Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mechanism-based enzyme inactivator, alanine racemase, S-adenosylhomocysteine hydrolase, D-amino acid aminotransferase, gamma-aminobutyric acid aminotransferase, arginine decarboxylase, aromatase, L-aromatic amino acid decarboxylase, dihydrofolate reductase, dihydroorotate dehydrogenase DNA polymerase I, dopamine beta-hydroxylase, histidine decarboxylase, beta-lactamase, monoamine oxidase, ornithine decarboxylase, serine proteases, testosterone 5 alpha-reductase, thymidylate synthetase, xanthine oxidase.
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PMID:The potential use of mechanism-based enzyme inactivators in medicine. 306 67

A plasmid that consists of an 812-base-pair segment containing the replication origin of plasmid ColE1 and of a 1240-base-pair segment containing a beta-lactamase gene has been constructed. The plasmid DNA has three principal sites where transcription is initiated in vitro. One is located in the ColE1 segment 555 nucleotides upstream from the origin. Most transcription from this site extends past the origin; some of the transcripts form hybrids spontaneously with the template at their 3' portions. Cleavage of these transcripts by RNase H generates 3' termini at the origin region. When DNA polymerase I is included in the reaction along with RNA polymerase and RNase H, dAMP or dCMP is added directly onto the cleaved RNA molecules, most of which retain the intact 5' terminus. The addition of a deoxyribonucleotide to the cleaved RNA can be regarded as the first step of ColE1 DNA synthesis. Once it has served as a primer, the RNA is eliminated from the product by RNase H.
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PMID:Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. 615 50

Short single-stranded gaps in circular DNA molecules can be generated enzymatically, often at predetermined sites. These can serve as targets for in vitro mutagenesis procedures that result in alterations in nucleotide sequence within or very near the gap. Deamination of unpaired cytosine residues with sodium bisulfite has been used to induce mutations in the BglI restriction site of SV40 DNA and within defined regions of the beta-lactamase gene on pBR322. A new method of induction of mutations at gaps, called "gap misrepair," has been developed; it was used to cause changes at the HindIII and C1aI restriction sites on pBR322 DNA. Gap misrepair reactions using DNA polymerase I of Micrococcus luteus in the presence of T4 DNA ligase and three of the four deoxynucleoside triphosphates yielded all three possible substitutions for adenine and cytosine residues in the DNA.
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PMID:Single-stranded gaps as localized targets for in vitro mutagenesis. 628 91

We describe a procedure by which the codon (AGC) for the active-site serine-70 of pBR322 beta-lactamase (penicillinase, penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) is altered to that for cysteine (TGC). The pertinent nucleotide bases, A-G-C-A, positions 410-413, of pBR322 are excised by treating a limited HgiAI digest of pBR322 with the 3' leads to 5' exonuclease of T4 DNA polymerase. The new sequence, T-G-C-A, is inserted in two steps. First, the Kpn I molecular linker d(T-G-G-T-A-C-C-A) is ligated into the gap described above. The internal sequence G-T-A-C is then excised enzymatically with Kpn I and T4 DNA polymerase and the molecule is recircularized. This mutant gene, which codes for a thiol-beta-lactamase, confers on Escherichia coli K-12 hosts an ampicillin resistance that is reduced compared with that given by pBR322 yet is greater than that of E. coli lacking any intact beta-lactamase gene. Cell-free extracts of E. coli strains hosting the thiol-beta-lactamase gene possess a p-chloromercuribenzoate-sensitive beta-lactamase activity.
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PMID:Thiol-beta-lactamase: replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue. 681 41

Here, we describe assembly PCR as a method for the synthesis of long DNA sequences from large numbers of oligodeoxyribonucleotides (oligos). The method, which is derived from DNA shuffling [Stemmer, Nature 370 (1994a) 389-391], does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process. A 1.1-kb fragment containing the TEM-1 beta-lactamase-encoding gene (bla) was assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (nt) in length. The synthetic gene was PCR amplified and cloned in a vector containing the tetracycline-resistance gene (TcR) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the TcR colonies were ApR, making this approach a general method for the rapid and cost-effective synthesis of any gene. We tested the range of assembly PCR by synthesizing, in a single reaction vessel containing 134 oligos, a high-molecular-mass multimeric form of a 2.7-kb plasmid containing the bla gene, the alpha-fragment of the lacZ gene and the pUC origin of replication. Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli, yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies.
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PMID:Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. 759 Mar 20

The beta-lactamase-producing Asia-type plasmid pJD4 of Neisseria gonorrhoeae is a 7.4-kb, broad-host-range plasmid. It is part of a family of plasmids which are structurally related yet vary in size, found in both N. gonorrhoeae and Haemophilus ducreyi. Branch-point analysis by electron microscopy indicates that pJD4 carries three clustered but distinguishable origins of replication, which we named ori1, ori2, and ori3. Although pJD4 belongs to incompatibility (Inc) group W, it also carries a silent IncFII determinant which is expressed when ori2 and ori3 are absent. The Africa-type plasmid pJD5, a naturally occurring deletion derivative of pJD4, carries only ori1, belongs to the IncFII group, and, in contrast to pJD4, requires DNA polymerase I (Pol I) for replication. Plasmids constructed from pJD4 which lack ori1 but carry ori2 and ori3 do not require Pol I and are incompatible with IncW plasmids, suggesting that the ori2 or ori3 region contains the IncW determinant. We have cloned a replication initiation protein (RepB) that is necessary for ori2 and ori3 to function. This Rep protein is distinct from RepA, which is necessary for ori1. Thus, pJD4 is unique because it is the smallest plasmid characterized containing three origins of replication and two unique Rep proteins.
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PMID:Multiple origins and replication proteins influence biological properties of beta-lactamase-producing plasmids from Neisseria gonorrhoeae. 1154 7

The fidelity of DNA replication by Escherichia coli DNA polymerase I (pol I) was assessed in vivo using a reporter plasmid bearing a ColE1-type origin and an ochre codon in the beta-lactamase gene. We screened 53 single mutants within the region Val(700)-Arg(712) in the polymerase active-site motif A. Only replacement of Ile(709) yielded mutator polymerases, with substitution of Met, Asn, Phe, or Ala increasing the beta-lactamase reversion frequency 5-23-fold. Steady-state kinetic analysis of the I709F polymerase revealed reductions in apparent K(m) values for both insertion of non-complementary nucleotides and extension of mispaired primer termini. Abolishment of the 3'-5' exonuclease activity of wild-type pol I increased mutation frequency 4-fold, whereas the combination of I709F and lack of the 3'-5' exonuclease yielded a 400-fold increase. We conclude that accurate discrimination of the incoming nucleotide at the polymerase domain is more critical than exonucleolytic proofreading for the fidelity of pol I in vivo. Surprisingly, the I709F polymerase enhanced mutagenesis in chromosomal DNA, although the increase was 10-fold less than in plasmid DNA. Our findings indicate the feasibility of obtaining desired mutations by replicating a target gene at a specific locus in a plasmid under continuous selection pressure.
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PMID:In vivo mutagenesis by Escherichia coli DNA polymerase I. Ile(709) in motif A functions in base selection. 1160 76

We present a system for random mutagenesis in Escherichia coli for the evolution of targeted genes. To increase error rates of DNA polymerase I (Pol I) replication, we introduced point mutations in three structural domains that govern Pol I fidelity. Expression of error-prone Pol I in vivo results in strong mutagenesis of a target sequence encoded in a Pol I-dependent plasmid (8.1 x 10-4 mutations per bp, an 80,000-fold increase), with a preference for plasmid relative to chromosome sequence. Mutagenesis is maximal in cultures maintained at stationary phase. Mutations are evenly distributed and show a variety of base pair substitutions, predominantly transitions. Mutagenesis extends at least 3 kb beyond the 400-500 nt reportedly synthesized by Pol I. We demonstrate that our error-prone Pol I can be used to generate enzymes with distinct properties by generating TEM-1 beta-lactamase mutants able to hydrolyze a third-generation lactam antibiotic, aztreonam. Three different mutations contribute to aztreonam resistance. Two are found in the extended-spectrum beta-lactamases most frequently identified in clinical isolates, and the third (G276R) has not been previously described. Our system of targeted mutagenesis in E. coli should have an impact on enzyme-based applications in areas such as synthetic chemistry, gene therapy, and molecular biology. Given the structural conservation between polymerases, this work should also provide a reference for altering the fidelity of other polymerases.
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PMID:Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I. 1290 25

Serratia spp. are an important cause of hospital-acquired infections and outbreaks in high-risk settings. Twenty-one patients were infected or colonized over a nine-month period during 2001-2002 on a neonatal unit. Twenty-two isolates collected were examined for antibiotic susceptibility, beta-lactamase production and genotype. Random-amplified polymorphic DNA polymerase chain reaction and pulsed-field gel electrophoresis revealed that two clones were present. The first clone caused invasive clinical infection in four babies, and was subsequently replaced by a non-invasive clone that affected 14 babies. Phenotypically, the two strains also differed in their prodigiosin production; the first strain was non-pigmented whereas the second strain displayed pink-red pigmentation. Clinical features suggested a difference in their pathogenicity. No environmental source was found. The outbreak terminated following enhanced compliance with infection control measures and a change of antibiotic policy. Although S. marcescens continued to be isolated occasionally for another five months of follow-up, these were sporadic isolates with distinct molecular typing patterns.
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PMID:An outbreak of Serratia marcescens on the neonatal unit: a tale of two clones. 1705 99

Isolated of multidrug resistance Pseudomonas aeruginosa (MDRP) that the receptivity pattern of the antimicrobial suscepti respectively resembled isolated from clinical specimens (sputum) in two patients of each internal medicine ward in Kitasato University East Hospital for two days from September 18 and 20, 2004. Both of bacteria were formed small colonies of a smooth-type on dollargalluskey improvement-type BTB agar plates, and the judgment of ClassB (metallo)-beta-lactamase by biochemical properties and disk diffusion method sodium mercaoto-acetic acid (SMA) was mutually corresponding. Moreover, it was same serotype C according to the serotype, and it was confirmed that it was the same bacterial strain from the molecular epidemiology analysis by Random amplified polymorphic DNA polymerase chain reaction (Random amplified polymorphic DNA polymerase chain reaction: RAPD). From the investigation of clinical backgrounds of two patients who isolated bacterial strains, September 18, 2004. 10 : 20 a.m., and 10 : 40 a.m., other chances that can become with contact infection in this hospital, except conducted X-Ray or roentgenograph of the chest and abdomen of Portable X-ray device continuously done by one radiation technician was not seen. Because it had turned out that a radiation technician who had taken charge had been neglecting the hand washing at the time of each X-Ray or roentgenograph, it was guessed the case with nosocomial infection by contact infection occurred via specific radiation technician.
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PMID:[Nosocomial infection by multidrug-resistant Pseudomonas Aeruginosa (MDRP) presumably spread a radiation technician]. 1662 93


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