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)

The yeast Kluyveromyces lactis synthesizes a beta-galactosidase (EC 3.2.1.32) which is inducible by lactose. We have isolated the gene that codes for this enzyme using recombinant DNA techniques. K. lactis DNA was partially digested with the restriction endonuclease Eco R1 and joined to Eco R1-digested pBR322 plasmid DNA using DNA ligase. ligase. A lac-mutant of Escherichia coli lacking the structural gene for beta-galactosidase was transformed with ligated DNA. Three lac+ transformants containing recombinant plasmids were selected. Two of the plasmids (pK15 and pK17) contain four Eco R1-K. lactis DNA fragments having molecular weights of 2.2, 1.4, 0.55 and 0.5 x 10(6) daltons. The other plasmid (pK16) lacks the smallest fragment. E. coli carrying any of these plasmids produce beta-galactosidase activity that has a sedimentation coefficient and immunological determinants that are nearly identical to K. lactis beta-galactosidase and distinctly different from E. coli beta-galactosidase. DNA-DNA hybridization studies show that the four Eco R1 fragments in pK15 hybridize to K. lactis but not to E. coli DNA.
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PMID:Molecular cloning and expression in E. coli of a yeast gene coding for beta-galactosidase. 10 Feb 26

Previous studies have described a partially defined system for the DNA-directed in vitro synthesis of beta-galactosidase (Kung, H.F., Redfield, B., Treadwell, B.V., Eskin, B., Spears, C., and Weissbach, H. (1977) J. Biol. Chem. 252, 6889-6894). An Ehrlich ascites extract was shown in these in vitro studies to acylate Escherichia coli tRNA with 13 amino acids, and the ascites extract was used in place of the corresponding 13 E. coli aminoacyl-tRNA synthetases. The present studies indicate that the ascites extract is supplying an additional protein factor, besides the aminoacyl-tRNA synthetases, that stimulates the DNA-directed synthesis of beta-galactosidase. The protein factor has been highly purified and may be functioning by protecting mRNA against degradation. In addition, NAD or T4 DNA ligase stimulates the synthesis of beta-galactosidase in the partially defined system.
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PMID:DNA-directed in vitro synthesis of beta-galactosidase. Purification and characterization of stimulatory factors in an ascites extract. 11 1

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

Vaccinia virus gene SalF 15R potentially encodes a polypeptide of 63 kD which shares 30% amino acid identity with S. pombe and S. cerevisiae DNA ligases. DNA ligase proteins can be identified by incubation with alpha-(32P)ATP, resulting in the formation of a covalent DNA ligase-AMP adduct, an intermediate in the enzyme reaction. A novel radio-labelled polypeptide of approximately 61 kD appears in extracts from vaccinia virus infected cells after incubation with alpha-(32P)ATP. This protein is present throughout infection and is a DNA ligase as the radioactivity is discharged in the presence of either DNA substrate or pyrophosphate. DNA ligase assays show an increase in enzyme activity in cell extracts after vaccinia virus infection. A rabbit antiserum, raised against a bacterial fusion protein of beta-galactosidase and a portion of SalF 15R, immune-precipitates polypeptides of 61 and 54 kD from extracts of vaccinia virus-infected cells. This antiserum also immune-precipitates the novel DNA ligase-AMP adduct, thus proving that the observed DNA ligase is encoded by SalF 15R.
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PMID:Vaccinia virus encodes a polypeptide with DNA ligase activity. 258 53

UV-irradiation of stationary phase cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe leads to a 9-fold and 90-fold increase in transcript levels from the respective DNA ligase genes CDC9 and CDC17, whereas exponential cells show only 3-fold and 2-fold increases. Induction of CDC9 after MMS treatment and gamma-irradiation was also observed by using a CDC9-lacZ translational fusion and assaying for beta-galactosidase. Surprisingly, irradiation of S. cerevisiae induces only a 50% increase in DNA ligase itself, probably reflecting the extremely high in vivo stability of the enzyme. The UV-induction of ligase may be part of a "fail-safe" mechanism which, together with the enzyme stability, ensures adequate supplies of this essential enzyme.
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PMID:Induction of yeast DNA ligase genes in exponential and stationary phase cultures in response to DNA damaging agents. 332 47

A recombinant plasmid for expression of full-length human DNA ligase I (phLig-I) was constructed in a plasmid/phage chimeric vector, pTD-T7N, which was derived from pUC118 by oligonucleotide-directed mutagenesis. The insert contained a 2757-base pair coding sequence for a whole human DNA ligase I and an extra ACC codon adjacent to the ATG initiation codon. This ACC codon was required for achieving high levels of expression of full-length DNA ligase I in Escherichia coli strain BL21. The recombinant plasmid, which was designed to exploit the T7 late promoter and the ATG initiation codon for beta-galactosidase was transfected into E. coli BL21 cells that express T7 RNA polymerase. The recombinant clone produced relatively high levels of DNA ligase I with a molecular mass of 130 kDa, as estimated by SDS-polyacrylamide gel electrophoresis. The DNA ligase was purified to near-homogeneity by the two-step column chromatographic procedure from BLphLig-I cells that had been induced with isopropyl beta-D-thiogalactoside. The specific activity, chromatographic behavior, kinetic properties, molecular mass, and antigenicity of the recombinant human DNA ligase I were indistinguishable from those of purified mammalian DNA ligase I. Metabolically labeling experiments with 32P(i) indicate that the recombinant DNA ligase I was present as an enzyme-AMP reaction intermediate, but not as a phosphoprotein, in the E. coli cells.
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PMID:Expression of active human DNA ligase I in Escherichia coli cells that harbor a full-length DNA ligase I cDNA construct. 822 62

It was demonstrated previously that a deoxyribophosphodiesterase (dRpase) activity is associated with the DNA repair enzyme exonuclease I, and that this activity is stimulated by the addition of the E. coli single-stranded DNA-binding protein (Ssb). This activity catalyzes the release of deoxyribose-phosphate groups at apurinic/apyrimidinic (AP) sites in the DNA that have been cleared by the action of an AP endonuclease. We have now used the yeast two-hybrid system to demonstrate that a protein-protein interaction occurs between exonuclease I and Ssb. When the E. coli ssb gene was fused in frame to the DNA-activating domain of the GAL4 transcriptional activator and the exonuclease I gene was fused in frame to the DNA-binding domain, a functional GAL4 transcriptional activator was produced as determined by growth of yeast on selective medium and the measurement of beta-galactosidase activity. We have also demonstrated that Ssb can stimulate the dRpase activity of exonuclease I using double-stranded bacteriophage M13 DNA containing several strand interruptions at incised AP sites. These results suggest that Ssb may be required for efficient base-excision repair in bacteria.
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PMID:Protein-protein interactions between the Escherichia coli single-stranded DNA-binding protein and exonuclease I. 861 28

Protein engineers have widely adopted directed evolution as a design algorithm, but practitioners have not come to a consensus about the best method to evolve protein molecular recognition. We previously used DNA shuffling to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with increased beta-galactosidase activity. Epistatic (synergistic) mutations in amino acids 557, 566, and 568, which are part of an active site loop, were identified in that experiment (Matsumura, I., and Ellington, A. D. (2001) J. Mol. Biol. 305, 331-339). Here we show that site saturation mutagenesis of these residues, overexpression of the resulting library in E. coli, and high throughput screening led to the rapid evolution of clones exhibiting increased activity in reactions with p-nitrophenyl-beta-d-xylopyranoside (pNP-xyl). The xylosidase activities of the 14 fittest clones were 30-fold higher on average than that of the wild-type GUS. The 14 corresponding plasmids were pooled, amplified by long PCR, self-ligated with T4 DNA ligase, and transformed into E. coli. Thirteen clones exhibiting an average of 80-fold improvement in xylosidase activity were isolated in a second round of screening. One of the evolved proteins exhibited a approximately 200-fold improvement over the wild type in reactivity (k(cat)/K(m)) with pNP-xyl, with a 290,000-fold inversion of specificity. Sequence analysis of the 13 round 2 isolates suggested that all were products of intermolecular recombination events that occurred during whole plasmid PCR. Further rounds of evolution using DNA shuffling and staggered extension process (StEP) resulted in modest improvement. These results underscore the importance of epistatic interactions and demonstrate that they can be optimized through variations of the facile whole plasmid PCR technique.
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PMID:Rapid evolution of beta-glucuronidase specificity by saturation mutagenesis of an active site loop. 1506 62

Bacteriophage phiYeO3-12 is a T7/T3-related lytic phage that naturally infects Yersinia enterocolitica serotype O:3 strains by using the lipopolysaccharide O polysaccharide (O antigen) as its receptor. The phage genome is a 39,600-bp-long linear, double-stranded DNA molecule that contains 58 genes. The roles of many of the genes are currently unknown. To identify nonessential genes, the isolated phage DNA was subjected to MuA transposase-catalyzed in vitro transposon insertion mutagenesis with a lacZ' gene-containing reporter transposon. Following electroporation into Escherichia coli DH10B and subsequent infection of E. coli JM109/pAY100, a strain that expresses the Y. enterocolitica O:3 O antigen on its surface, mutant phage clones were identified by their beta-galactosidase activity, manifested as a blue color on indicator plates. Transposon insertions were mapped in a total of 11 genes located in the early and middle regions of the phage genome. All of the mutants had efficiencies of plating (EOPs) and fitnesses identical to those of the wild-type phage when grown on E. coli JM109/pAY100. However, certain mutants exhibited altered phenotypes when grown on Y. enterocolitica O:3. Transposon insertions in genes 0.3 to 0.7 decreased the EOP on Y. enterocolitica O:3, while the corresponding deletions did not, suggesting that the low EOP was not caused by inactivation of the genes per se. Instead, it was shown that in these mutants the low EOP was due to the delayed expression of gene 1, coding for RNA polymerase. On the other hand, inactivation of gene 1.3 or 3.5 by either transposon insertion or deletion decreased phage fitness when grown on Y. enterocolitica. These results indicate that phiYeO3-12 has adapted to utilize Y. enterocolitica as its host and that these adaptations include the products of genes 1.3 and 3.5, DNA ligase and lysozyme, respectively.
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PMID:Nonessential genes of phage phiYeO3-12 include genes involved in adaptation to growth on Yersinia enterocolitica serotype O:3. 1568 5

Protein function can be engineered through iterated cycles of random mutagenesis and screening (directed evolution). Optimization of protein expression is essential for the development of sensitive and precise high throughput assays. Here we optimize the performance of a plasmid-borne Escherichia coli lacZ gene in two rounds of directed evolution. First, its promoter was "randomized" by whole plasmid polymerase chain reaction (PCR) and intra-molecular self-ligation. A genetically stable constitutive expression vector was isolated in an in vivo genetic selection. Second, the entire plasmid was randomly mutated in a slightly mutagenic long polymerase chain reaction. The PCR products were digested with a restriction enzyme, self-ligated by T4 DNA ligase and transformed into E. coli. The resulting library of beta-galactosidase (beta-gal) mutants consisted mostly ( approximately 80%) of hypomorphs, suggesting that the mutation rate was appropriate for directed evolution applications. We isolated and characterized 14 variants with increased activity in reactions with 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal). The purified protein derived from one clone exhibited a 100-fold improvement in k(cat) over its parent in reactions with para-nitrophenyl-beta-d-galactopyranoside (pNP-gal). This latter result clearly demonstrates the utility of whole plasmid mutagenic PCR for directed protein evolution.
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PMID:Whole plasmid mutagenic PCR for directed protein evolution. 1585 86


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