- PCR-RFLP and Sequence Analysis of the rDNA ITS Region in the Fusarium spp.
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Lee , Young-Mi , Yong-Keel Choi , Byung-Re Min
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J. Microbiol. 2000;38(2):66-73.
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To investigate the genetic relationship among 12 species belonging to the Fusarium section Martiella, Dlaminia, Gibbosum, Arthrosporiella, Liseola and Elegans, the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) were amplified with primer pITS1 and pITS4 using the polymerase chain reaction (PCR). After the amplified products were digested with 7 restriction enzymes, restriction fragment length polymorphism (RFLP) patterns were analyzed. The partial nucleotide sequences of the ITS region were determined and compared. Little variation was observed in the size of the amplified product having sizes of 550bp or 570bp. Based on the RFLP analysis, the 12 species studied were divided into 5 RFLP types. In particular, strains belonging to the section Martiella were separated into three RFLP types. Interestingly, the RFLP type of F. solani f. sp. piperis was identical with that of isolates belonging to the section Elegans. In the dendrogram derived from RFLP analysis of the ITS region, the Fusarium spp. examined were divided into two major groups. In general, section Martiella excluding F. solani f. sp. piperis showed relatively low similarity with the other section. The dendrogram based on the sequencing analysis of the ITS2 region also gave the same results as that of the RFLP analysis. As expected, 5.8S, a coding region, was highly conserved, whereas the ITS2 region was more variable and informative. The difference in the ITS2 region between the length of F. solani and its formae speciales excluding F. solani f. sp. piperis and that of other species was caused by the insertion/deletion of nucleotides in positions 143-148 and 179-192.
- Studies on the Inhibition of HIV Replication with a Number of RRE Decoy Derivatives
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Lee , Seong Wook
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J. Microbiol. 1998;36(4):308-315.
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RRE decoys are short RNA oligonucleotides corresponding to the HIV Rev response element (RRE) sequence, which protect cells from HIV replication by inhibiting the binding of the HIV regulatory protein Rev to the authentic HIV RRE region. Previously minimal RRE decoy containing the 13-nucleotide primary Rev binding domain of RRE was described to be a potent inhibitor of HIV in CEM cells. In this report, we analyzed and compared the ability of a series of RRE decoy derivatioves to inhibit HIV replication in CEM cells to develop increasingly effective RRE decoy. Using an improved tRNA cassette to express high level of RRE transcripts in cells, we found that a variant form of stem-loop II(SLII) binding domain of wild type RRE termed RRE40 was more potent than any other RRE decoys previously developed or tested here and protected all cells most effectively from HIV. RRE40 was previously selected in vitro which binds to Rev protein 10-fold better than wild type RRE. CEM cells expressing RRE40 decoy RNAE40 decoys inhibit HIV specifically by sequestering Rev binding to the authentic RRE target in HIV RNA and indicated that RRE40 RNA identified by using in vitro binding studies also binds Rev in cells. These obserbations have important implications for experiments involving optimization of clinical application of RNA decoy based gene therapy protocol against HIV.
- A plasmid vector faciliting gene expression in both yeast and mammalian cells
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Lee , Tae Ho
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J. Microbiol. 1997;35(2):149-151.
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A plasmid vector with combined features of yeast shuttle vector and mammalian expression vector was constructed to facilitate expression of cloned gene in both cell-types. All necessary elements required for plasmid maintenance and selection in E. coli, yeast and mammalian cells were size-economically arranged in this plasmid. The human cytomegalovirus (CMV) immediate early promoter and yeast GAL1 promoter were sequentially placed in front of the gene to be expressed. The synthetic splicing donor and acceptor sequences were inserted into the immediate upstream and downstream of the GAL1 promotor, allowing the CMV promotor to direct the expression of a given gene in mammalian cell environment by splicing out the interfering GAL1 promotor sequence. When the resulting vector containing LacZ as a gene was introduced into yeast and mammalian cells, both cells efficiently produced β-galactosidase, dimonstrating its dual host usage.
- Reorganization of chromatin conformation from an active to an inactive state after cessation of transcription
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Lee , Myeong Sok
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J. Microbiol. 1996;34(1):54-60.
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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.
- Induced Level of CIN2 Gene Transcripts in yeast by Cycloheximide Treatment
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Lee , Myeong Sok
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J. Microbiol. 1995;33(1):46-50.
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The interrelationship of expression of the two adjacent genes, HSP82 and CIN2 in Saccharomyces cerevisiae was investigated. The two genes are very close each other, such that the poly(A) addition site of the HSP82 gene is likely to be immediately followed by the promoter region of the CIN2 gene. Thus, transcription of the upstream HSP82 gene might affect expression of the CIN2 gene via promoter occlusion. Based on HSP82 and CIN2 mRNA analysis of the wild type, disruption mutant of the HAP82 gene, and conditional RNA polymerase II mutant strains, I conclude that CIN2 transcription is not affected by HSP82 transcription. However, surprisingly the level of CIN2 gene transcripts, but not HSP82, is heightened by cycloheximide treatment, presumably due to an increase in its stability. No change is observed in kinetics of HSP82 RNA induction by cycloheximide treatment.
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