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)

Using genetic and biochemical techniques, we have determined that beta-galactosidase in the yeast Kluyveromyces lactis is coded by the LAC4 locus. The following data support this conclusion: (1) mutations in this locus result in levels of beta-galactosidase activity 100-fold lower than levels in uninduced wild type and all other lac- mutants; (2) three of five lac4 mutations are suppressible by an unlinked suppressor whose phenotype suggests that it codes for a nonsense suppressor tRNA; (3) a Lac+ revertant, bearing lac4--14 and this unlinked suppressor, has subnormal levels of beta-galactosidase activity, and the Km for hydrolysis of o-nitrophenyl-beta, D-galactoside and the thermal stability of the enzyme are altered; (4) the level of beta-galactosidase activity per cell is directly proportional to the number of copies of LAC4; (5) analysis of cell-free extracts of strains bearing mutations in LAC4 by two-dimensional acrylamide gel electrophoresis shows that strains bearing lac4--23 and lac4--30 contain an inactive beta-galactosidase whose subunit co-electrophoreses with the wild-type subunit, while no subunit or fragment of the subunit is observable in lac4--8, lac14--14 or lac4--29 mutants; (6) of all lac4 mutants, only those bearing lac4--23 or lac4--30 contain a protein that cross-reacts with anti beta-galactosidase antibody, a finding consistent with the previous result; and (7) beta-galactosidase activity in several Lac+ revertants of strains carrying lac4--23 or lac4--30 has greatly decreased thermostability.
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PMID:Lac4 is the structural gene for beta-galactosidase in Kluyveromyces lactis. 680 Aug 77

A previously constructed trp-S-lacZ fusion encoding a hybrid protein with beta-galactosidase activity was subcloned from a multicopy plasmid onto a lambda vector. Single-copy lysogens of lambda trpS-lacZ were used to determine whether trpS was regulated in a manner similar to that of other aminoacyl-tRNA synthetases. trpS regulation was found to resemble that of the majority of synthetases, in that expression of the lysogen-encoded hybrid beta-galactosidase varied with growth rate; beta-galactosidase activity increased 2.5-fold as the generation time decreased from 150 to 37 min. This regulatory response was confirmed by DNA/RNA hybridization experiments, which also suggested that this form of metabolic regulation occurred at the transcriptional level. No alteration in the level of hybrid beta-galactosidase was observed, however, when cells were starved for tryptophan.
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PMID:Regulation of tryptophanyl-tRNA synthetase formation. 704

Plasmids encoding various external guide sequences (EGSs) were constructed and inserted into Escherichia coli. In strains harboring the appropriate plasmids, the expression of fully induced beta-galactosidase and alkaline phosphatase activity was reduced by more than 50%, while no reduction in such activity was observed in strains with non-specific EGSs. The inhibition of gene expression was virtually abolished at restrictive temperatures in strains that were temperature-sensitive for RNase P (EC 3.1.26.5). Northern blot analysis showed that the steady-state copy number of EGS RNAs was several hundred per cell in vivo. A plasmid that contained a gene for M1 RNA covalently linked to a specific EGS reduced the level of expression of a suppressor tRNA that was encoded by a separate plasmid. Similar methods can be used to regulate gene expression in E. coli and to mimic the properties of cold-sensitive mutants.
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PMID:Artificial regulation of gene expression in Escherichia coli by RNase P. 747 48

A base substitution of G to U was constructed at position 529 in Escherichia coli 16S rRNA. The U529 mutant ribosomes were functional and present on polysomes but were highly error prone and caused a progressive loss of cell viability. They displayed elevated levels of readthrough of stop codons and frameshifting, and an increase in thermal sensitivity of beta-galactosidase, suggestive of missense errors. These results demonstrate that the university conserved G529 is involved in tRNA selection at the A site during protein synthesis.
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PMID:A mutation at the universally conserved position 529 in Escherichia coli 16S rRNA creates a functional but highly error prone ribosome. 748 93

The requirements for efficient translation termination are incompletely understood. Since the local context surrounding stop codons can influence the efficiency of translation termination, premature termination codons introduced by random mutation may not always terminate at the optimal efficiencies expected of naturally occurring stop codons. To investigate whether this could result in physiologically significant levels of read through, we examined the suppression of premature translation termination mutations within a sequence motif of the yeast Ste6 protein (Ste6p) that is highly conserved among members of the ATP-binding cassette (ABC) transporter family. The human cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in individuals with the disease cystic fibrosis, is also a member of this protein family. The mutations examined in Ste6p were chosen because a premature termination codon at the corresponding residue of CFTR has previously been reported to cause less severe pulmonary involvement than some missense mutations, suggesting that low level suppression of this stop codon could be occurring. Our results indicate that these premature stop codons in Ste6p can be suppressed at frequencies as high as 10%. Characterization of this phenomenon using a beta-galactosidase read through assay system showed that a limited sequence context surrounding this site contained information that was sufficient to cause suppression of translation termination. Amino acid sequence analysis of the full-length translation products produced by read through of an amber codon demonstrated that termination suppression was mediated by near-cognate tRNA mispairing that resulted in the insertion of tyrosine, lysine, or tryptophan.
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PMID:Premature translation termination mutations are efficiently suppressed in a highly conserved region of yeast Ste6p, a member of the ATP-binding cassette (ABC) transporter family. 751 33

4-Nitroquinoline 1-oxide (4NQO) is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. We recently determined the mutation spectrum induced by the ultimate metabolite of 4NQO, acetoxy-4-aminoquinolone 1-oxide in the M13lacZ'/E. coli lacZ delta M15 alpha-complementation assay. Our data suggested that dGuo-C8-AQO induces (per se or via AP sites) G to Pyr transversions. Here we report our study on 4NQO mutagenesis in monkey cells. 4NQO lesions were induced in vitro on a single-stranded (ss) DNA shuttle vector carrying the supF tRNA gene. This vector was able to replicate both in mammalian cells and in bacteria. The mutations induced in monkey cells were screened by the white/blue beta-galactosidase activity assay in E. coli. We took advantage of the peculiar feature of ss supF DNA in which the extent of secondary structure may be a function of the temperature, with the dependence of the 4NQO-specific adduct spectrum on DNA secondary structure. We reasoned that mutational spectra derived from damage induced in the presence (20 degrees C) or absence (70 degrees C) of DNA secondary structure should be different. The result of sequencing a total of 89 induced and spontaneous mutants confirmed that the spectra are statistically different. These data suggest that the two 4NQO guanine adducts may induce different mutations.
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PMID:Mutation spectrum of 4-nitroquinoline 1-oxide-damaged single-stranded shuttle vector DNA transfected into monkey cells. 751 38

Four Escherichia coli operons, the leuV operon which encodes tRNA(1Leu), the leuX operon which encodes tRNA(6Leu), the metT operon which encodes tRNA(3Leu), and the argT operon which encodes tRNA(1Leu), were examined for the stringent response induced by serine hydroxamate and for growth rate-dependent regulation. In nuclease protection assays, the leuV operon displayed the stringent response in response to leucine starvation, analog inhibition, and growth of a temperature-sensitive leucyl-tRNA synthetase mutant at nonpermissive temperatures. The leuV operon also exhibited the stringent response in multicopy plasmids. The promoters of all four leucyl operons were fused to the gene for beta-galactosidase and inserted into the chromosome by using bacteriophage lambda. All except the leuX promoter displayed growth rate-dependent regulation, consistent with the recent report that the concentration of tRNA(6Leu) actually decreases as growth rate increases. The leuV promoter fused to the beta-galactosidase gene showed a decrease in efficiency in the presence of extrachromosomal copies of rRNA genes. All chromosomal tRNA genes examined showed decreased transcriptional activity following a stringent response, but the leuX gene responded to a lesser extent (3-fold versus 10-fold or more) than the others. Primer extension analysis of this promoter showed little if any response to serine hydroxamate treatment, suggesting that multiple levels of control may exist or that promoter context effects are important in regulation.
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PMID:In vivo regulatory responses of four Escherichia coli operons which encode leucyl-tRNAs. 768 Mar 41

The leuV promoter which produces tRNA(1Leu) in Escherichia coli has been extensively mutagenized in order to determine the effects of altered sequences on promoter efficiency (strength) and on growth-rate-dependent regulation (GDR). Each mutant promoter was ligated with a beta-galactosidase reporter gene into the chromosome of a host cell by phage lambda lysogenization. Reporter gene activities were measured for cells growing in selected media at various growth rates. Sequences which flank the -10 consensus region, when altered, caused remarkable up-promoter effects, increasing efficiency in some cases almost 10-fold. One up mutation which had five successive T residues in the 'discriminator' region completely abolished GDR, whereas several mutations with single base changes in the discriminator had little or no effect on GDR. Another mutation which changed one base in the -35 region to bring it to consensus increased promoter strength 18-fold and sharply reduced GDR. Chimaeric promoters in which segments of leuV were replaced by segments of the his operon showed that only when the discriminator of leuV is replaced by the his discriminator was GDR-disturbed. All upstream sequences which were replaced by his sequences had little effect on GDR. Overall, there appeared to be little correlation between promoter efficiency and GDR.
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PMID:Mutagenesis and functional analysis of the Escherichia coli tRNA(1Leu) promoter. 768 Apr 11

Almost all spontaneous point mutations found on a single-stranded shuttle vector after its transfection and replication in monkey cells were located at cytosine residues. In order to understand this very specific type of targeting we have studied the possible implication of uracil residues in the induction of these spontaneous mutations. The single-stranded shuttle vector pCF3A carrying the supF tRNA gene as a mutagenesis target has been allowed to replicate in mammalian COS7 cells, mutations being screened in bacteria using the beta-galactosidase assay. Progenies from untreated DNA and DNA treated with the uracil-DNA glycosylase prior to transfection were analyzed to determine the amount and classes of mutations. While spontaneous mutation frequency was 9.7 x 10(-4) for control DNA, single-stranded vector treated with the E. coli uracil-DNA glycosylase exhibited a reduced mutation frequency of about 30%. The abolished mutations were mainly confined to the cytosine to thymine transitions for which a decrease by a factor of 5 was indeed observed. This finding fits well with the fact that it is usually admitted that uracil pairs with adenine, indicating therefore that approximately 30% of spontaneous mutations observed in our experimental conditions and 80% of C to T transitions may be due to the presence of uracil instead of cytosine.
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PMID:Implication of uracil in spontaneous mutagenesis on a single-stranded shuttle vector replicated in mammalian cells. 768 84

Saccharomyces cerevisiae transcription factor IIIC/tau is a multisubunit DNA-binding protein that plays key roles in tRNA and 5 S rRNA gene activation. Subunit composition, stoichiometry, and in vivo phosphorylation of TFIIIC/tau factor were investigated using factor prepared from strains carrying modified forms of TFC1, the gene encoding the 95-kDa TFIIIC/tau subunit (tau 95). Using an epitope-tagged TFC1 as well as a TFC1-lacZ fusion, TFIIIC was shown to contain a single 95-kDa subunit, which was localized by electron microscopy into tau A, the A block-binding domain of TFIIIC/tau. Three 35S-labeled polypeptides (at 138, 131, and 91 kDa) coimmunoprecipitated with a tau 95-beta-galactosidase fusion protein. The coprecipitation of the 91-kDa polypeptide makes it a likely subunit of the factor. Immunoprecipitation from 32P-labeled extracts revealed that three of the subunits (138, 131, and 95 kDa), but not the 91-kDa component, are phosphorylated in vivo.
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PMID:On the subunit composition, stoichiometry, and phosphorylation of the yeast transcription factor TFIIIC/tau. 768 37


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