Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The authors report on the activity of Hinf I restriction endonuclease on human fixed metaphase chromosomes. Experiments performed by digesting chromosomes just after harvesting or after ageing in methanol-acetic acid displayed a different pattern of digestion on metaphases, since only aged preparations showed gaps on heterochromatic regions of chromosomes 1, 9 and 16 and C-like bands on other chromosomes. In this view, the authors suggest that structural modifications of the DNA, induced by acid fixation, can influence Hinf I activity on fixed metaphase chromosomes.
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PMID:Hinf I restriction endonuclease digestion on human fixed metaphase chromosomes. 222 98

A simple and reliable method for the recovery of specific fragments of DNA from agarose gels is presented. The electroelution of the DNA onto the NENSORB cartridge matrix with the subsequent elution of the bound DNA by a methanol (50% v/v) wash has been shown to result in the quantitative recovery of the restriction fragment. Of importance is the fact that the DNA purified by this procedure is a viable substrate for further digestion by a second restriction endonuclease. The method does not require either phenol extraction or extensive desalting of the sample.
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PMID:Affinity chromatographic procedure for the quantitative recovery of DNA fragments from agarose gels. 301 50

The restriction endonuclease BstNI markedly reduced the extent of Giemsa staining of the C-band regions of methanol/acetic acid-fixed mouse chromosomes air-dried onto glass slides. The enzyme reduced the amount of hybridizable satellite DNA correspondingly, indicating that its effects can be attributed to cutting satellite DNA into fragments short enough to be removed from fixed chromosomes.
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PMID:Mechanism of endonuclease banding of chromosomes. 609 19

Sclerosing hemangioma of the lung remains poorly understood, and it is still unclear whether this lesion is neoplastic or not. It consists of two major cell types, pale cells and cuboidal cells. We analyzed the clonality of each cell types from six female cases of surgically resected sclerosing hemangioma. The pale cells and cuboidal cells were separated by microdissection from methanol-fixed sections, and DNA was extracted for clonal analysis based on an X-chromosome-linked polymorphic marker, the human androgen receptor (HUMARA) gene or the phosphoglycerate kinase (PGK) gene. The HUMARA and PGK genes were found to be amplified with or without digestion by the methylation-sensitive restrictive endonuclease HpaII. Five of six cases were informative. Pale cells and cuboidal cells showed the same monoclonality in all of the informative cases, whereas the control cells showed a polyclonal pattern. Our results demonstrated that sclerosing hemangioma is caused by monoclonal expansion of cells, confirming that it is a neoplasia. Moreover, the present data indicate that both pale cells and cuboidal cells are derived from the same cell.
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PMID:Monoclonality of both pale cells and cuboidal cells of sclerosing hemangioma of the lung. 954 67

Atypical adenomatous hyperplasia (AAH) of the lung has been postulated as a possible precursor lesion of bronchioloalveolar carcinoma (BAC). The clonality of AAHs from seven female patients was analyzed to determine whether AAH is a monoclonal expansion. All AAHs were identified in lungs surgically resected for BAC. The clonality of the BAC and bronchiolar metaplasia in each case was also analyzed. Approximately 500 cells in each lesion were precisely microdissected from methanol-fixed sections. Adjacent normal lung tissue was collected as a normal control. DNA was extracted for clonal analysis based on an X-chromosome-linked polymorphic marker, the human androgen receptor gene (HUMARA). HUMARA was found to be amplified with or without previous digestion by the methylation-sensitive restriction endonuclease Hpa II. Five cases were informative. All 10 AAHs and 7 BACs obtained from the informative cases showed monoclonality, whereas the control cells showed polyclonality. Three different AAH lesions in a single case showed both possible patterns of monoclonality. BAC and contiguous AAH showed identical monoclonality in two cases. Two lesions of bronchiolar metaplasia, which was considered reactive, were polyclonal. Our results demonstrated the monoclonal nature of AAH, and this finding suggests that AAH is a precursor of BAC or a preneoplastic condition.
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PMID:Monoclonality of atypical adenomatous hyperplasia of the lung. 991 39

Adenosquamous carcinoma of the lung is a subset of pulmonary carcinomas, and comprises less than 4% of lung carcinomas. Its histogenesis remains unclear. The clonality of adenosquamous carcinoma from four female patients was analyzed to determine whether the clonality between the squamous cell and adenocarcinomatous components coincides. Each lesion was precisely microdissected from methanol-fixed sections. Adjacent normal lung tissue was collected as a normal control. DNA was extracted for clonal analysis based on an X-chromosome-linked polymorphic marker, the human androgen receptor gene (HUMARA). HUMARA was found to be amplified with or without previous digestion by the methylation-sensitive restriction endonuclease HpaII. All four cases were informative. Squamous cell and adenocarcinomatous components showed identical monoclonal patterns in all four patients. In one case, only the squamous cell carcinomatous component showed loss of heterozygosity of the HUMARA locus. The results suggest that the squamous cell and adenocarcinomatous components originate from the same cell.
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PMID:Clonal analysis of adenosquamous carcinoma of the lung. 1062 36

The present investigation describes the construction of a genetically engineered single chain antibody (scFv) against the rat transferrin receptor (OX26), and demonstrates that this scFv antibody can be fully processed and expressed as a soluble secreted molecule in the methylotrophic yeast Pichia pastoris. Restriction endonuclease sites located at both 5'- and 3'-flanking regions of OX26 coding region in the prokaryote pOPE-OX26 vector were engineered to incorporate yeast compatible restriction endonuclease sites (i.e. EcoRI and SmaI or AvrII). The modified OX26 cDNA was subcloned into the Pichia expression vectors pPIC9 and pHIL-S1. An OX26 scFv high producer clone [GS115 His+ Mut+ (pPIC-OX26 SacI)] was isolated and used for large-scale production and characterization. Because the engineered scFv contains both a c-myc tag and a (His)5 tail, the OX26 scFv was purified to homogeneity by immobilized metal affinity chromatography. The identity of the OX26 scFv was confirmed by Western blot analyses with both anti c-myc and anti poly-His antibodies. Minor immunoreactive bands corresponding to hyperglycosylated and partially processed alpha-factor leader prosequence were also detected in the purified OX26 scFv, and these contaminants were markedly reduced when the expression of the OX26 scFv was performed in minimal methanol medium buffered with phosphate at pH = 7. The present investigation suggests that this expression system may be useful for the production of anti-receptor single chain antibodies that can be used as brain drug delivery vectors.
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PMID:Cloning and expression in Pichia pastoris of a genetically engineered single chain antibody against the rat transferrin receptor. 1132 66

The pachytene bivalents with high-resolution multiple G bands of zebrafish were obtained after the treatment with alkaline hypotonic solution and high concentration of chloroform fixative solution. When comparing six group chromosomes from different pachytene specimens, the characteristic and the number of bands were well matched. In order to systematize this technique and get stable result, we summarize the preparation procedure of the zebrafish bivalents. The 6-month-old to one-year-old zebrafish whose spermary appears ivory-white and opaque, is good material. The whole testis should be treated with hypotonic solution for 1.5 approximately 2 h at room temperature. Then, the testes were fixed for 20 min in high chloroform fixative solution (chloroform: methanol: acetic acid, 3: 6:1), and fixed in Carnoy's solution (methanol: acetic acid, 3:1) for two times. In addition, with the treatment of restriction endonuclease Alu I directed in situ nick translation, we successfully obtained well-resolved restrictive endonuclease banding of zebrafish bivalents, which was considered as G-like band patterns. The aging of the specimen is also important factor, should let them dry at room temperature for one week. The application of these methods in cytogenetics research of zebrafish and other fish can be expected. Construction of the steady technique system to prepare high resolution banding bivalents and idiogram of zebrafish is the basement to found stable and accurate framework for physical map.
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PMID:[Advanced methods of preparing pachytene bivalents and high resolution multiple bands of zebrafish (Danio rerio)]. 1547 7

Cysticercosis is caused by the metacestode form of Taenia solium-Cysticercus cellulosae and it causes great economic losses and threatens the people's health. There are some problems on how to control cysticercosis, in order to resolve the key problem that the native antigen to diagnose and prevent cysticercosis is very limited and is not satisfied, Pichia pastoris Expression System was used to express recombinant P2 protein. The interested P2 gene was got by digesting the pGEM - P2 vector using restriction endonuclease, then it was inserted into the secretory pPIC9K Pichia pastoris expression vector and transformed into E. coli. Positive recombinant plasmids were selected sequenced and named pPIC9K-P2 and it was linearized by Sal I and Bgl II, then the linear DNA transfored into Pichia pastoris GS115 by electroporation. The recombinant expression vector pPIC9K - P2 integrated into GS115 via homologous recombination between the transforming DNA and regions of homology within the genome. The transformants were screened for multicopy recombinants using G418 and were distinguished for Mut phenotypes by MD and MM. Two different phenotypes were generated-HIS+ MUT+ (Methanol utilization plus) and HIS+ MUT(S) (Methanol utilization slow). PCR analysis of the multicopy recombinants indicated that the P2 gene was integrated within the genome of pichia Pastoris. The multicopy recombinants were named GS115/pPIC9K - P2HIS+ MUT+ and GS115/pPIC9K-P2HIS+ MUT(S), both HIS+ MUT+ and HIS+ MUT(S) were induced with methanol. The results of SDS-PAGE and Western blot demonstrated that the culture supernatant of the induced Pichia pastoris contained P2 protein which was accumulated up to 33 % of total proteins in the culture supernant and its molecular weight is 12.6kD. The results of the clinical study indicated that the expression P2 protein could be used to diagnose human cysticercosis and swine cysticercosis as diagnosis antigen.
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PMID:[Expression of phosphoprotein P2 of Cysticercus cellulosae in Pichia pastoris and its application]. 1596 58

HC-pro gene of Watermelon Mosaic virus was obtained by RT-PCR was 1371bp in length. It was cloned into pPI(9K, then the eucaryotic recombinant expression plasmid pPIC9K-WHC was constructed. After being linearized with restriction endonuclease Sal I , the recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation. The high copy transformants with Mut+ /His+ phenotype were selected by RT-PCR and screening on G418, MD and MM medium. Induced by methanol for 5 days, the culture supernatant was analyzed by SDS-PAGE, the results showed that a specific protein with a molecular weight of about 66 kD was expressed. Western blot analysis proved that the expression protein could specifically bind to HC-Pro polyclonal antibody. Far western blot analysis proved that the expression protein could bind to coat protein, given support to "bridge" hypothesis that HC-Pro help aphid transmission of non-persistent viruses.
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PMID:[Research on secretion expression in Pichia pastoris and function of the HC-pro gene of watermelon mosaic virus]. 1825 44


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