• Journal of Microbiology and Biotechnology
• /
• 제25권11호
• /
• pp.1827-1834
• /
• 2015

The SMG1 lipase from Malassezia globosa is a newly found mono- and diacylglycerol (DAG) lipase that has a unique lid in the loop conformation that differs from the common alpha-helix lid. In the present study, we characterized the contribution of three residues, L103 and F104 in the lid and F278 in the rim of the binding site groove, on the function of SMG1 lipase. Site-directed mutagenesis was conducted at these sites, and each of the mutants was expressed in the yeast Pichia pastoris, purified, and characterized for their activity toward DAG and p-nitrophenol (pNP) ester. Compared with wild-type SMG1, F278A retained approximately 78% of its activity toward DAG, but only 11% activity toward pNP octanoate (pNP-C8). L103G increased its activity on pNP-C8 by approximately 2-fold, whereas F104G showed an approximate 40% decrease in pNP-C8 activity, and they both showed decreased activity on the DAG emulsion. The deletion of 103-104 retained approximately 30% of its activity toward the DAG emulsion, with an almost complete loss of pNP-C8 activity. The deletion of 103-104 showed a weaker penetration ability to a soybean phosphocholine monolayer than wild-type SMG1. Based on the modulation of the specificity and activity observed, a pNP-C8 binding model for the ester (pNP-C8, N102, and F278 form a flexible bridge) and a specific lipid-anchoring mechanism for DAG (L103 and F104 serve as "anchors" to the lipid interface) were proposed.

• Asian Pacific Journal of Cancer Prevention
• /
• 제16권18호
• /
• pp.8177-8186
• /
• 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.

• BMB Reports
• /
• 제37권2호
• /
• pp.153-158
• /
• 2004

The acid-labile subunit (ALS) is known to interact with the IGF binding protein (IGFBP) in the presence of insulin-like growth factors (IGFs). Studies, however, indicate that ALS forms a doublet with IGFBP3, independent of IGFs. To characterize the structural domain required for the IGF-free ALS-IGFBP3 interaction, seven recombinant human IGFBP3 mutants were generated: three deletion mutants and four site-specific mutants that had altering N-terminal regions of IGFBP3. ALS and IGFBP3 mRNAs were co-injected into Xenopus oocytes, and their products were cross-linked and immunoprecipitated using antisera against ALS or IGFBP3. Among the deletion mutants, the mutant of D40 (deleted in 11-40th amino acids) exerted no effect in the interaction with ALS, while D60 (${\Delta}11$-60) demonstrated a moderate reduction. D88 (${\Delta}11$-88), however, showed a significant decrease. In the case of site-specific mutants, the mutation that alterated the IGF binding site (codons 56 or 80) exerted a significant reduction in the interaction, whereas codons 72 or 87 showed no significant change in the interaction with ALS. The stability of the ALS-IGFBP3 interaction was analyzed according to a time-dependent mode. Consistent with the binding study, mutants on the IGF binding sites (56 or 80) consistently show a weakness in the ALS-IGFBP3 interaction when compared to the mutants that covered the non-IGF binding sites (72 or 87). This study suggests that the N-terminal of IGFBP3, especially the IGF binding site, plays an important role in interacting with ALS as well as in stabilizing the dual complex, independent of IGFs.

• Journal of Microbiology
• /
• 제37권4호
• /
• pp.248-255
• /
• 1999

We have analyzed the MRE3/REC114 gene of Saccharomyces cerevisiae, previously detected in isolation of mutants defective in meiotic recombination. We cloned the MRE3/REC114 gene by complementation of the meiotic recombination defect and it has been mapped to chormosome XIII. The DNA sequence analysis revealed that the MRE3 gene is identical to the REC114 gene. The upstream region of the MRE3/REC114 gene contains a T_4C site, a URS (upstream repression sequence) and a TR (T-rich) box-like sequence, which reside upstream of many meiotic genes. Coincidentally, northern blot analysis indicated that the three sizes of MRE3/REC114 transcripts, 3.4, 1.4 and 1.2 kb, are induced in meiosis. A less abundant transcript of 1.4 kb is detected in both mitotic and meiotic cells, suggesting that it is needed in mitosis as well as meiosis. To examine the role of the MRE3/REC114 gene, we constructed mre3 disruption mutants. Strains carrying an insertion or null deletion of the MRE3/REC114 gene showed slow growth in nutrient medium and the doubling time of these cells increased approximately by 2-fond compared to the wild-type strain. Moreover, the deletion mutant (${\delta}$mre3) displayed no meiotically induced recombination and no viable spores. The mre3/rec114 spore lethality can be suppressed by spo13, a mutation that causes cells to bypass reductional division. The double-stranded breaks (DSBs) which are involved in initiation of meiotic recombination were not detected in the analysis of meiotic chromosomal DNA from the mre3/rec114 disruptant. From these results we suggest that the MRE3/REC114 gene product is essential in normal growth and in early meiotic stages involved in meiotic recombination.

• Journal of Genetic Medicine
• /
• 제15권2호
• /
• pp.64-71
• /
• 2018

Purpose: Overgrowth syndromes are conditions that involve generalized or localized areas of excess growth. In this study, the clinical, molecular, and genetic characteristics of Korean patients with overgrowth syndrome were analyzed. Materials and Methods: We recruited 13 patients who presented with overgrowth syndrome. All patients fulfilled inclusion criteria of overgrowth syndrome. Analysis of the clinical and molecular investigations of patients with overgrowth syndrome was performed retrospectively. Results: Among the 13 patients with overgrowth syndrome, 9 patients (69.2%) were found to have molecular and genetic causes. Among the seven patients with Sotos syndrome (SS), two had a 5q35microdeletion that was confirmed by fluorescent in situ hybridization. In two patients with SS, intragenic mutations including a novel mutation, c.5993T>A (p.M1998L), were found by Sanger sequencing. One patient had one copy deletion of NDS1 gene which was confirmed by multiplex ligation-dependent probe amplification. Among five patients with Beckwith-Wiedemann syndrome, three had aberrant imprinting control regions; 2 hypermethylation of the differentially methylated region of H19, 1 hypomethylation of the differentially methylated region of Kv. In one patient displaying overlapping clinical features of SS, a de novo heterozygous deletion in the chromosomal region 7q22.1-22.3 was found by single nucleotide polymorphism-based microarray. Conclusion: Considering high detection rate of molecular and genetic abnormalities in this study, rigorous investigations of overgrowth syndrome may be an important tool for the early diagnosis and genetic counseling. A detailed molecular analysis of the rearranged regions may supply the clues for the identification of genes involved in growth regulation.

• Molecules and Cells
• /
• 제20권3호
• /
• pp.385-391
• /
• 2005

As a first step towards identifying genes involving in the signal transduction pathways mediating rice blast resistance, we isolated 3 mutants lines that showed enhanced susceptibility to rice blast KJ105 (91-033) from a T-DNA insertion library of the japonica rice cultivar, Hwayeong. Since none of the susceptible phenotypes co-segregated with the T-DNA insertion we adapted a map-based cloning strategy to isolate the gene(s) responsible for the enhanced susceptibility of the Hwayeong mutants. A genetic mapping population was produced by crossing the resistant wild type Hwayeong with the susceptible cultivar, Nagdong. Chi-square analysis of the $F_2$ segregating population indicated that resistance in Hwayeong was controlled by a single major gene that we tentatively named Pi-hy. Randomly selected susceptible plants in the $F_2$ population were used to build an initial map of Pi-hy. The SSLP marker RM2265 on chromosome 2 was closely linked to resistance. High resolution mapping using 105 $F_2$ plants revealed that the resistance gene was tightly linked, or identical, to Pib, a resistance gene with a nucleotide binding sequence and leucine-rich repeats (NB-LRR) previously isolated. Sequence analysis of the Pib locus amplified from three susceptible mutants revealed lesions within this gene, demonstrating that the Pi-hy gene is Pib. The Pib mutations in 1D-22-10-13, 1D-54-16-8, and 1C-143-16-1 were, respectively, a missense mutation in the conserved NB domain 3, a nonsense mutation in the 5th LRR, and a nonsense mutation in the C terminus following the LRRs that causes a small deletion of the C terminus. These findings provide evidence that NB domain 3 and the C terminus are required for full activity of the plant R gene. They also suggest that alterations of the resistance gene can cause major differences in pathogen specificity by affecting interactions with an avirulence factor.

• 대한수의학회지
• /
• 제52권4호
• /
• pp.223-230
• /
• 2012

The worldwide distribution and continuing genetic mutation of avian influenza virus (AIV) has been posed a great threat to human and animal health. A comparison of 3 isolates of AIV H9N2, A/chicken/Korea/KBNP-0028/00 (H9N2) (KBNP-0028), A/chicken/Korea/SNU8011/08 (H9N2) (SNU 8011) and an inactivated oil vaccine strain A/chicken/Korea/01310/01 (H9N2) (01310), was performed. The former 2 AIVs were isolated from field cases before and after the application of an inactivated H9N2 vaccine in 2007, respectively. The antigenic relationship, viral shedding, tissue tropism and genetic analysis were examined. The comparison of virus shedding from the cloaca and the oropharynx revealed that both isolates were more frequently isolated from the upper respiratory tract (90~100%) 1 day post inoculation (DPI) compared with isolation 5 DPI from gastrointestinal tracts (10~60%). Moreover, the isolate KBNP-0028 were recovered from all organs including bone marrow, brain and kidneys, indicating higher ability for broad tissue dissemination than that of SNU 8011. KBNP-0028 replicated earlier than other strains and with a higher titer than SNU 8011. In full-length nucleotide sequences of the NA gene and a partial sequence of the HA gene of SNU 8011, we found that there might be significant changes in tissue tropism, virus replication and genetic mutation in AIV H9N2 isolates.

• Journal of Microbiology and Biotechnology
• /
• 제26권4호
• /
• pp.700-709
• /
• 2016

The mitogen-activated protein kinase HOG1 (high-osmolarity glycerol response pathway) plays a crucial role in the response of yeast to hyperosmotic shock. Trichosporonoides oedocephalis produces large amounts of polyols (e.g., erythritol and glycerol) in a culture medium. However, the effects of HOG1 gene knockout and environmental stress on the production of these polyols have not yet been studied. In this study, a To-HOG1 null mutation was constructed in T. oedocephalis using the loxP-Kan-loxP/Cre system as replacement of the targeted genes, and the resultant mutants showed much smaller colonies than the wild-type controls. Interestingly, compared with the wild-type strains, the results of shake-flask culture showed that To-HOG1 null mutation increased erythritol production by 1.44-fold while decreasing glycerol production by 71.23%. In addition, this study investigated the effects of citric acid stress on the T. oedocephalis HOG1 null mutants and the wild-type strain. When the supplementation of citric acid in the fermentation medium was controlled at 0.3% (w/v), the concentration of erythritol produced from the wild-type and To-HOG1 knockout mutant strains improved by 18.21% and 21.65%, respectively.

• Journal of Microbiology and Biotechnology
• /
• 제20권2호
• /
• pp.415-424
• /
• 2010

The purpose of this study was to investigate the relationship between the global regulatory mechanism known as quorum sensing and expression of virulence factors in Escherichia coli O157:87. A nonpolar luxS deletion was introduced into the chromosome of strain CI03J, a human clinical isolate from South Korea, to create the ${\Delta}luxS$ mutant strain ML03J. Phenotypic characterization of wild-type and mutant strains demonstrated that ML03J had no obvious growth or metabolic defects on 0.2% glucose LB medium, produced a functionally defective flagellum, and could not utilize sorbose; the biological significance of sorbose utilization is unknown. Omics-based analysis revealed the involvement of LuxS in the transcriptional activation of several flagella/chemotaxisrelated genes (flhD; fliA, C, D, S, Z; and cheA, Y, Z), repression of glutamate-dependent acid resistance genes (gadAB), and expression of virulence factors including Shiga toxin, hemolysin, and SepD within the LEE pathogenicity island.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 2001년도 추계학술대회 및 정기총회
• /
• pp.108-108
• /
• 2001

Axin2/Conductin/Axil and its ortholog Axin are negative regulators of the Wnt signaling pathway, which promote the phosphorylation and degradation of ${\beta}$-catenin. While Axin is expressed ubiquitously, Axin2 mRNA was seen in a restricted pattern during mouse embryogenesis and organogenesis. Because many sites of Axin2 expression overlapped with those of several Wnt genes, we tested whether Axin2 was induced by Wnt signaling. Endogenous Axin2 mRNA and protein expression could be rapidly induced by activation of the Wnt pathway, and Axin2 reporter constructs, containing a 5.6 kb DNA fragment including the promoter and first intron, were also induced. This genomic region contains eight Tcf/LEF consensus binding sites, five of which are located within longer, highly conserved non-coding sequences. The mutation or deletion of these Tcf/LEF sites greatly diminished induction by ${\beta}$-catenin, and mutation of the Tcf/LEF site T2 abolished protein binding in an electrophoretic mobility-shift assay. These results strongly suggest that Axin2 is a direct target of the Wnt pathway, mediated through Tcf/LEF factors. The 5.6 kb genomic sequence was sufficient to direct the tissue specific expression of d2EGFP in transgenic embryos, consistent with a role for the Tcf/LEF sites and surrounding conserved sequences in the in vivo expression pattern of Axin2. Our results suggest that Axin2 participates in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal.