thioflavin T (ThT) fluorescence, which is specifically intercalated into the G-quadruplex, emitting strong fluorescence. Sensitivity of tandem gene amplification assay for detection of the EGFR exon 19-del from gDNA was as low as 3.6 pg, and mutant gDNA present in the pooled normal plasma was readily detected as low as 1% fraction. Hence, fluorometric detection of low-abundance EGFR exon 19 deletion mutation using tandem gene amplification may be applicable to clinical diagnosis of NSCLC patients with appropriate TKI treatment.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2015년도 추계학술대회 및 정기총회
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• pp.28-28
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• 2015

Asexual reproduction or conidiation in aspergilli is a primary mean to produce their progenies that is environmentally and genetically controlled tightly. Previously, intensive researches in the model fungus Aspergillus nidulans disclosed some genes playing important roles in asexual and sexual development. Among them, one gene encoding a putative helix-loop-helix (HLH) transcription factor, named ndrA, has been isolated and characterized as a downstream regulator of developmental master regulator NsdD. By using comparative genome search of A. niduans NdrA protein, its orthologues have been identified in A. fumigatus and A. flavus, respectively (AfudrnA and AfldrnA). Deletion of the ndrA genes in both Aspergillus species made them unable to produce the conidia yet abundant production of sclerotia in A. flavus. Complementation of ndrA deletion strains by intact ndrA ORFs has restored the conidiation as in the control strains. In A. fumigatus, ndrA deletion also resulted in loss of conidiation phenotype. Northern analyses showed that the ndrA genes in both Aspergillus species are highly expressed at the early stage of the conidiation. Interestingly, the ndrA genes were found to be necessary for the proper expression of brlA genes. Antifungal sensitivity test revealed that the ndrA genes might be responsible for the sensitivity or resistance to some antifungal agents. However, ndrA deletion did not greatly influence the growth in both strains. And the A. flavus ndrA gene did not affect the aflatoxin production. Taken together, ndrA genes in Aspergillus species could be an important positive regulator of conidiation under the regulation of the nsdD gene yet upstream of the brlA gene.

• 생명과학회지
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• 제16권3호
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• pp.454-458
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• 2006

출아효모인 Sacharomyces cerevisiae S288C균주를 이용한 효모의 게놈이 완성된 후 S. cerevisiae는 다양한 연구 모델로 이용되어져 왔다. 현재까지 효모를 이용한 기능 유전체학 측면에서의 연구는 laboratory strainin인 S288C 균주 또는 그 유래의 균주들이다. 그러나 자연에서 분리된 효모 또는 산업적으로 이용되어지고 있는 S. cerevisiae의 유전학 측면에서의 연구는 낮은 포자형성률 및 형질전환률, 그리고 S288C 균주와의 게놈상의 상이성 때문에 거의 이루어지지 않고 있다. 여기서 우리 연구진은 자연에서 분리된 Saccharomyces cerevisiae KNU5377 균주를 이용하여 random spore analysis를 통해 MATa 및 $MAT{\alpha}$ 타입의 각각의 haploid cell을 분리 후 이미 보고된 KanMX module를 가지고 round PCR기법에 의한 short flanking homology 기법을 이용하여 전사조절인자인 HSF1 유전자가 치환된 변이주를 구축할 수 있었다. 덧붙여, 모든 유전자에 이 기법을 적용할 수는 없다는 것을 확인하였다. 앞으로 이 변이주를 통해 기능 유전체학적인 측면에서 이 유전자의 스트레스와의 관련성을 연구하고자 한다.