• Korean Journal of Microbiology
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• v.55 no.3
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• pp.199-205
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• 2019

Yeast two-hybrid (Y2H) technique has been used to study protein-protein interactions, but its application particularly to a large-scale analysis of protein interaction networks, is limited by the fact that the technique is labor-intensive, based on scoring colonies on plate. Here, we develop a new reporter for the measurement of the protein-protein interactions by flow cytometry. The yeast harboring interacting proteins can also be enriched by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). When two interacting proteins are present in the same yeast cell, a reporter protein containing 10 tandem repeats of c-myc epitope becomes localized on the surface of the cell wall, without affecting cell growth. We successful measured the surface display of c-myc epitope upon interacting p53 with SV40 T antigen by flow cytometry. Thus, the newly developed Y2H assay based on the display of c-myc repeat on yeast cell wall could be used to the simultaneous analysis of multiple protein-protein interactions without laborious counting colonies on plate.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.183-188
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• 2002

The ohmic heating of foods for sterilization provides a shorter come-up time compared to conventional thermal processes. The electric fields as well as the heat generated by ohmic heating facilitate germicidal effects. In the present study, the effect of ohmic heating on the structure and permeability of the cell membrane of yeast cells, Saccharomyces cerevisae, isolated from Takju (a traditional Korean rice-beer), was investigated. The ohmic heating was found to translocate intracellular protein materials out of the cell wall, and the amount of exuded protein increased significantly as the electric field increased from 10 to 20 V/cm. As higher frequencies were applied, more materials were exuded. Compared to conventional heating, more amounts of proteins and nucleic acids were exuded when these cells were treated with ohmic heating. The molecular weights of the major exuded proteins ranged from 14 kDa to 18 kDa, as analyzed by Tricine-SDS PAGE. A TEM study also confirmed the leakage of cellular materials, thus indicating irreversible damage to the cell wall by ohmic heating. It was, therefore, concluded that the electric fields generated by ohmic heating induced electroporation, causing irreversible damage to the yeast cell wall and promoting the translocation of intracellular materials.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.223-228
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• 1997

A DNA fragment containing the gene for cell wall hydrolase of alkalophilic Bacillus subtilis BL-29 was cloned into E. coli JM109 using pUC18 as a vector. A recombinant plasmid, designated pCWL45B, was contained in the fragment originating from the alkalophilic B. subtilis BL-29 chromosomal DNA by Southern hybridization analysis. The nucleotide sequence of a 1.6-kb HindIII fragment containing a cell wall hydrolase-encoding gene was determined. The nucleotide sequence revealed an open reading frame (ORF) of 900 bp with a concensus ribosome-binding site located 6 nucleotide upstream from the ATG start codon. The primary amino acid sequence deduced from the nucleotide sequence revealed a putative protein of 299 amino acid residues with an M.W. of 33, 206. Based on comparison of the amino acid sequence of the ORF with amino acid sequences in the GenBank data, it showed significant homology to the sequence of cell wall amidase of the PBSX bacteriophage of B. subtilis.

• Food Science and Preservation
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• v.2 no.1
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• pp.185-193
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• 1995

This paper was investigated the changes of the cell wall components, enzyme activities during ripening of jujuba fruits for elucidating the softening metabolism of jujuba fruits. Firmness were decreased during ripening. Moisture content did not show any notable cahanges until ripening stage but they decreased a little In overripe jujuba fruits. Polygalacturonase activities were not detected at nature green stage and $\beta$-galactosidase activities were until turning stage. But polygalacturonase activities in ripening and overripening were 51.31 and 100.72 units/100g-fr, wt. respectively. $\beta$-galactosidase activities were 16.05 and 182.55units/100g-fr. wt. in the same stages. The content of water-soluble protein was increased in overripening. Stage the contents of cell wall and alcohol-insoluble material were. decraesed during maturation, but water-soluble material was increased. The pectin and alkali-soluble hemicellulose were increased until ripening stage, but decreased in overripe jujube fruits. The total pectin and insoluble pectin during ripening, but decreased in overripe jujuba fruits.

• Korean Journal of Veterinary Research
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• v.25 no.2
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• pp.149-153
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• 1985

Salmonella pullorum strain W was serially passaged on the brain heart infusion agar containing acrdine orange(AO) as a concentration of 100 mcg/ml. S. pullorum AO60 and S. pullorum AO150, which were subcultured 60 and 150 passages on AO media, were examined for permeability barrier function of the cell wall. AO60 and AO150 were appeared to be decreased in susceptibility against hydrophobic substances such as crystal violet, chloramphenicol and rifamycin, which might be resulted from the changes of permeability barrier function of the cell wall. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacterial protein, the protein profiles of AO6O and AO150 didn't differ significantly from W, but increased amount of the band of MW 140,000-145,000 was confirmed. And [G+C] contents of DNA in AO60 and A0150 were decreased than that of W.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.508-514
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• 2001

A stuructural gene (ycl) encoding novel yeast cell wall hydrolase, YCL, was cloned from alkalophilic Bacillus alcalophilus subsp. YB380 by PCR, and transformed into E. coli JM83. Based on the N-terminal and internal amino acid sequences of the enzyme, primers were designed for PCr. The positive clone that harbors 1.8 kb of the yeast cell wall hydrolase gene was selected by the colony hybridization method with a PCR fragment as a probe. According to the computer analysis, this gene contained a 400-base-paired N-terminal domain of the enzyme. Based on nucletide homology of the cloned gene, a 850 bp fragment was amplified and the C-terminal domain of the enzyme was sequenced. With a combination of the two sequences, a full nucleotide sequence for YCL was obtained. This gene, ycl, consisted of 1,297 nucleotides with 27 nucleotides with 27 amino acids of signal sequence, 83 redundant amino acids of prosequence, and 265 amino acids of the mature protein. This gene was then cloned into the pJH27 shuttle vector and transformed into the Bacillus subtilis DB104 to express the enzyme. It was confirmed that the expressed cell wall hydrolase that was produced by Bacillus subtilis DB104 was the same as that of the donor strain, by Western blot using polyclonal antibody (IgY) prepared from White Leghorn hen. Purified yeast cell wall hydrolase and expressed recombinant protein showed a single band at the same position in the Western blot analysis.

• KSBB Journal
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• v.7 no.3
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• pp.209-215
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• 1992

The aims of this sutdy was to investgate the action mechanism of polycation on the $\beta$-glucan synthetase II (GS II) related to cell wall synthesis in suspension cultured carrot cells. In the suspension cultured cells treated with poly-L-Iysine($12{\mu}M$) and poly-L-ornithine ($12{\mu}M$) having ploycationic nature, GS II activity increased about 40% and 50% than that of the control respectively. And similar response was observed when ATP and NaF were treated. On the other hand, ploy-L-lysine and ploy-L-ornithine did nor affect the membrane permeability. Phorbol-12-myrlstate-13-acetate (TPA), activator of protein klnase, increased about 35% and 1-(5-isoquinolinyl sulfonyl)-2-methyl-piperrazine (H-7), inhibitor of protein kinase, decreased about 30% of GSII activity than that of control. These results suggest that polycation plays a role in the cell wall synthesis by increasing GS II activity through phosphorylation.

• ALGAE
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• v.31 no.4
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• pp.379-390
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• 2016

When a multinucleate cell of Bryopsis plumosa was collapsed by a physical wounding, the extruded protoplasm aggregated into numerous protoplasmic masses in sea water. A polysaccharide envelope which initially covered the protoplasmic mass was peeled off when a cell membrane developed on the surface of protoplast in 12 h after the wounding. Transmission electron microscopy showed that the protoplasmic mass began to form a continuous cell membrane at 6 h after the wounding. The newly generated cell membrane repeated collapse and rebuilding process several times until cell wall developed on the surface. Golgi bodies with numerous vesicles accumulated at the peripheral region of the rebuilding cell at 24 h after the wounding when the cell wall began to develop. Several layers of cell wall with distinctive electron density developed within 48-72 h after the wounding. Proteome profile changed dramatically at each stage of cell rebuilding process. Most proteins, which were up-regulated during the early stage of cell rebuilding disappeared or reduced significantly by 24-48 h. About 70-80% of protein spots detected at 48 h after the wounding were newly appeared ones. The expression pattern of 29 representative proteins was analyzed and the internal amino acid sequences were obtained using mass spectrometry. Our results showed that a massive shift of gene expression occurs during the cell-rebuilding process of B. plumosa.

• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.1977-1988
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• 2015

β-1,3-glucanosyltransferases play essential roles in cell wall biosynthesis in yeast. Kluyveromyces lactis has six putative β-1,3-glucanosyltransferase genes. KlGAS1-1 and KlGAS1-2 are homologs of Saccharomyces cerevisiae gene GAS1. RT-qPCR indicated the transcription level of KlGAS1-1 was significantly reduced while heterologous protein (thermostable xylanase B) secretion was enhanced during medium optimization. To evaluate if these two events were related, and to improve xylanase B secretion in K. lactis, we constructed KlGAS1-1 and KlGAS1-2 single deletion strains and double deletion strain, respectively. KlGAS1-1 gene deletion resulted in the highest xylanase B activity among the three mutants. Only the double deletion strain showed morphology similar to that of the GAS1 deletion mutant in S. cerevisiae. The two single deletion strains differed in terms of cell wall thickness and xylanase B secretion. Transcription levels of β-1,3-glucanosyltransferase genes and genes related to protein secretion and transport were assayed. The β-1,3-glucanosyltransferase genes displayed transcription complementation in the cell wall synthesis process. KlGAS1-1 and KlGAS1-2 affected transcription levels of secretion- and transport-related genes. Differences in protein secretion ratio among the three deletion strains were associated with changes of transcription levels of secretion- and transport-related genes. Our findings indicate that KlGAS1-1 deletion is an effective tool for enhancing industrial-scale heterologous protein secretion in K. lactis.

• Journal of Plant Biology
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• v.22 no.1_2
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• pp.35-43
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• 1979

The present investigation has been made to study the deficiency symptoms of boron on the formation of cell wall and the development of the individual components of the orchid cell wall. Analytical samples were taken from two sources; one from the individual orchid plants started from an apical meristem culture followed by the generation of the protocorm-like body which was developed into a plant, the other from the plant cultivated in water for 30 days. The amount of boron in the cultrues were controlled and the deficiency symptoms were observed under theelectron microscope, optical microscope with samples taken from the zones of elongation of leaves and compared the dry weight of cell walls and finally the various fractions of the cell wall components. The following results were obtained: (1) The growth of roots and leaves was hampered in the boron deficient plants. (2) In the boron-deficient leaves a severe necrosis and cracks were developed in the tissue of zone of elongation besides the decrease in growth. (3) under the electorn microscope the cell walls of boron-deficient plants showed rough undulated structures unlike the smooth control cell walls. (4) the dry weight of total cells and cell walls of boron deficient plants were higher than the control plants. (5) In the boron deficient plant the amout of pectin and hemicellulose isolated from cell walls were higher and the amount of protein was lower than the controlled plots.