• 생명과학회지
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• 제10권4호
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• pp.422-430
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• 2000

In order to search for the effects of Shine-Dalgarno (SD) sequence and nucleotide sequence of spacer region (SD-ATG) on bGH expression, oligonucleotides containing random SD sequences and a spacer region were chemically synthesized. The distance between SD region and initiation codon (ATG) was fixed to 9 nucleotides in length. The expression vectors have been constructed using pT7-1 vector containing a T7 promoter. Positive clones were screened with colony hybridization and named pT7A or pT7B plasmid series. The selected clones were confirmed by DNA sequencing and finally, 19 clones having various SD combinations were obtained. When bovine growth hormone was induced by IPTG in E. coli BL21(DE3), all cells harboring these plasmids produced a detectable level of bGH in western blot analysis. However, various SD sequences did not affect on bGH expression, indicating that the sequences of SD and the spacer region did not sufficiently destabilize mRNA secondary structure of bGH gene. Therefore, these results indicate that the disruption of mRNA secondary structure might be a major factor for regulating bGH expression in the translational initiation process.

• Asian-Australasian Journal of Animal Sciences
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• 제25권8호
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• pp.1055-1062
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• 2012

Two SNPs, i.e. L127V and T172M, of bovine growth hormone (GH) causing the presence of GH gene haplotypes A, B, and C was previously shown to alter intramuscular fatty acid (FA) composition in Japanese Black (JB) heifers. To determine the SNP effect on somatotropic hormone concentration and lipogenesis, we measured plasma GH, insulin, and insulin-like growth factor-1 (IGF-1) concentrations. We also measured mRNA levels of fatty acid synthase (FASN), stearoyl-coA desaturase (SCD), and sterol regulatory element binding proteins-1 (SREBP-1) and FA composition in diaphragm tissues. Heifers with genotype CC had the lowest plasma insulin concentration and FASN and SCD mRNA levels among genotypes. FASN mRNA levels in haplotype A tended to positively correlate with saturated FA (SFA) content and negatively correlated with C18:2 and unsaturated FA (USFA) contents. SCD mRNA levels in haplotype A positively correlated with monounsaturated FA (MUFA) contents and negatively correlated with C18:0 content. They also tended to positively correlate with C16:1, C18:1, and USFA contents and USFA/SFA ratio and negatively correlate with SFA content. Taken together, GH gene polymorphism affects the lipogenic genes expression levels and their relationships with fatty acid compositions in diaphragm tissues of JB heifers at 31 months of age.

• Asian-Australasian Journal of Animal Sciences
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• 제20권4호
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• pp.501-510
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• 2007

The present study was conducted to establish primary bovine muscle satellite cell (MSC) culture conditions and to investigate the effects of various steroid hormones on transcription of the genes involved in muscle cell proliferation and differentiation. Of three different types of proteases (type II collagenase, pronase and trypsin-EDTA) used to hydrolyze the myogenic satellite cells from muscle tissues, trypsin-EDTA treatment yielded the highest number of cells. The cells separated by hydrolysis with type II collagenase and incubated on gelatin-coated plates showed an enhanced cell attachment onto the culture plate and cell proliferation at an initial stage of cell growth. In this study, the bovine MSCs were maintained in vitro up to passage 16 without revealing any significant morphological change, and even to when the cells died at passage 21 with decreased or almost no cell growth or deformities. When the cells were incubated in a steroid-depleted environment (DMEM(-)/10% CDFBS (charcoal-dextran stripped FBS)), they grew slowly initially, and were widened and deformed. In addition, when the cells were transferred to an incubation medium containing steroid (DMEM(+)/10% FBS), the deformed cells resumed their growth and returned to a normal morphology, suggesting that steroid hormones are crucial in maintaining normal MSC morphology and growth. The results demonstrated that treatments with 19-nortestosterone and testosterone significantly increased AR gene expression (p<0.05), implying that both testosterone and 19-nortestosterone bind with AR and that the hormone bound-AR complex up-regulates the genes of its own receptor (AR) plus other genes involved in satellite cell growth and differentiation in bovine muscle.

• Journal of Animal Science and Technology
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• 제54권3호
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• pp.219-226
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• 2012

Gender specificity in muscle growth and development is well known. Genesis of muscle is dependent on proliferation and differentiation potential of resident myogenic satellite cells (MSCs) present in muscle fibers. Multipotential capacity of forming myocyte, osteocyte, and adipocyte like cell makes MSCs a unique stem cell. To understand the molecular mechanism involved in determination of muscle quality due to difference in hormone concentration of different gender of animals, MSCs were isolated from bovine skeletal muscle and cultured in male, female, and castrated serum supplemented media. DNA microarray used consisted of 24,000 spots with 70 mer oligo in each spot. A total of 88 genes were up-regulated and 551 genes were down-regulated by more than two fold. Among up-regulated gene, 33, 34, and 21 genes were found up-regulated in cells grown in male, female, and castrated serum, respectively. Interestingly, male serum showed 4, female 11 and castrated male showed 4 genes expressed highly in each gender. Further study on the highly up-regulated gene may unfold the mystery of gender specificity found in muscle development. Also, the identification of differentially expressed genes in gender-specific serum will add information on infrastructure of bovine genome research.

• Journal of Animal Science and Technology
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• 제44권6호
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• pp.693-700
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• 2002

한우 종모우에 있어 소 성장호르몬 유전자의 출현빈도를 알아보고 소 성장호르몬 다형과 정액성상과의 관계를 살펴보기 위하여 실시하였다. 한우 종모우 109두의 소 성장호르몬 유전자를 Alu I 제한효소로 처리하여 PCR-RFLP로 분석하였다. Leucine(Leu)과 valine(Val) 유전자의 출현빈도는 각각 0.88과 0.12이었다. 소 성장호르몬 VV 유전자형을 지닌 한우 종모우가 다른 유전자형을 지닌 종모우보다 정액성상(정액량, 정자농도, 총정자수)이 떨어지는 경향을 보였지만 소 성장호르몬의 유전자형이 정액성상에 유의적인 영향은 미치지 못하였다. 채취순번에 따른 영향에 있어서도 VV 유전자형을 지닌 종모우가 다른 유전자형을 지닌 종모우보다 정액성상이 떨어지는 경향을 보였지만 유의적인 차이는 나타내지는 못하였으며 다만 년도에 따른 영향에 있어 1998년도 VV 유전자형을 지닌 종모우가 다른 유전자형을 지닌 종모우보다 총정자수에 있어 유의적으로 적게 나타났다(P<0.05). 전체적으로 소 성장호르몬 유전자의 VV 유전자형을 지닌 종모우의 정액성상이 다른 유전자를 지닌 종모우의 정액성상보다 낮은 경향을 보였지만, 조사한 109두의 한우 종모우 중 VV 유전자형을 지닌 종모우가 1두로 분석되어 이 종모우의 정액성상만을 이용하여 소 성장호르몬 유전자다형이 정액성상에 미치는 영향을 살펴보았기에 종모우 선발시 기초자료로 이용하기에는 추가적인 시험이 필요할 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제18권11호
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• pp.1548-1551
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• 2005

The leptin, an anti-obesity protein, is a hormone protein expressed and secreted mainly from adipocyte tissue, and involved in regulation of body weight, food intake and energy metabolism. In an effort to discover polymorphism(s) in genes whose variant(s) might be implicated in phenotypic traits of growth, we have sequenced exons and their boundaries of leptin gene including 1,000 bp upstream of promoter region with twenty-four unrelated Korean cattle. Fifty-seven sequence variants were identified: fourteen in 5' flanking region, twenty-seven in introns, eight in exons, and eight in 3' flanking region. By pair-wise linkage analysis among polymorphisms, ten sets of SNPs were in absolute linkage disequilibrium (LD) (|D'| = 1 and $r^2$ = 1). Among variants identified, thirty-six SNPs were newly identified, and twenty-one SNPs, which were reported in other breeds, were also confirmed in Korean cattle. The allele frequencies of variants were quite different among breeds. The information from SNPs of bovine leptin gene could be useful for further genetic studies of this gene.

• Asian-Australasian Journal of Animal Sciences
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• 제18권5호
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• pp.723-727
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• 2005

Ghrelin is a novel growth-hormone-releasing acylated peptide, which has been purified and identified in rat stomach. In the present study, the full-length sequence of bovine ghrelin cDNA was cloned by RT-PCR. The bovine ghrelin cDNA sequence derived in the present study included a 348 bp open reading frame and a 137 bp 3'UTR. The putative amino acid sequence of bovine prepro-ghrelin consisted of 116 amino acids, which contained the 27-amino acid ghrelin. The sequence analysis of the bovine ghrelin gene revealed that an intron existed between Gln$^{13}$ and Arg$^{14}$ of ghrelin. This exon-intron boundary matched the GT-AG rule of the splicing mechanism. Compared with rats, which have two tandem CAG sequences in the 3'end of intron, bovine ghrelin genome has only one CAG sequence. Therefore, although rats can produce 28 amino acid-ghrelin and 27 amino acid-des-Gln$^{14}$-ghrelin by alternative splicing, ruminant species, including bovines, might be able to produce only one type of ghrelin peptide, des-Gln$^{14}$-ghrelin. The influence of aging on plasma ghrelin concentration was also examined. Plasma ghrelin concentration increased after birth to approximately 600 days of age, and then remained constant.

• 한국가축번식학회지
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• 제25권4호
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• pp.381-388
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• 2001

본 연구는 돼지에서 성장호르몬과 결합되는 부위에 변이를 유도하여 결합력이 향상된 성장호르몬 결합단백질을 획득하기 위하여 수행되었다. 돼지의 지방으로부터 얻은 성장호르몬 수용체 RNA 내 성장호르몬 결합단백질 부분을 756 bp의 cDNA로 전향하고 클로닝한 후 site-directed mutagenesis 방법을 이용하여 26과 122번째 아미노산을 변이시켰다. 26번째 아미노산은 성장 호르몬과의 결합에 관련이 있다고 알려져 있는 돼지 성장호르몬 수용체 외막에 존재하는 다섯 군데의 N-linked glycosylation 부위와 가까이 위치한 부분이고, 122번째 아미노산은 소에서의 결합부위로 알려져 있다. 이렇게 변이를 유도한 성장호르몬 결합 단백질을 pET-32(c) 발현벡터에 삽입시키고 과발현시켰고 이를 정제하여 30 kDa의 변이를 유도한 성장호르몬 결합 단백질을 얻었다. 이러한 방법으로 성장호르몬 결합 단백질을 성장기에 있는 세포나 동물에 주입한다면 보다 향상된 성장을 볼 수 있을 것으로 사료된다.

• Journal of Microbiology
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• 제44권1호
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• pp.64-71
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• 2006

To investigate the effects of the nucleotide sequences in Shine-Dalgarno (SD) and the spacer region (SD-ATG) on bovine growth hormone (bGH) gene expression, the expression vectors under the control of the T7 promoter (pT7-7 vector) were constructed using bGH derivatives (bGH1 & bGH14) which have different 5'-coding regions and were induced in E. coli BL21 (DE3). Oligonucleotides containing random SD sequences and a spacer region were chemically synthesized and the distance between the SD region and the initiation codon were fixed to nine bases in length. The oligonucleotides were annealed and fused to the bGH1 and bGH14 cDNA, respectively. When the bGH gene was induced with IPTG in E. coli BL21(DE3), some clones containing only bGH14 cDNA produced considerable levels of bGH in the range of $6.9\%\;to\;8.5\%$ of total cell proteins by SDS-PAGE and Western blot. Otherwise, the bGH was not detected in any clones with bGH1 cDNA. Accordingly, the nucleotide sequences of SD and the spacer region affect on bGH expression indicates that the sequences sufficiently destabilize the mRNA secondary structure of the bGH14 gene. When the free energy was calculated from the transcription initiation site to the +51 nucleotide of bGH cDNA using a program of nucleic acid folding and hybridization prediction, the constructs with values below -26.3 kcal/mole (toward minus direction) were not expressed. The constructs with the original sequence of bGH cDNA also did not show any expression, regardless of the free energy values. Thus, the disruption of the mRNA secondary structure may be a major factor regulating bGH expression in the translation initiation process. Accordingly, the first stem-loop among two secondary structures present in the 5'-end region of the bGH gene should be disrupted for the effective expression of bGH.

• BMB Reports
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• 제31권2호
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• pp.123-129
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• 1998

Foreign proteins, $endo-{\beta}-1,4-glucanase$ of Bacillus subtilis, preS1+S2 region of hepatitis B virus large surface antigen, human ${\beta}_2-adrenergic$ receptor ($h{\beta}_{2}AR$), and bovine growth hormone (bGH) were expressed in Saccharomyces cerevisiae and secreted into the medium. These proteins were expressed using the alcohol dehydrogenase I (ADH1) promoter of Saccharomyces cerevisiae and secreted by signal sequence of the 97 K killer toxin gene of doublestranded linear DNA plasmid (pGKL1) of S. cerevisiae. All these proteins underwent severe modifications; in particular, N-glycosylation in the case of $endo-{\beta}-1,4-glucanase$, $h{\beta}_2AR$, and preS1+S2. Seventy four percent of the expressed $endo-{\beta}-1,4-glucanase$ was secreted into the culture medium. Highly modified proteins were detected in the culture medium and in the cell. Expressed $h{\beta}_2AR$, which has seven transmembrane domains, remained in the cell. The degrees of secretion and modification and the states of proteins in the culture medium and in the cell were quite different. These results indicated that the nature of the protein has a critical role in its secretion and modifications.