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)

A plasmid clone that suppresses galactose toxicity in a gal7 yeast strain has been isolated from a multicopy genomic DNA library. Molecular analysis revealed that the region responsible for the suppression of galactose toxicity corresponds to the ORF YPR030w, which was named MRG19. A CEN-based plasmid carrying the above ORF was unable to suppress the toxicity. Galactokinase activity was substantially reduced in cell extracts obtained from transformants bearing multiple copies of MRG19. Multiple copies of MRG19 were also able to suppress galactokinase expression driven by the CYC1 promoter but not the TEF1 promoter. Multiple copies of MRG19 could not suppress GAL1-driven galactokinase expression in a gal80 strain. However, MRG19-mediated suppression of CYC1-driven galactokinase expression was independent of GAL80 function. These results imply that multiple copies of MRG19 suppress galactokinase expression probably at the level of transcription. In agreement with this idea, multiple copies of MRG19 also suppress beta-galactosidase expression driven by the GAL1 promoter in a GAL80-dependent manner. Disruption of MRG19 leads to an increase in the cell density at stationary phase in synthetic complete medium. MRG19 encodes a previously uncharacterised 124-kDa protein that shows no sequence homology to any known proteins.
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PMID:Multiple copies of MRG19 suppress transcription of the GAL1 promoter in a GAL80-dependent manner in Saccharomyces cerevisiae. 1066 72

Studies of N-ethyl-N-nitrosourea (ENU)-induced mutagenesis with a tyrosine auxotroph of Escherichia coli revealed a new type of revertant. This mutant strain was interesting because: (i) it was not a true revertant of the nonsense (ochre) defect nor a tRNA suppressor mutation; and (ii) it was induced by ENU to greater extent in a UmuC-defective host. Genetic mapping located the probable mutation to a region of the E. coli chromosome containing a newly described gene called tas. To investigate this mutation, the upstream region of the tas gene from both wild-type and mutant cells was cloned into a promoterless lacZ expression vector and recombined onto a lambda bacteriophage. Recombinant bacteriophage were inserted into the bacterial chromosome and beta-galactosidase (betaGal) assays were performed. These assays revealed an almost three-fold greater expression of betaGal from the mutant DNA than from the wild-type DNA. Sequence analysis of the region directly upstream of the tas gene revealed a G:C to A:T transition at base number 2263 (numbering based on GenBank Accession #AE000367), located within a potential promoter site. Further sequencing indicated no other mutations within the 1454bp region analyzed; however, there were several nucleotide differences seen in our B/r strain of E. coli, when compared with the published E. coli K-12 sequence. A total of 10 base differences were discovered; one in mutH, six within a potential open reading frame (ORF-o237) and three in non-coding regions. Yet, none of the changes altered the predicted amino acid sequences. These results provide evidence of a mechanism for increased expression of the novel gene tas and support the neutral drift hypothesis for the evolution of DNA sequences.
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PMID:Identification of a mutation causing increased expression of the tas gene in Escherichia coli FX-11. 1147 Apr 87

A cysE gene encoding a serine acetyltransferase (SAT) potentially involved in the biosynthesis of cysteine was identified approximately 4 kb upstream of the previously described aapJQMP gene cluster that encodes an amino acid permease in Rhizobium leguminosarum strain 3841. The gene exhibits >40% identity to the family of SATs containing N-terminal extensions that have been described for other bacteria and plants. The ORF has three possible translation initiation sites which potentially encode polypeptides of 311, 277 and/or 259 amino acid residues, respectively. All three ORFs complemented the cysE mutation in an Escherichia coli cysteine auxotroph, strain JM39. Insertion of Tn5-lacZ into cysE in the genome of R. leguminosarum (strain RU632) lowered SAT activity in crude extracts by >95%. However, RU632 was not a cysteine auxotroph, which suggests that R. leguminosarum possesses some redundancy in cysteine biosynthesis. Additional copies of cysE could not be detected in the genome when the R. leguminosarum cysE gene was used as a hybridization probe. Therefore it is possible that R. leguminosarum possesses an alternative pathway for cysteine biosynthesis which avoids O-acetylserine. Strain RU632 was unaffected in its ability to nodulate Pisum sativum, and the nodules were effective for N(2) fixation (measured by C(2)H(2) reduction). Transcriptional activity of cysE was determined by measuring the beta-galactosidase arising from cysE::Tn5-lacZ fusions. Maximal levels of expression were observed during early exponential growth and were not influenced by the level of sulphur (supplied as sulphate). However, transcription was repressed by approximately twofold in ammonium-grown, as opposed to glutamate-grown, cultures. Repression by ammonium was not seen in a strain defective for ntrC.
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PMID:Evidence for redundancy in cysteine biosynthesis in Rhizobium leguminosarum RL3841: analysis of a cysE gene encoding serine acetyltransferase. 1153 95

It was reported recently that changing the TIR (translational initiation region) of lambda N gene resulted in the increasing expression of lambda N gene and it was regulated at translational level. According to the alignment, the leader sequence of lambda N gene had three parts: a code region for ORF lambda N, the upstream sequence of ORF lambda N and 17 bp of spacer between ORF lambda N and downstream of lambda N gene. ORF lambda N is an open reading frame located at upstream of lambda N gene coding a peptide of 19 amino acids. To study the mechanism of regulation of lambda N gene expression, three serials of plasmids with mutant leader region of lambda N gene were constructed. (1) pMC1403-XT, in which the start codon or the partial code of ORF lambda N was connected with lacZ to obtain the ORF lambda N-lacZ fusion gene and in which the ORF lambda N-lacZ expression was under the control of a strong trp/lac promoter; (2) The ORF lambda N mutants in which the termination codon TAA was introduced into ORF lambda N at different positions by site-directed mutagenesis to preterminate the ORF lambda N on plasmid pMC1403N; (3) Mutants in which a deletion was located at upstream ORF lambda N in the ORF lambda N mutants above. The results obtained from determination of the beta-galactosidase activity in the transformants harboring the different plasmids showed that the ORF lambda N-lacZ expression was suppressed by the ORF lambda N code region and the lambda N expression was increased in both the second and third serials of mutants. At the same time the results from RNA-DNA dot hybridization showed that the lambda N gene expression was regulated at translational level. Therefore it was predicted that the reason of relatively low expression for lambda N gene in pMC1403N was due to the low efficiency of ORF lambda N translation. There are two ways to increase the expression of lambda N gene. One is to preterminate the translation of ORF lambda N at a suitable position to decrease the ORF lambda N effects on downstream lambda N gene translation; the other is to change the upstream sequence of ORF lambda N to improve its translation efficiency.
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PMID:[lambda N gene expression regulated by the leader sequence]. 1155 54

We have isolated a C. albicans gene, named FCR3 (for fluconazole resistance 3), based upon its ability to suppress the FCZ hypersusceptibility of a Saccharomyces cerevisiae mutant strain (JY312) lacking the transcription factors Pdr1p and Pdr3p. The FCR3 ORF (1200 bp) encodes a 399 amino acid protein containing a basic leucine zipper (bZip) domain. Fcr3p displays the highest level of sequence homology with the S. cerevisiae Yap3p protein (34% identity, 45% similarity). We had previously shown that deletion of the PDR5 gene encoding a multidrug transporter completely abolished the ability of FCR3 to suppress the FCZ hypersusceptibility of JY312, suggesting that FCR3 confers FCZ resistance by activating PDR5 expression. We show here that the beta-galactosidase activity of a PDR5 promoter-lacZ construct in JY312 is increased two-fold upon FCR3 overexpression, demonstrating that FCR3 regulates PDR5 at the transcriptional level. We also show that FCR3 overexpression not only suppresses the hypersusceptibility of JY312 to 4-nitroquinoline-N-oxide (4-NQO) but also confers higher levels of resistance to this compound as compared to the wild-type KY320 strain. Since PDR5 is not involved in 4-NQO resistance, this result indicates that FCR3 can also activate the transcription of other genes that can confer 4-NQO resistance. Finally, Northern blot analysis indicates that FCR3 encodes a single 2.4 kb RNA transcript in C. albicans, suggesting that the FCR3 mRNA contains long 5' and/or 3' untranslated regions. The nucleotide sequence of the FCR3 gene has been deposited at GenBank under Accession No. AF342983.
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PMID:Functional isolation of the Candida albicans FCR3 gene encoding a bZip transcription factor homologous to Saccharomyces cerevisiae Yap3p. 1156 Dec 89

brlA is a primary regulator of asexual development in Aspergillus nidulans. Activation of brlA is necessary and sufficient for conidiophore development. It is known that brlA produces two overlapping transcripts, designated brlAalpha and brlAbeta. We found that expression of brlA is subject to complex regulation, in that activation of the two brlA transcripts is regulated at different levels. While brlAalpha is regulated at the transcriptional level, brlAbeta is regulated at both the transcriptional and translational levels. brlAalpha expression requires both abaA and brlA, but overexpression of brlAbeta can induce brlAalpha in an abaA mutant. brlAbetamuORF, a short ORF located upstream of the brlA initiator codon, regulates expression of brlA by damping translation of the brlAbeta ORF, and translational repression of brlA expression prevents premature development in A. nidulans. Transcriptional control of brlAbeta is apparently independent of BrlA. In order to understand better the transcriptional control of brlAalpha and brlAbeta, we have made 5' deletions in the essential approximately 2-kb upstream control sequences that regulate brlAbeta transcription and fused them to the E. coli lacZ reporter gene. Various deletions in this region resulted in only minor changes in the regulation of beta-galactosidase expression. The results of the deletion experiments indicate that there are probably several cis-acting control sequences involved in the regulation of brlAbeta. As a complementary approach, we fused various fragments of the 2034-bp brlAbeta and 754-bp brlAalpha control sequences to an otherwise inactive amdS::lacZ fusion, in order to search for regions that are sufficient to place the reporter under developmental control. We identified two approximately 600-bp brlAbeta fragments extending from -2901 to -2293 and -967 to -414, respectively, and a approximately 150-bp brlAalpha segment from -271 to -127, that confer activity on the inactive amdS promoter. brlA is overexpressed in an abaA null mutant and one site for abaA-dependent repression is apparently located in the -742 to -414 brlAbeta fragment. This indicates that abaA-mediated repression of brlA expression occurs through control of brlAbeta, but apparently involves a mechanism that does not require AbaA binding to brlA(p) sequences, because there are no AbaA binding sites in this region.
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PMID:Complex control of the developmental regulatory locus brlA in Aspergillus nidulans. 1168 68

Previous studies employing two-dimensional gel electrophoresis and N-terminal protein sequencing have shown elevated synthesis of the enzyme methionine sulfoxide reductase (MsrA) in Staphylococcus aureus in response to cell-wall-active antibiotics. In the present study, the S. aureus msrA gene was cloned, overexpressed, purified as His-tagged MsrA and shown to have methionine sulfoxide reductase activity. The transcription of msrA was studied by assaying beta-galactosidase activity in an msrA promoter::lacZ fusion strain and by Northern blot analysis. Transcription of msrA was increased by oxacillin; but not by a variety of other stresses including H2O2. Northern blot analysis revealed that the size of the msrA transcript was 2.3 kb, considerably larger than the 531 nt msrA ORF. The msrA transcription start site was mapped 25 nt upstream of the msrA start codon. Computer analysis from database sequences indicated at least three additional ORFs downstream of msrA. The deduced amino acid sequences of two of these three ORFs showed significant sequence homologies to PilB, and enzyme IIA of the phosphotransferase system, respectively. The third ORF could not be identified by homology searches. Northern blot hybridization with probes specific to the msrA downstream region indicated that the S. aureus msrA was transcribed as part of a polycistronic message. Interestingly, purified S. aureus PilB was shown to possess approximately approximately 28-fold higher methionine sulfoxide reductase activity than the MsrA. An insertional knockout mutation in the first gene of this operon resulted in increased susceptibility of the mutant to H2O2 compared to the parent strain, but not to oxacillin.
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PMID:Molecular characterization of a chromosomal locus in Staphylococcus aureus that contributes to oxidative defence and is highly induced by the cell-wall-active antibiotic oxacillin. 1170 Mar 54

To extend the tools available for biochemical and genetical analysis in the fission yeast Schizosaccharomyces pombe we have investigated the development of gene reporter systems using the secreted alpha-galactosidase encoded by the Sz. pombe ORF SPAC869.07c (CAB60017), which we propose naming Mel1p to reflect its structural and functional similarity to MEL1p in Saccharomyces cerevisiae. The alpha-galactosidase activity can be monitored in liquid assays and converted the colourless substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside (X-alpha-gal) into an insoluble blue product that was suitable for semi quantitative plate-based assays; colonies expressing the highest levels of alpha-galactosidase developed the most intense blue colour. Unlike assays based on beta-galactosidase, the Sz. pombe colonies develop the blue colouration under normal growth conditions, avoiding the need to replicate colonies to fresh plates for analysis. It is therefore suitable for screening large numbers of colonies. To illustrate the use of mel1 as a reporter we linked expression to the sxa2 gene promoter to provide a convenient readout for signalling through the pheromone response pathway. The sxa2 > mel1 strain identified constitutively active Mam2 pheromone receptors from a randomly mutagenised library. There was an approximate correlation between the intensity of the blue colour developed by each mutant colony and its level of constitutive activity and we identified a subset of mutants with low constitutive activity that could not have been isolated by a previous screen using nutritional selection. The mel1 alpha-galactosidase activity identified and characterised in this study can be easily adapted to provide a gene reporter for many biological processes and is a new addition to the research tools available in Sz. pombe.
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PMID:Development of a semi-quantitative plate-based alpha-galactosidase gene reporter for Schizosaccharomyces pombe and its use to isolate a constitutively active Mam2. 1558 May 93

An Escherichia coli model system was developed to estimate the capacity of the integrase of the Drosophila melanogaster retrotransposon gypsy (mdg4) for precise excision of the long terminal repeat (LTR) and, hence, the entire gypsy. The gypsy retrotransposon was cloned in the form of a PCR fragment in the pBlueScript II KS+ (pBSLTR) vector, and the region of the second open reading frame (INT ORF2) of this element encoding integrase was cloned under the lacZ promoter in the pUC19 vector and then recloned in pACYC184 compatible with pBSLTR. The LTR was cloned in such a manner that its precise excision from the recombinant plasmid led to the restoration of the nucleotide sequence and the function of the ORF of the lacZ gene contained in the vector; therefore, it was detected by the appearance of blue colonies on a medium containing X-gal upon IPTG induction. Upon IPTG induction of E. coli XL-1 Blue cells obtained by cotransformation with plasmids pACCint and pBSLTR on an X-gal-containing medium, blue clones appeared with a frequency of 1 x 10(-3) to 1 x 10(-4), the frequency of spontaneously appearing blue colonies not exceeding 10(-9) to 10(-8). The presence of blue colonies indicated that that the integrase encoded by the INT ORF2 (pACYC 184) fragment was active. After the expression of the integrase, it recognized and excised the gypsy LTR from pBSLTR, precisely restoring the nucleotide sequence and the function of the lacZ gene, which led to the expression of the beta-galactosidase enzymatic activity. PCR analysis confirmed that the LTR was excised precisely. Thus, the resultant biplasmid model system allowed precise excisions of the gypsy LTR from the target site to be detected. Apparently, the gypsy integrase affected not only the LTR of this mobile element, but also the host genome nucleotide sequences. The system is likely to have detected only some of the events occurring in E. coli cells. Thus, the integrase of gypsy is actually capable of not only transposing this element by inserting DNA copies of the gypsy retrotransposon to chromosomes of Drosophila, but also excising them, gypsy is excised via a precise mechanism, with the original nucleotide sequence of the target site being completely restored. The obtained data demonstrate the existence of alternative ways of the transposition of retrotransposons and, possibly, retroviruses, including gypsy (mdg4).
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PMID:[Precise excision of long terminal repeats of the gypsy (mdg4) retrotransposon of Drosophila melanogaster detected in Escherichia coli cells is explained by its integrase function]. 1732 85

A novel transglycosylating beta-galactosidase was purified from Enterobacter agglomerans B1. It was a homodimer of approximately 248 kDa. The optimal pH and temperature for oNPGal hydrolysis were 7.5-8.0 and 37-40 degrees C, respectively. The K(m) values for oNPGal and lactose were 0.06 and 114 mM, respectively. The enzyme produced galacto-oligosaccharides in a 38% yield at the lactose concentration of 12.5% (w/v). When using oNPGal as donor, the enzyme was able to catalyze glycosyl transfer to a series of acceptors, including hexose, pentose, beta- or alpha-disaccharides, hexahydroxy alcohol, cyclitol, and aromatic glycosides. This suggested the enzyme to be a potential synthetic tool for preparing galactose-containing chemicals. The gene encoding this enzyme was cloned by degenerate PCR and TAIL-PCR. It revealed an ORF of 3090 nucleotides encoding a 1029 amino-acid protein, which had been expressed in Escherichia coli. Transferase activities in both recombinant and natural enzymes were similar.
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PMID:A novel beta-galactosidase capable of glycosyl transfer from Enterobacter agglomerans B1. 1733 32


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