• Journal of Microbiology
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• v.44 no.1
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• pp.29-34
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• 2006

The purpose of this study was to develop molecular identification method for medical mushrooms and their preparations based on the nucleotide sequences of nuclear large subunit (LSD) rDNA. Four specimens were collected of each of the three representative medicinal mushrooms used in Korea: Ganoderma Incidum, Coriolus versicolor, and Fomes fomentarius. Fungal material used in these experiments included two different mycelial cultures and two different fruiting bodies from wild or cultivated mushrooms. The genomic DNA of mushrooms were extracted and 3 nuclear LSU rDNA fragments were amplified: set 1 for the 1.1-kb DNA fragment in the upstream region, set 2 for the 1.2-kb fragment in the middle, and set 3 for the 1.3-kb fragment downstream. The amplified gene products of nuclear large subunit rDNA from 3 different mushrooms were cloned into E. coli vector and subjected to nucleotide sequence determination. The sequence thus determined revealed that the gene sequences of the same medicinal mushroom species were more than $99.48\%$ homologous, and the consensus sequences of 3 different medicinal mushrooms were more than $97.80\%$ homologous. Restriction analysis revealed no useful restriction sites for 6-bp recognition enzymes for distinguishing the 3 sequences from one another, but some distinctive restriction patterns were recognized by the 4-bp recognition enzymes AccII and HhaI. This analysis was also confirmed by PCR-RFLP experiments on medicinal mushrooms.

• BMB Reports
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• v.28 no.5
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• pp.402-407
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• 1995

Single-run Partial cDNA sequencing was conducted on 1,592 randomly selected human fetal liver cDNA clones of Korean origin to isolate novel genes related to liver functions. Each partial cDNA sequence determined was analyzed by comparing it with the databases. GenBank, Protein Information Resource (PIR) and SWISS-PROT Protein Sequence Data Bank. From a set of 1.592 cDNA clones reported here, 1,433 (90.0% of the total) were informative cDNA sequences. The other 159 clones were identified as DNA sequences which had originated from the cloning vector. Among 1,433 informative partial cDNA sequences, 851 (59.3%) clones were revealed to be identical to known human genes. These known genes have been classified into 225 different kinds of genes. In addition, 340 clones (23.7%) showed various degrees of homology to previously known human genes. Ninety four (6.6%) clones contained various repeated sequences. Twenty four (1.7%) partial cDNA sequences were found to have considerable homology to known genes from evolutionarily distant organism such as yeast, rice, Arabidopsis, mouse and rat, based on database matches, whereas 124 (8.7%) had no Significant matches. Human homologues to functionally characterized genes from different organisms could be classified as candidates for novel human genes of similar functions. Information from the partial cDNA sequences in this study may facilitate the analysis of genes expressed in human fetal liver.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1359-1367
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• 2005

The gene encoding Pyrobaculum arsenaticum DNA polymerase (Par DNA polymerase) was cloned and sequenced. The gene consists of 2,361 bp coding for a protein with 786 amino acid residues. The deduced amino acid sequence of Par DNA polymerase showed a high similarity to archaeal family B-type DNA polymerases (Group I), and contained all of the motifs conserved in the family B-type DNA polymerases for $3'{\rightarrow}5'$ exonuclease and polymerase activities. The Par DNA polymerase gene was expressed under the control of the T7lac promoter on the expression vector pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RP. The expressed enzyme was purified by heat treatment, and Cibacron blue 3GA and $Hirap^{TM}$ Heparin HP column chromatographies. The optimum pH of the purified enzyme was 7.5. The enzyme activity was activated by divalent cations, and was inhibited by EDTA and monovalent cations. The half-life of the enzyme at $95^{\circ}C$ was 6 h. Par DNA polymerase possessed associated $3'{\rightarrow}5'$ proofreading exonuclease activity, which is consistent with its deduced amino acid sequence. PCR experiment with Par DNA polymerase showed an amplified product, indicating that this enzyme might be useful in DNA amplification and PCR-based applications.

• Journal of Microbiology and Biotechnology
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• v.18 no.10
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• pp.1634-1640
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• 2008

This study was focused on the screening of valuable genetic resources, such as promoters from metagenome, and describes a promoter trapping system with a bidirectional probe concept, which can select promoters or operons from various biological resources including metagenomic DNA. A pair of reporters, GFP and DsRed, facing the opposite direction without promoters, is an effective system that can function regardless of the direction of inserted promoters. The feasibility of this system was tested for the isolation of constitutively expressed promoters in E. coli from a soil metagenome, resulting in a potential pool of various promoters for practical application. The analyses of structural organization of the trapped genes demonstrated that constitutively expressible promoters in E. coli were broadly distributed within the metagenome, and suggested that some promoters were useful for the construction of expression vectors. Based on these observations, three constitutive promoters were employed in the expression vector system and their potentials for practical application were evaluated in terms of expression level, protein solubility, and effects on host growth.

• Interdisciplinary Bio Central
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• v.4 no.4
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• pp.14.1-14.6
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• 2012

Splice site prediction in DNA sequence is a basic search problem for finding exon/intron and intron/exon boundaries. Removing introns and then joining the exons together forms the mRNA sequence. These sequences are the input of the translation process. It is a necessary step in the central dogma of molecular biology. The main task of splice site prediction is to find out the exact GT and AG ended sequences. Then it identifies the true and false GT and AG ended sequences among those candidate sequences. In this paper, we survey research works on splice site prediction based on support vector machine (SVM). The basic difference between these research works is nucleotide encoding technique and SVM kernel selection. Some methods encode the DNA sequence in a sparse way whereas others encode in a probabilistic manner. The encoded sequences serve as input of SVM. The task of SVM is to classify them using its learning model. The accuracy of classification largely depends on the proper kernel selection for sequence data as well as a selection of kernel parameter. We observe each encoding technique and classify them according to their similarity. Then we discuss about kernel and their parameter selection. Our survey paper provides a basic understanding of encoding approaches and proper kernel selection of SVM for splice site prediction.

• Journal of Microbiology and Biotechnology
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• v.13 no.3
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• pp.457-462
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• 2003

The complete nucleotide sequence of a plasmid, pMG1, isolated from Bifidobacterium longum MG1 has been determined. This plasmid, composed of 3,862 base pairs with 65.1% of G+C content. harbors two major open reading frames (ORF) encoding putative proteins of 29 kDa (ORF I) and 71 kDa (ORF II). ORF I showed relatively high amino acid sequence homology with replication proteins of other plasmids from Gr Im-positive and -negative bacteria. Upstream of ORF I, four sets of tandem repeat sequences resembling the iteron structure of related plasmids were found. S1 endonuclease treatment and Southern blot analysis revealed that pMG1 accumulates single-stranded DNA (ssDNA) intermediate, which indicate i the rolling circle replication (RCR) mechanism of this plasmid. Homology search indicated that ORF II encodes plasmid mobilization protein, and the presence of highly conserved oriT sequence in the upstream of this gene supported this assumption. RT-PCR showed that only ORF I is expressed in vivo. Based on these results, pMG 1 was exploited to construct a shuttle vector, pBES2. It was successfully transformed into Bifidobacterium and maintained stably.

• The Korean Journal of Malacology
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• v.26 no.2
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• pp.185-188
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• 2010

The Web-based the genus Haliotis SNP database was constructed on the basis of Intel Server Platform ZSS130 dual Xeon 3.2 GHz cpu and Linux-based (Cent OS) operating system. Haliotis related sequences (2,830 nucleotide sequences, 9,102 EST sequences) were downloaded through NCBI taxonomy browser. In order to eliminate vector sequences, we conducted vector masking step using cross match software with vector sequence database. In addition, poly-A tails were removed using Trimmest software from EMBOSS package. The processed sequences were clustered and assembled by TGICL package (TIGR tools) equipped with CAP3 software. A web-based interface (Haliotis SNP Database, http://www.haliotis.or.kr) was developed to enable optimal use of the clustered assemblies. The Clustering Res. menu shows the contig sequences from the clustering, the alignment results and sequences from each cluster. And also we can compare any sequences with Haliotis related sequences in BLAST menu. The search menu is equipped with its own search engine so that it is possible to search all of the information in the database using the name of a gene, accession number and/or species name. Taken together, the Web-based SNP database for Haliotis will be valuable to develop SNPs of Haliotis in the future.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1583-1590
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• 2006

T4 endonuclease V (T4 endo V) [EC 3. 1. 25. 1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a cyclobutane pyrimidine dimer DNA glycosylase (CPD glycosylase) and an apyrimidic/apurinic endonuclease (AP lyase). T4 denV (414 bp cDNA) encoding T4 en do V (138 amino acid) was synthesized and expressed using either an expression vector, pTriEx-4, in E. coli or a baculovirus AcNPV vector, pBacPAK8, in insect cells. The recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed from bacteria was purified using one-step affinity chromatography with a HiTrap Chelating HP column and used to make rabbit anti-His-Tag/T4 endo V polyclonal antibody for detection of recombinant T4 endo V (rT4 endo V) expressed in insect cells. In the meantime, the recombinant baculovirus was obtained by cotransfection of BacPAK6 viral DNA and pBP/T4 endo V in Spodoptera frugiperda (Sf21) insect cells, and used to infect Sf21 cells to overexpress T4 endo V protein. The level of rT4 endo V protein expressed in Sf21 cells was optimized by varying the virus titers and time course of infection. The optimal expression condition was set as follows; infection of the cells at a MOI of 10 and harvest at 96 h post-infection. Under these conditions, we estimated the amount of rT4 endo V produced in the baculovirus expression vector system to be 125 mg/l. The rT4 endo V was purified to homogeneity by a rapid procedure, consisting of ion-exchange, affinity, and reversed phase chromatographies, based on FPLC. The rT4 endo V positively reacted to an antiserum made against rHis-Tag/T4 endo V and showed a residual nicking activity against CPD-containing DNA caused by UV. This is the first report to have T4 endo V expressed in an insect system to exclude the toxic effect of a bacterial expression system, retaining enzymatic activity.

• Biomedical Science Letters
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• v.2 no.2
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• pp.231-240
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• 1996

Human blood clotting (coagulation) factor 9 cDNA which codes for 461 amino acid has been cloned by screening human fetal liver cDNA library using PCR. This 1.4 kb cDNA spanning from the ATG initiation codon to the TAA termination codon was cloned into bacterial .expression vector pGEX-2T, generating pGEX-F9 plasmid. The plasmid pGEX-F9 expresses about 73 kDa GST (Glutathione S-transferase)-Factor 9 fusion protein when introduced into E. coli. Western blot analysis using polyclonal antibody raised against human factor 9 confirmed this fusion protein contains factor 9 protein. The level of GST-factor 9 expression was about 20% of total protein and the purification of fusion protein was efficiently achieved by using GST agarose bead based on one step purification protocol.

• YAKHAK HOEJI
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• v.45 no.1
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• pp.108-115
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• 2001

We have recently reported the development of a high efficiency expression vector, pCK, which can drive a high level of gene expression in mouse skeletal muscle. In this study, we tested the therapeutic potential of pCK expressing human VEGF165, pCK-VEGF in the rabbit ischemic hind limb model. To determine the optimal dose of plasmid DNA, various concentrations of pCK-CAT were injected into the muscle of a rabbit hind limb and the levels of CAT activity were determined. It was found that the expression level of the exogenously added gene became stable between 250 and 1,000 $\mu$g. Based on this result, we tested whether intramuscular transfer of 500$\mu$g of pCK-VEGF could actually modulate collateral vessel development in a rabbit ischemic hind limb model. It was found that relative to the control group injected with the pCK lacking the VEGF sequence, single intramuscular doses (500$\mu$g) of pCK-VEGF produced statistically significant augmentation of collateral vessels as determined by the angiographic vessel count, maximal blood flow by Doppler flowmeter and the number of capillaries by histology. These results suggest that a single 500$\mu$g-delivery of pCK-VEGF is potent enough to induce sufficient angiogenic activity and achieve therapeutic benefit on this rabbit model.