• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.224-224
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• 2017

In plants, cysteine(Cys) is decisive for protein and glutathione that acts as an indispensable sulfur grantor for methionine and many other sulfur containing secondary products. Cys formation is involved in the consecutive two reactions using two enzymes-serine acetyl transferase (SAT) and O-acetylserine (thiol)lyase (OASTL) and appeared in plant cytosol, chloroplast and mitochondria. OASTL is able to produce mimosine with 3-hydroxy-4-pyridone (3H4P) in lieu of $H_2S$ for Cys. In this report, we describe the first time cloning, purification and characterization of cytosolic(cy)OASTL from M. pudica and its expression in Escherichia coli and try to find out the cross link between this OASTL and the mimosine formation and to elucidate the metabolic role of cy-OASTL in M. pudica. The purified recombinant protein was 34.7 KDa. The optimum reaction pH and temperature was 6.5 and $50^{\circ}C$, respectively. The Michaelis constant (Km) and the Vmax value of the enzyme was $252{\pm}25{\mu}M$ and $57{\pm}3{\mu}M\;cysteine\;min^{-1}\;{\mu}g\;protein^{-1}$ for sulfide and $159{\pm}21{\mu}M$ and $58{\pm}2.4{\mu}M\;cysteine\;min^{-1}\;{\mu}g\;protein^{-1}$ for OAS subsequently. After cleaving the His-tag, we tried to observe cy-OASTL to form mimosine with appropriate substrate but it was not successful. It may be concluded that cy-OASTL of the present study is only Cys specific, not mimosine.

• International Journal of Industrial Entomology and Biomaterials
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• v.4 no.2
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• pp.155-162
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• 2002

Cytochrome P45O (CYP) gene has been known to play one of the most important roles in metabolizing the exogenous materials. In insect, CYP is particularly known to detoxify toxic materials by adding oxygen molecule to the hydrophobic region of the materials. Thus, CYP-dependent metabolism is associated with the adaptation of insect to host plant chemicals. This in turn is known to be one of the driving forces for CYP diversification. In the present study, we cloned seven gene fragments of CYP 4 (CYP4) family from the midgut of the beet armyworm, Spodoptera exigua, through RT.PCT, Sequence analysis of the product showed the gene fragment to contain an open reading frame of ~150 amino acids, consisted of ~450 bp. The cloned gene fragments contained typical, conserved regions found in CYP4 family. Pairwise comparison of the deduced amino acid sequences among seven clones ranged in divergence from 0% to 52.86% and resulted in five distinct clones. The other two clones were identical or differ by one amino acid respectively to the corresponding clone, although each differed by ten nucleotides. Analysis of correlation between GenBank-registered, full length CYP4 and the cloned fragments resulted in statistically significant relationship ($r^{2}$ = 0.96085; p < 0.001), suggesting utility of the partial sequences as such full-length sequences. Phylogenetic analysis of the clones with GenBank-registered insect and mammal CYP4 family sequences by parsimony and several distance methods subdivided the clones into two groups: tones belonging to CYP4S and the others to CYP4M families.

• Journal of Sericultural and Entomological Science
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• v.35 no.2
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• pp.129-133
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• 1993

The 44Md plasmid of Bacillus thruingiensis var. kurstaki HD-1(B. t k HD-1) was partially digested with Sau3AI and the fragments were cloned into E. coli HB101 on vector pBR322. Of 2, 950 clones with a recombinant pBR322, only one clone KC1 was determined to have the gene for crystal toxic proteins from the 44Md plasmid of B. t k HD-1 at the BamHI site of pBR322. The recombinant pBR322 was named pKC1 and its molecular size was 12kb. The KC1 produced a protein which was toxic to the silkworm and antigenically similar to the crystal toxic protein of B. t k HD-1. Also, electrophoretic mobility of the KC1 protein was apparently the same as that of the crystal toxic protein of B. t k HD-1.

• Journal of Aquaculture
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• v.9 no.1
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• pp.93-100
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• 1996

Previously, as an effort to make an autonomously replicating expression vector in fish, an ARS (autonomously replicating sequence) was cloned from MAR (matrix attachment region) of Misgurnus mizolepis. The DNA fragment composed of 443 base pairs contains ARS core consensus sequences, topoisomerase II consensus sequences, and A or T box sequences which are homologous to the known consensus sequences originated from other organisms. The clond ARS, as other DNA replication origins, contains inverted repeat sequences and several potential hairpin loop structures. These consensus sequences and hairpin structures may serve as recognition signals for regulatory proteins of DNA replication initiation.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.1
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• pp.20-24
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• 2015

Insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-1 (IGFBP-1) play a pivotal role in regulating cellular hypoxic response. In this study, we cloned and characterized the genes encoding IGF-1 and IGFBP-1 to improve the current knowledge on their roles in highland Bos grunniens (Yak). We also compared their expression levels in the liver and kidney tissues between yaks and lowland cattle. We obtained full-length 465 bp IGF-1 and 792 bp IGFBP-1, encoding 154 amino acids (AA) IGF-1, and 263 AA IGFBP-1 protein, respectively using reverse transcriptase-polyerase chain reaction (RT-PCR) technology. Analysis of their corresponding amino acid sequences showed a high identity between B. grunniens and lowland mammals. Moreover, the two genes were proved to be widely distributed in the examined tissues through expression pattern analysis. Real-time PCR results revealed that IGF-1 expression was higher in the liver and kidney tissues in B. grunniens than in Bos taurus (p<0.05). The IGFBP-1 gene was expressed at a higher level in the liver (p<0.05) of B. taurus than B. grunniens, but it has a similar expression level in the kidneys of the two species. These results indicated that upregulated IGF-1 and downregulated IGFBP-1 are associated with hypoxia adaptive response in B. grunniens.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.3
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• pp.421-427
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• 2012

The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Milk is presently the most mature system for production of therapeutic proteins from a transgenic animal. Specifically, ${\beta}$-casein is a major component of cow, goat and sheep milk, and its promoter has been used to regulate the expression of transgenic genes in the mammary gland of transgenic animals. Here, we cloned the porcine ${\beta}$-casein gene and analyzed the transcriptional activity of the promoter and intron 1 region of the porcine ${\beta}$-casein gene. Sequence inspection of the 5'-flanking region revealed potential DNA elements including SRY, CdxA, AML-a, GATA-3, GATA-1 and C/EBP ${\beta}$. In addition, the first intron of the porcine ${\beta}$-casein gene contained the transcriptional enhancers Oct-1, SRY, YY1, C/EBP ${\beta}$, and AP-1, as well as the retroviral TATA box. We estimated the transcriptional activity for the 5'-proximal region with or without intron 1 of the porcine ${\beta}$-casein gene in HC11 cells stimulated with lactogenic hormones. High transcriptional activity was obtained for the 5'-proximal region with intron 1 of the porcine ${\beta}$-casein gene. The ${\beta}$-casein gene containing the mutant TATA box (CATAAAA) was also cloned from another individual pig. Promoter activity of the luciferase vector containing the mutant TATA box was weaker than the same vector containing the normal TATA box. Taken together, these findings suggest that the transcription of porcine ${\beta}$-casein gene is regulated by lactogenic hormone via intron 1 and promoter containing a mutant TATA box (CATAAAA) has poor porcine ${\beta}$-casein gene activity.

• BMB Reports
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• v.34 no.4
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• pp.322-328
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• 2001

Excision and amplification of pre-determined, large genomic segments (taken directly from the genome of a natural host, which provides an alternative to conventional cloning in foreign vectors and hosts) was explored in human cells. In this approach, we devised a procedure for excising a large segment of human genomic DNA, the iNOS gene, by using the Cre/loxP system of bacteriophage P1 and amplifying the excised circles with the EBNA-1/oriP system of the Epstein-Barr virus. Two loxP sequences, each of which serves as a recognition site for recombinase Cre, were integrated unidirectionally into the 5'-UTR and 3'-UTR regions of the iNOS gene, together with an oriP sequence for conditional replication. The traps-acting genes cre and EBNA-1, which were under the control of a tetracycline responsive $P_{hcmv^*-1}$ promoter, were also inserted into the 5'-UTR and 3'-UTR regions of the iNOS gene, respectively, by homologous recombination. The strain carrying the inserted elements was stably maintained until the excision and amplification functions were triggered by the induction of cre and EBNA-1. Upon induction by doxycycline, Cre excised the iNOS gene that was flanked by two ZoxP sites and circularized it. The circularized iNOS gene was then amplified by the EBNA-1/oriP-system. With this procedure, approximately a 45.8-kb iNOS genomic fragment of human chromosome 17 was excised and successfully amplified in human cells. Our procedure can be used effectively for the sequencing of unclonable genes, the functional analysis of unknown genes, and gene therapy.

• BMB Reports
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• v.36 no.6
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• pp.586-592
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• 2003

Four putative promoters of the temperate mycobacteriophage L1 were cloned by detecting the $\beta$-galactosidase reporter expression in E. coli transformants that carried L1 specific operon-fusion library. All of the four L1 promoters were also found to express differentially in the homologous environment of mycobacteria. Of the four promoters, two were suggested to be the putative early promoters of L1 since they express within 0 to 10 min of the initiation of the lytic growth of L1. One of the putative early promoters showed a relatively better and almost identical activity in both E. coli and M. smegmatis. By a sequence analysis, we suggest that the L1 insert that contained the stronger early promoter possibly carries two convergent E. coli $\sigma^{70}$-like L1 promoters, which are separated from each other by about 300 nucleotides. One of them is the early promoter of L1 as it showed a 100% similarity with the early $P_{left}$ promoter of the homoimmune phage L5. The second promoter, designated P4, was suggested for its appreciable level of reporter activity in the absence of the -10 element of the $P_{left}$ equivalent of L1. By analyzing most of the best characterized mycobacteriophages-specific promoters, including the L1 promoter P4, we suggest that both the -10 and -35 hexamers of the mycobacteriophage promoters are highly conserved and almost similar to the consensus -10 and -35 hexamers of the E. coli $\sigma^{70}$ promoters.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.12
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• pp.1670-1674
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• 2001

To understand molecular mechanisms that regulate deposition and release of intramuscular fat, a fasting-induced clone was identified by differential screening from cDNA library of adipose tissues of Korean cattle. The clone had a total length of 1,319 nucleotides coding for 334 amino acids. It was identified as one encoding L-lactate dehydrogenase H chain (LDH-B). Comparison of the deduced amino acid sequences of bovine LDH-B with those of pig, human, rat, and mouse showed 98%, 98%, 97%, and 96% identity, respectively. Food deprivation for 48 h increased mRNA levels of LDH-B gene in adipose tissues of Korean cattle compared to fed- and 6 h refed- tissues. The expression of obese mRNA was examined for individual adipose tissue from several fat depots. Fasting induced expression of LDH-B gene in subcutaneous adipose tissues, but it did not affect expression levels in abdominal, perirenal and intramuscular tissues. Results demonstrate that induction of LDH-B gene during fasting may represent a metabolic shift from anaerobic state to aerobic predominance in fasted adipose tissues and that its responses to fasting are different among several adipose tissues.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.12
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• pp.1702-1709
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• 2016

The reproductive function of G-protein subunit Galphaq (GNAQ), a member of the G protein alpha subunit family, has been extensively studied in humans and rats. However, no data is available on its status in ruminants. The objectives of this study were to evaluate the expression pattern of the GNAQ in the testis and epididymis of sheep by polymerase chain reaction (PCR). The mRNA expression levels were detected by real-time fluorescent quantitative PCR, and cellular localization of GNAQ in the testis and epididymis was examined by immunohistochemistry. Additionally, GNAQ protein was qualitatively evaluated via western blot, with the results indicating that similarities between GNAQ mRNA levels from sheep was highly conserved with those observed in Bos taurus and Sus scrofa. Our results also indicated that GNAQ exists in the caput and cauda epididymis of sheep, while GNAQ in the testis and epididymis was localized to Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells, spermatid, principal cells, and epididymis interstitial cells. The concentrations of GNAQ mRNA and protein in the caput and cauda epididymis were significantly greater than those observed in the corpus epididymis (p<0.01) and testis (p<0.05). Our results indicated that GNAQ exists at high concentrations in the caput and cauda epididymis of sheep, suggesting that GNAQ may play an important role in gonad development and sperm maturation.