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)

A novel approach to oligonucleotide-mediated, site-directed in vitro mutagenesis is described that allows for the efficient generation of sequence modifications on double-stranded substrates without the need for subcloning into special vectors. Site-directed deletions as well as point mutations were introduced into the genes encoding human tissue plasminogen activator (tPA) and the Bacillus amyloliquefaciens alpha-amylase gene using lambda exonuclease to enzymatically degrade DNA 5' to 3' in order to generate a single-stranded template in the immediate vicinity of the oligonucleotide annealing site. The mutagenizing oligonucleotide, used both to redefine the 5' end of the molecule and to introduce base changes, was annealed to the single-stranded target sequence producing substrates for both the exonucleolytic and polymerizing activities of DNA polymerase Klenow fragment. Resolution of the resultant heteroduplex by Escherichia coli resulted in the generation of the desired deletion point mutation in the tPA sequence with an efficiency of 38% as determined by differential hybridization and 32% as determined by restriction analysis, with final verification by sequence data. As a further test of the method, two point mutations were introduced simultaneously with the desired sequence deletion into the Bacillus amyloliquefaciens alpha-amylase gene, generating a Pst I restriction site at the junction of the DNA encoding the signal peptide and the mature enzyme with an efficiency of 0.3% as determined by sequence data of hybridization-positive/Pst I-positive clones. The lambda exonuclease procedure is designed for use in situations where site-directed deletions must be introduced efficiently alone or with single or double point mutations.
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PMID:Use of lambda exonuclease for efficient oligonucleotide-mediated site-directed deletion and point mutation of double-stranded DNA. 295 1

We have developed a method useful for immunohistochemical studies by combining tissue fixation with buffered neutral formalin and polyester wax embedding. Buffered neutral formalin fixation preserves cell and tissue fine structure, and also the antigenicity of unstable enzymes. Polyester wax embedding makes possible thin serial sections of various tissues and preserves antigenicities for at least 6 months. We have demonstrated using this technique the localization of alpha-amylase in mouse salivary gland, parietal-cell specific antigen in mouse glandular stomach, and DNA polymerase alpha and beta in chick tissue.
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PMID:Polyester wax embedding and sectioning technique for immunohistochemistry. 620 37

The alpha-amylase gene (amy) of Streptomyces lividans TK24 was cloned in an amylase deficient mutant strain S. lividans M2. The cloned gene contained an open reading frame (ORF) of 2757 nucleotides (919 amino acids) coding for a protein of 100 kDa. Sequencing of the amino terminus of the extracellular alpha-amylase protein revealed the presence of a signal peptide of 33 amino acid residues. The transcriptional initiation site was mapped by the primer extension method with T4 DNA polymerase and was found to be transcribed from an unique promoter. The alpha-amylase protein produced by S. lividans was larger than those derived from other origins. It also contained the four common conserved regions characteristic of other alpha-amylase proteins.
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PMID:Cloning and characterization of an alpha-amylase gene from Streptomyces lividans. 848 50

A substantial proportion of the streptococcal species found in dental plaque biofilms are able to interact with the abundant salivary enzyme alpha-amylase. These streptococci produce proteins that specifically bind amylase. An important plaque species, Streptococcus mitis, secretes a 36-kDa amylase-binding protein into the extracellular milieu. Proteins precipitated from S. mitis NS51 cell culture supernatant by the addition of purified salivary amylase were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a membrane, and a prominent 36-kDa band was cut from the membrane and sequenced to yield the N-terminal amino acid sequence DSQAQYSNGV. Searching the S. mitis genome sequence database revealed a single open reading frame containing this sequence, and the gene was amplified by the S. mitis genomic DNA polymerase chain reaction. The coding region of this open reading frame, designated amylase-binding protein C (AbpC), was cloned into an Escherichia coli expression vector and the recombinant AbpC (rAbpC) was purified from the soluble fraction of the E. coli cell lysate. Purified AbpC was found to interact with immobilized amylase, confirming AbpC as a new streptococcal amylase-binding protein.
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PMID:Identification and characterization of amylase-binding protein C from Streptococcus mitis NS51. 2033 2

The present investigation demonstrates the potential of 2-alkylmalonic acid amphiphile as inhibitor of metalloenzymes like Taq DNA polymerase and alpha-amylase. A dose-dependent inhibition of Taq DNA polymerase was observed when a polymerase chain reaction (PCR) was performed in the presence of amphiphiles while in the case of alpha-amylase the inhibition was found to be independent of the inhibitor concentration. Control experiments revealed that both the chelating as well as the amphiphilic nature of the inhibitor was essential for enzyme inhibition. The fluorescence intensity and lifetime of alpha-amylase were also found to decrease in the presence of the amphiphiles. Steady-state fluorescence quenching studies suggested that removal of the metal ion from the enzyme leads to a decrease in the solvent accessibility of tryptophans, indicating change in the tertiary structure of the protein. It is proposed that removal of metal ion from the active sites of the enzyme by the amphiphilic compound possibly leads to disruption of the native conformation of the enzyme which is responsible for loss of its activity.
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PMID:2-Alkylmalonic acid: amphiphilic chelator and a potent inhibitor of metalloenzyme. 2066 18