Journal of Microbiology

Journal Article

A Method for Physical Analysis of Recombination Intermediates in Saccharomyces cerevisiae: Kiwon Rhee , Hyungseok Choi , Keun P. Kim , Jeong H. Joo; J. Microbiol. 2023;61(11):939-951. Published online December 11, 2023; DOI: https://doi.org/10.1007/s12275-023-00094-w

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Abstract: Meiosis is a process through which diploid cells divide into haploid cells, thus promoting genetic diversity. This diversity arises from the formation of genetic crossovers (COs) that repair DNA double-strand breaks (DSBs), through homologous recombination (HR). Deficiencies in HR can lead to chromosomal abnormality resulting from chromosomal nondisjunction, and genetic disorders. Therefore, investigating the mechanisms underlying effective HR is crucial for reducing genome instability. Budding yeast serves as an ideal model for studying HR mechanisms due to its amenability to gene modifications and the ease of inducing synchronized meiosis to yield four spores. During meiosis, at the DNA level, programmed DSBs are repaired as COs or non-crossovers (NCOs) through structural alterations in the nascent D-loop, involving single-end invasions (SEIs) and double-Holliday junctions (dHJs). This repair occurs using homologous templates rather than sister templates. This protocol, using Southern blotting, allows for the analysis and monitoring of changes in DNA structures in the recombination process. One-dimensional (1D) gel electrophoresis is employed to detect DSBs, COs, and NCOs, while twodimensional (2D) gel electrophoresis is utilized to identify joint molecules (JMs). Therefore, physical analysis is considered the most effective method for investigating the HR mechanism. Our protocol provides more comprehensive information than previous reports by introducing conditions for obtaining a greater number of cells from synchronized yeast and a method that can analyze not only meiotic/mitotic recombination but also mitotic replication.

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

Dysregulation of KSHV Replication by Extracts from Carthamus tinctorius L.: Han Lee , Hyosun Cho , Myoungki Son , Gi-Ho Sung , Taeho Lee , Sang-Won Lee , Yong Woo Jung , Yu Su Shin , Hyojeung Kang; J. Microbiol. 2013;51(4):490-498. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-3282-7

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Abstract: Carthamus tinctorius L. (CT) is traditionally used to reduce ailments from diseases of the musculoskeletal system and connective tissue and diseases of blood circulation and the cardiovascular system. Flower extracts from CT are known to have antibacterial activity, anti-inflammatory activity, and to inhibit tumor promotion in mouse skin carcinogenesis. In order to discover new antiviral agents from CT extracts, we tested whether CT extracts contain antiviral activity against gammaherpesvirus infection. This study demonstrated that treatment with CT extracts disrupted KSHV latency in the viral-infected host cells, iSLK-BAC16. n-Hexane and EtOH fractions of CT extracts critically affected at least two stages of the KHSV life-cycle by abnormally inducing KSHV lytic reactivation and by severely preventing KSHV virion release from the viral host cells. In addition to the effects on KSHV itself, CT extract treatments induced cellular modifications by dysregulating cell-cycle and producing strong cytotoxicity. This study demonstrated for the first time that CT extracts have antiviral activities that could be applied to development of new anti-gammaherpesviral agents.

NOTE] Kaposi’s Sarcoma-Associated Herpesvirus Infection of Endothelial Progenitor Cells Impairs Angiogenic Activity In Vitro: Seungchul Yoo , Sil Kim , Seungmin Yoo , In-Taek Hwang , Haewol Cho , Myung-Shin Lee; J. Microbiol. 2011;49(2):299-304. Published online May 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0408-7

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Abstract: A recent study reported that endothelial progenitor cells (EPCs) are one of the reservoirs of Kaposi’s sarcoma associated herpesvirus (KSHV). Although EPCs are closely linked to angiogenesis and vasculogenesis, little is known about the angiogenic potential of KSHV in EPCs. In this study, we used EPCs isolated from human umbilical cord blood to show that early infection by KSHV in vitro impaired the neovascularization of EPCs in matrigel. Our results suggest that KSHV may disrupt the angiogenic potential of EPCs and that the disseminated infection of KSHV could be associated with EPC dysfunction.

Kaposi’s Sarcoma-Associated Herpesvirus Viral Protein Kinase Interacts with RNA Helicase A and Regulates Host Gene Expression: Jae Eun Jong , Junsoo Park , Sunmi Kim , Taegun Seo; J. Microbiol. 2010;48(2):206-212. Published online May 1, 2010; DOI: https://doi.org/10.1007/s12275-010-0021-1

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Abstract: RNA helicase A (RHA) containing the DExH motif is a human homolog of maleless protein that regulates expression of genes located in the Drosophila X chromosome during dosage compensation. RHA exerts helicase activity that unwinds double-stranded RNA and DNA to a single-strand form. The protein acts as a bridging factor mediating interactions of CBP/p300 and RNA pol II, and consequently affects gene expression. Kaposi’s sarcoma-associated herpesvirus (KSHV) is a member of the γ-herpesvirus subfamily that causes several disorders. The majority of herpesviruses commonly encode predicted viral protein kinases. KSHV open reading frame 36 (ORF36) codes for protein kinase domains, and functions as a serine/threonine protein kinase. KSHV ORF36 is classified as a late gene, as it is expressed during lytic replication and localized in the nuclei of KSHV-infected cells. Recent studies show that viral protein kinase (vPK) interacts with cellular proteins. In this study, we determined the cellular localization of vPK in KSHVinfected BCBL-1 cells using confocal microscopy. Proteomic analysis indicates that cellular proteins interacted with vPK, and co-immunoprecipitation reactions further reveal interactions between vPK and RHA. Moreover, KSHV vPK appeared to regulate the transcriptional activation of Cre promoter, and plays an important role in cellular transcription of RHA.

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