• Journal of Microbiology and Biotechnology
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• 제22권10호
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• pp.1336-1342
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• 2012

In Burkholderia pseudomallei, the pathogen that causes melioidosis, the gene cluster encoding the capsular polysaccharide, is located on chromosome 1. Among the 19 capsular genes in this cluster, wzm has not been thoroughly studied. To study the function of wzm, we generated a deletion mutant and compared it with the wild-type strain. The mutant produced less biofilm in minimal media and was more sensitive to desiccation and oxidative stress compared with the wild-type strain, indicating that wzm is involved in biofilm formation and membrane integrity. Scanning electron microscopy showed that the bacterial cells of the mutant strain have more defined surfaces with indentations, whereas cells of the wild-type strain do not.

• Journal of Interdisciplinary Genomics
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• 제5권1호
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• pp.1-4
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• 2023

Kleefstra syndrome is caused by chromosome 9q34.3 deletion or heterozygous mutations in the Euchromatin Histone Methyl Transferase 1 (EHMT1) gene. The prevalence is estimated 1:25,000 to 1:35,000. Intellectual disability, distinctive facial features, hypotonia in childhood can be accompanied. The spectrum of Kleefstra syndrome includes behavioral/psychiatric problems, hearing and visual impairments, seizures, congenital heart defects, genitourinary defects, and obesity. Therefore, it is necessary to understand the pathophysiology and various manifestation of Kleefstra syndrome and discussing with a multidisciplinary team will help diagnose and treat Kleefstra syndrome patients.

• Clinical and Experimental Reproductive Medicine
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• 제30권3호
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• pp.203-206
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• 2003

Objectives: Controversial arguments exists on both the case for and against on the accumulation of mitochondrial DNA (mtDNA) deletion in association to tissue and age. The debate continues as to whether this mutation is a major contributor to the phenotypic expression of aging and common degenerative diseases or simply a clinical insignificant epiphenomenon. The objective of this study was to determine whether the accumulation of mtDNA deletion is correlated with age-related and tissue-specific variation. Materials and Methods: One hundred and fifty-seven tissues from blood, ovary, uterine muscle, and abdominal muscle were obtained from patients ranging in age from 31$\sim$60 years. After reviewing the clinical reports, patients with mitochondrial disorder were excluded from this study. The tissues were obtained at gynecological surgeries with the consent of the patient. Total DNA isolated from blood, ovary, uterine muscle, and abdominal muscle was amplified by two rounds of PCR using two pairs of primers corresponding to positions 8225-8247 (sense), 13551-13574 (antisense) for the area around deleted mtDNA and 8421-8440 (sense), 13520-13501 (antisense) for nested PCR product. A statistical analysis was performed by $x^2$-test. Results: About 0% of blood, 94.8% of ovary, 71.4% of uterine muscle, and 86.1% abdominal muscle harbored mtDNA deletion. When we examined the proportion of deleted mtDNA according to age deletion rate was 90% of ovary, 63.6% of uterine muscle, 77.7% of abdominal muscle in thirties and 100% of all tissue in fifties. Conclusion: The findings of this study suggest that the mtDNA deletion is varied in tissue-specific pattern and increases with aging.

• The Plant Pathology Journal
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• 제24권1호
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• pp.1-7
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• 2008

Fusarium verticillioides (teleomorph Gibberella moniliformis) is associated with maize worldwide causing ear rot and stalk rot, and produces fumonisins, a group of mycotoxins detrimental to humans and animals. While research tools are available, our understanding of the molecular mechanisms associated with fungal virulence and fumonisin biosynthesis in F. verticillioides is still limited. One of the restraints that hampers F. verticillioides gene characterization is the fact that homologous recombination (HR) frequency is very low (<2%). Screening for a true gene knock-out mutant is a laborious process due to a high number of ectopic integrations. In this study, we generated a F. verticillioides mutant (SF41) deleted for FvKU70, a gene directly responsible for non-homologous end-joining mechanism, with the aim of improving HR frequency. Here, we demonstrate that FvKU70 deletion does not impact key Fverticillioides phenotypes, e.g., development, secondary metabolism, and virulence, while dramatically improving HR frequency. Significantly, we also confirmed that a high percentage (>85%) of the HR mutant strains harbor a desired mutation with no additional copy of the mutant allele inserted in the genome. We conclude that SF41 is suitable for use as a type strain when performing high-throughput gene function studies in F. verticillioides.

• The Plant Pathology Journal
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• 제32권4호
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• pp.371-376
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• 2016

Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1416-1421
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• 2006

We identified a gene encoding the N-acetylmuramoyl L-alanine amidase (amiB) of Vibrio anguillarum, which catalyzes the degradation of peptidoglycan in bacteria. The entire open reading frame (ORF) of the amiB gene was composed of 1,722 nucleotides and 573 amino acids. The deduced amino acid sequence of AmiB showed a modular structure with two main domains; an N-terminal region exhibiting an Ami domain and three highly conserved, continuously repeating LysM domains in the C-terminal portion. An amiB mutant was constructed by homologous recombination to study the biochemical function of the AmiB protein in V. anguillarum. Transmission electron microscopy (TEM) revealed morphological differences, and that the mutant strain formed trimeric and tetrameric unseparated cells, suggesting that this enzyme is involved in the separation of daughter cells after cell division. Furthermore, inactivation of the amiB gene resulted in a marked increase of sensitivity to oxidative stress and organic acids.

• Animal cells and systems
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• 제2권1호
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• pp.81-86
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• 1998

Recent identification and characterization of plant homeobox genes suggest that they play important roles in morphogenetic events. OSH1, one of the rice homeobox genes, is thought to be related to organ development since the changes of OSH1 gene expression cause morphological abnormalities of leaves by the ectopic expression and is expressed during early embryogenesis. In this experiment, the expression pattern of OSH1 was analyzedinmutants by in situ hybridization, and OSH1's potential as a molecular marker was explored. Region-specific expression of OSH1 during early embryogenesis shows that OSH1 could be used as a molecular marker for characterizing embryo mutants. Although several organless and shootless mutants showed normal expression of OSM1, some mutants exhibited abnormal expression patterns. In a minute organless cle1-1 embryo whose epidermis resembled morphologically the epithelium of scutellum, OSH1 expression was limited to a small basal region. This expression pattern suggests the gross deletion of the basal part. In a radicleless mutant, odm115, OSH1 expression was detected in a basal region instead of subcentral region of the ventral side. Together with other characteristics (short embryo and normal adventitious roots), odm115 was estimated to be derived from the deletion of basal region. Among five shootless mutants, three showed normal expression of OSH1. In the shl2 embryo, no expression of OSH1 was observed. In the shl1 embryo, however, OSH1 expression was extended to a dorsal side, indicating that SHL2 might be related to dorsoventral patterning. The above results of in situ hybrydization clearly indicate that OSH1 can be utilized as a marker for characterizing gene functions of embryo mutants.

• The Plant Pathology Journal
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• 제30권4호
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• pp.367-374
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• 2014

Genomes contain a large number of unique genes which have not been found in other species. Although the origin of such "orphan" genes remains unclear, they are thought to be involved in species-specific adaptive processes. Here, we analyzed seven orphan genes (MoSPC1 to MoSPC7) prioritized based on in planta expressed sequence tag data in the rice blast fungus, Magnaporthe oryzae. Expression analysis using qRT-PCR confirmed the expression of four genes (MoSPC1, MoSPC2, MoSPC3 and MoSPC7) during plant infection. However, individual deletion mutants of these four genes did not differ from the wild-type strain for all phenotypes examined, including pathogenicity. The length, GC contents, codon adaptation index and expression during mycelial growth of the four genes suggest that these genes formed during the evolutionary history of M. oryzae. Synteny analyses using closely related fungal species corroborated the notion that these genes evolved de novo in the M. oryzae genome. In this report, we discuss our inability to detect phenotypic changes in the four deletion mutants. Based on these results, the four orphan genes may be products of de novo gene birth processes, and their adaptive potential is in the course of being tested for retention or extinction through natural selection.

• Journal of Microbiology and Biotechnology
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• 제27권1호
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• pp.171-178
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• 2017

A series of novel effector molecules secreted by the type three secretion system (T3SS) of Shigella spp. have been reported in recent years. In this study, a proteomic approach was applied to study T3SS effectors systematically. First, proteins secreted by the S. flexneri wild-type strain after Congo Red induction were separated and identified using two-dimensional electrophoresis to display the relative abundance of all kinds of early effectors for the first time. Then, a gene deletion mutant of known virulence repressor (OspD1) and a gene overexpressed mutant of two known virulence activators (MxiE and IpgC) were constructed and analyzed to discover potential late effectors. Furthermore, the supernatant proteins of gene deletion mutants of two known translocators (IpaB and IpaD), which would constantly secrete effectors, were also analyzed. Among all of the secreted proteins identified in our study, IpaH1.4, IpaH_5, and IpaH_7 have not been reported before. These proteomics data of the secreted effectors will be valuable to understand the pathogenesis of S. flexneri.

• Molecules and Cells
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• 제40권6호
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• pp.426-433
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• 2017

Ephrin-A5 has been implicated in the regulation of brain morphogenesis and axon pathfinding. In this study, we used bacterial homologous recombination to express a LacZ reporter in various ephrin-A5 BAC clones to identify elements that regulate ephrin-A5 gene expression during mesencephalon development. We found that there is mesencephalon-specific enhancer activity localized to a specific +25.0 kb to +30.5 kb genomic region in the first intron of ephrin-A5. Further comparative genomic analysis indicated that two evolutionary conserved regions, ECR1 and ECR2, were present within this 5.5 kb region. Deletion of ECR1 from the enhancer resulted in disrupted mesencephalon-specific enhancer activity in transgenic embryos. We also found a consensus binding site for basic helix-loop-helix (bHLH) transcription factors (TFs) in a highly conserved region at the 3'-end of ECR1. We further demonstrated that specific deletion of the bHLH TF binding site abrogated the mesencephalon-specific enhancer activity in transgenic embryos. Finally, both electrophoretic mobility shift assay and luciferase-based transactivation assay revealed that the transcription factor Ascl1 bound the bHLH consensus binding site in the mesencephalon-specific ephrin-A5 enhancer in vitro. Together, these results suggest that the bHLH TF binding site in ECR1 is involved in the positive regulation of ephrin-A5 gene expression during the development of the mesencephalon.