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)

Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity. We have isolated cDNAs from the Trypanosomatidae Leishmania major and Trypanosoma cruzi capable of complementing the deficiency of exonuclease III and dUTPase in the Escherichia coli mutant BW286. This double mutant is non-viable at 37 degreesC due to an accumulation of non-repaired sites following excision of uracil from DNA. The genes were expressed as beta-galactosidase-AP endonuclease fusion proteins and as such are active in repair of AP sites in E. coli. The Trypanosoma and Leishmania sequences have unique N-termini containing sequences that correspond to probable nuclear transport signals, while the C-terminal domains exhibit pronounced similarity to exonuclease III. The L.major gene was overexpressed as a histidine-tagged protein and recombinant enzyme exhibited endonuclease activity on AP DNA in vitro. Furthermore, expression of the enzymes in AP endonuclease-deficient E.coli mutants conferred significant resistance to killing by methylmethane sulphonate and peroxides. This study constitutes one of the first descriptions of DNA repair enzymes in these pathogenic organisms where oxidative stress is an important mechanism of both drug-mediated and intracellular killing.
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PMID:Apurinic/apyrimidinic endonuclease genes from the trypanosomatidae leishmania major and Trypanosoma cruzi confer resistance to oxidizing agents in DNA repair-deficient Escherichia coli. 988 72

To examine the previously proposed retroregulation model of spc mRNA degradation, two strains of Escherichia coli B/r were used; one has wild-type spc and lac operons and the other has a lac operon deletion, a wild-type spc operon, and a Pspc-rplN-lacZ fusion operon lacking the normal control sites of the spc operon (rplN is the first gene in the spc operon of ribosomal proteins). The decay of rplN mRNA and of lacZ mRNA in these strains was determined during exponential growth at different rates and after transcript initiation was inhibited by the antibiotic rifampicin. Functional decay of lacZ mRNA was monitored by measurements of beta-galactosidase activity and chemical decay was monitored using probes complementary to rplN, rplX, and to the 5' and 3'-terminal sections of lacZ. Analysis of the data was based on the assumption that the decay involves an endonucleolytic cleavage that functionally inactivates the mRNA and that this is followed by exonucleolytic degradation of the cleavage products. The major conclusions were: (1) During exponential growth, lacZ mRNA of the lac operon was translated about twice as frequently as lacZ mRNA of the spc-lac fusion, and both kinds of lacZ mRNA were translated at an elevated rate in the presence of rifampicin. (2) For lacZ mRNA from the lac operon, the endonuclease inactivation reaction was not affected by rifampicin, but the exonuclease reaction was inhibited. (3) The decay of rplN mRNA from the spc operon was accelerated in the presence of rifampicin; the average life was estimated to be six minutes during exponential growth in LB medium, and 2.8 minutes in the presence of rifampicin. (4) The decay of the rplN section of mRNA from the spc-lac operon fusion was coupled to the decay of the downstream lacZ mRNA section and was strongly inhibited (i.e. partially blocked) in the presence of rifampicin. These results show that the decay of spc mRNA differs in some important aspects from the decay of lac mRNA and support the retroregulation model. Moreover, the results indicate that rifampicin can have a significant and selective impact on the kinetics of both mRNA translation and decay.
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PMID:Decay of rplN and lacZ mRNA in Escherichia coli. 1032 60

PpLSU3, a mobile group I intron found in the ribo-somal RNA genes of Physarum polycephalum, encodes the I-PpoI homing endonuclease. This enzyme represents one of the rare cases in nature where a protein is expressed from an RNA polymerase I transcript. Our previous results showed that the full length intron, but not a further processed species, is the messenger for I-PpoI, implying a role of the untranslated region (UTR) in gene expression. To study the function of the 3'-UTR in expression of the endonuclease and in splicing of the intron, we replaced the I-PpoI gene in PpLSU3 with the gene for the alpha-fragment of Escherichia coli beta-galactosidase, and then integrated this chimeric intron into all the chromosomal rDNA repeats of yeast. The resulting cells synthesized functional alpha-fragment, as evidenced by a complementation assay analogous to that used in E.coli. The beta-galactosidase activity thus provides an unusual and potentially valuable readout for Pol I transcription from chromosomal rDNA. This is the first example in which a eucaryotic homing endonuclease gene has been successfully replaced by a heterologous gene. Using deletion mutagenesis and a novel randomization approach with the alpha-fragment as a reporter, we found that a small segment of the 3'-UTR dramatically influences both splicing and protein expression.
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PMID:Functional alpha-fragment of beta-galactosidase can be expressed from the mobile group I intron PpLSU3 embedded in yeast pre-ribosomal RNA derived from the chromosomal rDNA locus. 1068 39

We have engineered a new vector that makes use of direct ligation for the generation of replication-defective recombinant adenovirus constructs. In the pAd5-Blue vector, unique yet common restriction endonuclease sites exist, that allow cloning in a directional manner of a gene of interest under control of a cytomegalovirus promoter, upstream of a simian virus 40 polyadenylation signal. The insertion of the new gene replaces the beta-galactosidase alpha gene fragment in the pAd5-Blue vector, allowing the identification of recombinants in bacterial culture by the selection of white colonies. Plasmid DNA from white colonies is digested with PacI and transfected into 293 cells, resulting in the generation of a homogenous population of adenovirus containing the gene of interest. The pAd5-Blue vector system does not rely on recombination either in mammalian or bacterial cells. Furthermore, because of compatible overhangs, the variety of restriction endonucleases that can be used to generate the inserted gene gives flexibility to the process for greater ease of use. The system is quick and straightforward, allowing the generation of recombinant adenoviruses within three weeks of obtaining an appropriate insert. This new vector should greatly enhance the ease and speed with which new recombinant adenovirus constructs can be made.
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PMID:pAd5-Blue: direct ligation system for engineering recombinant adenovirus constructs. 1173 12

For applications such as sequencing, transfection, and in vitro transcription, PCR products have to be subcloned into plasmids. Many strategies are used for cloning, blunt-end ligation or the incorporation of restriction endonuclease sites into PCR primers for appropriate vectors. However, the most convenient and direct method is T/A cloning. In this study, we developed two of the pGEM-7Zf(+) phagemid T-tail vectors using AhdI-restriction endonuclease sites, and these T vectors have all the features of pGEM-7Zf(+): f1 ori, T7, and SP6 RNA polymerase promoters, the alpha-peptide coding region of beta-galactosidase for X-gal blue/white color selection, the beta-lactamase gene for recombinant colony selection, and binding sites for pUC/M13 forward and reverse sequencing primers. These AhdI-containing phagemid vectors, pGEM-NJ105 and pGEM-NJ107, are useful for the easy and inexpensive preparation of T vectors and direct cloning of PCR products.
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PMID:Construction of two pGEM-7Zf(+) phagemid T-tail vectors using AhdI-restriction endonuclease sites for direct cloning of PCR products. 1238 33

Using a monoclonal antibody that specifically recognizes thymine glycol (Tg) in DNA, we measured the kinetics of the removal of Tg from the genomes of wild-type and repair gene mutant strains of Escherichia coli treated with hydrogen peroxide. Tg is rapidly and efficiently removed from the total genomes of repair-proficient cells in vivo and the removal of Tg is completely dependent on the nth gene that encodes the endonuclease III glycosylase. Hence, it appears that little redundancy in the repair of Tg occurs in vivo, at least under the conditions used here. Moreover, previous studies have found that nth mutants are not sensitive to killing by hydrogen peroxide but xth mutant strains (deficient in the major AP endonuclease, exonuclease III) are sensitive. We find that cell death correlates with the persistence of single-strand breaks rather than the persistence of Tg. We attempted to measure transcription-coupled removal of Tg in the lactose operon using the Tg-specific monoclonal antibody in an immunoprecipitation approach but were not successful in achieving reproducible results. Furthermore, the analysis of transcription-coupled repair in the lactose operon is complicated by potent inhibition of beta-galactosidase expression by hydrogen peroxide.
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PMID:Global genome removal of thymine glycol in Escherichia coli requires endonuclease III but the persistence of processed repair intermediates rather than thymine glycol correlates with cellular sensitivity to high doses of hydrogen peroxide. 1240 47

Circular concatemerization of the recombinant adeno-associated virus (rAAV) genome has been suggested as the predominant process facilitating long-term rAAV transduction in muscle. A recent study (S. Song, P. J. Laipis, K. I. Berns, and T. R. Flotte, Proc. Natl. Acad. Sci. USA 98:4084-4088, 2001) with SCID mice, which are defective in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), has suggested that DNA-PKcs regulates the removal of free rAAV vector ends in muscle tissue. In the present study, we have sought to evaluate whether a lack of DNA-PKcs activity reduces circularization of rAAV genomes in SCID muscle and whether such a reduction alters the directivity of heterodimerization. Consistent with the previous report, linear rAAV genomes and free vector ends were detected only in DNA-PKcs-deficient muscle by Southern blotting. Appreciable amounts of circular rAAV genomes were detected in both DNA-PKcs-deficient and wild-type muscle samples by Southern blotting and bacterial trapping experiments. The existence of double-D inverted terminal repeat circular intermediates in SCID and wild-type muscles was also supported by their sensitivity to T7 endonuclease I digestion. However, DNA-PKcs-deficient muscle did demonstrate a approximately 50% reduction in the abundance of rescued circular genomes, despite equivalent levels of single rAAV transduction seen in wild-type animals. Dual trans-splicing lacZ vectors were used to functionally evaluate directional head-to-tail intermolecular viral genome concatamerization in vivo. Although AAV genomes are processed differently in SCID and wild-type muscles, a comparable level of trans-splicing-mediated beta-galactosidase expression was observed in both strains, suggesting that both circular and linear AAV concatemers may have contributed to the trans-splicing-mediated transgene expression. In summary, we have shown that SCID skeletal muscle retains a fairly high capacity to form circular genomes, despite a significant increase in linear vector genomes. Furthermore, the alteration in equilibrium between circular and linear concatemer genomes caused by the lack of DNA-PKcs activity does not appear to significantly affect the efficiency of dual-vector gene expression from head-to-tail linear and/or circular heterodimers.
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PMID:Consequences of DNA-dependent protein kinase catalytic subunit deficiency on recombinant adeno-associated virus genome circularization and heterodimerization in muscle tissue. 1266 82

A novel T-vector was constructed that could be used for direct cloning and expression of PCR-amplified cDNA. The technique was based on the insertion into the parent vector of two endonuclease-Eam1105I restriction sequences spaced by an expression cassette of the full-length beta-galactosidase, which helped to improve cloning efficiency and to minimize the non-recombinant background of the T-vector when used to clone PCR products. Moreover, this method took advantage of the reconstitution of the rarest restriction sequence of MssI to enable directional cloning. These advantages make the T-vector suitable for high-throughput expression and analysis.
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PMID:Construction of a restriction-endonuclease-Eam1105I-generated T-vector for high-throughput cloning and expression. 1742 95

Type II restriction-modification (R-M) systems are composed of linked restriction endonuclease and modification methyltransferase genes and serve as barriers to horizontal gene transfer even though they are mobile in themselves. Their products kill host bacterial cells that have lost the R-M genes, a process that helps to maintain the frequency of the R-M systems in the viable cell population. Their establishment and maintenance in a bacterial host are expected to involve fine regulation of their gene expression. In the present study, we analyzed transcription of the modification gene and its regulation within the EcoRI R-M system. Northern blotting revealed that the downstream ecoRIM gene is transcribed as a monocistronic mRNA and as part of a larger bicistronic mRNA together with the upstream ecoRIR gene. Primer extension, RNase protection, and mutational analysis using lacZ gene fusions identified two overlapping promoters for ecoRIM gene transcription within the ecoRIR gene. Further mutational analysis revealed that two upstream AT-rich elements within the ecoRIR gene, "AATAAA" and "ATTATAAATATA," function as negative regulators of these promoters. Simultaneous substitution of these two elements resulted in a four-fold increase in beta-galactosidase activity and a five-fold increase in transcript levels as measured by RNase protection assay. RNA measurements of the ecoRIM transcript suggested that these elements decreased ecoRIM expression by interfering with transcription initiation of the ecoRIM promoters. Possible roles for these ecoRIM promoters and their negative regulators in the EcoRI R-M system are discussed.
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PMID:Regulation of the EcoRI restriction-modification system: Identification of ecoRIM gene promoters and their upstream negative regulators in the ecoRIR gene. 1761 69

The SOS response is an important mechanism which allows Escherichia coli cells to maintain genome integrity. Two key proteins in SOS regulation are LexA (repressor) and RecA (coprotease). The signal for SOS induction is generated at the level of a RecA filament. Depending on the type of DNA damage, a RecA filament is produced by specific activities (helicase, nuclease and RecA loading) of either RecBCD, RecF or a hybrid recombination pathway. It was recently demonstrated that RecA loading activity is essential for the induction of the SOS response after UV-irradiation. In this paper we studied the genetic requirements for SOS induction after introduction of a double-strand break (DSB) by the I-SceI endonuclease in a RecA loading deficient recB mutant (recB1080). We monitored SOS induction by assaying beta-galactosidase activity and compared induction of the response between strains having one or more inactivated mechanisms of RecA loading and their derivatives. We found that simultaneous inactivation of both RecA loading functions (in recB1080 recO double mutant) partially impairs SOS induction after introduction of a DSB. However, we found that the RecJ nuclease is essential for SOS induction after the introduction of a DSB in the recB1080 mutant. This result indicates that RecJ is needed to prepare ssDNA for subsequent loading of RecA protein. It implies that an additional type of RecA loading could exist in the cell.
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PMID:RecJ nuclease is required for SOS induction after introduction of a double-strand break in a RecA loading deficient recB mutant of Escherichia coli. 1844 87


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