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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M. tuberculosis complex. The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis. As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size. Southern blot analysis showed that both of them hybridized with the genomic DNA from M. tuberculosis and M. bovis but not with the DNA from other mycobacterial species. To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34. The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes. Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA. Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes. Restriction maps of some clones isolated were determined.
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PMID:Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes. 168 20

Adenovirus DNA polymerase (AdPol) contains three clusters of basic amino acids within the N-terminal 48 amino acids: RARR, which begins at amino acid 8, RRRVR, which begins at amino acid 25, and RARRRR, which begins at amino acid 41. These clusters are designated BS I, BS II, and BS III, respectively. (The amino acid codes are: R, arginine; A, alanine; V, valine.) Mutational analysis of these noncontiguous clusters showed that AdPol contains a novel organization of bipartite nuclear localization signals (NLS) that interact differentially to serve in the nuclear targeting of AdPol or of chimeric proteins in which AdPol is linked to Escherichia coli beta-galactosidase (beta-gal). The region containing BS I and BS II functioned interdependently as an NLS for the nuclear targeting of AdPol, for which BS III was dispensible. However, the region containing BS II and BS III constituted a second and more efficient bipartite NLS for the nuclear targeting of the AdPol-E. coli beta-gal fusion protein. Moreover, deletion or limited insertion of amino acids in the spacer region between BS II and BS III did not affect their nuclear targeting function for these fusion proteins. Chou-Fasman predictive analysis of protein secondary structure in the vicinity of the bipartite NLS sequences supports a model in which protein conformation in the spacer region may play an important role in bringing these clusters of basic amino acids into close proximity, allowing them to function as nuclear targeting signals for this class of nuclear proteins.
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PMID:Three basic regions in adenovirus DNA polymerase interact differentially depending on the protein context to function as bipartite nuclear localization signals. 177 81

We purified a mouse DNA repair enzyme having apurinic/apyrimidinic endonuclease, DNA 3'-phosphatase, 3'-5'-exonuclease and DNA 3' repair diesterase activities, and designated the enzyme as APEX nuclease. A cDNA clone for the enzyme was isolated from a mouse spleen cDNA library using probes of degenerate oligonucleotides deduced from the N-terminal amino acid sequence of the enzyme. The complete nucleotide sequence of the cDNA (1.3 kilobases) was determined. Northern hybridization using this cDNA showed that the size of its mRNA is about 1.5 kilobases. The complete amino acid sequence for the enzyme predicted from the nucleotide sequence of the cDNA (APEX nuclease cDNA) indicates that the enzyme consists of 316 amino acids with a calculated molecular weight of 35,400. The predicted sequence contains the partial amino acid sequences determined by a protein sequencer from the purified enzyme. The coding sequence of APEX nuclease was cloned into pUC18 SmaI and HindIII sites in the control frame of the lacZ promoter. The construct was introduced into BW2001 (xth-11, nfo-2) strain cells of Escherichia coli. The transformed cells expressed a 36.4-kDa polypeptide (the 316 amino acid sequence of APEX nuclease headed by the N-terminal decapeptide of beta-galactosidase) and were less sensitive to methyl methanesulfonate than the parent cells. The fusion product showed priming activity for DNA polymerase on bleomycin-damaged DNA and acid-depurinated DNA. The deduced amino acid sequence of mouse APEX nuclease exhibits a significant homology to those of exonuclease III of E. coli and ExoA protein of Streptococcus pneumoniae and an intensive homology with that of bovine AP endonuclease 1.
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PMID:cDNA and deduced amino acid sequence of a mouse DNA repair enzyme (APEX nuclease) with significant homology to Escherichia coli exonuclease III. 193 31

The Schizosaccharomyces pombe POL3 gene was isolated by sequence homology with a region of the Saccharomyces cerevisiae POL3 gene, the only gene sequenced to date encoding the catalytic subunit of eukaryotic DNA polymerase delta. The fission yeast POL3 gene contains a 52 base-pair (bp) intron and encodes a 3600 bp transcript the 5'-end of which is located 32 bp upstream from the initiation codon. The polypeptides predicted from budding and fission yeast POL3 genes share 52% of conserved amino acid residues and have a 60% identical central region. This structural conservation of the catalytic subunit of DNA polymerases delta is probably related to functional constraints. A portion of the most conserved region was used to raise antibodies against an S. pombe polymerase delta/beta-galactosidase fusion protein expressed in Escherichia coli. The purified antibodies recognized a 123,000 Da protein in S. pombe wild-type cell extracts and inhibited an aphidicolin-sensitive DNA polymerase activity that was distinct from DNA polymerase alpha. The antibodies also detected a 140,000 Da protein in extracts from different proliferating mammalian cells, indicating that the catalytic subunits of DNA polymerase delta are highly conserved between yeast and higher eukaryotes.
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PMID:Characterization of the POL3 gene product from Schizosaccharomyces pombe indicates inter-species conservation of the catalytic subunit of DNA polymerase delta. 196 Jul 23

Sequence studies of the adenovirus 2 genome have revealed the presence of a large open reading frame (ORF) from 22.9 to 14.2 map units that is believed to encode most of the adenovirus DNA polymerase (Ad Pol). An 838-base-pair fragment (19.6-17.3 map units) containing approximately 25% of this ORF has been cloned and expressed in a beta-galactosidase-chloramphenicol acetyltransferase (lacZ-CAT) expression vector under the control of the trp-lac hybrid promoter. This recombinant vector directed the synthesis of a 58-kDa lacZ-Ad Pol-CAT fusion protein that has CAT activity. This fusion protein was easily purified by affinity chromatography in which chloramphenicol, the substrate for CAT, was covalently bound to a matrix. Antisera were prepared against the purified 58-kDa lacZ-Ad Pol-CAT fusion protein and were found to react specifically with the 140-kDa Ad Pol by ELISA and immunoblot analysis. In addition, these antisera recognized 120- and 29-kDa polypeptides in immunoblot analysis of partially purified terminal protein precursor (pTP)-Ad Pol complex. The exact nature of the 120- and 29-kDa polypeptides is not known, but they may be breakdown products of Ad Pol. Although the lacZ-Ad Pol-CAT fusion protein is not active in any of the Ad Pol enzymatic reactions, antibody against the prokaryotic fusion protein should be useful for screening bacteria harboring plasmids that have been constructed to express the entire Ad Pol ORF.
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PMID:The 140-kDa adenovirus DNA polymerase is recognized by antibodies to Escherichia coli-synthesized determinants predicted from an open reading frame on the adenovirus genome. 258 Dec 53

A 1.4 kb region downstream of the DNA polymerase gene of Autographa californica nuclear polyhedrosis virus was sequenced. Two open reading frames (ORFs) were identified of 927 and 474 bases in length. The 927 base ORF encodes a 34.8K protein as determined by in vitro translation of both hybrid-selected RNA and RNA synthesized in vitro from a 927 base ORF template. The predicted amino acid sequence of the 34.8K polypeptide (p34.8) reveals a hydrophobic N terminus, two potential N-glycosylation sites, and potential sites for phosphorylation by casein kinase I and protein kinase C. The p34.8 gene has a strong codon usage bias which is strikingly different from that of the polyhedrin gene. The two 5' ends of the 927 base ORF transcripts initiate from an ATAAG sequence and a GTAAG sequence 11 and 87 bases upstream of the ATG codon respectively. A short upstream reading frame is present in the leader sequence of the longer RNA. The transcripts have multiple 3' ends; the most proximal endpoint correlates with a polyadenylation signal overlapping the translational termination codon of the 927 base ORF. Transcripts of the latter were not observed early in the infection cycle but appeared 6 h after infection and were maximally expressed at 12 to 24 h post-infection. The late nature of these transcripts was confirmed by their sensitivity to aphidicolin and cycloheximide, inhibitors of DNA replication and protein synthesis respectively. Attempts to construct viral mutants carrying a deletion of the p34.8 gene and fusion with the beta-galactosidase gene suggest that the former gene is essential for viral replication.
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PMID:Sequence, transcription and translation of a late gene of the Autographa californica nuclear polyhedrosis virus encoding a 34.8K polypeptide. 267 27

A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.
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PMID:Expression of active rat DNA polymerase beta in Escherichia coli. 304 24

A calf thymus cDNA expression library was constructed in the EcoRI site of lambda gt11 and probed with an antibody raised against calf thymus DNA polymerase alpha. Three classes of antibody-reactive clones were isolated. The largest class carried a 1.9 kilobase calf cDNA insert and expressed a 165-175 kilodalton beta-galactosidase:calf fusion protein which displayed DNA polymerase activity. The characteristic responses of the polymerase activity to alpha-specific inhibitors and antibodies identified the 1.9 kilobase cDNA as a sequence specifically derived from the structural gene encoding the pol alpha catalytic core.
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PMID:Cloning and expression of a cDNA encoding a catalytically active fragment of calf thymus DNA polymerase alpha. 309 17

We have localized a cDNA fragment that codes for human DNA polymerase-beta. Using somatic cell and in situ hybridization techniques, this cDNA was cloned by screening a human KM-3 cell cDNA library in lambda gt 11 for expression of fused beta-galactosidase-human DNA polymerase-beta proteins. We have mapped this human polymerase-beta gene to the short arm of chromosome 8 in the subregion 8p11----p12.
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PMID:Chromosome sublocalization of a cDNA for human DNA polymerase-beta to 8p11----p12. 337 50

A segment from the pre-s region of the woodchuck hepatitis virus (WHV) was inserted into an open reading frame vector allowing for the expression in Escherichia coli of viral determinants as part of a fusion protein. The bacterially synthesized fusion molecule contained eight amino acids from beta-galactosidase (beta-gal) at the N terminus, followed by 89 pre-s-encoded amino acids and 219 amino acids of chloramphenicol acetyltransferase (CAT) at the C terminus (beta-gal:pre-s:CAT). This tribrid protein was used to generate antiserum which had a significant titer to the viral portion of the fusion polypeptide. Anti-beta-gal:pre-s:CAT was used in Western blot analysis to identify viral proteins containing pre-s-encoded determinants. Antiserum to the tribrid molecule recognized four WHV polypeptides with molecular masses of 33, 36, 45, and 47 kilodaltons, each of which was also recognized by a monoclonal antibody to WHV surface antigen. Using the same anti-tribrid serum, we also identified analogous polypeptides from ground squirrel hepatitis virus. The antiserum was also used to immunoprecipitate virus particles containing endogenous DNA polymerase activity, indicating that pre-s determinants are found on the surface of mature virions. Based on previous computer studies and the location of pre-s-encoded molecules on the surface of virus particles, a role in hepadnavirus host cell entry is suggested for these polypeptides.
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PMID:Identification and localization of pre-s-encoded polypeptides from woodchuck and ground squirrel hepatitis viruses. 394 37


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