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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)

Previously, we identified a class of genes in Dictyostelium that are prespore cell-type specific in their expression in the multicellular aggregate and are inducible by cAMP acting through cell-surface cAMP receptors. In this paper, we report the cloning and analysis of the regulatory regions controlling the expression of one such gene that encodes a spore coat protein, SP60. By use of a fusion of the firefly luciferase gene and Escherichia coli lacZ [expresses beta-galactosidase (beta-gal)], we have identified cis-acting regions required for proper spatial and temporal expression in multicellular aggregates and for cAMP induction in shaking cell culture. Deletion analysis suggests that a CA-rich element (CAE) and surrounding sequences present three times within the 5'-flanking sequence are required for proper regulation. SP60-lacZ fusions that include all three of these regions express lacZ only in the posterior approximately 85% of migrating slugs (prespore zone). Studies show that SP60 is expressed during mid to late aggregation, and SP60-lacZ-positive cells are spatially localized as a doughnut-shaped ring within the forming aggregate. Cells within the skirt that surrounds the aggregate and that are still migrating into the aggregate do not stain. Sequential 5' deletions of CAEs and surrounding regions affect the expression level of SP60-luciferase in response to developmental signals and cAMP, as well as the spatial pattern of SP60-lacZ. Deletion of the first (most 5') of these regions restricts the spatial expression of SP60-lacZ fusions to the anterior of the prespore zone. When both the first and second regions are removed, the expression level drops, and the staining is restricted to the prespore/prestalk boundary. Furthermore, the staining pattern that is seen with these two deletions is present as a gradient from anterior to posterior within the prespore zone. Deletion of all three regions results in a loss of both cAMP and developmentally induced expression. These results suggest the presence of a gradient within the prespore zone that differentially affects the activity of promoters containing different numbers of response elements.
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PMID:A spatial gradient of expression of a cAMP-regulated prespore cell-type-specific gene in Dictyostelium. 216 44

Measurements of cyclic phosphodiesterase, or of beta-galactosidase in the case of cpdB'-'lacZ fusions, indicate that cpdB expression in both Escherichia coli and Salmonella typhimurium is modulated by carbon source availability, consistent with previous observations in Salmonella. Nucleotide sequence analysis and transcription mapping of both cpdB genes have revealed, in their 5' flanking regions, sequences with good similarity to consensus -10 and -35 regions and cyclic AMP-cyclic AMP receptor protein (cAMP-CRP) binding sites. Furthermore, they are strongly conserved in both organisms. Deletion analysis of an E. coli cpdB'-'lacZ fusion supports the identification of these elements, and a role for the cAMP-CRP binding site in modulating constitutive cyclic phosphodiesterase expression.
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PMID:Transcription and regulation of the cpdB gene in Escherichia coli K12 and Salmonella typhimurium LT2: evidence for modulation of constitutive promoters by cyclic AMP-CRP complex. 217 62

A plasmid carrying a CRP-dependent promoter fused to the lac structural genes was manipulated to construct a set of spacing mutants that have varying lengths between the CRP binding site and the -35 region. The lengths of the spacer were changed over 45 bp by inserting or deleting nucleotides. DNase I footprinting analysis revealed that the spacer length did not affect the binding of cAMP-CRP to the CRP site. The effect of the spacer length on transcription activation by cAMP-CRP was tested in vivo by beta-galactosidase and quantitative S1 assays with crp+ and delta crp cells harboring plasmids. Insertions or deletions of non-integral helical turns, which displace the CRP site onto the opposite face of DNA helix compared to the original promoter, eliminated completely the activation of transcription. In contrast, changing the spacer length by integral helical turns allowed the promoter to respond to CRP, although the degree of activation varied with the length of the spacer. We conclude that stereospecific positioning of CRP and RNA polymerase on the DNA helix is strictly required for CRP action. The data support a model that CRP stimulates transcription by directly contacting RNA polymerase.
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PMID:Helical phase dependent action of CRP: effect of the distance between the CRP site and the -35 region on promoter activity. 217 26

The adenylyl cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cAMP from ATP, and two RAS polypeptides, responsible for stimulation of cAMP synthesis by guanine nucleotides. We have obtained rabbit antibodies that recognize the CYR1 protein. Antibodies were raised against synthetic oligopeptides and against a recombinant beta-galactosidase/CYR1 fusion protein. These antibodies have allowed the identification of the CYR1 gene product as a 205 kDa protein. Treatment with trypsin (2 micrograms/ml) reduced the size of the CYR1 protein from 205 to 155 kDa and produced an activated enzyme which no longer responded to guanine nucleotides. This result is consistent with a model in which adenylyl cyclase activity is regulated by an inhibitory domain near the amino-terminus of the CYR1 protein. This model is further supported by the finding that adenylyl cyclase activity is also markedly elevated and unresponsive to guanine nucleotides in mutant yeast strains that express only the carboxy-terminal half of the CYR1 protein. Treatment with high trypsin concentrations (greater than 10 micrograms/ml) caused release of adenylyl cyclase activity from the membrane. Comparison of immunoreactive CYR1 fragments released by trypsin and membrane bound genetically altered proteins suggests that the CYR1 protein is attached to the membrane via a separate trypsin sensitive anchoring protein rather than via a membrane anchoring domain.
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PMID:Adenylyl cyclase in yeast: antibodies and mutations identify a regulatory domain. 218 89

The Escherichia coli lac promoter mutation Pr115, an A X T to T X A transversion at +1 (the transcription initiation site of the lac wild-type and lac UV5 promoters), creates a new "-10 region"-like sequence starting at +1. We show that this mutation activates a new RNA polymerase binding site (P115) that overlaps with, and is shifted 12 base-pairs downstream from, the wild-type RNA polymerase binding site (P1). Nuclease S1 mapping studies and RNA polymerase protection experiments in vitro indicate that, in the absence of CAP-cAMP, this new site is used preferentially over the P1 site. In vivo, beta-galactosidase assays of the Pr115 mutation in combination with mutations of the P1 "-35 region" demonstrate that the P1 -35 region sequences are not involved in the interaction between RNA polymerase and P115 in the absence of CAP-cAMP; therefore P115 is an independent binding site. The presence of CAP-cAMP in vivo stimulates polymerase binding and initiation at P1, which serves to block polymerase from binding at P115.
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PMID:Lactose promoter mutation Pr115 activates an overlapping promoter within the lactose control region. 241 53

lambda gt11 phages harboring five different cDNA fragments for the regulatory (R) subunit of Dictyostelium discoideum cAMP-dependent protein kinase (CAK) directed the synthesis of this protein in Escherichia coli cells. Crude bacterial extracts were probed with an antiserum against the Dictyostelium R subunit. The presence of specific epitopes for the R subunit in a given extract was compared with high-affinity cAMP-binding activity and with the ability to inhibit the catalytic (C) subunit through protein-protein interaction. The expression and the biochemical properties of these proteins were correlated with their cDNA nucleotide sequence. The results show that the Dictyostelium R subunit can be functionally expressed in E. coli cells either as a fusion protein with beta-galactosidase or as a nonfusion protein. In both cases, the products of cDNA clones containing the entire coding sequence retained high-affinity cAMP-binding activity and the capacity to interact with the catalytic subunit. One of the fusions, lacking the 94 N-terminal residues, failed to inhibit catalytic activity, although it bound cAMP with an affinity similar to that of the native R protein from D. discoideum.
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PMID:Expression and properties of the regulatory subunit of Dictyostelium cAMP-dependent protein kinase encoded by lambda gt11 cDNA clones. 245 May 71

On the basis of Escherichia coli DNA and vectors pBR322, pUC19, hybrid plasmids restoring Udp+ phenotype in the E. coli deletion (delta udp) mutant have been obtained. The udp gene is carried by a 8 kb PstI fragment (on the pUD2) and by a smaller 2.87 kb PstI-SalGI fragment from the PstI fragment (pUD7). The uridine phosphorylase level was 30 times higher in the cells containing hybrid plasmid as compared to the strain with chromosomal location of the udp gene. On the other hand, the measurements of uridine phosphorylase activity in the cytR- and cya- background indicate that expression of the cloned udp gene escapes partially negative control of the CytR repressor and positive control of cAMP--CRP complex. These data suggest that the 2.87 kb PstI--SalGI-fragment contains the intact udp gene which is transcribed from its own promoter. Increase in the activity of beta-galactosidase encoded by udp-lacZ fusion has been observed in the presence of pUD2 or pUD7, which was suggested to be the consequence of titration of CytR repressor molecules in the operator region of the cloned udp.
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PMID:[Cloning the uridine phosphorylase (udp) gene of Escherichia coli and its expression in recombinant plasmids]. 251 86

Analysis of the induction of expression of cea-lacZ fusions in cya and crp mutants showed that catabolite repression affects the kinetics of induction and the rate of induced synthesis. In a cya mutant, addition of cAMP reduced the induction lag and increased the amount of beta-galactosidase produced. The CRP-cAMP complex was found to bind to two sites 5' to the cea promoter, but deletion analysis showed that only one of these was involved in the control of cea. Deletion of this site resulted in a loss of the stimulatory effects of cAMP in a cya mutant.
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PMID:Interaction of the CRP-cAMP complex with the cea regulatory region. 254 Apr 17

Interaction of negative (CytR) and positive (cAMP-CRP) control in the promoter region of the uridine phosphorylase (udp) gene of Escherichia coli has been studied by using udp-lac operon fusions in which the structural lacZ gene is expressed from the wild type promoter udpP+ or from mutant promoters udpP1 and udpP18. The specific activity of beta-galactosidase was examined in these fusions in cytR+ and cytR- backgrounds after introduction of specific mutations in crp locus, crp* and crp(a) altering interaction of CRP protein with catabolite-sensitive promoters. The data obtained using crp* mutation confirm the proposed model of the udp gene regulation, according to which CytR repressor protein interferes with CRP binding site in the promoter-operator region of the udp gene and thereby prevents the positive action of cAMP-CRP complex on the udp expression. Additional data in favor of this model were obtained using crp(a) mutation which most probably alters the structure of CRP protein in such a way that it exhibits more high affinity to the udp promoter, as compared to the CytR repressor protein. Indeed, taken by itself, the crp(a) mutation did not lead to any increase in the expression of udpP+-lac fusion under the conditions of cAMP limitation (on glucose-grown cells), in spite of whether or not the CytR repressor was present. However, when combined with the ptsG mutation or when cells were grown on succinate medium, complete constitutive expression of udpP+-lac fusion is observed, even in the presence of the cytR gene product. The effect of the crp(a) mutation was virtually the same in strains harboring udpP1-lac fusion. These data are in accordance with suggestion that udpP1 is a mutation in the site of the promoter-operator region that responds to the cytR gene product, while the corresponding binding site for CRP protein is still unaltered in this mutant. On the other hand, the crp(a) mutation causes only slight alteration in the expression of udpP18-lac fusion, providing additional evidence that udpP18 mutation seems to comprise a modification of the promoter-operator region, where binding sites for CRP and CytR proteins overlap.
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PMID:[Interaction of negative (CytT) and positive (cAMP-CRP) regulation in the promoter region of the uridine phosphorylase (udp) gene in Escherichia coli K-12]. 266 21

A series of plasmids were constructed in which a promoter was introduced into a lac-based operon fusion vector. A perfectly symmetrical oligonucleotide of 22-bp corresponding to an idealized binding site for cAMP receptor protein (CRP) of E. coli was chemically synthesized. The synthetic CRP site was placed between the promoter and the lacZ structural gene with varying distances from the promoter. Specific binding of cAMP-CRP complex to the synthetic CRP site was shown by a gel retardation and a DNase I footprinting assays. Plasmid constructs were transformed into crp+ and crp- cells carrying a chromosomal deletion of the lac genes. The regulatory effect of the inserted CRP site was examined by comparing the beta-galactosidase activity and the levels of RNA transcript in two cells harboring the plasmids. We found a strong inhibitory effect of the CRP site in the presence of cAMP and CRP when it was placed close to the promoter. When the CRP site was placed far downstream of the promoter, a moderate repression of transcription was observed.
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PMID:Regulatory effect of a synthetic CRP recognition sequence placed downstream of a promoter. 284 30


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