• Animal cells and systems
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• 제11권2호
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• pp.175-182
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• 2007

The lysozyme II gene of cabbage butterfly Artogeia rapae was cloned from fat body of the larvae injected with E. coli and its nucleotide sequence was determined by the RACE-PCR. It has an open reading frame of 414 bp nucleotides corresponding to 138 amino acids including a signal sequence of 18 amino acids. The estimated molecular weight and the isoelectric point of the lysozyme II without the signal peptide were 13,649.38 Da and 9.11, respectively. The A. rapae lysozyme II (ARL II) showed the highest identity (81%) in the amino acid sequence to Manduca sexta lysozyme among other lepidopteran species. The two catalytic residues ($Glu^{32}$ and $Asp^{50}$) and the eight Cys residue motifs, which are highly conserved among other c-type lysozymes in invertebrates and vertebrates, are also completely conserved. A phylogenetic analysis based on amino acid sequences indicated that the ARL II was more closely related to M. sexta, Hyphantria cunea, Heliothis virescens, and Trichoplusia ni lysozymes. The ARL II gene was expressed in Spodoptera frugiperda 21 insect cells and the recombinant ARL II (rARL II) was purified from cell-conditioned media by cation exchange column chromatography and reverse phase FPLC. The purified rARL II was able to form a clear zone in lysoplate assay against Micrococcus luteus. The lytic activity was estimated to be 511.41 U/mg, 1.53 times higher than that of the chicken lysozyme. The optimum temperature for the lytic activity of the rARL II was $50^{\circ}C$, the temperature dependency of the absolute lytic activity of rARL II was higher than that of the chicken lysozyme at low temperatures under $65^{\circ}C$.

• Animal Systematics, Evolution and Diversity
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• nspc3호
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• pp.139-146
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• 1992

중합효소연쇄반응(PCR)을 이용한 클로닝 기법과 Taq 염기서열분석법을 사용하여 갑각류에 속하는 뿔물맞이게(Pugettia quadridens)(십각 목, 범배 아목, 게 하목)에 대한 18S 리보솜 RNA 유전자의 1차염기서열을 밝혔다. 본 종의 18S 리보솜 RNA 유전자는 십각류에 속하는 또 다른 종인 두드러기어리게(Oedignathus inermis)보다 46개가 짧은 1837개의 염기로 이루어져 있었다. 염기가 삽입되거나 결실된 부분을 고려하지 않았을때에 두 종간에 염기서열 유사도는 90.8%이었다. 염기서열이 가장 보존적인 부위는 1137-1206(70개) 분위로 이 부위에서는 두 종이 완전히 동일한 염기서열을 보이고 있었고, 변이가 연속적으로 가장 큰 부위는 46-55 부위였고 399-407 부위가 그 다음으로 많은 연속적 변이를 가지고 있었다. 18S 리보솜 RNA 유전자의 1차구조에 있어서 염기서열의 변이는 이 유전자 전체를 통해 고르게 분포하지 않았다.

• Animal cells and systems
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• 제13권4호
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• pp.447-452
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• 2009

We cloned the complete complementary DNA (cDNA) of a Pacific oyster (Crassostrea gigas) cytochrome P450 (CYP450)-related protein using rapid amplification of cDNA ends (RACE). The cDNA included a 1470 bp open reading frame that began with the first ATG codon at position 103 bp and ended with a TAG stop codon at position 1573 bp (GenBank accession EF451959). The sequence had all major functional domains and characteristics of previously characterized CYP450 molecules, including the heme-binding region (FGVGRRRCVG) and putative arginine codon (R) integral to enzymatic function. An NCBI/GenBank database comparison to other CYP450 genes revealed that the deduced C. gigas CYP450 amino acid sequence is similar to that of mouse (Mus musculus) CYP450 2D/II (28%, accession AK078880), rabbit (Oryctolagus cuniculus) CYP450 2D/II (28%, AB008785), and white-tufted-ear marmoset (Callithrix jacchus) CYP450 2D (28%, AY082602). Thus, although the C. gigas CYP450 we cloned appears to belong to the 2D type of the CYP450 group, it has low similarity to this type. CYP450 mRNA expression increased over 6 h in C. gigas gills at $30^{\circ}C$ and $10^{\circ}C$, and then decreased, indicating that CYP450 plays an important role in C. gigas exposed to water temperature changes. This finding can be used as a physiological index for Pacific oysters exposed to changing water temperatures.

• BMB Reports
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• 제40권1호
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• pp.22-28
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• 2007

MyoD, expressed in skeletal muscle lineages of vertebrate embryo, is one of muscle-specific basic helix-loop-helix (bHLH) transcription factors, which plays a key role in the determination and differentiation of all skeletal muscle lineages. In this study, a cDNA of grass carp MyoD was cloned and characterized from total RNA of grass carp embryos by RT-PCR. The full-length cDNA of grass carp MyoD is 1597 bp. The cDNA sequence analysis reveals an open reading frame of 825 bp coding for a protein of 275 amino acids, which includes a bHLH domain composed of basic domain (1-84th amino acids) and HLH domain (98-142th amino acids), without signal peptide. Then the MyoD cDNA of grass carp was cloned to yeast expression vector pPICZ$\alpha$A and transformed into P. pastoris GS115 strain, the recombinant MyoD protein with a molecular weight of about 31KD was obtained after inducing for 2d with 0.5% methanol in pH 8.0 BMGY medium, and the maximum yield was about 250 mg/L in shaking-flask fermentation. The results were expected to benefit for further studies on the crystal structure and physiological function of fish MyoD.

• International Journal of Industrial Entomology and Biomaterials
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• 제3권2호
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• pp.121-126
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• 2001

A cDNA encoding a cathepsin D homologue was cloned from a cDNA library of the mulberry longicorn beetle, Apriona germari. Sequence analysis of the cDNA encoding the cathepsin D homologue of A. germari revealed that the 1,158 bp cDNA has an open reading frame of 386 amino acid residues. The deduced protein sequence of the A. germari cathepsin D homologue shows high homology with cathepsin D in insects, Aedes aegypti (68.2% amino acid similarity) and Drosophila melanogaster (67.2% amino acid similarity). Two aspartic residues and six cystein residues in the A. germari cathepsin D homologue are present at identical locations in all of the other catepsins D. Unlike cathepsins D in two insect species, A. gemari cathepsin D homologue appears to have two putative glycosylation sites, rather than one. Phylogenetic analysis revealed the A. germari cathepsin D homologue is more closely related to insect cathepsins D than to the other animal cathepsins D. Northern blot analysis suggests that A. germari cathepsin D homologue gene is expressed in most if not all, body tissues.

• Asian-Australasian Journal of Animal Sciences
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• 제16권7호
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• pp.1071-1086
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• 2003

Reproductive biotechnologies continue to be developed for genetic improvement of both river and swamp buffalo. Although artificial insemination using frozen semen emerged some decades back, there are still considerable limitations. The major problem appears to be the lack of efficient methods for estrus detection and timely insemination. Controlled breeding experiments in the buffalo had been limited and similar to those applied in cattle. Studies on multiple ovulation and embryo transfer are essentially a replica of those in cattle, however with inherent problems such as lower number of primordial follicles on the buffalo ovary, poor fertility and seasonality of reproduction, lower population of antral follicles at all stages of the estrous cycle, poor endocrine status and a high incidence of deep atresia in ovarian follicles, the response in terms of transferable embryo recovery has remained low with 0.51 to 3.0 per donor and pregnancy rates between 15 to 30%. In vitro production of buffalo embryos is a valid alternative to recovery of embryos by superovulation. This aspect received considerable attention during the past decade, however the proportion of embryos that develops to the blastocyst stage is still around 25-30% and hence the in vitro culture procedures need substantial improvement. Embryo cryopreservation procedures for direct transfer post thaw need to be developed for bubaline embryos. Nuclear transfer and embryo cloning is a technique that has received attention in various species during recent years and can be of immense value in buffaloes as they have a low rate of embryo recoveries by both in vitro and in vivo procedures. Gender pre-selection, genome analysis, gene mapping and gene transfer are a few of the techniques that have been studied to a limited extent during recent years and are likely to be included in future studies on buffaloes. Very recently, reproductive biotechnologies have been applied to feral buffaloes as well, but the results obtained so far are modest. When fully exploited they can play an important role in the preservation of endangered species.

• Asian-Australasian Journal of Animal Sciences
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• 제19권9호
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• pp.1257-1261
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• 2006

Pleiomorphic adenoma gene-like1 (PLAGL1) encodes a zinc-finger (ZF) protein with seven ZFs of the C2H2-type which is a regulator of apoptosis and cell cycle arrest, and also regulates the secretion of insulin. In both human and mouse, PLAGL1 is a candidate gene for tumor suppressor and transient neonatal diabetes mellitus (TNDM). In this study, a 2,238 bp fragment covering the complete coding region was obtained and deposited to GenBank (accession number: DQ288899). The reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that PLAGL1 was expressed almost equally in heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, uterus and ovary. Comparing the sequences of Large White and Meishan pigs, a C-T transition in exon 6 was found. The polymorphism could be detected by TaqI and was genotyped in five purebreds (Large White, Landrace, Meishan, Tongcheng and Bamei). Association analysis was performed between the polymorphism and carcass traits in 276 pigs of a "Large White${\times}$Meishan" F2 resource population. As a consequence, significant associations of the genotypes with shoulder backfat thickness (SFT) and internal fat rate (IFR) were observed. Pigs with TT genotype had low SFT and high IFR compared with TC or CC genotypes.

• Asian-Australasian Journal of Animal Sciences
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• 제24권6호
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• pp.834-843
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• 2011

Bacterial diversity was studied using PCR-DGGE, cloning and sequencing. DNA was isolated from digesta samples from stomach, ileum and colon of 28 weaned piglets (Large White${\times}$Mong Cai) fed dry control feed, naturally fermented liquid feed (FE) and a liquid diet with inclusion of rice distiller's residue feed. General bacterial diversity was described using DGGE analysis of the V3 region of 16S rDNA. The microbial populations in the stomach and the ileum were considerably influenced by the diet, while only marginal effects were observed in the colon. There was a large variation of the microbial flora in the stomach between individuals fed non-fermented diets. In contrast, animals fed diet FE had a more uniform microbial flora in the stomach and the ileum compared to the other diets. In total 47 bands from the DGGE profiles were cloned. In stomach, most frequently lactic acid bacteria were found. Feeding diet FE resulted in the occurrence of Pediococcus species in stomach and ileum. In pigs fed the other diets, Lactobacillus gallinarum, Lactobacillus johnsonii and Lactobacillus fermentum were found in stomach and ileum. Most of the sequences of bands isolated from colon samples and several from ileum matched to unknown bacteria, which often grouped within Prevotellaceae, Enterobacteriaceae, Bacteroidaceae and Erysipelotrichaceae. This study demonstrates that fermented liquid feed affects bacterial diversity and the specific microflora in stomach and ileum, which provides a potential to modulate the gut microflora with dietary means to increase the abundance of beneficial bacteria and improve piglets' health.

• Journal of Dairy Science and Biotechnology
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• 제29권1호
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• pp.49-54
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• 2011

담즙산 분해효소(Bile salt hydrolase, EC 3.5.1.24) 활성은 담즙산의 카르복실기와 결합되어 있는 아미노기(glycine or taurine)와의 amide 결합을 끊는 작용을 하며, 이 효소 활성은 사람이나 동물의 장내 미생물들에서 널리 분포하고 있다. 노인 분변으로부터 분리한 여러 균주의 Enterococcus faecalis중에서 BSH activity가 가장 높은 CU30-2를 선발하였다. BSH 유전자를 pET22b expression vector에 클로닝하여 Escherichia coli BL21(DE3) Gold를 이용하여 단백질을 발현하였다. 6x His-tag이 있는 BSH 효소를 $Ni^+$-NTA agarose column을 이용하여 분리 정제하였고, 6가지의 다른 담즙산염을 이용하여 기질 특이성을 비교하였다. E. faecalis CU30-2의 BSH 효소는 glycine이 결합된 담즙산염에 대한 효소활성이 taurine이 결합된 것에 대한 활성보다 약 50배 정도 높게 나타났다. 이 효소의 최적 pH와 온도는 각각 7.0과 40$^{\circ}C$로 확인되었다.

• Reproductive and Developmental Biology
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• 제37권1호
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• pp.1-8
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• 2013

In order to investigate genetic stability and gene expression profile after cloning procedure, two groups of cloned pigs were used for swine leukocyte antigen (SLA) gene nucleotide alteration and microarray analyses. Each group was consist of cloned pigs derived from same cell line (n=3 and 4, respectively). Six SLA loci were analyzed for cDNA sequences and protein translations. In total, 16 SLA alleles were identified and there were no evidence of SLA nucleotide alteration. All SLA sequences and protein translations were identical among the each pig in the same group. On the other hand, microarray assay was performed for profiling gene expression of the cloned pigs. In total, 43,603 genes were analyzed and 2,150~4,300 reliably hybridized spots on the each chip were selected for further analysis. Even though the cloned pigs in the same group had identical genetic background, 18.6~47.3% of analyzed genes were differentially expressed in between each cloned pigs. Furthermore, on gene clustering analysis, some cloned pigs showed abnormal physiological phenotypes such as inflammation, cancer or cardiomyopathy. We assumed that individual environmental adaption, sociality and rank in the pen might have induced these different phenotypes. In conclusion, the results of the present study indicate that SLA locus genes appear to be stable following SCNT. However, gene expressions and phenotypes between cloned pigs derived from the same cell line were not identical even under the same rearing conditions.