Journal of Microbiology

Journal Articles

Deletion of IRC19 Causes Defects in DNA Double-Strand Break Repair Pathways in Saccharomyces cerevisiae: Ju-Hee Choi, Oyungoo Bayarmagnai, Sung-Ho Bae; J. Microbiol. 2024;62(9):749-758. Published online July 12, 2024; DOI: https://doi.org/10.1007/s12275-024-00152-x

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Abstract: DNA double-strand break (DSB) repair is a fundamental cellular process crucial for maintaining genome stability, with homologous recombination and non-homologous end joining as the primary mechanisms, and various alternative pathways such as single-strand annealing (SSA) and microhomology-mediated end joining also playing significant roles under specific conditions. IRC genes were previously identified as part of a group of genes associated with increased levels of Rad52 foci in Saccharomyces cerevisiae. In this study, we investigated the effects of IRC gene mutations on DSB repair, focusing on uncharacterized IRC10, 19, 21, 22, 23, and 24. Gene conversion (GC) assay revealed that irc10Δ, 22Δ, 23Δ, and 24Δ mutants displayed modest increases in GC frequencies, while irc19Δ and irc21Δ mutants exhibited significant reductions. Further investigation revealed that deletion mutations in URA3 were not generated in irc19Δ mutant cells following HO-induced DSBs. Additionally, irc19Δ significantly reduced frequency of SSA, and a synergistic interaction between irc19Δ and rad52Δ was observed in DSB repair via SSA. Assays to determine the choice of DSB repair pathways indicated that Irc19 is necessary for generating both GC and deletion products. Overall, these results suggest a potential role of Irc19 in DSB repair pathways, particularly in end resection process.

Development of a Novel Korean H9‑Specific rRT‑PCR Assay and Its Application for Avian Influenza Virus Surveillance in Korea: Mingeun Sagong , Yong-Myung Kang , Na Yeong Kim , Eun Bi Noh , Gyeong-Beom Heo , Se-Hee An , Youn-Jeong Lee , Young Ki Choi , Kwang-Nyeong Lee; J. Microbiol. 2023;61(10):929-936. Published online November 27, 2023; DOI: https://doi.org/10.1007/s12275-023-00088-8

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Since the 2000s, the Y439 lineage of H9N2 avian influenza virus (AIV) has been the predominant strain circulating in poultry in Korea; however, in 2020, the Y280 lineage emerged and spread rapidly nationwide, causing large economic losses. To prevent further spread and circulation of such viruses, rapid detection and diagnosis through active surveillance programs are crucial. Here, we developed a novel H9 rRT-PCR assay that can detect a broad range of H9Nx viruses in situations in which multiple lineages of H9 AIVs are co-circulating. We then evaluated its efficacy using a large number of clinical samples. The assay, named the Uni Kor-H9 assay, showed high sensitivity for Y280 lineage viruses, as well as for the Y439 lineage originating in Korean poultry and wild birds. In addition, the assay showed no cross-reactivity with other subtypes of AIV or other avian pathogens. Furthermore, the Uni Kor-H9 assay was more sensitive, and had higher detection rates, than reference H9 rRT-PCR methods when tested against a panel of domestically isolated H9 AIVs. In conclusion, the novel Uni Kor-H9 assay enables more rapid and efficient diagnosis than the “traditional” method of virus isolation followed by subtyping RT-PCR. Application of the new H9 rRT-PCR assay to AI active surveillance programs will help to control and manage Korean H9 AIVs more efficiently.

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Development and evaluation of a multiplex real-time RT-PCR assay for simultaneous detection of H5, H7, and H9 subtype avian influenza viruses
Se-Hee An, Na-Yeong Kim, Gyeong-Beom Heo, Yong-Myung Kang, Youn-Jeong Lee, Kwang-Nyeong Lee
Journal of Virological Methods.2024; 327: 114942. CrossRef

Functional characterization of HigBA toxin-antitoxin system in an Arctic bacterium, Bosea sp. PAMC 26642: Eunsil Choi , Ahhyun Huh , Changmin Oh , Jeong-Il Oh , Ho Young Kang , Jihwan Hwang; J. Microbiol. 2022;60(2):192-206. Published online February 1, 2022; DOI: https://doi.org/10.1007/s12275-022-1619-9

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Abstract

Toxin-antitoxin (TA) systems are growth-controlling genetic elements consisting of an intracellular toxin protein and its cognate antitoxin. TA systems have been spread among microbial genomes through horizontal gene transfer and are now prevalent in most bacterial and archaeal genomes. Under normal growth conditions, antitoxins tightly counteract the activity of the toxins. Upon stresses, antitoxins are inactivated, releasing activated toxins, which induce growth arrest or cell death. In this study, among nine functional TA modules in Bosea sp. PAMC 26642 living in Arctic lichen, we investigated the functionality of BoHigBA2. BohigBA2 is located close to a genomic island and adjacent to flagellar gene clusters. The expression of BohigB2 induced the inhibition of E. coli growth at 37°C, which was more manifest at 18°C, and this growth defect was reversed when BohigA2 was co-expressed, suggesting that this BoHigBA2 module might be an active TA module in Bosea sp. PAMC 26642. Live/dead staining and viable count analyses revealed that the BoHigB2 toxin had a bactericidal effect, causing cell death. Furthermore, we demonstrated that BoHigB2 possessed mRNA-specific ribonuclease activity on various mRNAs and cleaved only mRNAs being translated, which might impede overall translation and consequently lead to cell death. Our study provides the insight to understand the cold adaptation of Bosea sp. PAMC 26642 living in the Arctic.

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Evaluating the Contribution of the Predicted Toxin–Antitoxin System HigBA to Persistence, Biofilm Formation, and Virulence in Burkholderia pseudomallei
Itziar Chapartegui-González, Nittaya Khakhum, Jacob L. Stockton, Alfredo G. Torres, Igor E. Brodsky
Infection and Immunity.2022;[Epub] CrossRef
Chronicle of Research into Lichen-Associated Bacteria
Zichen He, Takeshi Naganuma
Microorganisms.2022; 10(11): 2111. CrossRef
Degradation of amoxicillin by newly isolated Bosea sp. Ads-6
Lei Yan, Ning Yan, Xi-Yan Gao, Ying Liu, Zhi-Pei Liu
Science of The Total Environment.2022; 828: 154411. CrossRef

Constantimarinum furrinae gen. nov., sp. nov., a marine bacterium isolated from saline volcanic rock aquifer (lava seawater) at Jeju Island, Republic of Korea: Sung-Hyun Yang , Hyun-Myung Oh , Mi-Jeong Park , Dongil Jang , Kae Kyoung Kwon; J. Microbiol. 2022;60(1):11-17. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1468-6

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Abstract

A Gram-stain-negative, aerobic, rod-shaped (0.3–0.5 × 1.0– 1.9 μm), non-motile marine bacterium designated as ALE3EIT was isolated from a saline volcanic rock aquifer (lava seawater) on Jeju Island, Republic of Korea. The 16S rRNA gene sequence analysis revealed that strain ALE3EIT showed high similarity to ‘Altibacter lentus’ JLT2010T (97.2%), followed by Marixanthomonas ophiurae KMM 3046T (94.5%). Growth was observed at 10–41°C (optimum, 30°C), at pH 6.0–8.5 (optimum, pH 7.5) and at 0.5–8% (optimum, 4.0%) NaCl. The predominant cellular fatty acids were iso-C15:0 (23.5%), iso-C16:0 (10.2%), iso-C16:0 3OH (10.5%), and iso-C17:0 3OH (16.8%). The DNA G + C contents was 40.4 mol%. The major respiratory quinone was MK-6. The major polar lipids were determined to be phosphatidylethanolamine, two unidentified glycolipids, and two unidentified aminolipids. Several phenotypic characteristics such as production of acetoin, activities of arginine dihydrolase and acid phosphatase, and utilization pattern of carbon sources differentiate strain ALE3EIT from ‘A. lentus’ JLT2010T. Activities of the lipase, trypsin, α- chymotrypsin and gelatinase and utilization pattern of carbon sources differentiate strain ALE3EIT from M. ophiurae KMM 3046T. The genome of strain ALE3EIT is 3.0 Mbp long and its ANI and AAI values against ‘A. lentus’ JLT2010T were 76.58 and 72.76, respectively, however, AAI values against members in other genera were lower than 72%. The phylogenomic tree inferred by PhyloPhlAn clearly differentiated the strain ALE3EIT together with strain JLT2010T from other genera in the Falvobacteriaceae. This polyphasic taxonomic data indicates that strain ALE3EIT should be identified as a novel species in the genus ‘Altibacter’, however, the name has not been validated. Therefore, the strain is classified as a novel genus and is proposed as Constantimarinum furrinae gen. nov., sp. nov. The type strain is ALE3EIT (= KCCM 43303T = JCM 33022T).

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Comparison of the anti-inflammatory effects of carotenoids-rich product from microalgal mass-cultured between natural seawater and magma seawater
Jayeon Cheon, Eun-A Kim, Nalae Kang, Taeho Kim, Lei Wang, Soo-Jin Heo, Seon-Heui Cha
Biocatalysis and Agricultural Biotechnology.2024; 62: 103420. CrossRef
Validation List no. 212. Valid publication of new names and new combinations effectively published outside the IJSEM
Aharon Oren, Markus Göker
International Journal of Systematic and Evolutionary Microbiology .2023;[Epub] CrossRef

Transposon insertion site sequencing (TIS) of Pseudomonas aeruginosa: Hongbaek Cho; J. Microbiol. 2021;59(12):1067-1074. Published online December 4, 2021; DOI: https://doi.org/10.1007/s12275-021-1565-y

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Abstract

Transposon insertion site sequencing (TIS) is a technique that determines the insertion profile of a transposon mutant library by massive parallel sequencing of transposon-genomic DNA junctions. Because the transposon insertion profile reflects the abundance of each mutant in the library, it provides information to assess the fitness contribution of each genetic locus of a bacterial genome in a specific growth condition or strain background. Although introduced only about a dozen years ago, TIS has become an important tool in bacterial genetics that provides clues to study biological functions and regulatory mechanisms. Here, I describe a protocol for generating high density transposon insertion mutant libraries and preparing Illumina sequencing samples for mapping the transposon junctions of the transposon mutant libraries using Pseudomonas aeruginosa as an example.

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Optimizing phage-based mutant recovery and minimizing heat effect in the construction of transposon libraries in Staphylococcus aureus
Sally W. Yousief, Nader Abdelmalek, Bianca Paglietti
Scientific Reports.2024;[Epub] CrossRef
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Meiyi Ge, Jian Luo, Yi Wu, Guobo Shen, Xi Kuang
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Optimization of Transposon Mutagenesis Methods in Pseudomonas antarctica
Sangha Kim, Changhan Lee
Microorganisms.2023; 11(1): 118. CrossRef
Construction of high-density transposon mutant library of Staphylococcus aureus using bacteriophage ϕ11
Wonsik Lee
Journal of Microbiology.2022; 60(12): 1123. CrossRef

Lactiplantibacillus plantarum LRCC5314 includes a gene for serotonin biosynthesis via the tryptophan metabolic pathway: Jiseon Jeong , Yunjeong Lee , Seokmin Yoon , Jong-Hwa Kim , Wonyong Kim; J. Microbiol. 2021;59(12):1092-1103. Published online December 4, 2021; DOI: https://doi.org/10.1007/s12275-021-1472-2

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Abstract

As the functions of probiotics within the same species may not be shared, it is important to analyze the genetic characteristics of strains to determine their safety and usefulness before industrial applications. Hence the present study was undertaken to determine functional genes, and beneficial activities of strain LRCC5314, a bacterial strain isolated from kimchi through comparative genomic analysis. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain LRCC5314 was a member of the species L. plantarum. Whole genome size of strain LRCC5314 was sequence was 3.25 Mb long, with a G + C content of 44.5 mol% and 3,031 predicted genes. Strain LRCC5314 could metabolize hexoses through homofermentation, which produces only lactic acid from hexoses. According to gene annotation, strain LRCC- 5314 contained genes of EPS production and CRISPR. Moreover, the strain contained genes that could encode a complete biosynthetic pathway for the production of tryptophan, which can be used as a precursor of serotonin. Notably, the tryptophan and serotonin activities strain LRCC5314 were higher than those of reference strains, L. plantarum ATCC 14917T, DSM 20246, DSM 2601, and ATCC 8014, which reach tryptophan amount of 0.784 ± 0.045 μM/ml in MRS broth and serotonin concentration of 19.075 ± 0.295 ng/ml in HT-22 cells. These findings indicated that L. plantarum LRCC5314 could provide a source for serotonin production and could be used as a functional probiotic for stress regulation.

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Ramya Balasubramanian, Elizabeth Schneider, Eoin Gunnigle, Paul D. Cotter, John F. Cryan
Neuroscience & Biobehavioral Reviews.2024; 158: 105562. CrossRef
Effect of postbiotic Lactiplantibacillus plantarum LRCC5314 supplemented in powdered milk on type 2 diabetes in mice
J.-H. Kim, W. Kwak, Y. Nam, J. Baek, Y. Lee, S. Yoon, W. Kim
Journal of Dairy Science.2024; 107(8): 5301. CrossRef
The role of pharmacomicrobiomics in HIV prevention, treatment, and women’s health
Erik C. Swanson, Christopher M. Basting, Nichole R. Klatt
Microbiome.2024;[Epub] CrossRef
Whole-Genome Sequence of Lactococcus lactis Subsp. lactis LL16 Confirms Safety, Probiotic Potential, and Reveals Functional Traits
Justina Mileriene, Jurgita Aksomaitiene, Kristina Kondrotiene, Tora Asledottir, Gerd Elisabeth Vegarud, Loreta Serniene, Mindaugas Malakauskas
Microorganisms.2023; 11(4): 1034. CrossRef
Probiotic Incorporation into Yogurt and Various Novel Yogurt-Based Products
Douglas W. Olson, Kayanush J. Aryana
Applied Sciences.2022; 12(24): 12607. CrossRef

Review

Potential of Bacillus velezensis as a probiotic in animal feed: a review: Fatima Khalid , Anam Khalid , Yuechi Fu , Qian Hu , Yunfang Zheng , Salman Khan , Zaigui Wang; J. Microbiol. 2021;59(7):627-633. Published online July 1, 2021; DOI: https://doi.org/10.1007/s12275-021-1161-1

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Abstract

Bacillus velezensis is a plant growth-promoting bacterium that can also inhibit plant pathogens. However, based on its properties, it is emerging as a probiotic in animal feed. This review focuses on the potential characteristics of B. velezensis for use as a probiotic in the animal feed industry. The review was conducted by collecting recently published articles from peer-reviewed journals. Google Scholar and PubMed were used as search engines to access published literature. Based on the information obtained, the data were divided into three groups to discuss the (i) probiotic characteristics of B. velezensis, (ii) probiotic potential for fish, and (iii) the future potential of this species to be developed as a probiotic for the animal feed industry. Different strains of B. velezensis isolated from different sources were found to have the ability to produce antimicrobial compounds and have a beneficial effect on the gut microbiota, with the potential to be a candidate probiotic in the animal feed industry. This review provides valuable information about the characteristics of B. velezensis, which can provide researchers with a better understanding of the use of this species in the animal feed industry.

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Research Support, Non-U.S. Gov't

Characterization of cell death in Escherichia coli mediated by XseA, a large subunit of exonuclease VII: Hyeim Jung , Junwei Liang , Yuna Jung , Dongbin Lim; J. Microbiol. 2015;53(12):820-828. Published online December 2, 2015; DOI: https://doi.org/10.1007/s12275-015-5304-0

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Abstract

Exonuclease VII (ExoVII) of Escherichia coli is a single strandspecific DNA nuclease composed of two different subunits: the large subunit, XseA, and the small subunit, XseB. In this study, we found that multicopy single-stranded DNAs (msDNAs), Ec83 and Ec78, are the in vivo substrates of ExoVII; the enzyme cuts the phosphodiester bond between the fourth and fifth nucleotides from the 5′ end. We used this msDNA cleavage to assess ExoVII activity in vivo. Both subunits were required for enzyme activity. Expression of XseA without XseB caused cell death, even though no ExoVII activity was detected. The lethality caused by XseA was rescued by surplus XseB. In XseA-induced death, cells were elongated and multinucleated, and their chromosomes were fragmented and condensed; these are the morphological hallmarks of apoptotic cell death in bacteria. A putative caspase recognition sequence (FVAD) was found in XseA, and its hypothetical caspase product with 257 amino acids was as active as the intact protein in inducing cell death. We propose that under ordinary conditions, XseA protects chromosome as a component of the ExoVII enzyme, but in some conditions, the protein causes cell death; the destruction of cell is probably carried out by the amino terminal fragment derived from the cleavage of XseA by caspase-like enzyme.

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Journal Article

A Novel Ribonuclease with Potent HIV-1 Reverse Transcriptase Inhibitory Activity from Cultured Mushroom Schizophyllum commune: Yong-Chang Zhao , Guo-Qing Zhang , Tzi-Bun Ng , He-Xiang Wang; J. Microbiol. 2011;49(5):803-808. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1098-x

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Abstract: A 20-kDa ribonuclease (RNase) was purified from fresh fruiting bodies of cultured Schizophyllum commune mushrooms. The RNase was not adsorbed on Affi-gel blue gel but adsorbed on DEAE-cellulose and CM-cellulose. It exhibited maximal RNase activity at pH 6.0 and 70°C. It demonstrated the highest ribonucleolytic activity toward poly (U) (379.5 μ/mg), the second highest activity toward poly (C) (244.7 μ/mg), less activity toward poly (A) (167.4 μ/mg), and much weaker activity toward poly (G) (114.5 μ/mg). The RNase inhibited HIV-1 reverse transcriptase with an IC50 of 65 μM. No effect on [3H-methyl]-thymidine uptake by lymphoma MBL2 cells and leukemia L1210 cells was observed at 100 μM concentration of the RNase. A comparison of RNases from S. commune and Volvariella volvacea revealed that they demonstrated some similarities in N-terminal amino acid sequence, optimum pH and polyhomoribonucleotide specificity. However, some differences in chromatographic behavior and molecular mass were observed.

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

Recombinant Expression and Purification of Functional XorII, a Restriction Endonuclease from Xanthomonas oryzae pv. oryzae: Dong Kyu Hwang , Jae-Yong Cho , Young Kee Chae; J. Microbiol. 2007;45(2):175-178.; DOI: https://doi.org/2515 [pii]

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Abstract: An endonuclease from Xanthomonas oryzae pathovar oryzae KACC 10331, XorII, was recombinantly produced in Escherichia coli using a T7 system. XorII was purified using a combination of ion exchange and immobilized metal affinity chromatography (IMAC). An optimized washing protocol was carried out on an IMAC in order to obtain a high purity product. The final amount of purified XorII was approximately 2.5 mg/L of LB medium. The purified recombinant XorII was functional and showed the same cleavage pattern as PvuI. The enzyme activity tested the highest at 25°C in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM dithiothreitol at a pH of 7.9.

Biochemical Quantitation of PM2 Phage DNA as a Substrate for Endonuclease Assay: Yoo Jin Joo , Hee-Ju Kim , Jae Yung Lee , Joon Kim; J. Microbiol. 2004;42(2):99-102.; DOI: https://doi.org/2038 [pii]

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Abstract: Bacteriophage PM2 has a closed circular form of double stranded DNA as a genome. This DNA from the phage is a useful source for nick-circle endonuclease assay in the fmol range. Due to difficulties in the maintenance of viral infectivity, storage conditions of the phage should be considered for the purification of PM2 DNA. The proper condition for a short-term storage of less than 2 months is to keep the PM2 phage at 4^oC; whereas the proper condition for a long-term storage of the PM2 phage for over 2 months is to keep it under liquid nitrogen in 7.5% glycerol. The optimal conditions for a high yield of phage progeny were also considered with the goal to achieve a successful PM2 DNA preparation. A MOI(Multiplicity Of Infection) of 0.03, in which the OD_600 of the host bacteria was between 0.3 and 0.5, turned out to be optimal for the mass production of PM2 phage with a burst size of about 214. Considerations of PM2 genome size, and the concentrations and radiospecific activities of purified PM2 DNA, are required to measure the endonuclease activity in the fmol range. This study reports the proper quantitation of radioactivity and the yield of purified DNA based on these conditions.

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.

Cloning and Sequencing of the rph Gene Encoding RNase PH from Legionella pneumophila: Se Jin Kim , Jong-Seok Lim , Nicholas P. Cianciotto , Yong-Kyung Choe; J. Microbiol. 1999;37(4):218-223.

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Abstract: Legionella pneumophila, the cause of Legionnaires disease, is able to survive intracellularily in eukaryotic cells such as monocytes, macorphages, and protozoan ogranisms. During protein biosynthesis, the rph gene encodes ribonuclease (RNase) PH which functions as a phosphorolytic nuclease that removes nucleotides following the CCA terminus of tRNA and as a nucleotidyl-transferase which adds nucleotides to the ends of RNA molecules by usingnucelside diohosphates as substrates.In this sutdy, the rph gene was screened in pUC19 library employing a DNA probe whcich was constructed from PCR based on a consensus pattern of multiple alignment of RNas PH. The encoded protein consists of 235 amino acid residues with a calculated molecualr weight of 26,112 Daltons. The RNase PH signature domains are completely conserved.

Expression and Characterization of the Human rpS3 in a Methylotrophic Yeast Pichia pastoris: Jae Yung Lee , Sang Oun Jung , BuHyun Youn , Oh Sik Kwon , Joon Kim; J. Microbiol. 2000;38(2):88-92.

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Abstract: A human ribosomal protein S3 (rpS3), which also functions as a DNA repair enzyme (UV endonuclease III), was expressed in a methylotrophic yeast, Pichia pastoris, and biochemically characterized. UV endonuclease activity was previously characterized, and this activity of mammalian rpS3 was found to be non-specific upon purification and storage. Under the Pichia expression system, the subcloned cDNA of the human rpS3 gene revealed a peptide of 42 kDa by SDS-PAGE and Western blot. The secreted form of human rpS3 rendered no endonuclease activity while the intracellular form showed UV specific endonuclease activity by the nick circle assay.

Mutational Analysis of an Essential RNA Stem-loop Structure in a Minimal RNA Substrate Specifically Cleaved by Leishmania RNA Virus 1-4 (LRV1-4) Capsid Endoribonuclease: Youngtae Ro , Jean L. Patterson; J. Microbiol. 2003;41(3):239-247.

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Abstract: The LRV1-4 capsid protein possesses an endoribonuclease activity that is responsible for the single sitespecific cleavage in the 5’ untranslated region (UTR) of its own viral RNA genome and the formation of a conserved stem-loop structure (stem-loop IV) in the UTR is essential for the accurate RNA cleavage by the capsid protein. To delineate the nucleotide sequences, which are essential for the correct formation of the stem-loop structure for the accurate RNA cleavage by the viral capsid protein, a wildtype minimal RNA transcript (RNA 5’ 249-342) and several synthetic RNA transcripts encoding pointmutations in the stem-loop region were generated in an in vitro transcription system, and used as substrates for the RNA cleavage assay and RNase mapping studies. When the RNA 5’ 249-342 transcript was subjected to RNase T1 and A mapping studies, the results showed that the predicted RNA secondary structure in the stem-loop region using FOLD analysis only existed in the presence of Mg2+ ions, suggesting that the metal ion stabilizes the stem-loop structure of the substrate RNA in solution. When point-mutated RNA substrates were used in the RNA cleavage assay and RNase T1 mapping study, the specific nucleotide sequences in the stem-loop region were not required for the accurate RNA cleavage by the viral capsid protein, but the formation of a stem-loop like structure in a region (nucleotides from 267 to 287) stabilized by Mg_2^+ ions was critical for the accurate RNA cleavage. The RNase T1 mapping and EMSA studies revealed that the Ca2+ and Mn2+ ions, among the reagents tested, could change the mobility of the substrate RNA 5’ 249-342 on a gel similarly to that of Mg_2^+ ions, but only Ca_2^+ ions identically showed the stabilizing effect of Mg_2^+ ions on the stem-loop structure, suggesting that binding of the metal ions (Mg_2^+ or Ca_2^+) onto the RNA substrate in solution causes change and stabilization of the RNA stem-loop structure, and only the substrate RNA with a rigid stem-loop structure in the essential region can be accurately cleaved by the LRV1-4 viral capsid protein.

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