Rat Insulin Genes: Construction of Plasmids Containing the Coding Sequences

Ullrich, Axel; Shine, John; Chirgwin, John M.; Pictet, Raymond; Tischer, Edmund; Rutter, William J.; Goodman, Howard M.

doi:10.1126/science.325648

Cited by 1,231 publications

(404 citation statements)

References 31 publications

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“…Blots were prehybridized and hybridized using ExpressHyb (Clontech) following the manufacturer's protocol. To analyse ADAM28 expression, RNA was isolated from mouse lung and epididymis [34]. To look for alternative transcripts [35], duplicate RNA samples (25 µg for each tissue) were examined by Northern blotting using a probe corresponding to bases 1246-1613 (encoding part of the metalloprotease-and disintegrin-like domains) or bases 2065-2410 (the region encoding the transmembrane and cytoplasmic domains).…”

Section: Northern Blot Analysismentioning

confidence: 99%

Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28

Howard

Maciewicz

Blobel

2000

Biochemical Journal

101

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The metalloprotease disintegrins are a family of membraneanchored glycoproteins with diverse functions in fertilization, myoblast fusion, neurogenesis and protein ectodomain shedding. Here we report a cDNA sequence, encoding a metalloprotease disintegrin, termed ADAM28 (' a disintegrin and metalloprotease 28 '), which was cloned from mouse lung. From protein sequence comparisons, ADAM28 is more closely related to snake venom metalloproteases (SVMPs) than to other ADAMs, and hence may cleave similar substrates to SVMPs, perhaps including components of the extracellular matrix. Northern blot analysis of selected mouse tissues revealed that ADAM28 is expressed highly and in alternatively spliced forms in the epididymis, suggesting a possible role in sperm maturation, and at lower levels in lung. The intracellular maturation of ADAM28 expressed in COS-7 cells resembles that of other ADAMs, in that ADAM28 is made as a precursor and processed to a mature form in a late Golgi

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Section: Northern Blot Analysismentioning

confidence: 99%

Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28

Howard

Maciewicz

Blobel

2000

Biochemical Journal

101

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“…For the purification of total RNA a modification of the method described by Glisin, Crkvenjakov & Byus (1974) and Ullrich et al (1977) was used.…”

Section: Purification Of Nucleic Acidsmentioning

confidence: 99%

Echinococcus multilocularis: molecular and immunochemical characterization of diagnostic antigen II/3-10

Felleisen

Gottstein

1993

Parasitology

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SUMMARYA recombinant Echinococcus multilocularis antigen (II/3-10), which had previously been shown to exhibit immunodiagnostic characteristics highly specific for human alveolar echinococcosis, and the corresponding native parasite antigen, were further characterized with immunochemical and molecular biological methods. Immunoblot analysis using a polyclonal antiserum raised in rabbits against the recombinant protein, and subsequent Northern hybridization analysis, revealed that the native antigen was expressed by E. multilocularis at the adult as well as at the metacestode stage. In metacestodes, the antigen was shown by using indirect immunofluorescence and the same antiserum to be localized within the germinal layer and membrane structures of developing protoscolices. Electrophoretic analyses revealed remarkable differences in the apparent molecular weight of the antigen under reducing and non-reducing conditions. In further immunoblot analyses, anti-II/3-10 antibodies identified the corresponding epitopes on bands with identical M T in all E. multilocularis isolates investigated (European, Asian and North American). By Southern hybridization analyses of the respective gene, phylogenetically related sequences were shown to be present in other helminth species such as E. granulosus and several Taenia spp. In the same respect, immunoblotting revealed that anti-II/3-10 antibodies reacted with antigens of different M r from various E. granulosus isolates and some other cestode species, indicating the presence of shared and thus potentially cross-reacting epitopes. The relevance of these findings for the immunodiagnostic performance of the recombinant antigen is discussed.

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“…After treatment with T4 DNA polymerase to flush the ends, the double-stranded cDNA molecules were size-fractionated to eliminate small-sized cDNA, which was less than 400 bp, to ensure successful adaptor addition and cloning. The resulting cDNA preparation was then ligated by EcoRI adaptors and cloned with T4 DNA ligase (Promega) into a plasmid pUC18 (Sangon, China), which had been cut with EcoRI (BRL) and treated with alkaline phosphatase (Takara, Japan) [31]. Transformation to E. coli DH5a was carried out by the calcium shock method described by Lederberg and Cohen [32,38].…”

Section: Constructions Of a Niger Cbx-209 Cdna Librarymentioning

confidence: 99%

Identification, characterization of levoglucosan kinase, and cloning and expression of levoglucosan kinase cDNA from Aspergillus niger CBX-209 in Escherichia coli

Zhuang

Zhang

2002

Protein Expression and Purification

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The first enzyme responsible for assimilating levoglucosan in Aspergillus niger CBX-209 was corroborated to be levoglucosan kinase that catalyzes the transfer of a phosphate group from ATP to levoglucosan to yield a glucose 6-phosphate in the presence of magnesium ion and ATP by FAB-mass spectrometric method combined with previous observations from HPLC and enzymological experiments. Levoglucosan kinase was purified to apparent homogeneity by using a combination of seven purification steps. SDS-PAGE revealed a single protein band of 56 KDa. It is a monomeric enzyme and maximal enzyme activity was measured at pH 9.3 and 30°C. This kinase is stable below 20°C at a quite broad pHs ranging from 6 to 10 and levoglucosan could protect the enzyme from thermal inactivation. Exclusive substrate specificity for levoglucosan suggested that not only the structure of the intramolecular glucosidic linkage but also the configuration of the pyranose frame would be specific for recognition by levoglucosan kinase. The K m values of this enzyme were 71.2 mM for levoglucosan and 0.25 mM for ATP, determined by double reciprocal plottings and ADP inhibited on the enzyme activity competitively with a Ki value of 0.20 mM. A cDNA library from A. niger was constructed in Escherichia coli DH5a. The library was screened for levoglucosan kinase gene on NCE selective medium and three positive recombinants were selected after a five day culture. Detection of activities of levoglucosan kinase in the cell extracts indicated that levoglucosan kinase gene (lgk) was expressed by the recombinant strain of E. coli DH5a.

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Rat Insulin Genes: Construction of Plasmids Containing the Coding Sequences

Cited by 1,231 publications

References 31 publications

Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28

Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28

Echinococcus multilocularis: molecular and immunochemical characterization of diagnostic antigen II/3-10

Identification, characterization of levoglucosan kinase, and cloning and expression of levoglucosan kinase cDNA from Aspergillus niger CBX-209 in Escherichia coli

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