• 한국수정란이식학회지
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• 제13권2호
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• pp.159-172
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• 1998

사람 성장호르몬 수용체(hGHR) cDNA는 PCR방법에 의하여 fagment로서 보고되어진 바 있으나, liver cDNA로 부터 전장을 cloning한 보고는 없는 실정으로 본 연구에서는 기능을 가진 약 4.6kbp의 cDNA hGHR을 cloning 하는데 성공하였다. 먼저 cloning하기 위하여 human liver mRNA와 human breast cancer tissue로부터 회수한 mRNA를 RT-PCR방법에 의하여 human cDNA library와 cloning에 필요한 probe를 제작하였다. human library mRNA는 GT-PCR방법에 의하여 증폭하여 증폭되어진 산물은 λZAP Vector를 이용하여 cDNA library를 구축하였고,screeing을 위하여 임 보고 되어진 hGHR fragment native sequence를 기초로 N-terminal부분의 primer를 설계하여 950bp의 probe를 얻는데 성공하였다. 이 probe를 이용하여 준비된 human liver cDNA library로부터 2.5$\times$10 6개의 plaque로부터 6개의 positive clone을 획득하였고, 이들중 poly Asignal인 "AATAAA"를 포함하고 있는 가장 긴 약 3.8kbp의 clone을 sequencing한 결과 open reading frame을 포함하고 있었으나, 5'부분의 결손되어 있었다. 그리하여 이 부분은 human breast cancer tissue로 부터 회수한 mRNA를 RT-PCR에 의하여 증폭하였고, sequencing결과 이미 보고되어진 native hGHR와 비교한 결과 하나의 nucleotide가 silent mutation으로 판명되었다.한편 human liver cDNA library로부터 cloning한 3.8cp의 positive clone의 5'end의 결손된 부분에 silent mutation된 PCR 산물을 연결함으로써 native hGHR와 유사한 cDNA hGHR subcloning에 성공하였다. 이러한 cDNA hGHR의 clone이 function을 가지고 있는지를 검토하기 위하여 eukaryotic 발현 vector인 pCXN2에 의거 ligation한 후 chinese hamster ovary cell[CHO-KI]에 transfect를 실시하였다. Dexamethasone은 첨가하지 않고 hGH만의 존재하에서 이들 cell을 배양시키고 cell menbrane에서 발현 여부를 판정키 위하여 hGHR monocloual antibody를 사용하여 flow cytometery해석을 실시하는 한편 125I-hGH binding assay에 의하여 hGH binding activity를 측정하였다. 최종적으로 GH signal transduction의 target genedf으로 알려져 있는 serine protease inhibitor 2.1(Spi 2.1) gene의 promotor activity를 검토한 결과 hGHR을 transfect한 CHO Cell에 있어서 hGH의 농도에 의존적으로 증가되었다. 따라서 본 실험에서 cloning한 cDNA hGHR는 native hGHR와 같은 기능을 가지는 것으로 판명되었다.것으로 판명되었다.

• Animal cells and systems
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• 제5권3호
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• pp.231-236
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• 2001

SINE-R retroposons have been derived from human endogenous retrovirus HERV-K family and found to be hominoid specific. Both SINE-R retroposons and HERV-K family are potentially capable of affecting the expression of closely located genes. From cDNA library of human fetal brain, we identified seven SINE-R retroposons and compared them with sequences derived from GenBank database. The SINE-R retroposons from human feta1 brain showed 85∼97% sequence similarities with the human-specific retroposon SINE-R.C2. They also showed 88∼96% sequence similarities with the sequence of the schizo-cDNA clone that derived from postmortem frontal cortex tissue of a schizophrenic patient. Phylogenetic analysis using the neiqhbor-joining method revealed that the seven new SINE-R retroposons from cDNA library of the human feta1 brain have proliferated independently during human evolution. The data indicate that such SINE-R retroposons are expressed in human fetal brain and deserve further investigation as potential leads to understanding of neuropsychiatric diseases.

• 인지과학
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• 제1권2호
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• pp.361-376
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• 1989

카테콜아민 생합성에 관여하는 마지막 효소인 phenylethanolamine Nmethyltransferase는 norepinephrine 을 epinephrine으로 전환시키는 중요한 효소이다. PNMT효소의 발현은 epinephrine 신경세표의 발현에 필수적이다.따라서 PNMT 유전자를 크로닝하여 그 구조를 결정하고,유전자 발현연구를 하는 것은 상당히 중요한 일이다.그러나 최근에 저자가 bovine 및 human cDA 를 처음으로 분리하여 그 구조를 보고한 것 외에는 아직까지 인간과 백서 전체 genomic DNA 의 분리 보고는 없다.이에 저자들은 인간과 백서 PNMT유전자의 전체구조와 여러종(species)사이의 진화적인 관계를 규명하기 위해서 human 과 Rat genomic library 를 만들고,이 library 를 이용하여 bovine cDNA 를 probe로 13.1kb와 13.2kb길이의 인간과 백서의 genomic clone 을 분리 크리닝하는데 성공하여 유전자의 구조적 규명하였다.

• Animal cells and systems
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• 제5권2호
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• pp.133-137
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• 2001

Long terminal repeats (LTRs) of the human endogenous retrovirus K family (HERV-K) have been found to be coexpressed with sequences of genes closely located nearby. We examined transcribed HERV-K LTR elements in human brain tissue. Using cDNA synthesized from mRNA of the human brain, we performed PCR amplification and identified ten HERV-K LTR elements. These LTR elements showed a high degree of sequence similarity (92.4-99.7%) with the human-specific LTR elements. A phylogenetic tree obtained by the neighbor-joining method revealed that HERV-K LTR elements could be divided into two groups through evolutionary divergence. Some HERV-K LTR elements (HKL-B7, HKL-B8, HKL-B10) belonging to the group II from human brain cDNA were closely related to the human-specific HERV-K LTR elements. Our data suggest that HERV-K LTR element are active in the human brain; they could conceivably play a pathogenic role in human diseases such as psychosis.

• BMB Reports
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• 제32권5호
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• pp.502-505
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• 1999

cDNA fragments of ovine liver pyridoxal kinase were amplified by PCR using degenerate oligonucleotide primers derived from partial amino acids sequences of the enzyme. Using PCR products as probes, several overlapping cDNA clones were isolated independently from an ovine liver and a human brain cDNA library. The largest cDNA clone for each was selected for sequence analysis. The ovine liver cDNA encodes a polypeptide of 297 amino acid residues with Mr of 32,925, whereas the human clone is comprised of an open reading frame encoding 312 amino acid residues with Mr of 35,102. The deduced sequence of the human brain enzyme is completely identical to that of human testes cDNA recently reported (Hanna et al., 1997). The ovine enzymes have approximately 77% sequence identity with the human enzyme although the two sequences are completely different in the N-terminus comprising 32 residues. This result suggests that pyridoxal kinase is highly homologous in mammalian species.

• Journal of Microbiology and Biotechnology
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• 제12권3호
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• pp.508-513
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• 2002

Human endogenous retroviral long terminal repeats (LTRs) have been found to be coexpressed with sequences of genes located nearby. It has been suggested that the LTR elements have contributed to the structural change or genetic variation of human genome connected to various diseases. The HERV-W family has been identified in the cerebrospinal fluids and brains of individuals with schizophrenia. Using a cDNA library derived from a human brain, the HERV-W LTR elements were examined and five new LTR elements were identified. These elements were examined using a YAC clone panel from the Xq21.3 region linked to psychosis that was replicated on the Y chromosome after the separation of the chimpanzee and human lineages. Fourteen elements of the HERV-W LTR were identified in that region. Those LTR elements showed a high degree of sequence similarity ($91.8-99.5\%$) with previously reported HERV-W LTR. A phylogenetic tree obtained from the neighbor-joining method revealed that new HERV-W LTR elements were closely related to the AXt000960, AF072504, and AF072506 from the GenBank database. The data indicates that several copy numbers of the HERV-W LTR elements exist on the Xq21.3 region and are also expressed in the human brain. These LTR elements need to be further investigated as potential leads to neuropsychiatric diseases.

• BMB Reports
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• 제38권1호
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• pp.28-33
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• 2005

A gene coding a novel isoform of carbamyl phosphate synthetase I (CPS1) was cloned from a human testicular library. As shown by cDNA microarray hybridization, this gene was expressed at a higher level in human adult testes than in fetal testes. The full length of its cDNA was 3831 bp, with a 3149 bp open reading frame, encoding a 1050-amino-acid protein. The cDNA sequence was deposited in the GenBank (AY317138). Sequence analysis showed that it was homologous to the human CPS1 gene. The putative protein contained functional domains composing the intact large subunit of carbamoyl phosphate synthetase, thus indicated it has the capability of arginine biosynthesis. A multiple tissue expression profile showed high expression of this gene in human testis, suggesting the novel alternative splicing form of CPS1 may be correlated with human spermatogenesis.

• 대한의생명과학회지
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• 제2권2호
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• pp.231-240
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• 1996

인체 혈액 응고 9인자는 간에서 생성되며 461개의 아미노산으로 구성된 당 단백질이다. 따라서 인체 혈액 응고 9인자 cDNA를 찾기 위해 태아의 간(fetal liver) cDNA library를 PCR(Polymerase Chain reaction) 방법으로 screening하였으며, 그 결과 ATG개시 코돈으로부터 TAA종료 코돈까지 포함하는 1.4 kb의 9인자 cDNA를 찾았다. 또한 클론된 9인자 cDNA를 박테리아에서 발현시키기 위해 박테리아 발현 벡터인 pGEX-2T 플라스미드에 클로닝하므로써 pGEX-F9 플라스미드를 제조하였다. pGEX-F9로 형질전환된 E. coli에서 PGEX-F9의 발현을 유도하면 73 kDa 크기의 GST-factor9 융합 단백질이 다량생성되며 , 이 단백질이 혈액 응고 9인자 단백질을 함유하는 융합 단잭질임을 혈액 응고 9인자 항체를 이용한 Western blot으로 입증하였다. E. coli에서 발현된 GST-factor 9 융합 단백질은 전체 단백질의 약 20%를 차지하며 GST agarose bead를 이용한 one step purificarion 방법을 통해 GST-factor9 융합 단백질을 쉽게 분리 할 수 있다.

• Journal of Microbiology and Biotechnology
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• 제25권4호
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• pp.547-553
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• 2015

The yeast surface-displayed cDNA library has been used to identify unknown antigens. However, when unknown target antigens show moderate-to-low abundance, some modifications are needed in the screening process. In this study, a directional random-primed cDNA library was used to increase the number of candidates for the unknown antigen. To avoid the loss of target yeast clones that express proteins at a low frequency in the cDNA library, a comprehensive monitoring system based on magnetic-activated cell sorting, fluorescence-activated cell sorting, and immunofluorescence was established, and a small number of target yeast cells was successfully enriched. These results showed that our optimized method has potential application for identifying rare unknown antigens of the human monoclonal antibody.

• Archives of Pharmacal Research
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• 제20권5호
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• pp.459-464
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• 1997

The human liver cDNA clone UDPGTh2, encoding a liver UDP-glucuronosyltransferase (UDPGT) was isolated from a .gamma. gt 11 cDNA library by hybridization to mouse transferase cDNA clone, UDPGTm1. UDPGTh2 encoded a 529 amino acid protein with an amino terminus membrane-insertion signal peptide and a carboxyl terminus membrane-spanning region. There were three potential asparagine-linked glycosylation sites at residues 67, 68, and 315. In order to obtain UDPGTh2 protein encoded from cloned human liver UDP-glucuronosyltransferase cDNA, the clone was inserted into the pSVL vector (pUDPGTh2) and expressed in COS 1 cells. The presence of a transferase with Mr-52,000 in transfected cells cultured in the presence of $[^{35}S]$ methionine was shown by immunocomplexed products with goat antimouse transferase IgG and protein A-Sepharose and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The expressed UDPGT was a glycoprotein as indicated by electrophoretic mobility shift in Mr-3,000-4,000 when expressed in the presence of tunicamycin. The extent of glycosylation was difficult to assess, although one could assume that glycosyl structures incorporated at the level of endoplasmic reticulum were always the core oligosaccharides. Thus, it is likely that at least two moieties inserted can account for the shift of Mr-3,000-4,000. This study demonstrates the cDNA and deduced amino acid sequence of human liver UDP-glucuronosyltransferase cDNA, UDPGTh2.