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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Korean Society for Biochemistry and Molecular Biology
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Volume & Issues
Volume 30, Issue 6 - Nov 1997
Volume 30, Issue 5 - Sep 1997
Volume 30, Issue 4 - Jul 1997
Volume 30, Issue 3 - May 1997
Volume 30, Issue 2 - Mar 1997
Volume 30, Issue 1 - Jan 1997
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Cloning of the Genomic DNA Which Complements the Drug-Hypersensitivity of Saccharomyces cerevlsiae
Lee, Yun-Sik ; Park, Kie-In ;
BMB Reports , volume 30, issue 3, 1997, Pages 167~172
The yeast Saccharomyces cerevisiae, mutant CH117, shows a drug-hypersensitivity (dhs) to cycloheximide, bleomycin, actinomycin D, 5-fluorouracil. nystatin, nigericin and several other antibiotics. CH 117 was also temperature-sensitive (ts). being unable to grow at
and secreted more invertase and acid phosphatase into the medium than the parent yeast. CH117 grows very slowly and the cell shape is somewhat larger and more sensitive to zymolyase than the wild type cells. Light microscopic and electron microscopic observation also revealed abnormality of the mutant cell wall. These characteristics indicate that CH117 has a defect in an essential component of the cell surface and that the cell wall which performs barrier functions has become leaky in the mutant. We screened a genomic library of wild type yeast for clones that can complement the mutation of CH117. A plasmid, pCHX1, with an insert of 3.6 kilobases (kbs) could complement the dhs and ts of CH117. Deletion and subcloning of the 3.6 kb insert showed that a gene for the complementation of mutant phenotypes was located in 1.9 kbs Puvll-Hindlll fragment.
Isolation and Characterization of a cDNA for a Ribulose-1,5-Bisphosphate Carboxylase Small Subunit in Spinach
Jin, Yun-Hae ; Park, Yang-Seo ; Jeong, Ji-Na ; Cho, Tae-Ju ; Cho, Nam-Jeong ;
BMB Reports , volume 30, issue 3, 1997, Pages 173~176
We isolated a cDNA clone that encodes a ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) from spinach using a soybean rbcS cDNA as a probe. The small subunit consists of 180 amino acids including a transit peptide of 57 residues. Comparison of the amino acid sequence with those of other plant species shows a maximum of 70-80% identical residues. Southern blot analysis suggests the existence of multiple rbcS genes in the spinach genome. Northern blot analysis indicates that the rbcS gene is expressed predominantly in leaves and that the expression of the gene is induced by light.
Construction and Characterization of a Single-Chain Immunoglobulin
Kim, Youn-Kyu ; Choi, In-Hak ; Ryu, Chun-Jeih ; Hong, Hyo-Jeong ;
BMB Reports , volume 30, issue 3, 1997, Pages 177~181
We constructed a single-chain immunoglobulin in which the carboxyl end of the heavy chain variable domain is covalently joined to the amino terminus of the light chain variable domain via peptide linker and the carboxyl end of the light chain variable domain is linked to human
Fc region through the hinge region. The molecule was expressed in Chinese hamster ovary cells, assembled into a dimeric molecule and secreted into the culture medium. The dimeric molecule (2E11) was purified from the culture supernatant by affinity chromatography on Protein G-Sepharose column. The size of the unreduced or reduced protein was the expected molecular weight of approximately 120 or 60 kDa, respectively, as assessed by SDS-polyacrylamide gel electrophoresis. The antigen-binding affinity of 2E11 was almost the same as that of a native antibody counterpart (CS131A), suggesting that the single-chain immunoglobulin may function like a native antibody.
Antagonists of Phosphatidylinositol 3-Kinase Block Phosphorylation-Dependent Activation of the Leukocyte NADPH Oxidase in a Cell-Free System
Park, Jeen-Woo ;
BMB Reports , volume 30, issue 3, 1997, Pages 182~187
The NADPH oxidase of phagocytes catalyzes the reduction of oxygen to
at the expense of NADPH. The enzyme is dormant in resting neutrophils and becomes activated on stimulation. During activation,
, a cytosolic oxidase subunit, becomes extensively phosphorylated at a number of serines located between S303-S379. Oxidase activation can also be achieved by the addition of phosphorylated recombinant
by protein kinase C in the cell-free system in the presence of
. The cell-free activation is inhibited by wortmannin and LY294002. specific inhibitors of phosphatidylinositol 3kinase (PI 3-kinasel) These results indicate that PI 3-kinase may playa pivotal role in the activation of NADPH oxidase.
Lipid Peroxidation Product-Mediated DNA Damage and Mutagenicity
Koh, Young-Ho ; Yoon, Seon-Joo ; Park, Jeen-Woo ;
BMB Reports , volume 30, issue 3, 1997, Pages 188~193
Membrane lipid peroxidation processes yield products that may react with DNA to cause mutations. Lipid hydroperoxides from linoleic acid in the presence of transition metal ions caused strand breaks in plasmid DNA. DNA damage induced by reactive aldehydes known to be produced by decomposition of lipid hydroperoxides, such as 4-hydroxynonenal or rnalondialdehyde, was repaired by endonucleases and exonuclease III which resulted in the increase of single strand breaks in DNA. Lipid hydroperoxides as well as malondialdehyde and 4-hydroxynonenal also caused mutations in the pUC18 lacZ' gene when measured as a loss of
. In conclusion. the lipid peroxidation could be an important intermediary event in DNA damage and mutation by oxidative stress.
Overexpression of Protein Kinase
Restores Mitogenic Responses of Enterocytic Differentiated Colon Carcinoma Cells to Diacylglycerol and Basic FGF
Lee, Han-Soo ;
BMB Reports , volume 30, issue 3, 1997, Pages 194~199
Previous studies have shown that the HD3 human enterocytic differentiated colon carcinoma cell lines having low
activity did not respond to diacylglycerol and basic FGF by growth and by activation of pp57 MAP kinase, but undifferentiated cell lines exhibiting high
activity did. To confirm a role of
in colonocyte mitogenesis, derivatives of HD3 cell line that stably overexpress a full-length of cDNA encoding the
isoform of human PKC were generated. The abundance and activity of
in two of the these cell lines, PKC3 and PKC8 were much higher than those in the C1 control cell line that carries the vector lacking the
insert. Following exposure to diacylglycerol or basic FGF, proliferation of PKC3 and PKC8 cells increased about 50%; but this effect was not seen with the control C1 cells. Also, in contrast to the control cells, the
cells displayed activation of pp57 MAP kinase when treated with diacylglycerol and basic FGF as undifferentiated cell lines did. These results provide direct evidence that
which plays a key role in mitogenic responses of colon carcinoma cells to diacylglycerol and basic FGF is down-regulated in enterocytic differentiation of colon cells.
Expression of Replication-Independent Chicken H3.3 Histone Gene without Introns
Son, Seung-Yeol ; Hong, Bum-Shik ;
BMB Reports , volume 30, issue 3, 1997, Pages 200~204
We eliminated introns from replication independent chicken H3.3 histone gene using a H3.3 cDNA clone and a genomic H3.3 clone. After introduction into Rat 3 cells, we observed its pattern of expression by analyzing mRNA from different phases of the cell cycle. Even without introns, the H3.3 gene was expressed constitutively at a low level throughout the cell cycle. This indicates that the introns in the H3.3 gene are not responsible for the cell cycle-independent expression of the gene. This result contradicts previous reports that suggested their importance in cell cycle regulated expression. We believe that other regions of the gene, promoter, coding region, and/or 3'-end of the gene, are involved in its expression pattern.
A 100 kDa Protein Binding to bHLH Family Consensus Recognition Sequence of RAT p53 Promoter
Lee, Min-Hyung ; Park, Sun-Hee ; Song, Hai-Sun ; Lee, Kyung-Hee ; Park, Jong-Sang ;
BMB Reports , volume 30, issue 3, 1997, Pages 205~210
p53 tumor suppressor plays an important role in the regulation of cellular proliferation. To identify proteins regulating the expression of p53 in rat liver, we analyzed p53 promoter by electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay. We found that a protein binds the sequence CACGTG, bHLH consensus sequence in rat p53 promoter. Southwestern blotting analysis with oligonucleotides containing this sequence shows that the molecular weight of the protein is 100 kDa. This size is not compatible with the bHLH family such as USF or c-Myc/Max which is known to regulate the expression of the human and mouse p53 gene. Therefore this 100 kDa protein may be a new protein regulating basal transcription of rat p53. We purified this 100 kDa protein through sequence-specific DNA affinity chromatogaphy.
Synthesis and Photoaffinity Labeling of 3'(2')-O-(p-azidobenzoyl) ATP
Shin, Seung-Jin ; Lee, Woo-Kyoung ; Park, Jong-Sang ;
BMB Reports , volume 30, issue 3, 1997, Pages 211~215
A photoactive analog of ATP, 3'(2')-O-(p-azidobenzoyl)-adenosine 5-triphosphate (AB-ATP) was synthesized by chemically coupling N-hydroxysuccinimidyl-4-azidobenzoate (NHS-AB) and ATP. The utility of AB-ATP as an effective active-site-directed photoprobe was demonstrated using catalytic subunit of protein kinase A as a model enzyme. Photoincorporation of AB-ATP was saturated with apparent dissociation constant of
and protected completely by
of ATP. When the enzyme was covalently modified by photolysis in the presence of saturating amounts of photoprobe, about 60% inhibition of enzyme activity was observed. These results demonstrate that AB-ATP has potential application as a probe to characterize ATP-binding proteins including protein kinases.
Induced Change in DNA Methylation of Fusarium oxysporum f. sp. niveum due to Successive Transfer
Kim, Dae-Hyuk ;
BMB Reports , volume 30, issue 3, 1997, Pages 216~221
Changes in pathogenicity of old and successively-cultured isolates of Fusarium oxysporum f. sp. niveum have been observed and the concept that such cultures will become attenuated is generally accepted. However, the genetic basis for this phenomenon has not been studied. In an effort to identify a DNA marker closely linked to variations, DNA methylation was investigated both before and after the successive transfers of F. o. f. sp. niveum isolates on artificial media. A sector of mycelium in F. o. f. sp. niveum race 2 isolate (TXXID) which showed variation in pigmentation and colonial morphology occurred after 18 successive weekly transfers on potato dextrose agar (PDA). The sector characteristics were stable and did not change after more successive transfers. It was shown that DNA methylation preexists in ribosomal RNA gene (rDNA) of F. o. f. sp. niveum and that additional changes in DNA methylation occurred during successive culturing.
Purification and Characterization of Protein Phosphatase 2C from Rat Liver
Oh, Joung-Sook ; Hwang, In-Seong ; Choi, Myung-Un ;
BMB Reports , volume 30, issue 3, 1997, Pages 222~228
Protein phosphatase 2C (PP2C) is one of the four major serine/threonine phosphatases which is dependent on
for its activity. PP2C was purified from rat liver cytosol and its characteristics were investigated. The substrate employed for routine assay was
phosphorylated by PKA. The purification process involved DEAE chromatography, ammonium sulfate fractionation, phenyl sepharose chromatography, sephacryl 5-200 gel filtration, and histone agarose chromatography. The SDS-PAGE of PP2C showed one major single protein band at a position corresponding to a molecular mass of 43 kd and the purification fold was 637. The enzyme showed a pH optimum of 8 and
. However, when the substrate was changed to
, the pH optimum was shifted to 7 and
was essential to the enzyme activity and okadaic acid did not exert any inhibitory effect on the enzyme. To examine residue in the active site of PP2C effects of some protein-modifying reagents were tested.
Synthesis and Biological Characterization of Indolicidin Analogues
Lim, Yong-Beom ; Pyun, Jae-Chul ; Park, Jong-Sang ;
BMB Reports , volume 30, issue 3, 1997, Pages 229~233
Indolicidin has been known to have a broad spectrum of antimicrobial activities against Gram negative and positive bacteria. Its eight analogues were chemically synthesized. The analogue design was based on the analysis of sequence to elucidate the role of some residues in the antibacterial mechanism of indolicidin. Bactericidal activities were assayed against Escherichia coli and Proteus vulgaris, and the membrane perturbing abilities of the peptides were assayed using a dye containing liposome. Among the eight analogues,
showed enhanced antibacterial activities. These results suggest that proline and cationic residues are important in the bactericidal activity of indolicidin. We tried to describe the antimicrobial mechanism of indolicidin with these results.
Quantitation of Hepatitis C Viral RNA Using Direct CRT-PCR
Park, Young-Suk ; Lee, Kyung-Ok ; Oh, Moon-Ju ; Chai, Young-Gyu ;
BMB Reports , volume 30, issue 3, 1997, Pages 234~236
Chronic hepatitis C virus (HCV) infection is associated with the rapid development of cirrhosis and hepatocellular carcinoma. It has been reported that the amount of HCV RNA may be correlated with the progression of hepatitis and may be a prognostic marker for treatment of HCV patients. The direct detection of HCV RNA by reverse transcription-polymerase chain reaction (RT-PCR) is widely used to determine the presence of circulating virions. The most relevant limit of this approach is the lack of quantitative information about the viral titer. In the present study, we developed the method for HCV quantitation using competitive reverse transcription (CRT)-PCR using the deleted HCV standard. The serially diluted standard was added in titrated amounts to the target HCV RNA. The mixture was then reverse transcribed and amplified in the same reaction tube. The methods were evaluated using over 110 HCV-PCR positive samples in Koreans. About 59% of the samples were judged to contain
copies of HCV RNA in 1 ml of serum.