• 한국연초학회지
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• 제23권2호
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• pp.133-140
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• 2001

PAP-I cDNA was synthesized from total RNA of Phytolacca americana leaves by RT-PCR, and then subcloned to recombinant vector pBluescript II SK-. Using PCR with primers designed in our laboratory, we could get the 9 deletion mutant PAP-I cDNA fragments. The first of the fragments was deleted by 66bp from immature N-terminal and then the rest were deleted by 90bp sequentially. Sequentially deletion mutant PAP-I cDNAs were inserted to pAc55M, on down-stream of gall promoter. Recombinant pAc55M was transformed to yeast cells, psy1 and the cells were spreaded on SC_urn-/glucose plate media. Colonies on SC_ura-/glucose plate were streaked on the same position of SC_ura-/glucose and SC_ura-/galactose plate, and we selected colonies growing on both plates, which carry non-cytotoxic deleted mutant PAP-I cDNA. We selected 4 deletion mutant PAP-I cDNAs which have not cytotoxicity.

• Journal of Genetic Medicine
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• 제12권1호
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• pp.57-60
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• 2015

CHARGE syndrome (coloboma, heart defects, atresia choanae, retarded growth and development, genital hypoplasia, and ear abnormalities) is characterized by multiple malformations and is diagnosed using distinct consensus criteria. Mutations in the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7) are the major cause of CHARGE syndrome. Clinical features of CHARGE syndrome considerably overlap those of 22q11.2 deletion syndrome. Of these features, immunodeficiency and hypocalcemia are frequently reported in patients with 22q11.2 deletion syndrome but are rarely reported in patients with CHARGE syndrome. In this report, we have described the case of a patient with typical phenotypes of 22q11.2 deletion syndrome but without the proven chromosome microdeletion. Mutation analysis of CHD7 identified a pathogenic mutation (c.2238+1G>A) in this patient. To our knowledge, this is the first case of CHARGE syndrome with immunodeficiency and hypocalcemia in Korea. Our observations suggest that mutation analysis of CHD7 should be performed for patients showing the typical phenotypes of 22q11.2 deletion syndrome but lacking the proven chromosome microdeletion.

• Molecules and Cells
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• 제27권1호
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• pp.89-97
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• 2009

We investigate the molecular and cellular etiologies that underlie the deletion of the six amino acid residues (${\Delta}F323-Y328$; ${\Delta}FY$) in human torsin A (HtorA). The most common and severe mutation involved with early-onset torsion dystonia is a glutamic acid deletion (${\Delta}E$ 302/303; ${\Delta}E$) in HtorA which induces protein aggregates in neurons and cells. Even though ${\Delta}FY$ HtorA forms no protein clusters, flies expressing ${\Delta}FY$ HtorA in neurons or muscles manifested a similar but delayed onset of adult locomotor disability compared with flies expressing ${\Delta}E$ in HtorA. In addition, flies expressing ${\Delta}FY$ HtorA had fewer aberrant ultrastructures at synapses compared with flies expressing ${\Delta}E$ HtorA. Taken together, the ${\Delta}FY$ mutation in HtorA may be responsible for behavioral and anatomical aberrations in Drosophila.

• BMB Reports
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• 제29권5호
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• pp.411-416
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• 1996

In order to study the role of the protein shell in both iron uptake and iron core formation of ferritin, we constructed a deletion mutant of the ferritin gene and expressed the mutant gene in Escherichia coli, This mutant was obtained by introducing an amber mutation at position Pro-157 and a deletion of the 19 amino acid residues at the carboxy-terminus of the recombinant tadpole H-chain ferritin. The deleted amino acids correspond to E-helix forming the hydrophobic channel in the protein. E. coli harboring the plasmid pTHP157, which contains the deleted gene, was grown at $23^{\circ}C$ in the presence of 0.1 mM IPTG, and the induced protein appeared to be partly soluble. Nondenaturing polyacrylamide gel electrophoresis showed that the expressed mutant H-chains coassemble into holoprotein, suggesting that E-helix is not necessary for assembly of the subunits as reported for human H-chain ferritin. Its ability in iron core formation was proven in an Fe staining gel, the result disagreeing with the observation that the hydrophobic channel is necessary for iron core formation in human H-chain ferritin.

• 한국임상수의학회지
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• 제22권4호
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• pp.386-391
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• 2005

This study was done to produce a mutated protein inactivated cytotoxicity of Shigatoxin 2e (Stx2e) of E.coli O139 isolates by deletional mutagenesis of Stx2e A subunit gene encoding active-site cleft of enzymatic domain in ST2e holotoxin. Cytotoxicity of the toxoid expressed from the mutant Stx2e gene was compared with wild type Stx2e for development of vaccine candidate. A recombinant plasmid pED18 containing Stx2e gene ot E.coli O139 isolates was used to generate mutation plasmid. Deletion mutagenesis was conducted for Stx2e A subunit gene encoding enzymatically active domain by polymerase chain reaction (PCR) using ot designed primer to induce deletional mutation. DNA sequence analysis was confirmed that the pentamer (Typ 202- Ser 206) that lies within the proposed active-site cleft in the second region was completely deleted. A DNA fragment of 1.1 kb that encode the new mutant Stx2eA gene was inserted into plasmid pRSET vector digested with EcoRV-Hind III and named pEDSET The PEDSET was transformed in E. coli for expression of mutant protein and the protein was confirmed by SDS-PACE and Western-blotting. The protein expressed by the mutant was tested to confirm the reduction of cytotoxic activities on Vero cell using microcytotoxicity assay compared with wild type Stx2e, the cytotoxicity of deletional mutant protein was at least reduced by 3,000-fold on Vero cell.

• Clinical and Experimental Pediatrics
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• 제56권6호
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• pp.265-268
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• 2013

Wiskott-Aldrich syndrome (WAS) is an inherited X-linked disorder. The WAS gene is located on the X chromosome and undergoes mutations, which affect various domains of the WAS protein, resulting in recurrent infection, eczema, and thrombocytopenia. However, the clinical features and severity of the disease vary according to the type of mutations in the WAS gene. Here, we describe the case of a 4-year-old boy with a history of marked thrombocytopenia since birth, who presented with recurrent herpes simplex infection and late onset of eczema. Examination of his family history revealed that older brother, who died from intracranial hemorrhage, had chronic idiopathic thrombocytopenia. Therefore, we proceeded with genetic analysis and found a new deletion mutation in the WAS gene: c.858delC (p.ser287Leufs$^*21$) as a hemizygous form.

• BMB Reports
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• 제48권3호
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• pp.184-189
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• 2015

Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2) is known to protect neurons from neurodegeneration during ischemia/reperfusion injury. We recently reported that ROS-mediated oxidative stress promotes phosphorylation of endogenous SHP-2 in astrocytes and complex formation between caveolin-1 and SHP-2 in response to oxidative stress. To examine the region of SHP-2 participating in complex formation with caveolin-1, we generated three deletion mutant constructs and six point mutation constructs of SHP-2. Compared with wild-type SHP-2, binding of the N-SH2 domain deletion mutant of SHP-2 to p-caveolin-1 was reduced greatly, using flow cytometric competitive binding assays and surface plasmon resonance (SPR). Moreover, deletion of the N-SH2 domain of SHP-2 affected $H_2O_2$-mediated ERK phosphorylation and Src phosphorylation at Tyr 419 in primary astrocytes, suggesting that N-SH2 domain of SHP-2 is responsible for the binding of caveolin-1 and contributes to the regulation of Src phosphorylation and activation following ROS-induced oxidative stress in brain astrocytes.

• Journal of Microbiology and Biotechnology
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• 제4권4호
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• pp.245-249
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• 1994

An amino-terminal 14 amino acids deletion and three site-directed mutations were created to investigate the mechanism of induction and repression of MerR regulatory protein in R100 mer operon from gramnegative Shigella flexneri. The amino-terminal 14 amino acids deletion, Cysl17Ser, and Cys126Ser mutations abolished the inducibility of the mer operon and the Hisl18Ala mutation resulted in the reduction of inducibility (about 9.1 % remaining) in complementation experiment in the presence of $Hg^{2＋}$ at subtoxic level ($1\mu M$). The complementation experiment with $Hg^{2＋}$ absent showed that Hisl18Ala, Cys126Ser, and wild-type MerR could repress the operon but Cysl17Ser could not, and the amino-terminal deletion mutant could neither induce nor repress the R100 mer operon.

• 대한수의학회지
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• 제37권3호
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• pp.541-546
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• 1997

VT2(Verotoxin-2)의 효소활성 영역에 해당되는 두 영역의 아미노산들에 대하여, 첫번째 보존영역의 Glu167을 conservative point mutation 시키고, 두 번째 보존영역의 구성 아미노산 5개 전부를 deletion mutation 시켜, 각 변이주에서 독성의 감소 정도를 wild type과 비교한 결과 다음과 같은 성적을 얻었다. 1. pKSC101을 Eco RI과 Pst I으로 절단하여 940bp insert를 통일 제한효소로 절단한 M 13mp19에 삽입하여 pEP19RF를 구축하였다. 이를 이용하여 dU-SSDNA template를 제조하고, mutagenic primer를 annealing 하여 변이를 도입하였으며, 변이가 도입된 insert를 acceptor plasmid에 삽입시켜 각각 발현 플라스미드 pOEX와 pDEX를 구축하였다. 각각의 mutant 단백질을 발현시키기 위하여 pOEX와 pDEX를 JM109에 형질전환시켜 mutant 재조합 균주인 POMUT109와 DEMUT109를 작성하였다. 2. POMUT109와 DEMUT109를 IPTG 유도 발현시킨 배양상층액을 Vero cell에 대하여 세포독성을 시험한 결과 wild type에 비하여 POMUT109의 배양 여액에서는 2000배, DEMUT109의 배양여액에서는 적어도 3000배 이상의 세포독성을 감소시켰다.

• IMMUNE NETWORK
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• 제1권2호
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• pp.151-161
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• 2001

Background: Inactivation in p53 tumor suppressor gene through a point mutation and deletion is one of the most frequent genetic changes found in human cancer, with 50% of an incidence. This high rate of mutation mostly suggests that the gene plays a central role in the development of cancer and the mutations detected so far were found in exons 5 to 8. Mutation of p53 locus produced accumulation of abnormal p53 protein, and negative regulation of cell proliferation and transcriptional activation as a suppressor of transformation were lost. In addition, inhibition of its normal cellular function of wild-type by mutant is an important step in tumorigenesis. Method: 4 colon cancer cell lines (SNU C1, C2A, C4, C5) were examined for mutation in exons 5 to 8 of the p53 tumor suppressor gene by PCR-SSCP analysis and expression pattern by western blotting and immunoprecipitation. p53-mediated transactivation ability were examined by CAT assay and base substitution of p53 in SNU C2A cell were detected by DNA sequencing. Results: 1) SNU C2A cell and SNU C5 cell were detected mobility shifts each in exon 5 and exon 7 of p53 gene by the PCR-SSCP method, implicating being of p53 mutation. 2) 3 colon cancer cell lines (SNU C1, SNU C2A, SNU C5) expressed wild type and mutant type p53 protein. 3) In northern blot experiment, SNU C2A and SNU C5 cell expressed high level of p53 mRNA. 4) Results of p53-mediated transactivation in colon cancer cell lines by CAT assay represented only SNU C2A cell has transcriptional activity. 5) DNA sequencing in SNU C2A cell showed missense mutation in codon 179 of one allele, histidine to arginine and wild type p53 in the other allele. Conclusion: Colon cancer cell lines showed correlation with mutation in p53 gene and accumulation of abnormal p53 protein. Colon cancer cell SNU C2A retained p53-mediated transactivation as heterozygous p53 with one mutant allele in 179 codon and the other wild-type allele.