• Asian-Australasian Journal of Animal Sciences
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• v.18 no.4
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• pp.483-489
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• 2005

To understand the molecular mechanisms that regulate intramuscular fat deposition and its release, cDNA clones expressed in adipose tissues of Korean cattle were identified by differential screening from adipose tissue cDNA library. By partial nucleotide sequencing of 486 clones and a search for sequence similarity in NCBI nucleotide databases, 245 clones revealed unique clones. By a functional grouping of the clones, 14% of the clones were categorized to metabolism and enzyme-related group (stearoyl CoA desaturase, lactate dehydrogenase, fatty acid synthase, ATP citrate lyase, lipoprotein lipase, acetyl CoA synthetase, etc), and 6% to signal transduction/cell cycle-related group (C/EBP, cAMP-regulated phosphoprotein, calmodulin, cyclin G1, cyclin H, etc), and 4% to cytoskeleton and extracellular matrix components (vimentin, ankyrin 2, gelosin, syntenin, talin, prefoldin 5). The obtained 245 clones will be useful to study lipid metabolism and signal transduction pathway in adipose tissues and to study obesity in human. Some clones were subjected to full-sequencing containing open reading frame. The cDNA clone of bovine homolog of human prefoldin 5 gene had a total length of 959 nucleotides coding for 139 amino acids. Comparison of the deduced amino acid sequences of bovine prefoldin 5 with those of human and mouse showed over 95% identity. The cDNA clone of bovine homolog of human ubiquitin-like/S30 ribosomal fusion protein gene had a total length of 484 nucleotides coding for 133 amino acids. Comparison of the deduced amino acid sequences of bovine ubiquitin-like/S30 ribosomal fusion protein gene with those of human, rat and mouse showed over 97% identity. The cDNA clone of bovine homolog of human proteolipid protein 2 mRNA had a total length of 928 nucleotides coding for 152 amino acids. Comparison of the deduced amino acid sequences of bovine proteolipid protein 2 with those of human and mouse showed 87.5% similarity. The cDNA clone of bovine homolog of rat thymosin beta 4 had a total length of 602 nucleotides coding for 44 amino acids. Comparison of the deduced amino acid sequences of bovine thymosin beta 4 gene with those of human, mouse and rat showed 93.1% similarity. The cDNA clone of bovine homolog of human myotrophin mRNA had a total length of 790 nucleotides coding for 118 amino acids. Comparison of the deduced amino acid sequences of bovine myotrophin gene with those of human, mouse and rat showed 83.9% similarity. The functional role of these clones in adipose tissues needs to be established.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.6
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• pp.892-897
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• 2005

Macrophage colony-stimulating factor (M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocyte lineage. Total and 16 poly (A) mRNA of bovine M-CSF were isolated from healthy bovine peripheral mononuclear cells stimulated by phobol 12-myristste 13-acetate (TPA). The more compatible cultured mononuclear cells were 5${\times}$10/ml for RNA isolation. TPA-activated mononuclear cells increased the level of M-CSF-mRNA more than concanavalin A (Con A) and lipopolysaccharide (LPS). The optimal analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) for14 Macrophage colonystimulating factor (M-CSF) as a growth factor required for bovine M-CSF was denaturation at 94$^{\circ}C$ for 1 minute, annealing at 57$^{\circ}C$ for 1 minute, extension at 72$^{\circ}C$ for 1 minute for 30 cycles. The size of cDNA of bovine M-CSF by RT-PCR was 774 base pairs. A 774 base pairs cDNA encoding bovine M-CSF was synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). Ligated cDNA was transformed to competent cells and then plasmid isolation and digestion was performed. Molecular cloning and sequencing were performed for cDNA of bovine M-CSF. The size of cloned cDNA of bovine M-CSF was 774base pairs. The homology of base sequence and amino acid sequence was 88% and 86% compared with known human M-CSF, respectively. From a high degree of sequence similarity, the obtained cDNA of bovine M-CSF is thought be a specific gene of bovine M-CSF.

• Korean Journal of Veterinary Research
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• v.35 no.2
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• pp.287-295
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• 1995

Molecular cloning and nucleotide sequencing were undertaken for the RNA genome of gp53 antigenic region in cytopathic Singer strain of bovine viral diarrhea virus. The cloned cDNA was 939 nucleotides in length having a base composition of 31.0% A, 19.6% C, 25.5% G and 24.0% T. The sequence was corresponded to approximately 77.8%(817 bases) of predicted gp53 region and 122 bases after 3'end of gp53 region in the Singer strain when compared with NADL strain of known sequence. A single open reading frame was found in the sequence of 2nd frame and was deduced as encoding 312 amino acids.

• Journal of Veterinary Science
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• v.21 no.6
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• pp.73.1-73.10
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• 2020

Background: Bovine papilloma is a neoplastic disease caused by bovine papillomaviruses (BPVs), which were recently divided into 5 genera and at least 24 genotypes. Objectives: The complete genome sequence of BPV type 15 (BPV Aks-02), a novel putative BPV type from skin samples from infected cows in Southern Xinjiang China, was determined by collecting warty lesions, followed by DNA extraction and amplicon sequencing. Methods: DNA was analyzed initially by polymerase chain reaction (PCR) using the degenerate primers FAP59 and FAP64. The complete genome sequences of the BPV Aks-02 were amplified by PCR using the amplification primers and sequencing primers. Sequence analysis and phylogenetic analysis were performed using bio-informatic software. Results: The nucleotide sequence of the L1 open reading frame (ORF) of BPV Aks-02 was 75% identity to the L1 ORF of BPV-9 reference strain from GenBank. The complete genome consisted of 7,189 base pairs (G + C content of 42.50%) that encoded 5 early (E8, E7, E1, E2, and E4) and 2 late (L1 and L2) genes. The E7 protein contained a consensus CX₂CX₂₉CX₂C zinc-binding domain and a LxCxE motif. Among the different members of this group, the percentages of the complete genome and ORFs (including 5 early and 2 late ORFs) sequence identity of BPV Aks-02 were closer to the genus Xipapillomavirus 1 of the Xipapillomavirus genus. Phylogenetic analysis and sequence similarities based on the L1 ORF of BPV Aks-02 revealed the same cluster. Conclusions: The results suggest that BPV type (BPV Aks-02) clustered with members of the Xipapillomavirus genus as BPV 15 and were closely related to Xipapillomavirus 1.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.417-421
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• 2004

A normal prion protein (PrPc) is converted to a protease resistant isoform (PrPsc) by an apparent self-propagating activity in bovine spongiform encephalopathies (BSE), which is a neurodegenerative disease. The cDNA encoding bovine PrP open reading frame (ORP) in Korean cattle was cloned by polymerase chain reaction (PCR). The cloned cDNA had a length of 795 base pairs which coded for a protein of 264 amino acid residues with a calculated molecular mass of 28.6 kDa. Identities of 90, 90, 79 and 78% on nucleotide and 94, 94, 84, and 84% on amino acid sequence were shown to PrP genes from sheep, goat, human, and mouse, respectively. The cloned DNA was ligated into the pQE30 expression vector and transformed into E. coli M15. The PrP was expressed by induction with isopropyl-$\beta$-D-thiogalactoside (IPTG) and purified on the Ni-NTA affinity column. High specific activities of the recombinant PrP were observed in the fraction of pH 5.8 eluate and showed a molecular mass of-29 kDa on SDS-PAGE and Western blot analysis.

• Parasites, Hosts and Diseases
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• v.40 no.1
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• pp.59-64
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• 2002

A Cryptosporidium parvum sporozoite and oocyst λgt11 cDNA library was screened with a hyperimmune rabbit serum that was developed against insoluble fragments of ultrasonicated oocysts. A clone named Cp22.4.1 encoding a protein of 231 amino acids with 4 zinc-finger domains characterized by a Cys-X2-Cys-X4-His-X4-Cys motif was isolated and characterized. There was a complete match between the sequencing data of the coding region of Cp22.4.1 and the corresponding gene at chromosomal level. Cloning in a pBAD-TOPO-TA expression vector permitted to evaluate the antigenicty of the recombinant His-tagged antigen. This antigen was recognized by 2 out of 5 sera from Cruptosporidium immune calves and not by sera from parasite naive animals.

• Animal Bioscience
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• v.37 no.5
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• pp.929-943
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• 2024

Objective: The present study was executed to explore the molecular mechanism of fibroblast growth factor 10 (FGF10) gene in bovine adipogenesis. Methods: The bovine FGF10 gene was overexpressed through Ad-FGF10 or inhibited through siFGF10 and their negative control (NC) in bovine adipocytes, and the multiplicity of infection, transfection efficiency, interference efficiency were evaluated through quantitative real-time polymerase chain reaction, western blotting and fluorescence microscopy. The lipid droplets, triglycerides (TG) content and the expression levels of adipogenic marker genes were measured during preadipocytes differentiation. The differentially expressed genes were explored through deep RNA sequencing. Results: The highest mRNA level was found in omasum, subcutaneous fat, and intramuscular fat. Moreover, the highest mRNA level was found in adipocytes at day 4 of differentiation. The results of red-oil o staining showed that overexpression (Ad-FGF10) of the FGF10 gene significantly (p<0.05) reduced the lipid droplets and TG content, and their down-regulation (siFGF10) increased the measurement of lipid droplets and TG in differentiated bovine adipocytes. Furthermore, the overexpression of the FGF10 gene down regulated the mRNA levels of adipogenic marker genes such as CCAAT enhancer binding protein alpha (C/EBPα), fatty acid binding protein (FABP4), peroxisome proliferator-activated receptor-γ (PPARγ), lipoprotein lipase (LPL), and Fas cell surface death receptor (FAS), similarly, down-regulation of the FGF10 gene enriched the mRNA levels of C/EBPα, PPARγ, FABP4, and LPL genes (p<0.01). Additionally, the protein levels of PPARγ and FABP4 were reduced (p<0.05) in adipocytes infected with Ad-FGF10 gene and enriched in adipocytes transfected with siFGF10. Moreover, a total of 1,774 differentially expressed genes (DEGs) including 157 up regulated and 1,617 down regulated genes were explored in adipocytes infected with Ad-FGF10 or Ad-NC through deep RNA-sequencing. The top Kyoto encyclopedia of genes and genomes pathways regulated through DEGs were the PPAR signaling pathway, cell cycle, base excision repair, DNA replication, apoptosis, and regulation of lipolysis in adipocytes. Conclusion: Therefore, we can conclude that the FGF10 gene is a negative regulator of bovine adipogenesis and could be used as a candidate gene in marker-assisted selection.

• Journal of Animal Science and Technology
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• v.48 no.2
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• pp.169-190
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• 2006

Researches in livestock are currently actively progressing to improve economically important traits using DNA markers. In cattle, the candidate genes have been selected based on their known functions in the target QTL (quantitative trait locus) region in order to identify QTN (quantitative trait nucleotide) for improving productivities. In this review, molecular genetic studies for the meat related traits, one of the major determinant of market prices, have been fully described. Also recent emerging microarray technique for identifying candidate genes in cattle has been discussed. In case of microarray, cDNA microarrays have been replaced to oligoarrays in order to minimize the experimental errors in cattle. Since the first draft of bovine genome sequences was appeared in the public domain, more markers in relation to the quantitative traits will be discovered in a short period of time and genes affecting difficult-to-measure traits, such as disease resistance, can also be selected for marker assisted selection in near future.

• BMB Reports
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• v.36 no.6
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• pp.545-551
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• 2003

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The $K_m$ and $V_{max}$ values for $NAD^+$ were 0.1 mM and $1.08\;{\mu}mol/min/mg$, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - $100\;{\mu}M$, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.82-82
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• 2003

Offspring have been produced from somatic cells in a number of species. This biotechnology introduced a new phenomenon in reprogramming and differentiation of somatic cell, namely totipotency. However, birth of oversized calves and perinatal abnormalities such as increased gestation length, lack of spontaneous parturition, higher incidence of dystocia, and reduced perinatal viability of offspring are frequently observed in pregnancies of cloned bovine fetuses. Disturbance of feto-placenta has been proposed as likely causes for abnomal growth. However. Little is known the mechanism responsible for the perinatal problems. Therefore, we focused on gestation length in somatic cell nuclear recipient cows. To solve this issues, placental tissues of control and cloned bovine were obtained by a cesarean section (C-section) before 5 days of paturition. Total RNA from control and cloned bovine placenta was extractd by TRIzol reagent. GeneFishing DEG kits (Seegene) were used to identify differentially expression genes. Total RNA (3 ug) were synthesized by M-MLV reverse transcriptase (200 u/ul) with 10 uM dT-annealing control primer (ACP1) at 42C for 90 min. Then, first-strand cDNA (50 ng) was amplified using the 5 uM arbitary ACP (1-20) and 10 uM dT-ACP2 primers. Some specific expression genes were amplified, Now, we are cloning and sequencing. These finding strongly can be support to solve the problems for parturition delay in nuclear transfer cows, suggest that placenta specific proteins are key indicators for the aberration of gestation and placental function in cows.