Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Nucleotide sequence analysis of the Escherichia coli chromosomal DNA inserted in the plasmid pLC33-5 of the Clarke and Carbon library [Clarke & Carbon (1976) Cell 9, 91-99] revealed the existence of the gene, fda, encoding the Class II (metal-dependent) fructose 1,6-bisphosphate aldolase of E. coli. The primary structure of the polypeptide chain inferred from the DNA sequence of the fda gene comprises 359 amino acids, including the initiating methionine residue, from which an Mr of 39,146 could be calculated. This value is in good agreement with that of 40,000 estimated from sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified dimeric enzyme. The amino acid sequence of the Class II aldolase from E. coli showed no homology with the known amino acid sequences of Class I (imine-forming) fructose 1,6-bisphosphate aldolases from a wide variety of sources. On the other hand, there was obvious homology with the N-terminal sequence of 40 residues already established for the Class II fructose 1,6-bisphosphate aldolase of Saccharomyces cerevisiae. These Class II aldolases, one from a prokaryote and one from a eukaryote, evidently are structurally and evolutionarily related. A 1029 bp-fragment of DNA incorporating the fda gene was excised from plasmid pLC33-5 by digestion with restriction endonuclease HaeIII and subcloned into the expression plasmid pKK223-3, where the gene came under the control of the tac promoter. When grown in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside, E. coli JM101 cells transformed with this recombinant expression plasmid generated the Class II fructose 1,6-bisphosphate aldolase as approx. 70% of their soluble protein. This unusually high expression of an E. coli gene should greatly facilitate purification of the enzyme for any future structural or mechanistic studies.
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PMID:Cloning, sequence analysis and over-expression of the gene for the class II fructose 1,6-bisphosphate aldolase of Escherichia coli. 264 77

A ColE1 plasmid from the Clarke and Carbon collection [Clarke, L. & Carbon, J. (1976) Cell 9, 91-99] that contains a 14.4-kilobase Escherichia coli DNA insert complements the rnc-105 mutation, which destroys the activity of the RNA-processing enzyme RNase III. This insert and smaller restriction endonuclease fragments derived from it were cloned into the plasmid pBR329. A number of these recombinant plasmids complemented the rnc-105 mutation in a recA genetic background. The smallest cloned fragment that compensated for the rnc-105 mutation was 1.3 kilobase in size. This fragment led to the synthesis of two polypeptides. One of these polypeptides was 25,300 daltons and corresponded in size to the subunit of RNase III. Fragments cloned in opposite orientations led to synthesis of RNase III, indicating that the cloned fragments contained an endogenous promoter. Extracts of an rnc+ E. coli strain containing an rnc+ plasmid had at least 10 times more RNase III activity than did an analogous strain containing the pBR329 plasmid.
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PMID:Molecular cloning of the gene for the RNA-processing enzyme RNase III of Escherichia coli. 298 17

Based on the rationale that Escherichia coli cells harboring plasmids containing the pnt gene would contain elevated levels of enzyme, we have isolated three clones bearing the transhydrogenase gene from the Clarke and Carbon colony bank. The three plasmids were subjected to restriction endonuclease analysis. A 10.4-kilobase restriction fragment which overlapped all three plasmids was cloned into the PstI site of plasmid pUC13. Examination of several deletion derivatives of the resulting plasmid and subsequent treatment with exonuclease BAL 31 revealed that enhanced transhydrogenase expression was localized within a 3.05-kilobase segment. This segment was located at 35.4 min in the E. coli genome. Plasmid pDC21 conferred on its host 70-fold overproduction of transhydrogenase. The protein products of plasmids carrying the pnt gene were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes from cells containing the plasmids. Two polypeptides of molecular weights 50,000 and 47,000 were coded by the 3.05-kilobase fragment of pDC11. Both polypeptides were required for expression of transhydrogenase activity.
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PMID:Cloning and expression of the transhydrogenase gene of Escherichia coli. 388 96

Si+ hybrid ColE1 plasmids of the Clarke-Carbon collection (Clarke, C., and Carbon, J. (1976) Cell 9, 91-99) which eliminate the sn-glycerol 3-phosphate growth requirement of a mutant of Escherichia coli with a Km defect in sn-glycerol-3-phosphate acyltransferase (plsB) were identified. Marked overproduction of a plasmid-encoded sn-glycerol-3-phosphate acyltransferase with a wild type Km in a host plsB- background indicates that the hybrid plasmids carry a structural gene for this enzyme. In addition, all of these plasmids suppress the phenotype of a mutation in a second locus involved in phospholipid biosynthesis, dgk (diglyceride kinase), and one of them also bears the dnaB structural gene. Diglyceride kinase activity is also overproduced in these strains. The linkage of plsB, dgk and dnaB loci was confirmed by transduction analysis which demonstrated the clockwise gene order malB, dnaB, dgk, plsB, and uvrA near Minute 91 on the E. coli linkage map. This is in contrast to the previously reported co-transduction of plsB with dctA near Minute 78 (Cronan, J. E., Jr., and Bell, R. M. (1974) J. Bacteriol., 120, 227-233). Recloning of restriction endonuclease fragments and in vitro mutagenesis have localized the dgk, and plsB loci to a 2.2-megadalton DNA segment, and have demonstrated that diglyceride kinase and sn-glycerol-3-phosphate acyltransferase activities reside in separate polypeptides. Availability of these clones and mutationally altered derivatives has allowed the identification of a single polypeptide (Mr = 83,000) corresponding to the sn-glycerol-3-phosphate acyltransferase and purification of this membrane-bound enzyme to near homogeneity (Larson, T. J., Lightner, V. A., Green, P. R., Modrich, P., and Bell, R. M. (1980) J. Biol. Chem. 255, 9421-9426). The size of the plsB polypeptide indicates that a major fraction of the DNA segment to which this gene has been localized is involved in coding for the sn-glycerol-3-phosphate acyltransferase.
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PMID:Membrane phospholipid synthesis in Escherichia coli. Cloning of a structural gene (plsB) of the sn-glycerol-3-phosphate acyl/transferase. 625 Oct 87

Plasmids containing the chromosome region of Escherichia coli encoding phoM, whose product is a positive regulator of alkaline phosphatase expression, were isolated from the Clarke and Carbon plasmid bank. A 9.9-kilobase EcoRI fragment of plasmid pLC17-39 (subcloned into pBR322) was able to complement both phoM and thrB mutations. Restriction endonuclease analysis and in vitro mutagenesis of the hybird plasmids enabled the localization of the phoM gene locus to 3 kilobases of the cloned chromosomal fragment. The phoM gene product was identified, with maxicell techniques, as a protein with an approximate molecular weight of 55,000. A phoM-lacZ protein fusion was constructed by using a plasmid carrying the phoM gene and a derivative of phage lambda, lambda plac Mu2. Restriction endonuclease analysis of the plasmid carrying the fusion indicated that phoM is transcribed in a clockwise direction on the circular E. coli chromosome. Analysis of strains bearing the fusion on a multiple-copy plasmid or integrated at the lambda attachment site of the chromosome indicated that the synthesis of the phoM gene product was unaffected by phosphate limitation of growth. The expression of the phoM gene was studied in strains with mutations in genes encoding effectors of the pho regulon. A threefold increase in phoM expression was seen in a phoU strain in comparison with the wild-type strain.
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PMID:Identification of the phoM gene product and its regulation in Escherichia coli K-12. 633 29

A transformant Escherichia coli colony bank [Clarke, L. & Carbon, J. (1976) Cell 9, 91-99] has been screened for hybrid ColE1 plasmids carrying the genes for D-mannitol utilization. Two of the plasmids, pLC11-7 and pLC15-48, were shown to contain the mannitol operon, which includes the structural genes for the mannitol-specific enzyme II of the phosphotransferase system and mannitol-1-phosphate dehydrogenase. One E. coli strain harboring plasmid pLC15-48 overproduced mannitol-1-phosphate dehydrogenase activity 4- to 5-fold. However, there was no corresponding increase in mannitol enzyme II activity. Plasmid pLC15-48 was shown to direct the synthesis of two polypeptides in E. coli minicells in the presence of cyclic AMP and mannitol. The larger (Mr = 60,000) was membrane bound and was specifically precipitated by antibody directed against purified mannitol-specific enzyme II. The smaller (Mr = 40,000) was soluble and had an electrophoretic mobility indistinguishable from that of the major component in a partially purified mannitol-1-phosphate dehydrogenase preparation. These data are consistent with previous genetic studies of the mannitol locus and confirm an independent conclusion [Jacobson, G. R., Lee, C. A. & Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 249-252] that mannitol enzyme II consists of a single type of polypeptide chain that has a Mr of 60,000. The plasmid pLC15-48 DNA was characterized by mapping of restriction endonuclease cleavage sites.
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PMID:Plasmid-directed synthesis of enzymes required for D-mannitol transport and utilization in Escherichia coli. 680 48

Three genes (mglA, mglB, and mglC) required for active transport of substrate by the methylgalactoside permease were identified in a hybrid ColE1-DNA plasmid isolated from a clone (pLC3-14) of the Clarke-Carbon bank of Escherichia coli genes. A 4.6-kilobase DNA fragment obtained from pLC3-14 was cloned into the plasmid vector pBR322. The presence of the three mgl genes in the resultant plasmid, pMG3, was verified by genetic complementation and biochemical analysis of mgl mutants transformed with pMG3 DNA. Derivatives of pMG3 containing deletions in each mgl gene were constructed; restriction endonuclease mapping and functional analysis of these plasmids allowed us to physically locate the mgl genes within the inserted plasmid DNA and also to identify a heretofore unknown protein component of the transport system. Expression of these plasmids in vivo resulted in the specific synthesis of three major proteins of apparent molecular weight of 19,000, 36,000, and 52,000. The 36,000-dalton protein is the galactose-binding protein previously identified as the mglB product. The 19,000-dalton protein maybe the product of mglD, a regulatory gene mapping outside of the mgl gene cluster. The 52,000-dalton protein is a new permease component which we have identified here as the mglA product based on the observation that pMG6, a plasmid with a 0.6-kilobase mglA deletion, failed to encode for this protein but produced a truncated polypeptide showing a reduction in molecular weight comparable to the extent of the deletion. In bacteria bearing an mglA+, B-, C+ plasmid (Pmg4), the 52,000-dalton protein is located to a large extent (73%) in the membrane fraction.
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PMID:Identification of the mglA gene product in the beta-methylgalactoside transport system of Escherichia coli using plasmid DNA deletions generated in vitro. 680 87

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