• Title/Summary/Keyword: Cloning and Expression

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The spatio-temporal expression analysis of parathyroid hormone like hormone gene provides a new insight for bone growth of the antler tip tissue in sika deer

  • Haihua Xing;Ruobing Han;Qianghui Wang;Zihui Sun;Heping Li
    • Animal Bioscience
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    • v.37 no.8
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    • pp.1367-1376
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    • 2024
  • Objective: Parathyroid hormone like hormone (PTHLH), as an essential factor for bone growth, is involved in a variety of physiological processes. The aim of this study was to explore the role of PTHLH gene in the growth of antlers. Methods: The coding sequence (CDS) of PTHLH gene cDNA was obtained by cloning in sika deer (Cervus nippon), and the bioinformatics was analyzed. The quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the differences expression of PTHLH mRNA in different tissues of the antler tip at different growth periods (early period, EP; middle period, MP; late period, LP). Results: The CDS of PTHLH gene was 534 bp in length and encoded 177 amino acids. Predictive analysis results revealed that the PTHLH protein was a hydrophilic protein without transmembrane structure, with its secondary structure consisting mainly of random coil. The PTHLH protein of sika deer had the identity of 98.31%, 96.82%, 96.05%, and 94.92% with Cervus canadensis, Bos mutus, Oryx dammah and Budorcas taxicolor, which were highly conserved among the artiodactyls. The qRT-PCR results showed that PTHLH mRNA had a unique spatio-temporal expression pattern in antlers. In the dermis, precartilage, and cartilage tissues, the expression of PTHLH mRNA was extremely significantly higher in MP than in EP, LP (p<0.01). In the mesenchyme tissue, the expression of PTHLH mRNA in MP was significantly higher than that of EP (p<0.05), but extremely significantly lower than that of LP (p<0.01). The expression of PTHLH mRNA in antler tip tissues at all growth periods had approximately the same trend, that is, from distal to basal, it was first downregulated from the dermis to the mesenchyme and then continuously up-regulated to the cartilage tissue. Conclusion: PTHLH gene may promote the rapid growth of antler mainly through its extensive regulatory effect on the antler tip tissue.

Characteristic of the Human Cloning Motif in Feature 〈Blueprint〉 (영화 〈블루프린트〉 속에 투영된 인간복제 모티프의 특성)

  • Jung, Won-Sik
    • The Journal of the Korea Contents Association
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    • v.11 no.1
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    • pp.123-133
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    • 2011
  • Rapid development of life sciences about human cloning in real world is reflected alternately ahead and behind in the movies and it brings up the conversation topics about the scenario of diverse human life in the near future. This study is the consideration of the characteristic of the human cloning motif in movies, focused on the feature film which consider human cloning a philosophical point of view. The emergence and evolution of somatic cell cloning conclude the problem of philosophical anthropology. And identity and un-naturality of human clone boils down to desire and dilemma of character in . The dramatize to the exclusion of genre's attribution and the setting of twofold relationship between original and clone as well as real mother and daughter prove the critical mind of as stated above. This study also reported it reinforces the characteristic of the human cloning motif in movies that plotting with parallel narrative and the consistent attitudes with various cinematic expression and techniques.

Molecular cloning, expression and characterization of a novel feruloyl esterase enzyme from the symbionts of termite (Coptotermes formosanus) gut

  • Chandrasekharaiah, Matam;Thulasi, Appoothy;Bagath, M.;Kumar, Duvvuri Prasanna;Santosh, Sunil Singh;Palanivel, Chenniappan;Jose, Vazhakkala Lyju;Sampath, K.T.
    • BMB Reports
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    • v.44 no.1
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    • pp.52-57
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    • 2011
  • Termites play an important role in the degradation of dead plant materials and have acquired endogenous and symbiotic cellulose digestion capabilities. The feruloyl esterase enzyme (FAE) gene amplified from the metagenomic DNA of Coptotermes formosanus gut was cloned in the TA cloning vector and subcloned into a pET32a expression vector. The Ft3-7 gene has 84% sequence identity with Clostridium saccharolyticum and shows amino acid sequence identity with predicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. The sequence analysis reveals that probably Ft3-7 could be a new gene and that its molecular mass was 18.5 kDa. The activity of the recombinant enzyme (Ft3-7) produced in Escherichia coli (E.coli) was 21.4 U with substrate ethyl ferulate and its specific activity was 24.6 U/mg protein. The optimum pH and temperature for enzyme activity were 7.0 and $37^{\circ}C$, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.

Cloning and Characterization of Porcine Uroplakin II Gene

  • D. N. Kwon;H. K. Shin;C. K. Hwang;D. W. Ok;Kim, J. H.
    • Proceedings of the KSAR Conference
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    • 2001.03a
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    • pp.19-19
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    • 2001
  • Mammalian urothelium undergoes unique membrane specialization by making the asymmetric unit membrane (AUM) that is covered with the apical cell surface during terminal differentiation. The AUM contains several major integral membrane proteins including uroplakin Ia, Ib, II and III. The genes for uroplakins have been cloned from humans and mice, but not from porcine. In this study, we report the cloning of the UPII genomic DNA, which codes for the full length open reading frame for the uroplakin II protein. The deduced amino acid sequence encodes of a hydrophobic NH$_2$-terminal peptide, a prosequence, and a mature protein. The prosequence contains three potential N-glycosylation sites and a RGRR cleavage site that may be involved in uroplakin II processing and maturation. Northern and immunohistochemistry analyses showed that the porcine UPII gene is only expressed in urothelium and that the protein was specifically localized in urothelial superficial cells. A 2kb of upstream in the promoter sequence contains multiple transcription factor binding sites, including GC-box, SPI, AP2, and GATA-box sites, but not for TATA or CAAT-box sequences. Comparison of the porcine UPII promoter sequence with that of the murine by MEME system presented two conserved motifs, suggesting a cis-acting regulatory role for the conserved sequences. Sequence homology between two species in motif A and B was 79% and 80% respectively, although their relative locations were different. During the gestation, mouse bladder at estrus stages and day 10 after parturition showed higher UPII expression, while showed lower expression at peri-implantation stage. Taken together, our results showed that the porcine UPII gene was expressed highly and specifically in the bladder urothelium and that steroid hormones for implantation changed the expression of UPII in the bladder, although the biological significance of UPII remains to be not determined.

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A Novel Integrative Expression Vector for Sulfolobus Species

  • Choi, Kyoung-Hwa;Hwang, Sungmin;Yoon, Naeun;Cha, Jaeho
    • Journal of Microbiology and Biotechnology
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    • v.24 no.11
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    • pp.1503-1509
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    • 2014
  • With the purpose of facilitating the process of stable strain generation, a shuttle vector for integration of genes via a double recombination event into two ectopic sites on the Sulfolobus acidocaldarius chromosome was constructed. The novel chromosomal integration and expression vector pINEX contains a pyrE gene from S. solfataricus P2 ($pyrE_{sso}$) as an auxotrophic selection marker, a multiple cloning site with histidine tag, the internal sequences of malE and malG for homologous recombination, and the entire region of pGEM-T vector, except for the multiple cloning region, for propagation in E. coli. For stable expression of the target gene, an ${\alpha}$-glucosidase-producing strain of S. acidocaldarius was generated employing this vector. The malA gene (saci_1160) encoding an ${\alpha}$-glucosidase from S. acidocaldarius fused with the glutamate dehydrogenase ($gdhA_{saci}$) promoter and leader sequence was ligated to pINEX to generate pINEX_malA. Using the "pop-in" and "pop-out" method, the malA gene was inserted into the genome of MR31 and correct insertion was verified by colony PCR and sequencing. This strain was grown in YT medium without uracil and purified by His-tag affinity chromatography. The ${\alpha}$-glucosidase activity was confirmed by the hydrolysis of $pNP{\alpha}G$. The pINEX vector should be applicable in delineating gene functions in this organism.