• BMB Reports
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• 제47권10호
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• pp.563-568
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• 2014

Human genome projects have enabled whole genome mapping and improved our understanding of the genes in humans. However, many unknown genes remain to be functionally characterized. In this study, we characterized human chromosome 4 open reading frame 34 gene (hC4orf34). hC4orf34 was highly conserved from invertebrate to mammalian cells and ubiquitously expressed in the organs of mice, including the heart and brain. Interestingly, hC4orf34 is a novel ER-resident, type I transmembrane protein. Mutant analysis showed that the transmembrane domain (TMD) of hC4orf34 was involved in ER retention. Overall, our results indicate that hC4orf34 is an ER-resident type I transmembrane protein, and might play a role in ER functions including $Ca^{2+}$ homeostasis and ER stress.

• 생명과학회지
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• 제17권9호통권89호
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• pp.1197-1203
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• 2007

세균인 Paenibacillus sp. DG-22의 유전체 DNA library가 제조되었으며, ${\beta}-xylosidase-$양성 클론이 형광기질인 $4-methylumbelliferyl-{\beta}-D-xylopyranoside$ $({\beta}MUX)$를 사용하여 확인되었다. 이 클론으로부터 재조합 플라스미드가 분리되었고 삽입된 4.3-kb 크기 DNA의 염기서열이 결정되었다. ${beta}-xylosidase$ 유전자는 분자량이 78.710 dal-ton이고 pI가 5.0인 701개의 아미노산을 암호화하는 2,106 염기쌍의 열린해독틀(ORF)로 구성되어있었다. xylA 유전자산물의 추론된 아미노산 서열은 과(family) 52에 속하는 클리코실 가수분해효소로 분류된 ${beta}-xylosidase$들과 상당한 유사성을 가지고 있었다. 이 xylA 유전자에 6개의 히스티딘-꼬리표를 붙이기 위해 pQE60 발현벡터에 다시 클로닝하였다. 재조합 ${beta}-xylosidase$ $(XylA-H_6)$가 열처리와 고정화금속친화성 크로마토그래피(IMAC)에 의해 순수하게 정제되었다. $XylA-H_6$ 효소의 최적 pH와 온도는 각각 pH 5.5-6.0과 $60^{\circ}C$이었다.

• Asian-Australasian Journal of Animal Sciences
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• 제15권4호
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• pp.576-584
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• 2002

Bovine PAS-4 is an integral membrane glycoprotein expressed in mammary epithelial cells. Complementary DNA (cDNA) cloning of PAS-4 was performed by reverse-transcriptase polymerase chain reaction (RT-PCR) with oligonucleotide probes based on it's amino terminal and internal tryptic-peptides. The cloned PAS-4 cDNA was 1,852 nucleotides (nt) long and its open reading frame (ORF) was encoded 1,413 base long. The deduced amino acid sequence indicated that PAS-4 consisted of 471 amino acid residues with molecular weight of 52,796, bearing 8 potential N-glycosylation sites and 9 cysteine residues. Partial bovine CD36 cDNA from liver also was sequenced and the homology of both nucleotide sequence was 94%. Most of the identical amino acid residues were in the luminal/extracellular domains. Contrary to PAS-4, bovine liver CD36 displays 6 potential N-glycosylation sites, which were located, except for those at positions 101 and 171, at same positions as PAS-4 cDNA. Cysteine residues of PAS-4 and CD36 were same at position and in numbers. Northern blot analysis showed that PAS-4 was widely expressed, although its mRNA steady-state levels vary considerably among the analyzed cell types. PAS-4 possessed hydrophobic amino acid segments near the amino- and carboxyl-termini. Two short cytoplasmic tails of the amino- and carboxyl-terminal ends constituted of a 5-7 and 8-11 amino acid residues, respectively.

• Journal of Microbiology and Biotechnology
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• 제9권1호
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• pp.113-118
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• 1999

The insulin-like growth factor (IGF) is found in all vertebrates and its type-II molecule is regarded as a fundamental embryonic growth factor during development. We have firstly identified, in this study, a cDNA clone corresponding to IGF-II (flIGF-II) from the adult brain of the teleost, Paralichthys olivaceus. We also examined the tissue expression of flIGF-II in several adult tissues by RT-PCR. The flIGF-II cDNA contained a complete ORF consisting of 215 amino acids and one stop codon. Its molecular characteristics appear to be similar to the previously identified IGF-II molecules, in which a common primary structure exhibiting B, C, A, D, and E domains is evidently observed. This cDNA clone seems to be cleaved at $Ala_{52}$ for the $NH_2$-end signal peptide and appears to produce a 98 amino acid-long E-peptide from the $Arg^{118}$. The functional B-D domain regions, therefore, include 65 amino acids and is able to encode a 7.4-kDa protein. The most prominent structural difference between IGF-I and IGF-II was that the D domain of IGF-II exhibits a two-codon-deleted pattern compared to the 8 amino acid-containing IGF-I. The insulin family signature in the A domain and six cysteins forming three disulfide bridges between the B and A domains were evolutionary-conserved from teleosts to mammalian IGF-II. Interestingly, the E-peptide region appears to provide a distinct hallmark between teleosts in amino acid composition. The flIGF-II shows 85.1% of sequence identity to salmon and trout, 90.6% to tilapia, and 98.4% to perch in amino acid level. In tissue expressions of IGF-II, it is very likely that flIGF-II has a significant expression in the adult brain. However, liver seems to be the main source for IGF-II production, and relatively low signals were observed in the adult muscle and kidney. Taken together, it would be concluded that the functional region for IGF-II mRNA is highly similar in phylogeny and is evolutionary, conserved as a mediator for the growth of vertebrates.

• Journal of Ginseng Research
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• 제42권4호
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• pp.455-462
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• 2018

Background: Ginsenoside Rh2 has been known to enhance the activity of immune cells, as well as to inhibit the growth of tumor cells. Although the repertoire of genes regulated by Rh2 is well-known in many cancer cells, the epigenetic regulation has yet to be determined, especially for comprehensive approaches to detect methylation changes. Methods: The effect of Rh2 on genome-wide DNA methylation changes in breast cancer cells was examined by treating cultured MCF-7 with Rh2. Pyrosequencing analysis was carried out to measure the methylation level of a global methylation marker, LINE1. Genome-wide methylation analysis was carried out to identify epigenetically regulated genes and to elucidate the most prominent signaling pathway affected by Rh2. Apoptosis and proliferation were monitored to examine the cellular effect of Rh2. Results: LINE1 showed induction of hypomethylation at specific CpGs by 1.6-9.1% (p < 0.05). Genome-wide methylation analysis identified the "cell-mediated immune response"-related pathway as the top network. Cell proliferation of MCF-7 was retarded by Rh2 in a dose-dependent manner. Hypermethylated genes such as CASP1, INSL5, and OR52A1 showed downregulation in the Rh2-treated MCF-7, while hypomethylated genes such as CLINT1, ST3GAL4, and C1orf198 showed upregulation. Notably, a higher survival rate was associated with lower expression of INSL5 and OR52A1 in breast cancer patients, while with higher expression of CLINT1. Conclusion: The results indicate that Rh2 induces epigenetic methylation changes in genes involved in immune response and tumorigenesis, thereby contributing to enhanced immunogenicity and inhibiting the growth of cancer cells.

• 한국잠사곤충학회지
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• 제52권1호
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• pp.45-51
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• 2014

면역 유도된 천잠(Antheraea yamamai) 유충에서 cecropin-A 유전자를 분리하였고 이 유전자를 Ay-CecA로 명명하였다. 전체 Ay-CecA cDNA 크기는 419 bp로 64개의 아미노산 잔기를 인코딩하는 195 bp ORF로 구성되어 있다. 천잠 CecA 유전자는 22개 잔기의 signal peptide, 4개 잔기의 propeptide 및 항균활성을 갖는 37개 잔기로 구성된 성숙 단백질(mature protein) 영역으로 구성되고 예상 분자량은 4046.81 Da으로 산출되었다. 천잠 CecA의 아미노산 서열은 다른 나비목 곤충에서 분리된 cecropin와 매우 높은 상동성(62 ~ 78%)을 나타냈다. Ay-CecA 유전자의 C말단에 기존에 보고된 곤충의 cecropin에서와 동일하게 C말단 아미드화를 위한 glycine 잔기가 존재하고 있다. 이 펩타이드의 항균활성을 검정하기 위해서 대장균 발현 시스템을 이용하여 활성이 있는 재조합 Ay-CecA 발현에 성공하였다. 발현 기주인 대장균에 대한 재조합 CecA 독성 중화를 위해서 불용성 단백질인 ketosteroid isomerase(KSI) 유전자를 CecA 유전자와 융합하였다. 융합 CecA-KSI 단백질은 대부분 불용성 단백질로 발현되었다. 발현된 융합단백질은 Ni-NTA immobilized metal affinity chromatography(IMAC)에 의해서 정제하였으며 CNBr 반응을 통하여 재조합 CecA를 절단하여 용출하였다. 최종적으로 양이온 교환 chromatography 과정을 통하여 CecA를 순수 정제하였다. 정제된 재조합 Ay-CecA는 그람음성균인 E. cori ML 35, Klebsiella pneumonia 및 Pseudomonas aeruginosa에 대해 매우 높은 항균활성을 나타냈었다. 따라서 본 연구 결과, 높은 항균활성 지닌 CecA는 천잠의 면역 반응에서 중요한 역할을 담당할 것으로 사료된다.

• Parasites, Hosts and Diseases
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• 제52권1호
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• pp.93-97
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• 2014

Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl ${\beta}$-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.

• 미생물학회지
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• 제52권1호
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• pp.110-115
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• 2016

남극 해양세균 Pseudoalteromonas sp. PAMC 21150에서 분리한 소형 플라스미드(small plasmid, pDK4)의 크기는 3,480bp이고 G+C 함량은 41.64%이며, 3개의 open reading frames(ORFs)을 포함하고 있다. 3개의 ORF는 replication initiation protein (RepA), conjugative mobilization protein (Mob), 그리고 기능이 밝혀지지 않은 단백질을 코팅하고 있다. PCR 반응으로 증폭한 pDK4를 Escherichia coli high-copy pUC19 클로닝 벡터에 삽입하여 fusion vector (pDOC153)를 제작하였고, pDOC 153에 chloramphenicol 저항성 유전자를 삽입하여 ampicillin/chloramphenicol 저항성 Pseudoalteromonas - Escherichia coli 셔틀 벡터(shuttle vector; 7,216 bp 크기; pDOC155)를 제작하였다. 북극 해양세균 P. issachenkonii PAMC 22718이 보유한 2개의 유전자(TonB-dependent receptor gene, chi22718_IV, and exochitinase gene, chi22718_III)를 pDOC155에 삽입하여 두 개의 pDOC155 변형체(pDOC158, pDOC165)를 제작하였다. pDOC158 혹은 pDOC165을 이용하여 triparental mating 방법에 의해 플리스미드 미보유 해양세균인 Pseudoalteromonas sp. PAMC 22137를 형질전환하였다. PCR을 이용한 유전자 증폭실험을 통해서, pDOC158와 pDOC165에 삽입된 유전자들은 Pseudoalteromonas sp. PAMC 22137와 E. coli $DH5{\alpha}$ 내에서 안정적으로 유지되는 것을 확인하였다. 위의 결과는 셔틀 벡터 pDOC155는 Pseudoalteromonas spp. 유래 유전자들을 다른 Pseudoalteromonas spp. 세포 안으로 전달할 수 있는 새로운 유전자 전달시스템으로 이용될 수 있음을 보여주었다.