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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Journal of Microbiology and Biotechnology
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Journal DOI :
The Korean Society for Applied Microbiology and Biotechnology
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Volume & Issues
Volume 15, Issue 6 - Dec 2005
Volume 15, Issue 5 - Oct 2005
Volume 15, Issue 4 - Aug 2005
Volume 15, Issue 3 - Jun 2005
Volume 15, Issue 2 - Apr 2005
Volume 15, Issue 1 - Feb 2005
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Dark Hydrogen Production by a Green Microalga, Chlamydomonas reinhardtii UTEX 90
SIM SANG JUN ; GONG GYEONG TAEK ; KIM MI SUN ; PARK TAl HYUN ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1159~1163
The production of hydrogen by Chlamydomonas reinhardtii UTEX 90, a marine green alga, was performed under dark fermentation. The effects of initial nitrogen and phosphorus concentration on the cell growth and the production of hydrogen and organic substances were investigated. In the growth stage, the maximum dry cell weight (DCW) was 3 g/l when the initial ammonium concentration was 15 mM. In the dark fermentation, the maximum hydrogen production was
DCW when the initial nitrogen concentration was 7.5 mM. The nitrogen concentration had a greater effect on organic compound and hydrogen production than the phosphorus concentration during the dark fermentation. An investigation of the duration of dark fermentation showed that, at least until three days, dark fermentation should be prolonged for maximum hydrogen production.
Characteristics of Microbial Biosurfactant as an Antifungal Agent Against Plant Pathogenic Fungus
YOO DAL-SOO ; LEE BAEK-SEOK ; KIM EUN-KI ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1164~1169
Characteristics of sophorolipid and rhamnolipid were evaluated as antifungal agents against plant pathogenic fungi. Eight percent of mycelial growth of plant pathogen (Phytophthora sp. and Pythium sp.) was inhibited by 200 mg/l of rhamnolipid or 500 mg/l of sophorolipid, and zoospore motility of Phytophthora sp. decreased by
at 50 mg/l of rhamnolipid and
at 100 mg/l of sophorolipid. The effective concentrations for zoospore lysis were two times higher than those of zoospore motility inhibition. The highest zoospore lysis was observed with Phytophthora capsici;
lysis at 100 mg/I of di-rhamnolipid or lactonic sophorolipid, showing the dependency of structure on the lysis. In the pot test, the damping-off disease incidence ratio decreased to
of control value at 2,000 mg/l sophorolipid and rhamnolipid, respectively. These results showed the potential of microbial glycolipid biosurfactants as an effective antifungal agent against damping-off plant pathogens.
Monitoring of Microorganisms Added into Oil-Contaminated Microenvironments by Terminal-Restriction Fragment Length Polymorphism Analysis
JUNG SEONG-YOUNG ; LEE JUNG-HYUN ; CHAI YOUNG-GYU ; KIM SANG-JIN ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1170~1177
Terminal-restriction fragment length polymorphism (T-RFLP) analysis was used to monitor inoculated oil-degrading microorganisms during bioremedial treatability tests. A pair of universal primers, fluorescently labeled 521F and 1392R, was employed to amplify small subunit rDNA in order to simultaneously detect two bacterial strains, Corynebacterium sp. IC10 and Sphingomonas sp. KH3-2, and a yeast strain, Yarrowia lipolytica 180. Digestion of the 5'-end fluorescence/labeled PCR products with HhaI produced specific terminal-restriction fragments (T-RFs) of 185 and 442 bases, corresponding to Corynebacterium sp. IC10 and Y. lipolytica 180, respectively. The enzyme NruI produced a specific T-RF of 338 bases for Sphingomonas sp. KH3-2. The detection limit for oildegrading microorganisms that were inoculated into natural environments was determined to be
of the total microbial count, regardless of the background environment. When three oil-degrading microorganisms were released into oil-contaminated sand microenvironments, strains IC10 and 180 survived for 35 days after inoculation, whereas strain KH3-2 was detected at 8 days, but not at 35 days. This result implies that T-RFLP could be a useful tool for monitoring the survival and relative abundance of specific microbial strains inoculated into contaminated environments.
Prediction on the Stability of Spray-Dried Lactobacillus reuteri KUB-AC5 by Arrhenius Equation for Long-Term Storage
KORAKOCH HAMSUPO ; SUKYAI PRAKIT ; LOISEAU GERARD ; NITISINPRASERT SUNEE ; MONTET DIDIER ; WANCHAITANAWONG PENKHAE ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1178~1182
Survival of thermotolerant Lactobacillus reuteri KUB-AC5 in
(w/v) skim milk was found to be
after spray drying by using a pilot scale spray dryer with inlet temperature at
and outlet temperature at
. The ability of dried cell to produce antimicrobial activity was not affected by the spray drying. The model system for predicting viability of spray-dried L. reuteri KUB-AC5 during long-term storage was established, based on the Arrhenius equation, and verified by experimental data, because the viability of cells during storage can be correlated with storage temperature. The viability during storage at
declined more rapidly than that storage at
Enzymatic Methanolysis of Castor Oil for the Synthesis of Methyl Ricinoleate in a Solvent-Free Medium
YANG JUNG-SEOK ; JEON GYU-JONG ; HUR BYUNG-KI ; YANG JI-WON ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1183~1188
Several lipases of commercial grade were screened to catalyze the methanolysis of castor oil, and an immobilized Candida antarctica (Novozym 435) had the highest activity among the lipases tested. To enhance the yield of methyl ricinoleate, several reaction parameters were optimized. The optimum temperature was
, and the original water content of lipase was sufficient to maintain the activity of lipase, and additional water supplied inhibited the methanolysis of castor oil. Because the lipase was deactivated by methanol, the reaction was tested by three-step addition of 1 molar equivalent of methanol to the oil. However, the oil was not completely converted to its methyl esters. The final reaction mixture using 3 molar equivalents of methanol to the oil consisted of
diricinoleate, and trace triricinoleate at the equilibrium state. The yield of methyl ricinoleate was
at 6 molar ratio of methanol to the oil with 300g of castor oil and 6g of immobilized Candida antarctica at
within 24 h.
Cloning and Characterization of Cyclohexanol Dehydrogenase Gene from Rhodococcus sp. TK6
CHOI JUN-HO ; KIM TAE-KANG ; KIM YOUNG-MOG ; KIM WON-CHAN ; JOO GIL-JAE ; LEE KYEONG-YEOLL ; RHEE IN-KOO ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1189~1196
The cyclohexanol dehydrogenase (ChnA), produced by Rhodococcus sp. TK6, which is capable of growth on cyclohexanol as the sole carbon source, has been previously purified and characterized. However, the current study cloned the complete gene (chnA) for ChnA and its flanking regions using a combination of a polymerase chain reaction (PCR) based on the N-terminal amino acid sequence of the purified ChnA and plaque hybridization from a phage library of Rhodococcus sp. TK6. A sequence analysis of the 5,965-bp DNA fragment revealed five potential open reading frames (ORFs) designated as partial pte (phosphotriesterase), acs (acyl-CoA synthetase), scd (short chain dehydrogenase), stp (sugar transporter), and chnA (cyclohexanol dehydrogenase), respectively. The deduced amino acid sequence of the chnA gene exhibited a similarity of up to
with members of the short-chain dehydrogenase/reductase (SDR) family. The chnA gene was expressed using the pET21 a(+) system in Escherichia coli. The activity of the expressed ChnA was then confirmed (13.6 U/mg of protein) and its properties investigated.
luxS and smcR Quorum-Sensing System of Vibrio vulnificus as an Important Factor for In Vivo Survival
SHIN NA-RI ; BAEK CHANG-HO ; LEE DEOG-YONG ; CHO YOUNG-WOOK ; PARK DAE-KYUN ; LEE KO-EUN ; KIM KUN-SOO ; YOO HAN-SANG ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1197~1206
Vibrio vulnificus is an opportunistic pathogen that causes a septicemia and expresses numerous virulence factors, in which luxS and smcR are genes encoding for components responsible for quorum-sensing regulation. In the present study, null mutants were constructed with lesions in each or both of these two genes from the V. vulnificus Vv
Z strain, which is a lacZ
and chloramphenicol/streptomycin-resistant derivative of the wild-type ATCC29307 strain, and their phenotypes related to virulence were compared with those of the parental cells.
and histopathological findings of luxS-, smcR-, or luxS- smcR- deficient mutant were not different from those of the parent strain, a lacZ-deficient streptomycin-resistant strain in mice. However, time of death in mice was delayed, and numbers of bacteria survived in bloodstream after intraperitoneal injection in mice were decreased by mutation, especially luxS and smcR double mutant (VvSR
ZSR). These phenomena were supported by increased serum sensitivity and delayed bacterial proliferation in both murine blood and iron-restricted medium. These results suggest that the luxS and luxR homologous genes in V. vulnificus could playa role in bacterial survival in host by enhancing proliferation and adjusting to changed environment.
Styrene Degradation in a Polyurethane Biofilter Inoculated with Pseudomonas sp. IS-3
KIM JAISOO ; RYU HEE WOOK ; JUNG DONG JIN ; LEE TAE HO ; CHO KYUNG-SUK ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1207~1213
In a search for bacteria capable of degrading styrene better than previously isolated strains, bacterium IS-3 was isolated from activated sludge and found to be most closely related to Pseudomonas sp. Styrene degradation by this strain was tested in liquid cultures and polyurethane-packed biofilters. In liquid cultures, the rate of styrene degradation by this bacterium increased from 24.93 to
for an initial mass range from 8.7 to
. The maximum styrene elimination capacity was 580-635
h at a space velocity (SV) of 50-200/h. The critical elimination capacities guaranteeing
removal of the input styrene were determined to be 635, 170, and 38
h, respectively, at SVs of 50, 100, and 200/h. Kinetic analysis revealed that the maximum styrene elimination velocity (
) for this biofilter was 1,000 g/m
h, and the saturation constant (
) was 454 ppmv. Together, these results suggest that a polyurethane biofilter containing Pseudomonas sp. IS-3 could have potential practical applications for the effective removal of styrene gas.
Inactivation of mutS Leads to a Multiple-Drug Resistance in Pseudomonas putida ATCC12633
KIM JEONG-NAM ; LEE SUNG-JAE ; LEE HO-SA ; RHIE HO-GUN ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1214~1220
Decreased porin-mediated outer membrane penetration of hydrophilic antibiotics is a common mechanism of antibiotic resistance in Gram-negative bacteria. This study was undertaken to determine whether a null mutation in Pseudomonas putida would suppress porin synthesis, and therefore reduce the susceptibility of the organism to streptomycin, norfloxacin, and tetracycline. Inverse PCR amplification and double-stranded DNA sequencing were used to identify chromosomal genes carrying TnphoA'-1 inserts. Genome database available was used to identify putative homologue genes, one of which encodes protein with homology to domains of the MutS of P. putida, suggesting a crucial role in the multidrug resistance. Increased resistance to streptomycin, norfloxacin, and tetracycline might be due to accumulation of compensatory mutations. Either no growth or slow growth was observed in P. putida KH1027 when grown in minimal medium containing gluconate, glucose, or citrate; however, it is not clear whether the growth patterns contributed to the multidrug resistance.
Influence of Electric Potential on Structure and Function of Biofilm in Wastewater Treatment Reactor : Bacterial Oxidation of Organic Carbons Coupled to Bacterial Denitrification
NA BYUNG KWAN ; SANG BYUNG IN ; PARK DAE WON ; PARK DOO HYUN ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1221~1228
Carbon electrode was applied to a wastewater treatment system as biofilm media. The spatial distribution of heterotrophic bacteria in aerobic wastewater biofilm grown on carbon electrode was investigated by scanning electron microscopy, atomic force microscopy, and biomass measurement. Five volts of electric oxidation and reduction potential were charged to the carbon anode and cathode of the bioelectrochemical system, respectively, but were not charged to electrodes of a conventional system. To correlate the biofilm architecture of bacterial populations with their activity, the bacterial treatment efficiency of organic carbons was measured in the bioelectrochemical system and compared with that in the conventional system. In the SEM image, the biofilm on the anodic medium of the bioelectrochemical system looked intact and active; however, that on the carbon medium of the conventional system appeared to be shrinking or damaging. In the AFM image, the thickness of biofilm formed on the carbon medium was about two times of those on the anodic medium. The bacterial treatment efficiency of organic carbons in the bioelectrochemical system was about 1.5 times higher than that in the conventional system. Some denitrifying bacteria can metabolically oxidize
, coupled to reduction of
was produced from the cathode in the bioelectrochemical system by electrolysis of water but was not so in the conventional system. The denitrification efficiency was less than
in the conventional system and more than
in the bioelectrochemical system. From these results, we found that the electrochemical coupling reactions between aerobic and anaerobic reactors may be a useful tool for improvement of wastewater treatment and denitrification efficiency, without special manipulations such as bacterial growth condition control, C/N ratio (the ratio of carbon to nitrogen) control, MLSS returning, or biofilm refreshing.
Mature HIV-like Particles Produced from Single Semliki Forest Virus-Derived Expression Vector
KIM EUN ; POO HAR-YOUNG ; SUNG MOON-HEE ; KIM CHUL-JOONG ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1229~1239
Human immunodeficiency virus-like particles (HIVVLPs) with native conformations similar to that of the wild-type virion could be valid candidates for vaccine development. To this end, we used a Semliki Forest Virus (SFV) expression system to produce HIV- VLPs containing high quantities of native envelope proteins. Here, we described a single SFV replicon containing the HIV gagpol and env genes under the control of separate subgenomic promoters. Mature VLPs incorporating the Gag and Env proteins were detected in the supernatant of replicon-expressing cells by Western blot analysis. The HIV-VLPs showed the expected molecular density (1.14-1.18 g/ml) on a
sucrose gradient; the particles were 100-120 nm in diameter and Env proteins were observed on their surfaces by immunogold electron microscopy. RT-PCR analysis of VLP-associated RNAs in mature HIV-VLPs revealed two SF V-derived RNA species (full-length and subgenomic). Immunization studies in Balb/c mice showed that these HIV-VLPs were capable of inducing both HIV-specific antibodies and cell-mediated immune responses. Taken together, our results indicate that the SFV replicon system is useful for the production of HIV-VLPs, which may be valuable candidates for an HIV vaccine.
Elucidation of Copper and Asparagine Transport Systems in Saccharomyces cerevisiae KNU5377 Through Genome-Wide Transcriptional Analysis
KIM IL-SUP ; YUN HAE SUN ; SHIMISU HISAYO ; KITAGAWA EMIKO ; IWAHASHI HITOSHI ; JIN INGNYOL ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1240~1249
Saccharomyces cerevisiae KNU5377 has potential as an industrial strain that can ferment wasted paper for fuel ethanol at
[15, 16]. To understand the characteristics of the strain, genome-wide expression was performed using DNA microarray technology. We compared the homology of the DNA microarray between genomic DNAs of S. cerevisiae KNU5377 and a control strain, S. cerevisiae S288C. Approximately
of the genes in S. cerevisiae KNU5377 were identified with those of the reference strain. YHR053c (CUP1), YLR155c (ASP3), and YDR038c (ENA5) showed lower homology than those of S. cerevisiae S288C. In particular, the differences in the regions of YHR053c and YLR155c were confirmed by Southern hybridization, but did not with that of the region of YDR038c. The expression level of mRNA in S. cerevisiae KNU5377 and S288C was also compared: the 550 ORFs of S. cerevisiae KNU5377 showed more than two-fold higher intensity than those of S. cerevisiae S288C. Among the 550 ORFs, 59 ORFs belonged to the groups of ribosomal proteins and mitochondrial ribosomal proteins, and 200 ORFs belonged to the group of cellular organization. DIP5 and GAP1 were the most highly expressed genes. These results suggest that upregulated DIP5 and GAP 1 might take the place of ASP3 and, additionally, the sensitivity against copper might be contributable to the lowest expression level of copper-binding metallothioneins encoded by CUP 1a (YHR053c) and CUP1b (YHR055c) in S. cerevisiae KNU5377.
Structural and Molecular Characterization of Extracellular Polysaccharides Produced by a New Fungal Strain, Trichoderma erinaceum DG-312
JOO JI-HOON ; YUN JONG-WON ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1250~1257
Two groups of exopolysaccharides (designated as Fr-I EPS and Fr-II EPS) were isolated from the culture filtrate of new fungal strain Trichoderma erinaceum DG-312 by Sepharose CL-6B chromatography. The structures of the exopolysaccharides were investigated using gas chromatography (GC), Fourier transform-infrared (FT-IR) spectroscopy, GCMS analysis, and NMR. GC analysis indicated that Fr-I EPS was composed of mainly mannose (
) and galactose (
), whereas Fr-II EPS contained mannose (
), galactose (
), and glucose (
). In the anomeric region (
) of the FT-IR spectrum, both EPSs exhibited obvious characteristic absorption of
, indicating the existence of mannose. The spectra of
-configurations were assigned at 880 and
, respectively. The results of GC-MS analyses confirmed that both EPSs were complex heteropolysaccharides with a (
)-linked mannan backbone. The C-1 region that appeared in the
spectra of these EPSs indicated a typical anomeric carbon signal. The Fr-I EPS showed two anomeric carbon signals at 102.6 and 99.6 ppm, whereas the Fr-II EPS displayed four anomeric carbon signals at 102.5, 99.6, 98.5, and 94.3 ppm. The molecular characteristics of the EPSs were further investigated using a size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS) system. The SEC/MALLS system revealed that the average molar masses of the EPSs were
(Fr-I EPS) and
(Fr-II EPS) g/mol, and the molecular conformation of both EPSs in aqueous solution was random coils.
Inhibitory Effect of Astragali Radix on Matrix Degradation in Human Articular Cartilage
CHOI SOOIM ; PARK SO-RA ; HEO TAE-RYEON ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1258~1266
The present study was carried out in order to assess the protective effects of calycosin-7-O-
-D-glucopyranoside, isolated from Astragali radix (AR), on hyaluronidase (HAase) and the recombinant human interleukin-
)-induced matrix degradation in human articular cartilage and chondrocytes. We isolated the active component from the n-butanol soluble fraction of AR (ARBu) as the HAase inhibitor and structurally identified as calycosin-7-O-
-D-glucopyranoside by LC-MS, IR,
NMR analyses. The
of this component on HAase was found to be 3.7 mg/ml by in vitro agarose plate assay. The protective effect of ARBu on the matrix gene expression of immortalized chondrocyte cell line C28/I2 treated with HAase was investigated using a reverse transcription polymerase chain reaction (RT-PCR), and its effect on HAase and IL-
-induced matrix degradation in human articular cartilage was determined by a staining method and calculating the amount of degraded glycosaminoglycan (GAG) from the cultured media. Pretreatment with calycosin-7-O-
-D-glucopyranoside effectively protected human chondrocytes and articular cartilage from matrix degradation. Therefore, calycosin-7-O-
-D-glucopyranoside from AR appears to be a potential natural ant-inflammatory or antii-osteoarthritis agent and can be effectively used to protect from proteoglycan (PG) degradation.
Production of Soluble Human Granulocyte Colony Stimulating Factor in E. coli by Molecular Chaperones
PARK SO-LIM ; SHIN EUN-JUNG ; HONG SEUNG-PYO ; JEON SUNG-JONG ; NAM SOO-WAN ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1267~1272
The effects of coexpression of GroEL/ES and DnaK/DnaJ/GrpE chaperones on the productivity of the soluble form of human granulocyte colony stimulating factor (hG-CSF) in E. coli were examined. Recombinant hG-CSF protein was coexpressed with DnaK/DnaJ/GrpE or GroEL/ES chaperones under the control of the araB or Pzt-1 promoter, respectively. The optimal concentration of L-arabinose for the expression of DnaK/DnaJ/GrpE was found to be 1 mg/ml. When L-arabinose was added at
=0.2 (early-exponential phase), soluble hG-CSF production was greatly increased. In addition, it was observed that the DnaK/DnaJ/GrpE and GroEL/ES chaperones had no synergistic effects on preventing aggregation of hG-CSF protein. Consequently, by coexpression of the DnaK/DnaJ/GrpE chaperone, the signal intensity of the hG-CSF protein band in the soluble fraction of cell lysate was increased from
, and Western blot analysis also revealed about a 4-5-fold increase of production of soluble hG-CSF over the non-induction case of DnaK/DnaJ/GrpE.
Solubilization of Hardly Soluble Phosphates and Growth Promotion of Maize (Zea mays L.) by Penicillium oxalicum Isolated from Rhizosphere
SHIN WANSIK ; RYU JEOUNGHYUN ; CHOI SEUNGJU ; KIM CHUNGWOO ; GADAGI RAVI ; MADHAIYAN MUNUSAMY ; SESHADRI SUNDARAM ; CHUNG JONGBAE ; SA TONGMIN ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1273~1279
Penicillium oxalicum strain CBPS-3F-Tsa, an efficient phosphate solubilizing fungus, was evaluated for its production of organic acid in vitro and effect of inoculation on the growth promotion of Maize under greenhouse conditions. The fungus solubilized 129.1, 118.8, and 54.1 mg P/1 of tricalcium phosphate [
], aluminum phosphate (
),and ferric phosphate (
), respectively, after 72 h of incubation. Malic acid, gluconic acid, and oxalic acid were detected in the flasks supplemented with various phosphate sources [240, 146, 145 mM/1
, respectively] together with a large amount of malic acid followed by the other two. The effects of inoculation of P. oxalicum CBPS-3F-Tsa on maize plants were studied under pot culture conditions. P. oxalicum CBPS-3F-Tsa was inoculated to maize plants alone or together with inorganic phosphates in the form of fused phosphates (FP) and rock phosphates (RP). Inoculation of P. oxalicum CBPS-3F-Tsa increased the plant growth and N and P accumulation in plants, compared with control plants, and also had positive effects when applied with RP. The results of this study show that the fungus P. oxalicum strain CBPS-3F-Tsa could solubilize different insoluble phosphates by producing organic acids, particularly malic acid, and also improved the efficiency of RP applied to maize plants.
Comparative Analyses of Flavonoids for nod Gene Induction in Bradyrhizobium japonicum USDA110
RYU JI-YOUNG ; HUR HOR-GIL ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1280~1285
Using the nodY::lacZ fusion system in Bradyrhizobium japonicum USDA 110, 22 flavonoids, which have structurally different features, were tested to define the role of the substituted functional groups as an inducer or inhibitor for the nod gene expression. A functional ,group of 4'-OH on the B-ring and the double bond between 2-C and 3-C on the C ring were required to induce the nod gene expression in B. japonicum USDA 110. In the case of isoflavones, the 4'-methoxyl group, which blocks the open 4'-OH functional group, did not significantly lower inducing activity, as compared with isoflavones with 4'-OH. However, all flavonols tested, which have a 3-OH functional group on the C-ring, did not induce, but inhibited the nod gene expression. Flavone, 7-hydroxyflavone, and kaempferol (5,7,4'-trihydroxyflavonol) at
concentration significantly inhibited the nod gene expression induced by 7,4'-dihydroxyflavone. However, 7-hydroxy-4'-methoxyflavone at
concentration showed a synergistic effect with genistein and 7,4'-dihydroxyflavone on the induction activity.
Diversity of Root-Associated Paenibacillus spp. in Winter Crops from the Southern Part of Korea
CHEONG HOON ; PARK SOO-YOUNG ; RYU CHOONG-MIN ; KIM JIHYUN F. ; PARK SEUNG-HWAN ; PARK CHANG SEUK ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1286~1298
The genus Paenibacillus is a new group of bacilli separated from the genus Bacillus, and most of species have been isolated from soil. In the present study, we collected 450 spore-forming bacilli from the roots of winter crops, such as barley, wheat, onion, green onion, and Chinese cabbage, which were cultivated in the southern part of Korea. Among these 450 isolates, 104 Paenibacillus-like isolates were selected, based on their colony shape, odor, color, and endospore morphology, and 41 isolates were then finally identified as Paenibacillus spp. by 16S rDNA sequencing. Among the 41 Paenibacillus isolates, 23 were classified as P. polymyxa, a type species of the genus Paenibacillus, based on comparison of the 16S rDNA sequences with those of 32 type strains of the genus Paenibacillus from the GenBank database. Thirty-five isolates among the 41 Paenibacillus isolates exhibited antagonistic activity towards plant fungal and bacterial pathogens, whereas 24 isolates had a significant growth-enhancing effect on cucumber seedlings, when applied to the seeds. An assessment of the root-colonization capacity under gnotobiotic conditions revealed that all 41 isolates were able to colonize cucumber roots without any significant difference. Twenty-one of the Paenibacillus isolates were shown to contain the nifH gene, which is an indicator of
fixation. However, the other 20 isolates, including the reference strain E681, did not incorporate the nifH gene. To investigate the diversity of the isolates, a BOX-PCR was performed, and the resulting electrophoresis patterns allowed the 41 Paenibacillus isolates to be divided into three groups (Groups A, B, and C). One group included Paenibacillus strains isolated mainly from barley or wheat, whereas the other two groups contained strains isolated from diverse plant samples. Accordingly, the present results showed that the Paenibacillus isolates collected from the rhizosphere of winter crops were diverse in their biological and genetic characteristics, and they are good candidates for further application studies.
Development of Serum-Free Media for Primary Culture of Human Articular Chondrocytes
CHOI YONG SOO ; LIM SANG MIN ; LEE CHANG WOO ; KIM DONG-IL ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1299~1303
Human articular chondrocytes (HAC) were cultivated as a monolayer in a serum-free medium for primary culture (SFM-P). An optimized SFM-P provides
proliferation rate of that obtainable from primary and secondary chondrocyte cultures grown in a control medium with serum. The gradual decrease in the amounts of synthesized glycosaminoglycan and type II collagen was improved by coating the culture dishes with type IV collagen and fibronectin. A significant improvement in the expression of type II collagen and aggrecan mRNA could be achieved. In addition, the monolayer cultures showed better synthesis of the extracellular matrices than alginate-bead cultures in SFM-P.
Expression of Human Interleukin-ll and Granulocyte-Macrophage Colony-Stimulating Factor in Transgenic Plants
LEE BO-YE ; LEE JEONG-HYUN ; YOON HOON-SEOK ; KANG KYUNG HO ; KIM KYUNG-NAM ; KIM JAE-HONG ; KIM JU-KON ; KIM JEONG-KOOK ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1304~1309
The production of therapeutic proteins for human diseases in plants results in many economic benefits, including reduced risk of animal virus contamination, high yields, and reduced production and storage costs. Human cytokines, interleukin-11 (hlL-11) and granulocyte-macrophage colony-stimulating factor (hGM-CSF), cDNAs were introduced into rice or tobacco, using either the maize ubiquitin promoter or the 35S promoter. The primary hIL-11 transgenic rice plants exhibited stunted growth and a sterile phenotype, whereas the hIL-11 transgenic tobacco plants did not. This suggests that hIL-11 expression in rice disrupts the normal growth and development of the plant. The regeneration efficiency of rice calli transformed with hGM-CSF was found to be approximately a quarter of that seen with the hIL-11, suggesting that hGM-CSF expression is more deleterious to the regeneration of rice calli than is hIL-11. However, the surviving hGM-CSF transgenic rice plants exhibited a normal phenotype of growth. Therefore, it appears that only those transgenic rice lines that expressed the human cytokines in small quantities were able to survive the selection process.
Transfected HepG2 Cells for Evaluation of Catechin Effects on Alcohol-Induced CYP2E1 Cytotoxicity
LEE YOO-HYUN ; HO JIN-NYOUNG ; DONG MI-SOOK ; PARK CHANG-HWAN ; KIM HYE-KYUNG ; HONG BUMSHIK ; SHIN DONG-HOON ; CHO HONG-YON ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1310~1316
To evaluate the toxicological properties of human cytochrome P450 2E1 (CYP2E1) induced by ethanol and possible protective effects of various green tea catechins on alcohol-induced toxicity, transfected HepG2 cells that stably and constitutively express human CYP2E1 were established using the recombinant retroviral expression vector. Exposure of the CYP2E1-expressing HepG2 cells to high concentration of ethanol (200 mM) for 5 days resulted in a more than
increase of cytotoxicity, assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase (LDH) release, and reactive oxygen species (ROS) production, and loss of normal morphology, in comparison with HepG2 cells containing control vector. Treatment of the cells with various catechins increased cell viability by more than 2-fold. (-)-Epicatechin gallate and(-)-catechin gallate at the lowest concentration (
) attenuated cell death induced CYP2E1 by
. Therefore, the results showed that the catechins, including epimerized catechins, have strong protective effects against alcohol-induced CYP2E1 toxicity, and it is correlated with antioxidant effect.
Immobilization of Penicillium citrinum by Entrapping Cells in Calcium Alginate for the Production of Neo-Fructooligosaccharides
Lim, Jung-Soo ; Park, Seung-Won ; Lee, Jin-Won ; Oh, Kyeong-Keon ; Kim, Seung-Wook ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1317~1322
This work describes neo-fructooligosaccharides (neo-FOSs) production using the immobilized mycelia of Penicillium citrinum. Some critical factors were evaluated to optimize maximal production of neo-FOS. Optimal alginate and cell concentrations were determined to be
cell, respectively, by statistical analysis. The optimal concentration of
, which is related to bead stability, was determined to be 2 M. It was possible to increase the neo-FOS production by adding 15 units of glucose oxidase to the batch reaction. By co-immobilizing cells and glucose oxidase, neoFOS productivity increased
compared with the whole-cell immobilization process. Based on the results above, a co-immobilization technique was developed and it can be utilized for large-scale production.
Predictive Modeling for the Growth of Listeria monocytogenes as a Function of Temperature, NaCl, and pH
PARK SHIN YOUNG ; CHOI JIN-WON ; YEON JIHYE ; LEE MIN JEONG ; CHUNG DUCK HWA ; KIM MIN-GON ; LEE KYU-HO ; KIM KEUN-SUNG ; LEE DONG-HA ; BAHK GYUNG-JIN ; BAE DONG-HO ; KIM KWANG-YUP ; KIM CHEOL-HO ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1323~1329
A mathematical model was developed for predicting the growth kinetics of Listeria monocytogenes in tryptic soy broth (TSB) as a function of combined effects of temperature, pH, and NaCl. The TSB containing four different concentrations of NaCl (2, 4, 5, and
) was initially adjusted to six different pH levels (pH 5, 6, 7, 8, 9, and 10) and incubated at 4, 10, 25, or 37
. In all experimental variables, the primary growth curves were well fitted (
=0.982 to 0.998) to a Gompertz equation to obtain the lag time (LT) and specific growth rate (SGR). Surface response models were identified as appropriate secondary models for LT and SGR on the basis of coefficient determination (
=0.907 for LT, 0.964 for SGR), mean square error (MSE=3.389 for LT, 0.018 for SGR), bias factor (
B,=0.706 for LT, 0.836 for SGR), and accuracy factor (
=1.567 for LT, 1.213 for SGR). Therefore, the developed secondary model proved reliable predictions of the combined effect of temperature, NaCl, and pH on both LT and SGR for L. monocytogenes in TSB.
-NMR Spectroscopic Study of Polyhydroxyalkanoic Acid Degradation Kinetics in Bacteria
Oh, Jung-Sook ; Choi, Mun-Hwan ; Yoon, Sung-Chul ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1330~1336
Polyhydroxyalkanoic acid (PHA) inclusion bodies were analyzed in situ by
-nuclear magnetic resonance (
-NMR) spectroscopy. The PHA inclusion bodies studied were composed of poly(3-hydroxybutyrate) or poly(3hydroxybutyrate-co-4-hydroxybutyrate), which was accumulated in Hydrogenophaga pseudoflava, and medium-chain-length PHA (MCL-PHA), which was accumulated in Pseudomonas fluorescens BM07 from octanoic acid or 11-phenoxyundecanoic acid (11-POU). The quantification of the
-NMR signals was conducted against a standard compound, sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). The chemical shift values for the in vivo NMR spectral peaks agreed well with those for the corresponding purified PHA polymers. The intracellular degradation of the PHA inclusions by intracellular PHA depolymerase(s) was monitored by in vivo NMR spectroscopy and analyzed in terms of first-order reaction kinetics. The H. pseudoflava cells were washed for the degradation experiment, transferred to a degradation medium without a carbon source, but containing 1.0 g/l ammonium sulfate, and cultivated at
for 72 h. The in vivo NMR spectra were obtained at
for the short-chain-length PHA cells whereas the spectra for the aliphatic and aromatic MCL-PHA cells were obtained at
, respectively. For the H. pseudoflava cells, the in vivo NMR kinetics analysis of the PHA degradation resulted in a first-order degradation rate constant of 0.075/h (
=0.94) for the initial 24 h of degradation, which was close to the 0.050/h determined when using a gas chromatographic analysis of chloroform extracts of sulfuric acid/methanol reaction mixtures of dried whole cells. Accordingly, it is suggested that in vivo
-NMR spectroscopy is an important tool for studying intracellular PHA degradation in terms of kinetics.
Identification of the Vibrio vulnificus fexA Gene and Evaluation of its Influence on Virulence
JU HYUN-MOK ; HWANG IN-GYUN ; WOO GUN-JO ; KIM TAE SUNG ; CHOI SANG HO ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1337~1345
Vibrio vulnificus is the causative agent of foodborne diseases such as gastroenteritis and life-threatening septicemia. Microbial pathogenicity is a complex phenomenon in which expression of numerous virulence factors is frequently controlled by a common regulatory system. In the present study, a mutant exhibiting decreased cytotoxic activity toward intestinal epithelial cells was screened from a library of V. vulnificus mutants constructed by a random transposon mutagenesis. By a transposon-tagging method, an open reading frame, fexA, a homologue of Escherichia coli areA, was identified and cloned. The nucleotide and deduced amino acid sequences of the fexA were analyzed, and the amino acid sequence of FexA from V. vulnificus was
similar to those of AreA, an aerobic respiration control global regulator, from other Enterobacteriaceae. Functions of the FexA were assessed by the construction of an isogenic mutant, whose fexA gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in vitro and in mice. The disruption of fexA resulted in a significant alteration in growth rate under aerobic as well as anaerobic conditions. When compared to the wild-type, the fexA mutant exhibited a substantial decrease in motility and cytotoxicity toward intestinal epithelial cell lines in vitro. Furthermore, the intraperitoneal
of the fexA mutant was approximately
times higher than that of parental wild-type. Therefore, it appears that FexA is a novel global regulator controlling numerous genes and contributing to the pathogenesis as well as growth of V. vulnificus.
A Modified PCR-Directed Gene Replacements Method Using
-Red Recombination Functions in Escherichia coli
KIM SANG-YOON ; CHO JAE-YONG ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1346~1352
We have developed a modified gene replacement method using PCR products containing short homologous sequences of 40- to 50-nt. The method required
-Red recombination functions provided under the control of a temperature-sensitive CI857 repressor expressed from the
promoter in the presence of IPTG on an easily curable helper plasmid. The method promoted the targeted gene replacements in the Escherichia coli chromosome after shifting cultures of the recombinogenic host, which carries the helper plasmid, to
for 15 min. Since this method employs
-Red recombination functions expressed from the easily curable helper plasmid, multiple rounds of gene replacements in the E. coli chromosome would be possible. The procedures described herein are expected to be widely used for metabolic engineering of E. coli and other bacteria.
Quantitative Detection of Salmonella typhimurium Contamination in Milk, Using Real-Time PCR
JUNG SUNG JE ; KIM HYUN-JOONG ; KIM HAE-YEONG ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1353~1358
A rapid and quantitative real-time PCR was developed to target the invasion A (invA) gene of Salmonella spp. We developed quantitative standard curves based on plasmids containing the invA gene. Based on these curves, we detected Salmonella spp. in artificially contaminated buffered peptone water (BPW) and milk samples. We were able to determine the invA gene copy number per ml of food samples, with the minimum detection limit of
copies/ml of BPW and
copies/ml of milk. When applied directly to detect and quantify Salmonella spp. in BPW and milk, the present real-time PCR assay was as sensitive as the plate count method; however, copy numbers were one to two logs higher than the colony-forming units obtained by the plate count methods. In the present work, the real-time PCR assay was shown to significantly reduce the total time necessary for the detection of Salmonella spp. in foods and to provide an important model for other foodborne pathogens.
Cloning, Expression, and Characterization of a Family B-Type DNA Polymerase from the Hyperthermophilic Crenarchaeon Pyrobaculum arsenaticum and Its Application to PCR
SHIN HEA-JIN ; LEE SUNG-KYOUNG ; CHOI JEONG JIN ; KOH SUK-HOON ; LEE JUNG-HYUN ; KIM SANG-JIN ; KWON SUK-TAE ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1359~1367
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
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
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
was 6 h. Par DNA polymerase possessed associated
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.
Profiling Pyocins and Competitive Growth Advantages of Various Pseudomonas aeruginosa Strains
Heo YUN-JEONG ; KO KWAN SOO ; SONG JAE-HOON ; CHO YOU-HEE ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1368~1376
Pseudomonas aeruginosa produces a variety of bacteriocidal substances including pyocins that are active against the same species, but their physiological roles are relatively unknown. Here, we profiled the bacteriocidal activities in the culture supernatants of various P. aeruginosa isolates and describe the competitive growth advantages of strains PAO1 and PA14 over some strains including PAK, which are sensitive to their bacteriocidal activities. These findings suggest that the factors governing the production of pyocins and the resistance to them play important roles in controlling P. aeruginosa populations in its local environments.
Simple Method to Correct Gene-Specific Dye Bias from Partial Dye Swap Information of a DNA Microarray Experiment
KIM BYUNG SOO ; KANG SOO-JIN ; LEE SAET-BYUL ; HWANG WON ; KIM KUN-SOO ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1377~1383
In a cDNA microarray experiment using Cy3 and Cy5 as labeling agents, particularly for the direct design, cDNAs from some genes incorporate one dye more efficiently than the other, which is referred to as the gene-specific dye bias. Dye-swaps, in which two dyes are switched on replicate arrays, are commonly used to control the gene-specific dye bias. We developed a simple procedure to extract the gene-specific dye bias information from a partial dye swap experiment. We detected gene-specific dye bias by identifying outliers in an X-Y plane, where the X axis represents the average log-ratio from two sets of dye swap pairs and the Y axis exhibits the average log ratio of four forward labeled arrays. We used this information for detecting differentially expressed genes, of which the additionally detected genes were validated by real-time RT-PCR.
Analysis of Integrity of Killed Hantavirus Vaccine by Antigen-Capture Reverse Transcriptase PCR
HWANG KYUNG-A ; JOO YOUNG-RAN ; SHIN YOUNG-HAK ; PARK KEUN-YONG ; NAM JAE-HWAN ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1384~1387
Hantavax(R) is one of the killed Hantavirus vaccines, and is commercially available in South Korea. This vaccine was developed by inactivation of virus isolated from infected suckling mouse brain with formalin. Although Hantavax(R) can induce neutralizing antibodies in vaccinees, the strength of this induction and the duration of the humoral immune response are controversial issues. In this study, we studied the native conformation of the killed vaccine by antigen-capture reverse transcriptase polymerase chain reaction with patient and vaccinee sera containing neutralizing antibodies against Hantavirus. The results showed that Hantavax(R) could bind HTNV patient and vaccinee sera like live virus, suggesting that the integrity of the viral epitope is maintained in Hantavax(R) and induces the protective antibodies, even though the virus was inactivated with formalin.
-D-Glucans Production by Agaricus blazei Murill by Nitrogen Supplementation
NA JEONG-GEOL ; KIM HYUN-HAN ; CHUN GIE-TAEK ; CHANG YONG KEUN ; LEE SANG JONG ; CHUNG YEON HO ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1388~1391
Temporal changes of cell growth pattern and intracellular content of
-D-glucans were investigated with off-gas data in Agaricus blazei culture where glucose was intermittently fed. It was observed that the time point of carbon source depletion coincided with the point of sudden drop in the carbon dioxide evolution rate (CER), and that the sole supplementation of glucose was not enough to maintain active cell growth and glucan content. On the other hand, when yeast extract, a typical nitrogen source, was supplemented together with glucose when the CER suddenly dropped because of carbon source depletion, an active cell growth could be maintained until the end of the culture and the glucan content did not decrease with culture time, significantly enhancing glucan productivity.
Stability and Structural Change of cAMP Receptor Protein at Low and High cAMP Concentrations
GANG JONGBACK ; CHUNG HYE-JIN ; PARK GWI-GUN ; PARK YOUNG-SEO ; CHOI SEONG-JUN ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1392~1396
Proteolytic digestion and CD measurement of wild-type and mutant cyclic AMP receptor proteins (CRPs) were performed either in the presence or absence of cyclic nucleotide. Results indicated that transition of a structural change to the hinge region by the binding of cAMP to the anti site was required for the binding of cAMP to the syn site near the hinge region and, although the occupancy of cAMP in the anti site increased the protein stability, CRP adopted more a stable conformation by the binding of cAMP to the syn site.
High-Level Expression of Recombinant Human Bone Morphogenetic Protein-4 in Chinese Hamster Ovary Cells
PARK JUNHO ; YU SUNGRYUL ; YOON JAESEUNG ; BAEK KWANGHEE ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1397~1401
Bone morphogenetic protein-4 (BMP-4) is a signaling homodimeric molecule that acts as a morphogen to influence cell fate in a concentration-dependent manner. The limited supply of a pure preparation of BMP-4, due to very low level of their expression in vivo, makes it difficult not only to study the biological activities of BMPs, but also to use them as a clinical tool. For a large-scale production of BMP-4, human BMP-4 cDNA was expressed in Chinese hamster ovary (CHO) cells by a recently development vector system, which confers position-independent stable expression of the foreign genes. The CHO cell line expressing recombinant human BMP-4 (rhBMP-4) at the level of
could be obtained after stepwise selection with methotrexate. This level of expression is about 70 times higher than those previously reported. The partially processed form of BMP-4 as well as mature form could be detected, when the aliquots of culture media were analyzed by Western blot. The glycosylation pattern and biological activity of the rhBMP-4 were determined by glycosidase treatment and the induction rate of alkaline phosphatase in mouse osteoblastic cells.
In Vivo Antifungal Effects of Coptis japonica Root-Derived Isoquinoline Alkaloids Against Phytopathogenic Fungi
LEE CHI-HOON ; LEE HOI-JOUNG ; JEON JU-HYUN ; LEE HOI-SEON ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1402~1407
The fungicidal activities of Coptis japonica (Makino) extracts and their active principles were determined against Botrytis cineria, Erysiphe graminis, Phytophthora infestans, Puccinia recondita, Pyricularia grisea, and Rhizoctonia solani using a whole plant method in vivo, and compared with natural fungicides. The responses varied according to the plant pathogen tested. At 2,000 mg/l, the chloroform and butanol fractions obtained from methanolic extracts of C. japonica exhibited strong/moderate fungicidal activities against B. cinerea, E. graminis, P. recondita, and Py. grisea. Two active constituents from the chloroform fractions and one active constituent from the butanol fractions were characterized as isoquinoline alkaloids, berberine chloride, palmatine iodide, and coptisine chloride, respectively, using spectral analysis. Berberine chloride had an apparent
value of approximately 190, 80, and 50 mg/l against B. cinerea, E. graminis, and P. recondita, respectively; coptisine chloride had an
value of 210,20, 180, and 290 mg/l against B. cinerea, E. graminis, P. recondita, and Py. grisea, respectively; and palmatine iodide had an
value of 160 mg/l against Py. grisea. The isoquinoline alkaloids were also found to be more potent than the natural fungicides, curcumin and emodin. Therefore, these compounds isolated from C. japonica may be useful leads for the development of new types of natural fungicides for controlling B. cinerea, E. graminis, P. recondita, and Py. grisea in crops.
Re-Engineering of Carcinoembryonic Antigen RNA with the Group I Intron of Tetrahymena thermophila by Targeted Trans-Splicing
JUNG HEUNG-SU ; LEE SEONG-WOOK ;
Journal of Microbiology and Biotechnology, volume 15, issue 6, 2005, Pages 1408~1413
Elevated expression of carcinoembryonic antigen (CEA) has been implicated in various biological aspects of neoplasia such as tumor cell adhesion, metastasis, blocking of cellular immune mechanisms, and antiapoptosis function. Thus, the CEA could be an important target for anticancer therapy. In this study, we developed Tetrahymena group 1 intron-based trans-splicing ribozymes that can specifically target and replace CEA RNA. To this end, we first determined which regions of the CEA RNA were accessible to ribozymes by employing an RNA mapping strategy that was based on a trans-splicing ribozyme library. Next, we assessed the ribozyme activities by comparing the trans-splicing activities of several ribozymes that targeted different regions of the CEA RNA, and then the ribozyme that could target the most accessible site was observed to be the most active with high fidelity in vitro. Moreover, the specific trans-splicing ribozyme was found to react with and altered the target CEA transcripts in mammalian cells with high fidelity. These results suggest that the Tetrahymena ribozyme can be utilized to replace CEA RNAs in tumors with a new RNA-harboring anticancer activity, thereby hopefully reverting the malignant phenotype.