Journal of Microbiology

Research Support, Non-U.S. Gov'ts

Cloning, Annotation and Expression Analysis of Mycoparasitism-Related Genes in Trichoderma harzianum 88: Lin Yao , Qian Yang , Jinzhu Song , Chong Tan , Changhong Guo , Li Wang , Lianhai Qu , Yun Wang; J. Microbiol. 2013;51(2):174-182. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2545-7

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Abstract: Trichoderma harzianum 88, a filamentous soil fungus, is an effective biocontrol agent against several plant pathogens. High-throughput sequencing was used here to study the mycoparasitism mechanisms of T. harzianum 88. Plate confrontation tests of T. harzianum 88 against plant pathogens were conducted, and a cDNA library was constructed from T. harzianum 88 mycelia in the presence of plant pathogen cell walls. Randomly selected transcripts from the cDNA library were compared with eukaryotic plant and fungal genomes. Of the 1,386 transcripts sequenced, the most abundant Gene Ontology (GO) classification group was “physiological process”. Differential expression of 19 genes was confirmed by real-time RT-PCR at different mycoparasitism stages against plant pathogens. Gene expression analysis revealed the transcription of various genes involved in mycoparasitism of T. harzianum 88. Our study provides helpful insights into the mechanisms of T. harzianum 88-plant pathogen interactions.

cDNA Cloning of Korean Human Norovirus and Nucleotidylylation of VPg by Norovirus RNA-Dependent RNA Polymerase: Byung Sup Min , Kang Rok Han , Jung Ihn Lee , Jai Myung Yang; J. Microbiol. 2012;50(4):625-630. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2087-4

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Abstract: Norovirus, a member of the Caliciviridae family, is a major causative agent of gastroenteritis worldwide. The cDNA of the entire genome of human norovirus (HuNV) was cloned using the RNA extracted from the stool sample of a Korean patient. The RNA genome consists of 7,559 nucleotides, carries 3 open reading frames (ORFs), 5' and 3' noncoding regions, and a poly(A) tail at the 3' end. Phylogenic analysis of the nucleotide sequence indicated that it belongs to GII.4, the most dominant genogroup. To analyze RNA synthesis and nucleotidylylation of VPg by RNA-dependent RNA polymerase (RdRp), recombinant RdRp and VPg were expressed in Escherichia coli as His-tagged forms. The HuNV RdRp exhibited template and divalent cation-dependent RNA synthesis in vitro. The HuNV RdRp nucleotidylylated HuNV VPg but not murine norovirus (MNV) VPg, whereas MNV RdRp nucleotidylylated both MNV and HuNV VPg more efficiently than HuNV RdRp.

Gene Expression Analysis of Phanerochaete chrysosporium During the Transition Time from Primary Growth to Secondary Metabolism: Mingfeng Jiang , Xiao Li , Liang Zhang , Hong Feng , Yizheng Zhang; J. Microbiol. 2009;47(3):308-318. Published online June 26, 2009; DOI: https://doi.org/10.1007/s12275-008-0275-z

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Abstract: In order to identify the secondary metabolism-related genes of Phanerochaete chrysosporium growing under pure O2 and nitrogen-limited conditions, 2322 ESTs fragments originated from two suppression-subtractive libraries were analyzed using the cDNA microarray technique. Ten significantly upregulated and 22 significantly downregulated genes were identified in the 72 h cultured mycelia RNA samples (secondary metabolism). According to qPCR, 16 out of the 32 genes were expressed differently in secondary metabolism. Transcripts of secondary metabolism up-regulation genes exhibited homologies to aryl-alcohol dehydrogenase (SSh1554), ABC transporter gene (SSH624), chitinase (SSH963), heat shock protein (SSH1193), catalase (SSH317), cytochrome P450 (SSH331), glucosamine-6-phosphate isomerase (SSH611), and alkyl hydroperoxide reductase (SSH362) genes. Ninety-three genes could be classified by Eukaryotic Orthologous Groups (KOG). Among the genes assigned a function, gene expression patterns were different in both secondary metabolism and primary metabolism. In the group of “Cellular Processes and Signaling,” most of the genes were from the primary metabolism library. On the other hand, genes from the secondary metabolism library were found mainly in the “Information Storage” and “Processing and Poorly Characterized” groups. Based on the KOG functional assignments, six genes belong to the ubiquitin system, and all of them were from primary metabolism phase. The presence of the H2O2-relevant genes suggested that parts of the genes expressed in 72 h might be involved in the ligninolytic process during secondary metabolism of P. chrysosporium.

Generation of Infectious Transcripts from Korean Strain and Mild Mottle Strain of Potato Virus X: Sun Hee Choi , Ki Hyun Ryu; J. Microbiol. 2008;46(5):502-507. Published online October 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0078-2

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Abstract: Full-length cDNAs of two different strains of Potato virus X (PVX-Kr and PVX-Mo) have been directly amplified by long template reverse transcription polymerase chain reaction (RT-PCR) using the 5’-end primer containing a SP6 or T7 RNA promoter sequence and the virus-specific 3’-end primer, and then constructed in plasmid vectors. Capped in vitro transcripts from cloned full-length cDNAs as well as those RTPCR amplicons proved to be infectious systemically on tobacco plants. Symptom expression on tobacco plants from PVX-Mo transcripts was faster and severer than that from PVX-Kr. In replication stability test of transcripts derived from PVX clones, progeny viruses showed stable replication according to sequencing through passages. This highly infectious transcript system from the full-length cDNA clones for PVX can be useful for recombinant molecules for functional analysis of viral proteins in plant-virus interaction study as well as for expression of foreign protein in planta.

The Identification of a Novel Pleurotus ostreatus dsRNA Virus and Determination of the Distribution of Viruses in Mushroom Spores: Yeo Jin Kim , Ji Yeon Kim , Ji Hye Kim , Seon Mee Yoon , Young-Bok Yoo , Se Won Yie; J. Microbiol. 2008;46(1):95-99.; DOI: https://doi.org/10.1007/s12275-007-0171-y

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Abstract: Double-stranded RNAs and virus particles were identified in Pleurotus ostreatus strain Shin-Nong in Korea. Isometric virus particles with a diameter of 33 nm were purified, which are similar to other Pleurotus viruses reported previously. This strain contains 5 dsRNAs, 8.0, 2.5, 2.4, 2.0, and 1.8 kb in size. The virus particles contain 2 dsRNAs, designated RNA-1 (2.5 kb), and RNA-2 (2.4 kb) which is a typical pattern of Partitiviridae. A non-encapsidated dsRNA of about 8.0 kb also was identified. Partial cDNA from RNA-1 was cloned, and sequence analysis revealed that this gene codes for RdRp. The comparison of the sequence from partial cDNA clone showed 35% amino acid homology with the C-terminal end of the RdRp gene of Helicobasidum mompa virus and Rosalinia necatrix virus. Specific primers designed from the partial sequences successfully amplified RT-PCR product from the infected mycelium and a single spore culture. We used these primers to determine the pattern of distribution of viruses in spores. Of the 96 different single spore cultures generated from Shin-Nong strain, a specific RT-PCR product was identified in 25 cultures, indicating that about 26% of basidiospores contain viruses.

Journal Article

Molecular Characteristics of Two Laccase from the Basidiomycete Fungus Polyporus brumalis: Sun-Hwa Ryu , A-Young Lee , Myungkil Kim; J. Microbiol. 2008;46(1):62-69.; DOI: https://doi.org/10.1007/s12275-007-0110-y

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Abstract: Two laccase cDNAs, pblac1 and pblac2, were cloned from a white-rot fungus strain, Polyporus brumalis (KFRI 20912). The cloned cDNAs consisted of 1,829 bp and 1,804 bp, and their open reading frames encoded proteins of 520 and 524 amino acids, with calculated molecular masses of approximately 55.9 kDa and 56 kDa, respectively. The deduced amino acid sequences of each protein showed 70% similarity. The copper binding regions were conserved in both proteins, as in other fungal laccases. RT-PCR analysis revealed that the transcript levels of the two laccases increased progressively in shallow stationary culture liquid medium. The transcript level of each laccase was induced when the fungus was exposed to di-butyl phthalate (DBP), suggesting that the two laccases are involved in DBP degradation. The overexpression of the pblac1 gene was derived by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase, using a homologous system. The activity of laccase in the transformants was significantly higher than that of the wild type. The identification of these laccase cDNAs was a first step to characterize the molecular events related to the lignin degradation ability of this basidiomycetous fungus, as well as the degradation of many recalcitrant xenobiotics.

Cloning and sequence determination of α-tubulin, β-tubline and Flagellar Calmodulin cDNAs of Naegleria gruberi: Choi, Youn Jeong , Park, Hye Lee , Lee, Joo Hun; J. Microbiol. 1995;33(1):40-45.

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Abstract: Five cDNAs encoding two α-tubulins(α13 and α15), two β-tubulins(β5 and β5), and one flagellar calmodulin (Cal-1) were cloned from naegleria gruberi NB-1 and their nt sequences were determined. The α13(EMBL number X81049) and β1(EMBL number X81050) contained a complete open reading frame for α-tubulin and β-tubulin, respectively. The other three clones (α15, β5 and Cal-1) had a part of coding region and a 3’ untranslated region of the respective genes. The α13, β1 and Cal-1 had no homologous sequences in the coding regions and in the 3’ untranslated sequences. However, the α13 and β1 shared an eight nucleotide (AATACAAA) sequence in front of the respective initiation codons. The AATACAAA sequence was also found in N. gruberi strain NEG α-tubulin cDNA clone(αpT1) at the same position. Comparison of the α13 to the αpT1 revealed another stretch of identical sequence, which is 30 nts long, in the 5’ untranlated region.

Quantitative analysis of gene expression pattern in aspergillus nidulans mycelia by sequencing of 3'-directed cDNA clones: Park, Yoon Dong , Lee, Dong Whan , Lee, Seog Jae , Kim, Jong Hwa , Chae, Keon Sang; J. Microbiol. 1996;34(1):23-29.

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Abstract: Since sequencing of randomly selected cDNA clones has been known to be a powerful approach to obtain information on gene expression pattern in specific cells or tissues, we have analyzed a 3'-directed cDNA library of vegetative mycelia of A. nidulans by single-pass sequencing of hundreds of randomly selected clones. Sequencing of 292 cDNA clones yielded 209 gene signatures (GSs) probably representing highly or lesser expressed genes in the vegetative mycelia. Among the 209 GSs, 25 (79 cDNA clones) appeared more than once and 184 only once. One GS appeared at a highest frequency of 6 times, 2 GSs5 times, 4 GSs 4 times, a GSs 3 times and 16 GSs twice. About 6.6% GSs comprizing of 13 GSs showed alternative polyadenylation. Among 23 redundant GSs, three were common in both mycelia and sexual organs, and 22 were probably mycelia-specific. Out of 209 GSs, 36 were identified in GenBank showing of 70% or greater similaritis. Only six GSs were for A. nidulans genes, and 13 GSs were of DNA or genes encoding cytoplasmic or organellar proteins. This pattern is similar to those in the human HepG2 cell line and in human colonic mucosa, although very few genes for nuclear proteins and for protein synthesis were in A. nidulans.

cDNA cloning and expresion of human rotavirus outer capsid protein VP4: Kang, Seok W. , Yang, Jai M.; J. Microbiol. 1998;36(3):214-221.

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Abstract: cDNA for the VP4-coding RNA segment 4 of human rotavirus isolated from Korean patients Was synthesized and cloned (HRV-k41), and its nucleotide sequence was determined. Comparative analysis of the nucleotide sequence of JRV-k41 showed 90.6%, 86.6%, 74.6%, 66%, 70.1% and 65.4% homology to P1A[8](Wa), P1B[4](RV5), P2[6](1076), P3[9](AU1), P4[10](69M), and P[14](PA169) genotypes respectively. The deduced amino acid sequence homology of HRV-k41 to P1A[8](Wa), P1B[4](RV5), P2[6](1076), P3[9](AU1), P4[10](69M), and P[14](PA169) genotypes respectively. The deduced amino acid sequence homology of HRV-k41 to P1A[8](Wa), P1B[4](RV5), P2[6](1076), P3[9](AU1), P4[10](69M) and P[14](169) was 92.9% 89.7%. 75.8%, 64.1%, 70.6% and 64.3% respectively. These results suggest that HRV-k41 is closely related to the P1A genotype. Two trypsin cleavage sites (arginine 240 and arginine 246) and four cysteine residues (215, 317, 379, and 773) conserved in VP4 of all rotavirus strains were also found in JRV-k41. Similar to other virulent human rotaviruses, an additional trypsin cleavage site(lysine 245) was also detected in this strain. The cDNA of the VP4-coding RNA segment was cloned into pGEX-4T-3, an Escherichia coli expression vector, and it's expression was confirmed by Western-blot analysis.

Quantitative Analysis of Expressed Genes in Aspergillus Oryzae by Sequencing 3'-directed cDNA Clones: Hwang, Hyun Ah , Lee, Dong Whan , Kim, Jong Hwa , Lee, Tae Kyoo , Yang, Moon Sik , Chae, Keon Sang; J. Microbiol. 1998;36(2):111-117.

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Abstract: Sequence analysis of randomly selected 3'-directed cKNA clones has been known to be one of the most powerful methods of examining the genes highly expressed in a tissue or cell type. We constructed a 3'-directed cDNA libraty from Aspergillus oryzae mycelia, and sequenced 345 randomly selected 3'-directed cDNA clones. Determined nucleotide sequences, not shorter than 30nt, were compared with one other to generate gene signatures (GSs) and were then compared with GenBank entries to analyze sequence similarity to known genes. A GS for the most highly expressed gene appeared six times, one GS five times, five GSs four times, five GSs three times and 22 GSs twice. In total, 324 clones yielded 268 GSs consisting of 34 redundant GSs appeaning at least twice and 234 solitary ones. Forty-three GSs showed similarities ranging from 60% to 99% with known sequences from Genbank. A considerable number of A. oryzae GSs mateched those obtained from the sexual structures of A. nidulans suggests that A. oryzae may not be phylogentically distant from A. nidulans and that A. oryzae may have a sexual life cycle from the ancient period.

Cloning and Regulation of Schizosaccharomyces pombe Gene Encoding Ribosomal Protein S20: Yoon-Jong Lee , Kyunghoon Kim , Eun-Hee Park , Ki-Sup Ahn , Daemyung Kim , Chang-Jin Lim; J. Microbiol. 2001;39(1):31-36.

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Abstract: A cDNA clone encoding the ribosomal protein S20 has been isolated from the Schizosaccharomyces pombe cDNA library by colony hybridization. The insert contained in the original plasmid pYJ10 was transferred into shuttle vector pRS316 to generate plasmid pYJ11. The cDNA insert of plasmid pYJ11 contains 484 nucleotides and encodes a protein of 118 amino acids with a calculated mass of 13,544 daltons. The deduced amino acid sequence of S. pombe ribosomal protein S20 is very homologous with fruit fly, rat, and budding yeast counterparts. It is also homologous with Xenopus S22 ribosomal protein. S. pombe ribosomal protein S20 appears to be relatively hydrophobic except the C-terminal region. The 728 bp upstream region of the S20 gene was amplified from chromosomal DNA and transferred into the BamHI/EcoRI site of the promoterless b-galactosidase gene of the vector YEp357R, which resulted in fusion plasmid pYS20. The synthesis of b-galactosidase from the fusion plasmid appeared to be the highest in the mid-exponential phase. The S. pombe cells with the fusion plasmid grown at 35oC gave lower b-galactosidase activity than the cells grown at 30oC. Computer analysis showed the consensus sequence CAGTCACA in the upstream regions of various ribosomal protein genes in S. pombe, which would be involved in the coordinated expression of small ibosomal proteins.

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