• BMB Reports
• /
• v.33 no.6
• /
• pp.483-492
• /
• 2000

The Herpes simplex virus type 1 (HSV-1) strain F glycoprotein H (gH) gene in the pHLB-4 plasmid was recombinated into a baculovirus expression vector (lacZ-HcNPV) to construct a recombinant virus GH-HcNPV expressing gH. The sequences of gH and its expression were analyzed. The gH gene was located in the 6.41 kb BglII fragment. The open reading frame (ORF) of the gH gene was 2,517 bp and codes 838 amino acid residues. Insect cells infected with this recombinant virus synthesized a high level of the matured and gX-gH fusion protein with approximately 112 kDa. The fusion gH protein was localized on the membrane of the insect cells as seen by using immunofluorescence assay and accumulated in the cultured media by the SDS-PAGE and immunoprecipitation assays. The amino acid sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane sequence. Antibodies raised in mice to this recombinant protein recognized viral gH and neutralized the infectivity of HSV-1 in vitro. These results demonstrate that it is possible to produce a mature protein by gene transfer in eukaryotic cells, and indicate the utility of the HcNPV-insect cell system for producing and characterizing eukaryotic proteins. Furthermore, the neutralizing antibodies would appear to protect mice against HSV; accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.6 no.2
• /
• pp.144-149
• /
• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.3
• /
• pp.678-682
• /
• 2005

The promoter region of a carbon starvation gene isolated from Pseudomonas putida was cloned and analyzed for its potential use for in situ bioremediation and bioprocessing. We constructed a recombinant plasmid pMKD101 by cloning the 0.65 kb promoter region of the gene into the promoter proving vector, pMK301, which contains the lacZ for ${\beta}$-galactosidase activity as a reporter gene. pMKD101 was transformed into the wild-type P. putida MK1, resulting in P. putida RPD101, and analyzed for ${\beta}$-galactosidase activity under different culture conditions. When RPD101 was grown on the minimal medium plus $0.1\%$ glucose as a sole carbon source in batch cultures, ${\beta}$-galactosidase activity was found to be 3.2-fold higher during the stationary phase than during the exponential phase. In chemostat cultures, ${\beta}$-galactosidase activity was found to be 3.1-fold higher at the minimal growth rate (dilution rate=$0.05\;h^{-1}$) than at the maximal growth rate (dilution rate=$0.173;h^{-1}$). The results suggest that a carbon starvation promoter can be utilized to maximize the expression of a desired gene under nutrient limitation.

• Asian-Australasian Journal of Animal Sciences
• /
• v.20 no.9
• /
• pp.1342-1348
• /
• 2007

POU1F1 is a positive regulator for GH, PRL and TSH${\beta}$ and its mutations associate with production traits in ruminant animals. We described a DdeI PCR-RFLP method for detecting a silent allele in the goat POU1F1 gene: TCT (241Ser)>TCG (241Ser). Frequencies of $D_1$ allele varied from 0.600 to 1.000 in Chinese 801 goats. Significant associations of DdeI polymorphism with production traits were found in milk yield (*p<0.05), litter size (*p<0.05) and one-year-old weight (*p<0.05) between different genotypes. Individuals with genotype $D_1D_1$ had a superior performances when compared to those with genotype $D_1D_2$ (*p<0.05). Hence, the POU1F1 gene was suggested to the potential candidate gene for superior milk performance, reproduction trait and weight trait. Genotype $D_1D_1$, characterized by a DdeI PCR-RFLP detection, was recommended to geneticists and breeders as a molecular marker for better performance in the goat industry.

• Journal of the Korean Chemical Society
• /
• v.34 no.2
• /
• pp.130-135
• /
• 1990

The title compound (C13H17NO2S) is monoclinic, space group P21/a, with a = 13.756 (3), b = 9.310(4), c = 21.305(3) $\AA$, $\beta$ = 95.0。, Z = 8, V = 2718.11 $\AA$3, Dc = 1.23$g·{\cdot}cm-3$, (Mo k$\alpha$) = 0.71069$\AA$,$\mu$ = 2.18 cm-1, F(000) = 1071.86, T = 298, R = 0.085 for 2935 unique observed reflections with I >2.0$\sigma$(I). The structure was solved by direct methods. The C-H bond lengths and the methyl groups are fixed and refined as their ideal geometry by allowing to ride on the parent atoms. Both molecules A and B have almost same structures except for two terminal ethyl groups. The ethylene-like skeleton including the nitro group in one molecule is nearly perpendicular to the plane of the methylbenzene group and two ethyl groups form a cis-type structure which has the dfferent orientations between two molecules; in the molecule A, two terminal methyl groups being the opposite directional arrangement against the plane of its skeleton, while in the B, with the same directional structure from its plane. The molecules in the crystal are packed together by non-bonded van der Waals forces.

• Journal of Life Science
• /
• v.25 no.3
• /
• pp.257-262
• /
• 2015

The genetic variability of four species of the genus Zoysia collected from South Korea was analyzed using an inter-simple sequence repeat (ISSR) marker system. Polymerase chain reactions (PCR) with eight ISSR primers generated 86 amplicons, 76 (87.1%) of which were polymorphisms. The polymorphism information content (PIC) value of the ISSR marker system was 0.848. The percentage of polymorphic loci (P_p) ranged from 41.2% to 44.7%. Nei’s gene diversity (H) ranged from 0.149 to 0.186, with an average overall value of 0.170. The mean of Shannon’s information index (I) value was 0.250. Total genetic diversity values (H_T) varied between 0.356 (ISSR-1) and 0.418 (ISSR-16), for an average overall polymorphic loci of 0.345. Interlocus variation in within-species genetic diversity (H_S) was low (0.170). On a per-locus basis, the proportion of total genetic variation due to differences among species (G_ST) was 0.601. This indicated that about 60.1% of the total variation was among species. Thus, about 39.9 of genetic variation was within species. The estimate of gene flow, based on G_ST, was very low among species of the genus Zoysia (N_m = 0.332). The phylogenic tree showed three distinct groups: Z. macrostachya and Z. tenuifolia clades and other species were formed the separated clusters. In conclusion, the ISSR assay was useful for detecting genetic variation in the genus Zoysia, and its discriminatory power was comparable to that of other genotyping tools.

• Asian-Australasian Journal of Animal Sciences
• /
• v.21 no.5
• /
• pp.723-731
• /
• 2008

Immunomodulatory feed additives might offer alternatives to antimicrobial growth promoters in pig production. This experiment was designed to determine the effects of dietary galacto-mannan-oligosaccharide (GMOS) and chitosan oligosaccharide (COS) supplementation on the immune response in early-weaned piglets. Forty 15-day-old piglets (Duroc$\times$Landrace$\times$Yorkshire) with an average live body weight of $5.6{\pm}0.51kg$ were weaned and randomly assigned to 4 treatment groups that were fed maize-soybean meal diets containing either basal, 110 mg/kg of lincomycin, 250 mg/kg of COS or 0.2% GMOS, respectively, over a 2-week period. Another six piglets of the same age were sacrificed on the same day at the beginning of the study for sampling, in order to obtain baseline values. Interleukin (IL)-1${\beta}$gene expression in peripheral blood monocytes, jejunal mucosa and lymph nodes, as well as serum levels of IL-1${\beta}$ IL-2 and IL-6, IgA, IgG, and IgM, were evaluated for 5 pigs from each group at 15 and 28 days of age. The results indicate that weaning stress resulted in decreases in serum antibody and cytokine levels. Dietary supplementation with GMOS or COS enhanced (p<0.05) IL-1${\beta}$gene expression in jejunal mucosa and lymph nodes, as well as serum levels of IL-1${\beta}$ IL-2, IL-6, IgA, IgG and IgM compared to supplementation with lincomycin. These findings suggest that GMOS or COS may enhance the cell-mediated immune response in early-weaned piglets by modulating the production of cytokines and antibodies, which shows that GMOS or COS have different effects than the antibiotic on animal growth and health.

• Radiation Oncology Journal
• /
• v.21 no.1
• /
• pp.54-65
• /
• 2003

Purpose : To analyze the gene expression Profiles of uterine ceulcal cancer, and its variation after radiation therapy, with or without concurrent chemotherapy, using a CDNA microarray. Materials and Methods :Sixteen patients, 8 with squamous ceil carcinomas of the uterine cervix, who were treated with radiation alone, and the other 8 treated w14h concurrent chemo-radiation, were Included in the study. Before the starling of the treatment, tumor biopsies were carried out, and the second time biopsies were peformed after a radiation dose of 16.2$\~$27 Gy. Three normal cervix tissues were used as a control group. The microarray experiments were peformed with 5 groups of the total RNAs extracted individually and then admixed as control, pre-radiation therapy alone, during-radiation therapy alone, pre-chemoradiation therapy, and during-chemoradlation therapy. The 33P-iabeled CDNAS were synthesized from the total RNAs of each group, by reverse transcription, and then they were hybridized to the CDNA microarray membrane. The gene expression of each microarrays was captured by the intensity of each spot produced by the radioactive isotopes. The pixels per spot were counted with an Arrayguage, and were exported to Microsoft Excel The data were normalized by the Z transformation, and the comparisons were peformed on the Z-ratio values calculated. Results : The expressions of 15 genes, including integrin linked kinase (ILK), CDC28 protein kinase 2, Spry 2, and ERK 3, were increased with the Z-ratio values of over 2.0 for the cervix cancer tissues compared to those for the normal controls. Those genes were involved In cell growth and proliferation, cell cycle control, or signal transduction. The expressions of the other 6 genes, Including G protein coupled receptor kinase 5, were decreased with the Z-ratio values of below -2.0. After the radiation thorapy, most of the genes, with a previously Increase expressions, represented the decreased expression profiles, and the genes, with the Z-ratio values of over 2.0, were cyclic nucleotlde gated channel and 3 Expressed sequence tags (EST). In the concurrent chemo-radiation group, the genes involved in cell growth and proliferation, cell cycle control, and signal transduction were shown to have increased expressions compared to the radiation therapy alone group. The expressions of genes involved in anglogenesis (angiopoietln-2), immune reactions (formyl peptide receptor-iike 1), and DNA repair (CAMP phosphodiesterase) were increased, however, the expression of gene involved In apoptosls (death associated protein kinase) was decreased. Conclusion : The different kinds of genes involved in the development and progression of cervical cancer were identified with the CDNA microarray, and the proposed theory is that the proliferation signal stalls with ILK, and is amplified with Spry 2 and MAPK signaling, and the cellular mitoses are Increased with the increased expression oi Cdc 2 and cell division kinases. After the radiation therapy, the expression profiles demonstrated 4he evidence of the decreased cancer cell proliferation. There was no sigificant difference in the morphological findings of cell death between the radiation therapy aione and the chemo-radiation groups In the second time biopsy specimen, however, the gene expression profiles were markedly different, and the mechanism at the molecular level needs further study.

• Korean Journal of Veterinary Research
• /
• v.38 no.4
• /
• pp.909-917
• /
• 1998

Six different sequences types(A through E and H) and a subtype(Bl) of the small subunit ribosomal RNA(SSUrRNA) gene were found in bovine Theileria isolates from different areas of Korea and Japan. The sequences were aligned and three hypervariable regions were observed in the nucleotide position ranges 212~231, 261~270 and 632~690. Five of the Theileria isolates yielded sequence type A; these were the field isolates KCB, KCN, and KCJ, and the laboratory stock KLS, all from Korea, and a single isolate from Japan (JHS). This sequence type is identical to the SSUrRNA gene sequence listed for Theileria buffeli (GenBank Accession No. Z15106) from Marula, Kenya. The Korean field isolate KKB yielded only a single sequence type (B), but multiple sequence types were found in some isolates. For example, KCB and JHS isolates yielded both types A and B ; isolate KKW showed types B and H; isolate KCN showed types A, C, and D ; and isolate KCJ showed types A, B, E, and a subtype B1. Finding of the multiple sequences SSUrRNA gene sequences suggests that bovine Theileria isolates from both Korea and Japan may consist of mixed populations.

• Asian-Australasian Journal of Animal Sciences
• /
• v.19 no.9
• /
• pp.1257-1261
• /
• 2006

Pleiomorphic adenoma gene-like1 (PLAGL1) encodes a zinc-finger (ZF) protein with seven ZFs of the C2H2-type which is a regulator of apoptosis and cell cycle arrest, and also regulates the secretion of insulin. In both human and mouse, PLAGL1 is a candidate gene for tumor suppressor and transient neonatal diabetes mellitus (TNDM). In this study, a 2,238 bp fragment covering the complete coding region was obtained and deposited to GenBank (accession number: DQ288899). The reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that PLAGL1 was expressed almost equally in heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, uterus and ovary. Comparing the sequences of Large White and Meishan pigs, a C-T transition in exon 6 was found. The polymorphism could be detected by TaqI and was genotyped in five purebreds (Large White, Landrace, Meishan, Tongcheng and Bamei). Association analysis was performed between the polymorphism and carcass traits in 276 pigs of a "Large White${\times}$Meishan" F2 resource population. As a consequence, significant associations of the genotypes with shoulder backfat thickness (SFT) and internal fat rate (IFR) were observed. Pigs with TT genotype had low SFT and high IFR compared with TC or CC genotypes.