• 한국미생물·생명공학회지
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• 제16권6호
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• pp.463-467
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• 1988

L. casei YIT9018에서 자연적으로 발생하는 Lac$^-$ mutant의 발생빈도와 이러한 변이주의 특성을 검토하였다. 보존균주로부터 spontaneous Lac$^-$ mutant의 발생은 MRS 배지에서 0.5%, 10% 환원탈지유에서 0.29%, pure Lac$^+$ colony로부터의 발생은 MRS 배지에서 0.25%, 10% 환원탈지유에서 0.1%의 빈도로 나타났다. 분리된 spontaneous mutant는 대부분이 Lac$^-$ mutant였고 가끔 Lac-Gal-도 나타났다. Lac$^-$ Gal$^+$ mutant는 배양 4일째부터 partial revertant가 출현하였으나 Lac$^-$ Gal$^-$는 전혀 나타나지 않았으며 Lac$^-$ mutant는 wild type보다 우유를 응고시키는데 더 많은 시간을 요하였다.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2000년도 국제심포지움 및 추계학술대회
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• pp.45-67
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• 2000

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a mutagenic and carcinogenic heterocyclic amino found in cooked meat. The in vivo mutagenicity and hepatocarcinogenicity of MeIQx were examined in mice harboring the lacZ mutation reporter gene ($Muta^{TM}$ Mice) and bitransgenic mice over-expressing the c-myc oncogene. C57B1/$\lambda$lacZ and bitransgenic c-myc (albumin promoter)/$\lambda$lacZ mice were bred and weaned onto an AIN-76 based diet containing $0.06\%$ (w/w) MeIQx or onto control diet. After 30 weeks on diet, only male bitransgenic mice on MeIQx developed hepatocellular carcinoma ($100\%$ incidence) indicating that there was synergism between c-myc over-expression and MeIQx. By 40 weeks, hepatic tumor incidence was $100\%$ ($17\%$) and $44\%$ ($0\%$) in male c-myc/$\lambda$lacZ and C57B1/$\lambda$lacZ mice given MeIQx (or control) diet, respectively, indicating that either MeIQx or c-myc over-expression alone eventually induced hepatic tumors. At either time point, mutant frequency in the lacZ gene was at least 40-fold higher in MeIQx-treated mice than in control mice of either strain. These findings suggest that MeIQx-induced hepatocarcinogenesis is associated with MeIQx-induced mutations. Elevated mutant frequency in MeIQx-treated mice also occurred concomitant with the formation of MeIQx-guanine adducts as detected by the $^{32}P$-postlabeling assay. Irrespective of strain or diet, sequence analysis of the lacZ mutants from male mouse liver showed that the principal sequence alteration was a single guanine-base substitution. Adenine mutations, however, were detected only in animals on control diet. MeIQx-fed mice harboring the c-myc oncogene showed a l.4-2.6-fold higher mutant frequency in the lacZ gene than mice not carrying the transgene. Although there was a trend toward higher adduct levels in c-myc mice, MeIQx-DNA adduct levels were not significantly different between c-myc/$\lambda$lacZ and C57B1/$\lambda$lacZ mice after 30 weeks on diet. Thus, it appeared that factors in addition to MeIQx-DNA adduct levels, such as the enhance rate of proliferation associated with c-myc over-expression, may have accounted for a higher mutant frequency in c-myc mice. In the control diet groups, the lacZ mutant frequency was significantly higher in c-myc/$\lambda$lacZ mice than in 057B1/$\lambda$1acZ mice. The findings are consistent with the notion that c-myc over-expression is associated with an increase in mutagenesis. The mechanism for the synergistic effects of c-myc over-expression on MeIQx hepatocarcinogenicity appears to involve an enhancement of MeIQx-induced mutations.

• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.2046-2055
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• 2007

Wild-type V. vulnificus cannot grow using lactose as the sole carbon source or take up the sugar. However, prolonged culture of this species in media containing lactose as the sole carbon source leads to the generation of a spontaneous lactose-utilizing (LU) mutant. This mutant showed strong ${\beta}$-galactosidase activity, whereas the wild-type strain showed a barely detectable level of the activity. A mutant with a lesion in a gene homologous to the lacZ of E. coli in the bacterium no longer showed ${\beta}$-galactosidase activity or generated spontaneous LU mutants, suggesting that the lacZ homolog is responsible for the catabolism of lactose, but the expression of the gene and genes for transport of lactose is tightly regulated. Genetic analysis of spontaneous LU mutants showed that all the mutations occur in a lacI homolog, which is located downstream to the lacZ and putative ABC-type lac permease genes. Consistent with this, a genomic library clone containing the lad gene, when present in trans, made the spontaneous LU mutants no longer able to utilize lactose as the sole carbon source. Taken together with the observation that excessive amounts of exogenously supplemented possible catabolic products of lactose have negative effects on the growth and survivability of V. vulnificus, we suggest that V. vulnificus has evolved to carry a repressor that tightly regulates the expression of lacZ to keep the intracellular toxic catabolic intermediates at a sublethal level.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.84-91
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• 2006

A Saccharomyces cerevisiae pgs1 nulI mutant, which is deficient with phosphatidyl glycerol (PG) and cardiolipin (CL) biosynthesis, grows well on most fermentable carbon sources, but fails to grow on non-fermentable carbon sources such as glycerol, ethanol, and lactate. This mutant also cannot grow on galactose medium as the sole carbon source. We found that the incorporation of $[^{14}C]-galactose$, which is the first step of the galactose metabolic pathway (Leloir pathway), into the pgs 1 null mutant cell was extremely repressed. Exogenously expressed PGS1 (YCpPGS1) under indigenous promoter could completely restore the pgs1 growth defect on non-fermentable carbon sources, and dramatically recovered $[^{14}C]-galactose$ incorporation into the pgs1 mutant cell. However, PGS1 expression under the GALl promoter $(YEpP_{GAL1}-PGS1myc)$ could not complement pgs1 mutation, and the GAL2-lacZ fusion gene $(YEpP_{GAL2}-lacZ)$ also did not exhibit its $\beta-galactosidase$ activity in the pgs1 mutant. In wild-type yeast, antimycin $A(1\;{\mu}g/ml)$, which inhibits mitochondrial complex III, severely repressed not only the expression of the GAL2-lacZ fusion gene, but also uptake of $[^{14}C]-galactose$. However, exogenously expressed PGS1 partially relieved these inhibitory effects of antimycin A in both the pgs1 mutant and wild-type yeast, although it could not basically restore the growth defect on galactose by antimycin A. These results suggest that the PGSI gene product has an important role in utilization of galactose by Gal genes, and that intact mitochondrial function with PGS1 should be required for galactose incorporation into the Leloir pathway. The PGS1 gene might provide a clue to resolve the historic issue about the incapability of galactose with deteriorated mitochondrial function.

• Journal of Microbiology and Biotechnology
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• 제9권3호
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• pp.352-357
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• 1999

Enterobacter sp. B54 inhibited growth of the fungus Phytophthora capsici on potato dextrose agar (PDA). Three mutants with antifungal activities (denoted M54-47, M54-113, and M54-329) which were lost or increased, through Pl::Tn5 lac mutagenesis, were used to isolate genes responsible for fungal inhibition on PDA. Two clones were selected from the partially EcoR1-digested genomic library of the wild-type strain by probing with genomic flanking sequences of each mutant. We have isolated a 20-kb EcoR1 genomic DNA fragment from this strain that contains genes involved in hyphal growth inhibition of P. capsici on PDA. Subcloning and expression analysis of the above DNA fragment identified a 8-kb region which was necessary for antifungal activities. A 8-kb HindⅢDNA fragment covers three genomic loci inserted by Tn5 lac in each mutant. This suggested that all genes which are related to antifungal activities might be clustered in simple forms of at least 5-8 kb sizes.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.976-983
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• 2003

Seven Escherichia coli cells defective with aerobic growth were isolated by the insertion of ${\lambda}placMu53$, a hybrid bacteriophage of ${\lambda}$ and Mu, which created a transcriptional fusion to lacZY. These insertion mutant cells were tested on an XG ($5-bromo-4-chloro-3-indolyl-{\beta}-D-galactopyranoside$) medium for anaerobic expression of lacZ by fusion to a promoter. The chromosomal DNA from these strains were digested by EcoRI, and the EcoRI fragments that contained the fused gene and lacZ sequence were identified by Southern hybridization, using lacZ containing plasmid as a probe. The EcoRI fragment from each strain was cloned and sequenced. The sequence data were compared with the GenBank database. The mutated gene of three strains, CYT4, CYT5, and OS11, was found to be identical, and it was nrdAB that encoded ribonucleoside diphosphate reductase. The gene nrdAB was at min 50.5 on the Escherichia coli linkage map and 2,348,084 on the physical map, and is involved in hemAe-related reduction-oxidation reaction. OS6 and OS14 mutant strains had insertion at min 8.3 and the mutated gene was hemB. The hemB encodes 5-aminolevulinate dehydratase or porphobilinogen synthase. The OS3 mutant had insertion in cydB at min 16.6. The cydAB encodes cytochrome d oxidase. In the case of OS1, the fusion was made with sucA, the E1 component of ${\alpha}-ketoglutarate$ dehydrogenase.

• 한국미생물·생명공학회지
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• 제26권5호
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• pp.393-399
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• 1998

고추역병균(Phytophthora capsici)의 성장을 in vitro에서 저해하는 길항균 Bl4를 한국토양으로부터 분리 동정하여 Enterobacter속임을 밝혔고 Pl::Tn5 lac에 의해 transposon돌연변이를 유기하여 길항력 강화주와 약화주들의 염색체내에 Tn5 lac이 각기 상이한 위치에 무작위로 삽입되었음을 southern hybridization에 의해 확인하였다.

• BMB Reports
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• 제36권1호
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• pp.35-42
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• 2003

Heterocyclic amines (HCAs) are potent mutagens generated during the cooking of meat and fish, and several of these compounds produce tumors in conventional experimental animals. During the past 5 years or so, HCAs have been tested in a number of novel in vivo murine models, including the following: lacZ, lacI, cII, c-myc/lacZ, rpsL, and $gpt{\Delta}$ transgenics, $XPA^{-/-}$, $XPC^{-/-}$, $Msh2^{+/-}$, $Msh2^{-/-}$ and $p53^{+/-}$ knock-outs, Apc mutant mice ($Apc^{{\Delta}716}$, $Apc^{1638N}$, $Apc^{min}$), and $A33^{{\Delta}N{\beta}-cat}$ knock-in mice. Several of these models have provided insights into the mutation spectra induced in vivo by HCAs in target and non-target organs for tumorigenesis, as well as demonstrating enhanced susceptibility to HCA-induced tumors and preneoplastic lesions. This review describes several of the more recent reports in which novel animal models were used to examine HCA-induced mutagenesis and carcinogenesis in vivo, including a number of studies which assessed the inhibitory activities of chemopreventive agents such as 1,2-dithiole-3-thione, conjugated linoleic acids, tea, curcumin, chlorophyllin-chitosan, and sulindac.

• 환경생물
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• 제20권4호
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• pp.316-324
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• 2002

To investigate the mutational spectrum of laccase (Lac) genes (lac -A and lac -B) involved in degrading lignin which is the recalcitrant cell wall polymer, the genes of the Pleurotus ostreatus mutants induced by gamma ray radiation were amplified by PCR and were cloned. All partial lac-A genes of 4 mutants (PO-6, -7, -14 and -15) consisted of 1763 base pairs due to the deletion of two bases (491-nt and 492-nt) and addition of one base (875-nt) in 1764 base pairs of lac -A gene of PO-1. Totally 36 mutational hot spots were detected and 32 positions were mutated in all of those 4 mutants simultaneously. These mutations were predominantly A : T -> G : C transitions (40%). Putative amino acid sequences of lac -A genes of mutants have one simultaneous mutated residue (from Thr-44 to Ala-44). The 1764 bp of partial lac -B gene was cloned only in PO -5 mutant and contained 19 mutated bases. These mutations were predominantly G : C->A : T transitions (45%). Lac-B protein of PO-5 has two mutated residues of Glu-290 and His-363 from Ala-290 and Phe-363, respectively. The hyper-mutational positions were concentrated in specific regions of between 50-nt and 900-nt in lac genes. These results suggested that the mutational hotspots responded to gamma radiation could be in some genes, at least lac -A and lac -B of p. ostreatus.

• 한국환경과학회지
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• 제21권9호
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• pp.1139-1147
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• 2012

To find out the growth conditions for the maximum activity of nitrogenase which catalyzes nitrogen fixation in Rhodobacter sphaeroides, the promoter activities of nifA and nifH were analyzed and the results indicated that expression of both nifA and nifH was increased in response to deprivation of both O2 concentration and nitrogen source. The nifA mutant was constructed by deleting the gene to investigate the effect of NifA, the transcriptional regulator, on the nifH and nifA expression in R. sphaeroides. Analysis of expression of nif genes using the nifA::lacZ and nifH::lacZ fusions in the nifA mutant revealed that NifA acts as a positive activator for nifH and an autoregulator in its own expression. The promoter activities of nifA and nifH in the prrA mutant grown under anaerobic and ${NH_4}^+$-free conditions were derepressed, comparing with those of the wild-type grown under the same conditions, indicating that the prrA product acts as a positive regulator in expression of nifA and nifH.