• 한국미생물·생명공학회지
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• 제47권2호
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• pp.278-288
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• 2019

In the present investigation, we attempted to design a protocol to develop a hybrid protein with better bioremediation capacity. Using in silico approaches, a Hybrid Open Reading Frame (Hybrid ORF) is developed targeting the genes of microorganisms known for degradation of organophosphates. Out of 21 genes identified through BLAST search, 8 structurally similar genes (opdA, opd, opaA, pte RO, pdeA, parC, mpd and phnE) involved in biodegradation were screened. Gene conservational analysis categorizes these organophosphates degrading 8 genes into 4 super families i.e., Metallo-dependent hydrolases, Lactamase B, MPP and TM_PBP2 superfamily. Hybrid protein structure was modeled using multi-template homology modeling (3S07_A; 99%, 1P9E_A; 98%, 2ZO9_B; 33%, 2DXL_A; 33%) by $Schr{\ddot{o}}dinger$ software suit version 10.4.018. Structural verification of protein models was done using Ramachandran plot, it was showing 96.0% residue in the favored region, which was verified using RAMPAGE. The phosphotriesterase protein was showing the highest structural similarity with hybrid protein having raw score 984. The 5 binding sites of hybrid protein were identified through binding site prediction. The docking study shows that hybrid protein potentially interacts with 10 different organophosphates. The study results indicate that the hybrid protein designed has the capability of degrading a wide range of organophosphate compounds.

• 미생물학회지
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• 제38권4호
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• pp.230-230
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• 2002

Class Ⅲ chitin synthases in filamentous fungi are important for hyphal growth and differentiation of several filamentous fungi. A genomic clone containing the full gene encoding Chs4, a class Ⅲ chitin synthase in Penicillium chrysogenum, was cloned by PCR screening and colony hybridization from the genomic library. Nucleotide sequence analysis and transcript mapping of chs4 revealed an open reading frame (ORF) that consisted of 5 exons and 4 introns and encoded a putative protein of 915 amino acids. Nucleotide sequence analysis of the 5′flanking region of the ORF revealed a potential TATA box and several binding sites for transcription activators. The putative transcription initiation site at -716 position was identified by primer extension and the expression of the chs4 during the vegetative growth was confirmed by Northern blot analysis. Amino acid sequence analysis of the Chs4 revealed at least 5 transmembrane helices and several sites for past-transnational modifications. Comparison of the amino acid sequence of Chs4 with those of other fungi showed a close relationship between P chrysogenum and genus Aspergillus.

• 대한소아치과학회지
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• 제33권2호
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• pp.304-310
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• 2006

본 연구는 치주질환 주요 병인균주 중의 하나인 P. intermedia를 대상으로 하여, 이 균주에서의 hemin 결합 단백질 유전자를 분리하고 염기서열을 결정하기 위하여 수행되었다. 본 연구에서는 약 5,000개의 recombinant colony들을 스크리닝하여 hemin 결합 단백질 유전자를 포함하고 있는 것으로 여겨지는 1개의 클론(pHem1)을 확인하였다. Restriction enzyme mapping 결과 pHem1의 insert DNA의 크기는 약 2.5kb이었으며 P. intermedia chromosomal DNA 내에는 hemin 결합 단백질 유전자가 single copy로 존재하였고, transcript의 크기는 약 1.8kb이었다. 분리한 pHem1 유전자는 1개의 ORF를 가지고 있으며, ORF의 크기는 2,550bp로 약 850개의 아미노산 폴리펩타이드로 구성되어 있다. 또한, pHem1 유전자는 이미 밟혀진 다른 유전자들과 상동성을 보이지 않았다. 본 연구는 P. intermedia에서의 porphyrin생리 및 hemin 획득기전을 분자생물학적으로 규명하는데 있어 중요한 의의가 있으리라 사료된다. 향후 pHem1 유전자의 특성 분석 등이 수행되어야 할 것으로 여겨진다.

• Asian-Australasian Journal of Animal Sciences
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• 제25권6호
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• pp.758-763
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• 2012

As a member of a subclass of immunophilins, it is controversial that FKBP38 acts an upstream regulator of mTOR signaling pathway, which control the process of cell-growth, proliferation and differentiation. In order to explore the relationship between FKBP38 and mTOR in the Cashmere goat (Capra hircus) cells, a full-length cDNA was cloned (GenBank accession number JF714970) and expression pattern was analyzed. The cloned FKBP38 gene is 1,248 bp in length, containing an open reading frame (ORF) from nucleotide 13 to 1,248 which encodes 411 amino acids, and 12 nucleotides in front of the initiation codon. The full cDNA sequence shares 98% identity with cattle, 94% with horse and 90% with human. The putative amino acid sequence shows the higher homology which is 98%, 97% and 94%, correspondingly. The bioinformatics analysis showed that FKBP38 contained a FKBP_C domain, two TPR domains and a TM domain. Psite analysis suggested that the ORF encoding protein contained a leucine-zipper pattern and a Prenyl group binding site (CAAX box). Tissue-specific expression analysis was performed by semi-quantitative RT-PCR and showed that the FKBP38 expression was detected in all the tested tissues and the highest level of mRNA accumulation was detected in testis, suggesting that FKBP38 plays an important role in goat cells.

• Plant Biotechnology Reports
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• 제3권4호
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• pp.277-283
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• 2009

One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1 day, delay medium for 7 days, selection medium for $4{\times}14$ days, regeneration medium, and finally on germination medium. The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter-bar ORF-nos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with $4mg\;1^{-1}$ phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the $R_1$ generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2002년도 제9회 학술 발표회 프로그램과 논문초록
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• pp.49-49
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• 2002

Mutants blocked at the earliest stages of morphological development in Streptomyces species are called bld mutants. We have cloned bldB gene ORF from Slividans. Genomic Southern blot analysis for main strains S.lividans, S.seoulensis, S.coelicolor A3(2), and S.griseus indicated that bldB gene is conserved in all main Streptomyces strains.(omitted)

• BMB Reports
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• 제33권2호
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• pp.184-187
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• 2000

Prostaglandin $E_2$ ($PGE_2$) plays an important role in the regulation of various gastric functions, and the growth-inhibitory activities on tumor cells are studied in vitro and in vivo. Although the mechanisms have attracted many researchers in the past decade, the molecular mechanisms of cell cycle arrest, or induction of apoptosis by $PGE_2$, is unclear. We investigated the effects of $PGE_2$ on the growth of the human gastric carcinoma cell line SNU1 and genes that are regulated by $PGE_2$ and isolated them using differential display RT-PCR (DD RT-PCR). FACS analysis suggested that SNU1 cells were arrested at the G1 phase by $PGE_2$ treatment. This growth inhibitory effect was in a time- and dose-dependent manner. Treatment of SNU1 cells with $10\;{\mu}g/ml$ $PGE_2$, followed by DD RT-PCR analysis, revealed differently expressed bands patterns from the control. Among the differently expressed clones, we found an unidentified cDNA clone (HGP-27) overexpressed in $PGE_2$-treated cells. The full-length cDNA of HGP-27 was isolated using RACE, which consisted of a 30-nt 5'-noncoding region, a 891-nt ORF encoding the 296 amino acid protein, and a 738-nt 3'-noncoding region including a poly(a) signal. This gene was localized on the short arm of chromosome number 11. Using the Motif Finder program, a myb-DNA binding repeat signature was detected on the ORF region. The COOH-terminal half was shown to have similarity with the $NH_3$-terminal domain of thioredoxin (Trx). This relation between HGP-27 and Trx implied a potential role for HGP-27 in modulating the DNA binding function of a transcription factor, myb.

• Asian Pacific Journal of Cancer Prevention
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• 제15권21호
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• pp.9439-9444
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• 2014

Purpose: To identify prostate cancer lncRNAs using a pipeline proposed in this study, which is applicable for the identification of lncRNAs that are differentially expressed in prostate cancer tissues but have a negligible potential to encode proteins. Materials and Methods: We used two publicly available RNA-Seq datasets from normal prostate tissue and prostate cancer. Putative lncRNAs were predicted using the biological technology, then specific lncRNAs of prostate cancer were found by differential expression analysis and co-expression network was constructed by the weighted gene co-expression network analysis. Results: A total of 1,080 lncRNA transcripts were obtained in the RNA-Seq datasets. Three genes (PCA3, C20orf166-AS1 and RP11-267A15.1) showed a significant differential expression in the prostate cancer tissues, and were thus identified as prostate cancer specific lncRNAs. Brown and black modules had significant negative and positive correlations with prostate cancer, respectively. Conclusions: The pipeline proposed in this study is useful for the prediction of prostate cancer specific lncRNAs. Three genes (PCA3, C20orf166-AS1, and RP11-267A15.1) were identified to have a significant differential expression in prostate cancer tissues. However, there have been no published studies to demonstrate the specificity of RP11-267A15.1 in prostate cancer tissues. Thus, the results of this study can provide a new theoretic insight into the identification of prostate cancer specific genes.

• Journal of Veterinary Science
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• 제22권4호
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• pp.51.1-51.10
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• 2021

Background: Malignant catarrhal fever (MCF) is a highly fatal lymphoproliferative disease of cattle, deer, bison, water buffalo, and pigs caused by the gamma-herpesviruses alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2). Objectives: This study aimed to determine the prevalence of OvHV-2 in sheep, goats, cattle, and buffalo in Rawalpindi and Islamabad, Pakistan, by applying molecular and phylogenetic methods. Methods: Blood samples were aspirated from sheep (n = 54), goat (n = 50), cattle (n = 46) and buffalo (n= 50) at a slaughterhouse and several farms. The samples were subjected to heminested polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis of the OvHV-2 POL gene and the OvHV-2 ORF75 tegument protein gene. Results: The highest percentage of MCF positive samples was in sheep (13%), whereas goat, cattle, and buffalo had lower positive percentages, 11%, 9%, and 6.5%, respectively. Four OvHV-2-positive PCR products obtained from sheep samples were sequenced. The sequences obtained were submitted to the NCBI GenBank database (MK852173 for the POL gene; MK840962, MK852171, and MK852172 for the ORF75 tegument protein gene). Phylogenetic analysis revealed a close similarity of study sequences with those of worldwide samples. Conclusions: This study is the first cross-sectional study on the prevalence and molecular detection of OvHV-2 in apparently healthy cattle and buffalo that could be carrying OvHV-2 acquired from OvHV-2-positive sheep and goats. The results indicate that OvHV-2 is circulating in Pakistan. Further studies are needed to characterize OvHV-2 and elucidate further its prevalence.

• The Plant Pathology Journal
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• 제15권5호
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• pp.270-279
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• 1999

cDNAs complementary to the 3'-terminal regions of two potyvirus genomes were cloned and sequenced. The clone G7 contains one open reading frame (ORF) of 1,338 nucleotides and a 3' untranslated region (3'-UTR) of 403 nucleotides at the 3'-end excluding the 3'end poly(A) tail. The putative viral coat protein (CP) shows 55%-92% amino acid sequence homology to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.0 kb by Northern blot analysis. Five cDNA clones were screened out using GPV2 oligonucleotide as a probe. One of these clones, DEA72, which has a longest cDNA insert, contains one ORF of 1,459 nucleotides and a 3'-UTR of 590 nucleotides at the 3'-end excluding the 3'-end poly(A) tail. The putative viral CP shows 57%-88% amino acid sequence homologies to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.6 kb by Northern blot analysis. The results of immunoblot and Northern blot analyses suggest that almost all of the tested garlic plants showing mosaic or streak symptoms are infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus in variable degrees but rarely infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus. Immunoelectron microscopy using anti-DEA72 CP antibody shows that this potyvirus is about 750 nm long and flexuous rod shaped.