• The Plant Pathology Journal
• /
• v.21 no.1
• /
• pp.13-20
• /
• 2005

Chlorella viruses are large icosahedral, plaque-forming, dsDNA viruses that infect certain unicellular, chlorellalike green algae. The genomic DNA of over 300 kb contains many useful genes and promoters. Over 40 chlorella viruses have been isolated from fresh water in Korea since 1998. The viruses were amplified initially in chlorella strain NC64A, and pure isolates were obtained by repeated plaque isolation. SDS-PAGE analysis revealed similar but distinct protein patterns, both among the group of purified viruses and in comparison with the prototype chlorella virus PBCV-1. Digestions of the 330- to 350-kb genomic DNAs with 10 restriction enzymes revealed different restriction fragment patterns among the isolates. The tRNA-coding regions of 8 chlorella viruses were cloned and sequenced. These viruses contain 14-16 tRNA genes within a 1.2- to 2-kb region, except for the SS-1 isolate, which has a 1039-bp spacer in a cluster of 11 tRNA genes. Promoter regions of several early genes were isolated and their activities were analyzed in transformed chlorella. Some promoters showed stronger activity than commonly used CaMV 35S promoter and chlorella transformation vectors for heterologous protein are beings constructed using these promoters.

• BMB Reports
• /
• v.38 no.4
• /
• pp.457-467
• /
• 2005

Open reading frame (ORF) 3 on the Choristoneura fumiferana granulovirus (ChfuGV), located in the 11 kb fragment of the BamHI genomic bank encodes a predicted 32-kDa putative kinase protein. Bioinformatics analysis on the predicted amino acid sequence of ChfuGV PK-1 revealed the existence of 11 catalytic subdomains. Sequence analysis within the 5'-untranslated region (5'-UTR) of ChfuGV pk-1 indicates the presence of both putative early and late promoter motifs, indicating that pk-1 may be expressed throughout the infection cycle. Promoter sequence analysis reveals that pk-1 is deprived of a TATA box and appears instead to be regulated by other cis-acting transcriptional regulatory elements. Temporal transcription analysis by RT-PCR confirms the appearance of transcripts detected from 2 h p.i. until 72 h p.i. Northern blot hybridization characterizes pk-1 transcription as a 1.2 kb transcript. Homology comparisons reveal that ChfuGV PK-1 protein is most closely related to Phthorimaea operculalla granulovirus (PoGV) with 80% amino acid identity.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.1
• /
• pp.216-219
• /
• 2004

The effect of culture temperature on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) in recombinant E. coli was investigated. E. coli cell was cotransformed with two plasmids (pTCGT1 and pGroll) in which the cgt and groEL/ES genes are under the control of T7 promoter and pzt-1 promoter, respectively. When tetracycline (10 ng/ml) and IPTG (l mM) were added as inducers at the early-exponential phase (2 h) and mid-exponential phase (3h), respectively, the solubilization of the inclusion body CGTase was greatly dependent on the temperature of the culture. At low culture temperature of $25^\circ{C}$, 2- or 3-fold higher activity and specific activity were obtained over $37^\circ{C}$. SDS-PAGE analysis revealed that about 62% of CGTase in the total CGTase protein was found in the soluble fraction by applying overexpression of GroEL/ES chaperone and by cultivation of E. coli at $25^\circ{C}$, whereas 33% of CGTase was detected in the soluble fraction at $37^\circ{C}$. Therefore, the expression of GroEL/ES and cultivation at $25^\circ{C}$ greatly enhanced the soluble production of CGTase in E. coli.

• Journal of Microbiology
• /
• v.39 no.4
• /
• pp.314-320
• /
• 2001

Macrophages stimulated by lipopolysaccharide(LPS) from gram negative bacteria undergo activation of a group of immediate early genes including Rantes. The mouse Rantes gene promoter region contains an LPS rsponsive element(LPE) We detected 3 specific bands termed B1, B2 and 3 formed by the interaction of the LPE and proteins found in LPS-stimulated RAW 367.7 cells. An additional band B4 was determined to be an Ap-1 binding protein. The B1 band appears within 1 hour of LPS nuclear extracts from LPS-stimulation, and this protein kinase enhances B1 and formation. The B1 band can be converted to band B2/B3 by adding specific heparin column fraction purified Ser/Thr specific protein phosphatases PP-1 and PP-2A can stimulate the same conversion to about the same extent. Thus, the formation of the LRE sequence binding complex appears to be regulated by Ser/Thr protein kinase and one or more Ser/Thr specific phosphatases. At least four proteins are involved in the trgulation of the LRE-dependent Rants experssion: two binding factors that bind directly to the target sequences. and two factors that control their binding. The future purification and characterization of these binding pro-teins will reveal in detail the mechanism of Rantes gene activation after LPS stimulation.

• KSBB Journal
• /
• v.17 no.6
• /
• pp.577-581
• /
• 2002

In an attempt to produce recombinant proteins using the pollen enriched in some plant species, a 1.7 kb DNA encoding urease subunit B (UreB) amplified by PCR from Helicobacter pylori urease gene cluster in pH808 plasmid was cloned to be expressed under CaMV35S promoter in lily (Lilium longiflorum) pollen tubes elongated in vitro. Lily pollen at early germinating stage was transformed with the ureB DNA using Agrobacterium via vacuum infiltration and, incubated for a full pollen tube growth 16 - 24 h in the dark in the presence of kanamycin. DNA integration and expression in the transgenic pollen were analyzed by the standard molecular techniques and the results suggest that the pollen in vitro may be employed as a protein factory in a disposable fashion.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.3 no.1
• /
• pp.43-49
• /
• 2001

We have cloned and characterized an immediate early-1 gene, iel, which is activated immediately upon entrance of the viral genome into the cell nucleus, from Bombyx mori nuclear polyhedrosis virus (BmNPV) K1 strain. This gene encodes a protein 584 amino acids with a predicted molecular weight of 67 kDa. The promoter and coding regions of BmNPV-K1 ie1 showed high homology with Autographa californica nuclear polyhedrosis virus and BmNPV T3 strain. The BmNPV-K1 ie1 was different from amino acid sequence at 4 positions in BmNPV T3. The location of ie1 gene in the BmNPV-K1 genome was confirmed by Southern blot analysis and its expression patterns at the transcriptional level in the infected cells were confirmed by Nerthern hybridization analysis.

• BMB Reports
• /
• v.55 no.5
• /
• pp.232-237
• /
• 2022

The Wnt/β-catenin signaling plays crucial roles in early development, tissue homeostasis, stem cells, and cancers. Here, we show that RNF152, an E3 ligase localized to lysosomes, acts as a negative regulator of the Wnt/β-catenin pathway during Xenopus early embryogenesis. Overexpression of wild-type (WT) RNF152 inhibited XWnt8-induced stabilization of β-catenin, ectopic expression of target genes, and activity of a Wnt-responsive promoter. Likewise, an E3 ligase-defective RNF152 had repressive effects on the Wnt-dependent gene responses but not its truncation mutant lacking the transmembrane domain. Conversely, knockdown of RNF152 further enhanced the transcriptional responses induced by XWnt8. RNF152 morphants exhibited defects in craniofacial structures and pigmentation. In line with this, the gain-of-RNF152 function interfered with the expression of neural crest (NC) markers, whereas its depletion up-regulated NC formation in the early embryo. Mechanistically, RNF152 inhibits the polymerization of Dishevelled, which is key to Wnt signaling, in an E3 ligase-independent manner. Together, these results suggest that RNF152 controls negatively Wnt/β-catenin signaling to fine-tune its activity for NC formation in Xenopus embryo.

• Journal of Microbiology and Biotechnology
• /
• v.4 no.3
• /
• pp.183-190
• /
• 1994

Cell-free extracts prepared from cultured insect cells, Spodoptera. frugiperda, were analyzed for activation of early gene transcription of an insect baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The template DNA used for in vitro transcription assays contained promoter sites for the baculovirus genes that have been classified as immediate early ($\alpha$) or early genes. These genes are located in the HindIII-K/Q region of the AcNPV genome. Nuclei isolated from the AcNPV-infected Spodoptera frugiperda cells were also used for in vitro transcription analysis by RNase-mapping the labeled RNA synthesized from in vitro run-on reaction in the isolated nuclei. The genes studied by this technique were p26 and pl0 genes which were classified as delayed early and late gene, respectively. We found that transcription of the genes from the HindIII-K region was accurately initiated and unique in the whole cell extract obtained from uninfected cells, although abundance of the in vitro transcripts was reverse to that of in vivo RNA. With isolated nuclei transcription of the p26 gene was inhibited by $\alpha$-amanitin suggesting that the p26 gene was transcribed by host RNA polymerase II. However, transcription of the pl0 gene in isolated nuclei was not inhibited by $\alpha$-amanitin, but rather stimulated by the inhibitor. We also found that the synthesis of $\alpha$-amanitin-resistant RNA polymerase was begun before 6 hr p.i., the time point at which the onset of viral DNA replication as well as the appearance of a-amanitin-resistant viral transcripts were detected. These studies give us strong evidence to support the previous data that early genes of AcNPV were transcribed by host RNA polymerease III, while transcription of late genes was mediated at least by a novel $\alpha$-amanitin-resistant RNA polymerase.

• Journal of Life Science
• /
• v.14 no.1
• /
• pp.121-125
• /
• 2004

The synergistic effect of lowered incubation temperature and CroEL/ES expression on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) was studied in recombinant E. coli. pTCGTl and pGroll carrying the cgt and groEL/ES genes under the control of T7 promoter and pzt-I promoter, respectively, were co-introduced. Tetracycline (10 ng/ml) and IPTG (1 mM) were added at the early-exponential phase (2 hr) and mid-exponential phase (3 hr). Low temperature cultivation at $25^{\circ}C$ with groEL/ES expression improved the activity of CGTase by two fold, compared to $37^{\circ}C$ cultivation without chaperones. SDS-PACE analysis revealed that about 69% of CGTase in the total CGTase protein was found in the soluble fraction by overexpression of GroEL/ES and cultivation at$25^{\circ}C$, whereas 20% of CGTase was detected in the soluble fraction when E. coli was cultivated at $37^{\circ}C$ without chaperone. The amount of soluble CGTase from $25^{\circ}C$ culture with chaperone was 3.5-fold higher than that of $37^{\circ}C$ culture without chaperone. Therefore the expression of CroEL/ES and low temperature cultivation greatly enhanced the soluble production of CGTase in E. coli.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.31 no.4
• /
• pp.587-593
• /
• 1998

To elucidate anti-tumor promoter from seaweed, the anti-tumor promoting activity of Ecklonia stolonifera, Undaria pinnatifida and Laminaria japonica extracts were determined by Epstein-Barr virus (EBV)-early antigen (EA) induction caused by a tumor promoter, teleocidin B-4. The methanol extracts of seaweed were subsequently fractionated with diethyl ether, distilled water, chloroform and ethyl acetate. Among the solvent fractions tested, chloroform and ethyl acetate fraction of E. stolonifera showed a high anti-tumor promoting activity at the levels of 88.0 and $85.9\%$ by the addition of 20 ${\mu}g/m{\ell}$, respectively. To characterize anti-tumor promoters from solvent fractions of E. stolonifera, the effects of phenols, chlorophyll derivatives and carotenoids on the anti-tumor promoting activity were investigated. Phenols, such as bromophenol and phloroglucinol showed anti-tumor promoting activity of $57\~66\%$ at 20 ${\mu}g/m{\ell}$. Pigments, such as chlorophylls and carotenoids exerted high anti-tumor promoting activities. Chlorophyll a and pheophorbide a exhibited the activity of $77.4\%$ and $66.6\%$ at 5${\mu}M/m{\ell}$, respectively. The active compounds of carotenoids were tentatively identified as lutein and $\alpha-cryptoxanthin$ from the profiles of visible spectra and R_f value of their authentic compounds, and showed anti-tumor promoting activities of $76.9\%$ and $84.4\%$ at dose of 20 ${\mu}g/m{\ell}$, respectively.