Journal of Microbiology

Volume 34(1); March 1996

Restriction pattern of the nucleic acid of Synechococcus sp. cyanophage: Park, Jong Geun , Kim, Min , Choi, Yong Keel , Yoon, Sung Nyu; J. Microbiol. 1996;34(1):1-6.

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Abstract: The nucleic acid of Synechococcus sp. cyanophage was identified as double-stranded DNA by the result of digestion with enzymes such as exonucleases, DNase, and S1 nuclease, and by acridine orange staining. The cyanophage DNA was cleaved with several restriciton ehdonucleases such as ApaI, BamHI, Bg/II, HaeIII, Eco RI, HindIII, PstI, AND aPAI gave the clearest sets of bands on agarose gels and the fragment numbers for each were 12, 20, 29, 20, and 7, respectively. The sums of the size from Bam HI and PstI digestions were estimated approximately 227±4 kb, which are in agreement with the result of the pulsed field gel electrphoresis. This virus is thought to have the largest genosome among those of known cyanophages, which corresponds to the largest head of 90 nm when compared with the head sizes of cyanophages discovered since 1963.

Analysis of fusogenic activity of autographa californica nuclear polyhedrosis virus (AcNPV) gp64 envelope glycoprotein: Kim, Hee Jin , Yang, Jai Myung; J. Microbiol. 1996;34(1):7-14.

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Abstract: The baculovirus gp64 glycoprotein is a major component of the envelope of budded virus (BV) and has been shown that it plays an essential role in the infection process, especially virus-cell membrane fusion. We have cloned Autographa californica Nuclear Polyhedrosis Virus (AcNPV) gp64 protein were examined for membrane fusion activity by using a synchtium formation assay under various conditions. The optimal conditions required for inducing membrane fusion are 1) form pH 4.0 to 4.8 2) 15 min exposure of cells to acidic pH 3) at least 1㎍ of gp64 cloned plasmid DNA per 3 × 10^6 cells 4) and an exposure of cells to acidic pH at 72 h post-transfection. In order to investigate the role of hydrophobicity of the gp64 glycoprotein for the membrane fusion, the two leucine residues (amino acid position at 229 and 230) within hydrophobic region I were substituted to alanine by PCR-derived site-directed mutagenisis and the membrane fusion activity of the mutant was analysed. The gp64 glycoprotein carrying double alamine substitution mutation showed no significant difference in fusion activity. This result suggested that minor changes in hydrophobicity at the amino acid position 229 and 230 does not affect the acid-induced membrane fusion activity of the gp64 glycoprotein.

Characterization of promotor sequences for strong expression of groEx IN Escherichia coli.: Lee, Jung E. , Lim, Ssang T. , Aha, Tae I.; J. Microbiol. 1996;34(1):15-22.

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Abstract: The cloned X-bacterial gene (groEx) which is analogous to groE of E. Coli strongly expressed in E. coli when grown at the temperature 27℃ or higher without having to add any inducers. By S1-nuclease mapping, primer extension analysis and site directed mutagenesis, we found 4 promoters in the gene. Among them two promoters located at 5'-extended region of the gene are homologous to the promoters found in groE family of heat-shock genes ; they are , σ^32 factor-dependent P1 promotor and σ^70 factor-dependet P2 promoter. The other two promoters found within the coding region of groESx were P3, 5'-TTGGCG-(18 bases)-AATACT-3' and P4, 5'-TTGGCA-(19 bases)-TAAGT which overlapped within 49 bases. These unique intragenic σ^70-dependent promoters are the first to be cloned and characterized in groE analogous heat-shock genes so far. These P3 and P4 promoters appeared to be responsible for the strong expression of GroElx in X-bacteria in vivo.

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.

DNA Replication is not Required in Re-establishment of HMRE Silencer Function at the HSP82 Yeast Heat Shock Locus: Lee, See Woo , Gross, David S.; J. Microbiol. 1996;34(1):30-36.

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Abstract: We have examined the re-establishment of HIMRE mediated silencing function on the transcriptional activity of yeast heat shock gene HSP82. To test whether the onset of SIR repression can occur in growing cells in the presence of a potent inhibitor of DNA replication, HMRa/HSP82 strains with SIR4^- and SIR4S^+ genetic backgrounds were arrested in S phase by incubation of a culture in 200 mM hydroxyurea for 120 min. It was clear that following a 20 minute heat shock, silencing of the HMRa/HSP82 allele in cells pretreated with hydroxyurea does occur in a SIR4-dependen fashion, even though the kinetics of repression appears to be substantially delayed. We also have tested whether re- establishment of silencing at the HMR/hsp82 locus can occur in G1-arrested cells. Cell cycle arrest at G1 phase was achieved by treatment of early log a cell cultures with α-factor mating pheromone, which induces G1 arrest. The result suggests that passage through S phase (and therefore DNA replication) is nor required for re-establishing silencer-mediated repression at the HMNRa/HSP82 locus. Finally, to test whether de novo protein synthesis is required for re-establishment of silencer-mediated repression, cells were pretreated with cycloheximide (500 ㎍/㎖) 120 min. It was apparent that inhibiting protein synthesis delays, but does not prevent, re-establishment of silencer-mediated repression. Altogether, these results indicate that re-establishment of silencer-mediated repression is not dependent on the DNA replication and has no requirement for protein synthesis.

Phylogenetic analysis of pleurotus species based on the nuclear SSU rRNA sequences: Jeong, Jae Hoon , Kim, Eun Kyoung , Roe, Jung Hye; J. Microbiol. 1996;34(1):37-39.

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Abstract: The internal regions of nuclear small subunit rRNA from 6 plaeurotus species and 5 Pleurotus ostreatus strains were amplified by PCR and sequenced. The DNA sequences of 8 Pleurotus strains (P. ostreatus NFFA2, NFFA4501, NFFA4001, KFFA4001, KFCC11635, P florida, P. florida, P. sajor-cuju, P. pulmonarius, and P. spodoleucus) were idential, but P. cornucopiae differed from them in two bases out of 605 bases. However, phylogenetic analysis of the sequences by DNA-distance matrix and UPGMA methods showed that P. ostreatus NFFA2m1 and NFFA2m2, known as mutants of P. ostreatus NFFA2, belonged to anther group of Basidiomycotina, which is close to the genus Auricularia. The difference of the SSU rDNA sequences of P. cornucopiae from other Pleurotus species tested corresponds to the difference of mitochondrial plasmid type present in Pleurotus species as observed by Kim et al. (1993, Korean J. Microbiol. 31, 141-147).

Role of chromatin structure in HMRE mediated transcriptional repression of the HSP82 heat shock gene: Lee, See Woo , Gross, Davis S.; J. Microbiol. 1996;34(1):40-48.

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Abstract: We have examined the chromatin structure of the HMRE/HSP82 and HMRa/HSP82 allels using three complementary approaches : DNase I chromating footprinting, micrococcal nuclease (MNase) nucleosome-protected ladder assay, and an in vivo E. coli dam methylase accessibility assay. The footprinting results indicate that the promoter and silencer sequences are assembled into nucleoprotein complexes which exhibit no detectable change in structure, despite a 70-fold range in expression levels. In addition, the promoter region of the HMRa/HSP82 allele is cleaved randomly by MNase in all cases, indicating the absence of anonical nucleosomes over this region irrespective of SIR4 or heat-shock. Finally, no discernible difference in the accessibility of the HMRE/HSP82 locus to dam methylase in SIR4 vs. sir4 cells was seen, which again suggests that the chromatin structure of HMRE/HSP82 allele is identical regardless of SIR4. Altogether, our results indicate that in contrast to other observations of the silent mating-type loci, no discernible structural alteration is detected at either HMR/HSP82 allele regardless of SIR genetic background or transcriptional state of the gene.

Effects of K^+ lon on in vitro RNA splicing of T4 phage thymidylate synthase gene: Sung, Jung Suk , Park, In Kook; J. Microbiol. 1996;34(1):49-53.

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Abstract: The effects of K^+ ion on the activity of RNA splicing of T4 phage thymidylate synthase gene have been investigated. The splicing activity was stimulated within the range of 5 to 20 mM concentration of KCI. When the concentration of KCI in the splicing reaction was brought to 100 or 200 mM a small amount of the exonl-intron product (1, 4 kb) was formed with large proportion of primary RNA transcript not undergoing splicing. This observation strongly suggests that there may exist come kinds of interferences with transesterification at the first step of splicing. Overall it can be concluded that K^+ ion exhibits very unique roles in RNA splicing of td gene depending on its concentration.

Reorganization of chromatin conformation from an active to an inactive state after cessation of transcription: Lee , Myeong Sok; J. Microbiol. 1996;34(1):54-60.

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Abstract: Taking advantage of the heat inducible HSP82 gene in yeast, chromatin structure after transcription cessation was investigated. Alteration of chromating conformation within the HSP82 gene transcription unit into an active state has been shown to correlate with its transcriptional induction. It was thus of interest to examine whether the active chromatin state within the HSP82 mRNA analysis, the gene ceased its transcription within a few hours of cultivation at a normal condition after heat induction. In this condition, an active chromatin conformation in the HSP82 gene body was changed into an inactive state which was revealed by DNase I resistance and by typical nucleosomal cutting periodicity in the corresponding chromatin. These results thus ruled out the possibility of a long-term maintenance of the DNase I sensitive chromatin after transcription cessation. DNA replication may be a critical event for the chromatin reprogramming.

Infectious RNA viruses in the edible mushroom pleurotus spp: Park, Jeong Soo , Kim, Young Ho; J. Microbiol. 1996;34(1):61-67.

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Abstract: Double-stranded RNA (dsRNA) viruses and single-stranded RNA(ssRNA) viruses were detected in a strain of Pleurotus mushroom cultivated in a farm. Those fungal viruses were purified in the pH 6.0 or pH 7.2 using CsCI or Cs₂SO₄buoyant density centrifugation. Each viral particles were not completely separated at any trials. However, mushroom bacili-form virus contains a single major nucleic acid with 0.7 Kb ssRNA, which might code for 20 Kd viral capsid protein. The dsRNAs are encapsidatred into spherical-form viruses, whereas ssRNA viral genomes are encapsidated into two different sizes of bacili-form particles. A healthy-looking mushroom also contained some spherical-form viruses with dsRNAs. Laboratory strains of Pleurotus ostreatus and a cultivated strain of P. sajor-caju did not show any viral particles. Mushrooms with specific disease symptoms. however, contained at least four different sizes of spherical-form viruses. Thus, we concluded that a bacilli-form virus case a severe disease symptoms of abnormal on mushroom development.

Cloning and mulecular characterization of a nprX gene of bacillus subtilis NS15-4 encoding a neutral protease: Lee, Seung Hwan , Yoon, Ki Hong , Nam, Hee Sop , Oh, Tae Kwang , Lee, Seog Jae , Chae, Keon Sang; J. Microbiol. 1996;34(1):68-73.

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Abstract: An nprX gene of Bacillus subtilis NS15-4 encoding a neutral protease was cloned and its molecular characteristics were analyzed. The complete nucleotide sequence indicated that there is an open reading frame (0RF) possibly encoding 521 amino acid polypeptide. The ORF used all codons expected two cysteine and a proline having a codon bias index (CBI) of 0.09 in Escherichia coli. There were homologous sequences to the consensus sequence of -35 and -10 regions of E. coli promoters and to a Shine-Dalgarno (SD) sequence located 25 bp downstream of a major transcription initiation site. Moreover, there were also five minor transcription initiation sites at 6. 7. 8. 14 and 15 nt downstream of the major site. Northern blot analysis revealed the presence of about 1.8 kb mRNA transcript in E. coli having the nprX gene. The nucleotide sequence was identified in GenBank to be a gene for a neutral protease of B. subtilis with six nucleotide difference in the ORF region. The flanking regions of the NprX ORF showed much more differences form those of other neutral protease genes except the nprE gene of B. subtilis, which has the most homology to the nprX gene, and of which the flanking regions were identical to those of the nprX gene.

Construction of secretion vectors using the α-amylase signal sequence of bacillus subtilis NA64: Kim, Sung Il , Lee, Se Yong; J. Microbiol. 1996;34(1):74-81.

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Abstract: Two secretion vectors, pUBA240 and pUB340 were constructed by using the promoter and secretory signal region of the α-amylase gene from an α-amylase hyperproducing strain, Bacillus subtilis NA64. In this secretion vector system, various restriction enzyme sites are located immediately after the proregion of the α-amylase gene for easy replacement of various foreign structural genes. To evaluate this secretion vectors, the β-lactamase gene of pBR322 was used as a reporter gene. The expressed and biologically active β-lactamase was secreted into the culture broth from B. subtilis LKS86 transformants harboring each β-lactamase secreting plasmid, pUBAbla and pUBSble. In both cases, more than 92% of expressed β- lactamases were located idn the culture medium. The amount of the secreted β-lactamase was about 80% of the total secreted proteins in the culture medium.

Purification and characterization of purine nucleoside phosphorylase (PNP) in micrococcus luteus: Choi , Hye Seon; J. Microbiol. 1996;34(1):82-89.

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Abstract: Purine nucleoside phosphorylase (PNP) was purified in Micrococcus luteus (M. luteus) using streptomycin sulfate and amomonium sulfate fractionation, three times by a Sephadex G-100 gel filtration and a DEAE-Sephadex A-50 ion exchange chromatography. The enzyme was purified 72 folds with a 11% recovery and showed a single band in a nondenaturing gel electrophoresis. The M. W. of PNP turned out to be 1.35 × 10^5 dalton in G-150 gel filtration chromatography. The stability of the enzyme was increased by treatment with both substrates, MgCI₂or CaCI₂, but not significantly kcal/mol. M. luteus PNP catalyzed the phosphorolysis of inosine, deoxyinosine, guanosine and deoxyguanosine with the Km value of 1.5 × 10^-3 M, 3.0 × 10^-3 M, 5.0 × 10^-4 M, respectively. The enzyme was reacted with adenosine, 1-methylnosine and 1-methylguanosine as substrates, which were shown to be poor substrates for mammalian enzyme.

Purification of carbosymethyl cellulase from hybrid between aspergillus niger and penicillium verruculosum: Yang, Young Ki , Lee, Jung Sup , Park, Hyung Nam , Moon, Myung Nim , Kim, Hong Sub , Kim, Jong Se , Lim, Chae Young , Rhee, Young Ha; J. Microbiol. 1996;34(1):90-94.

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Abstract: The carboxymethyl cellulase (CMCase) was purified from the induced culture filtrate of hybrid TAPW15703 between Aspergillus niger and penicillium verruculosum made by nuclear transfer. The enzyme was purified 80 fold with an overall yield 17% from the culture medium by ammonium sulfate fractionation, Sephadex G-75 gel permeation chromatography, and DEAE-ion exchange column chromatography. The molecular weight of the CMCase has estimated to be 32,000 daltons on SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel permeation chromatography. The purified enzyme functions optimally at pH 4.0 and 40℃. The Km value for carbosymethyl cellulose was 68 mM. The enzyme activity was increased by the presence of Mg^2+ and Mn^2+.

Degradation of collagens, immunoglobulins, and other serum proteins by protease of salmonella schottmulleri and its toxicity to cultured cells: Na, Byoung Kuk , Kim, Moon Bo , Song, Chul Yong; J. Microbiol. 1996;34(1):95-100.

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Abstract: The effect of the extracellular protease of Salmonella schottmulleri on human serum constituents such as immunoglobulins, hemoglobin and lysozyme and tissue constituents such as fibronectin and collagens was investigated. This protease degraded collagens (type I and III), fibronectin and serum proteins such as human hemoglobin and lysozyme. Bovine serum albumin was degraded slightly. Thus, the present study suggested the possibility that this protease is not only played an important role in invasion of S. schottmulleri by degrading the constituent proteins such as collagens and fibronectin but also induced complications observed in septicemia and chronic infections by degrading the serum proteins. This protease is also capable of degrading defence-oriented humoral proteins, immunoglobulins (IgG and IgM). Furthermore, it is toxic to HEp-2 cells. These findings clarified the possible role of Salmonella protease as a virulence factor in the pathogenesis of Salmonella infections.

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