SEARCH · Search PubMed

Results for “Lac Operon”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources

PubMed1984

Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes.

We present an approach for determining the in vivo distribution of a protein on specific segments of chromosomal DNA. First, proteins are joined covalently to DNA by irradiating intact cells with UV light. Second, these cells are disrupted in detergent, and a specific protein is immunoprecipitated from the lysate. Third, the DNA that is covalently attached to the protein in the precipitate is purified and assayed by hybridization. To test this approach, we examine the cross-linking in Escherichia coli of RNA polymerase to a constitutively expressed, lambda cI gene, and to the uninduced and isopropyl beta-D-thiogalactoside (IPTG)-induced lac operon. As expected, the recovery of the constitutively expressed gene in the immunoprecipitate is dependent on the irradiation of cells and on the addition of RNA polymerase antiserum. The recovery of the lac operon DNA also requires transcriptional activation with IPTG prior to the cross-linking step. After these initial tests, we examine the distribution of RNA polymerase on the leucine operon of Salmonella in wild-type, attenuator mutant, and promoter mutant strains. Our in vivo data are in complete agreement with the predictions of the attenuation model of regulation. From these and other experiments, we discuss the resolution, sensitivity, and generality of these methods.

DNA, Bacterial↗

PubMed1992

Regulated expression of foreign genes in vaccinia virus under the control of bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor.

The gene encoding bacteriophage T7 RNA polymerase (T7gene1) was placed under the control of regulatory elements from the Escherichia coli lac operon to construct an inducible vaccinia virus expression system consisting entirely of prokaryotic transcriptional machinery. Regulated expression of T7 RNA polymerase was necessary to construct a stable recombinant vaccinia virus harboring a T7 promoter; otherwise, uncontrolled expression led to interference with endogenous virus replication. To this end, the gene encoding the repressor protein of the lac operon was fused to a viral early/late promoter so that it was expressed constitutively, and the lac operator was interposed between a viral major late promoter and T7gene1. Greater than 99% repression of T7 RNA polymerase, which was relieved approximately 80-fold in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), was obtained. An expression cassette containing a T7 promoter-controlled beta-galactosidase reporter gene was recombined into a different region of the viral genome containing T7gene1. A stable, double recombinant virus was isolated and grown to a high titer. In the absence of inducer, beta-galactosidase expression was substantially repressed. Addition of increasing amounts of IPTG induced expression of beta-galactosidase to the point of suppression of viral replication. This hybrid vaccinia virus system (Vac/Op/T7) has potential applications for the efficient bioproduction of a wide variety of gene products.

Base Sequence↗

PubMed1972

Dissociation of Lac messenger ribonucleic acid transcription from translation during recovery from inhibition of protein synthesis.

Studies were made of the synthesis of Lac messenger ribonucleic acid (mRNA) by Escherichia coli in the absence of protein synthesis and of the coupling of transcription of lac operon to translation. Lac mRNA was not synthesized in the presence of chloramphenicol, and its synthesis steadily decreased during K(+) deprivation and treatment with puromycin. Since under these conditions total mRNA synthesis is not inhibited it is suggested that the control of Lac mRNA is distinct from that which regulates total mRNA synthesis. Lac mRNA synthesized during recovery from K(+) starvation or from chloramphenicol inhibition is not translated into functional enzyme, suggesting translational control over beta-galactosidase synthesis.

Bacterial Proteins↗

PubMed1992

Mutations in topA interfere with the inducible expression of DNA damage response loci in Salmonella typhimurium.

Strains of Salmonella typhimurium deficient in topoisomerase I activity (topA mutants) are UV sensitive and non-mutable (Overbye and Margolin: J Bacteriol 146:170-178, 1981). Using lac-operon fusions to DNA damage inducible (din) loci we investigated whether these observations could be explained by an inability of topA strains to efficiently induce DNA damage responses. Mitomycin C (MMC)-induced expression of lac-operon fusions to uvrB and to a second SOS locus, din-9, was largely eliminated in topA bacteria. The inducible expression of several other din-fusions was also diminished. This inducibility defect was mimicked by growth of din-9 topA+ bacteria in media of high osmolarity, a condition that leads to increased DNA supercoiling. Inhibitors of DNA gyrase efficiently induced din-9 in topA bacteria. Together, these results suggest that the topA effect on din expression may be mediated at the level of DNA supercoiling. The sensitivities of a number of din-fusions to topA paralleled the degree to which they were repressed by excess LexA, suggesting that mutations in topA might influence LexA-operator interactions and/or increase lexA expression.

Bacterial Proteins↗

PubMed1983

Mechanism of F factor-enhanced excision of transposon Tn5.

The reversion of lac:: Tn5 insertion mutations was used to examine the control of excision of the kanamycin-resistance transposon Tn5 in Escherichia coli. Earlier work which showed that the fertility factor F enhances Tn5 excision had led another group to suggest that this is due to the product of a putative transposable element-specific "recombination" gene in the F factor which can act on Tn5 located anywhere in the genome. We show, however, that Tn5 is excised from sites in the lac operon of F'lac plasmids several orders of magnitude more efficiently than from the same sites in the chromosomes of F-, F+ or homozygous lac:: Tn5[F'lac:: Tn5] strains. Thus F enhances Tn5 excision, but only if F and Tn5 are in cis in the same DNA molecule. Bacterial crosses showed that transfer of F'lac:: Tn5 plasmids by conjugation stimulates Tn5 excision, and that transfer is frequent even within F' populations. These results suggest that the ability of F to enhance excision is the consequence of DNA transfer in conjugation.

Conjugation, Genetic↗

PubMed1998

Detection of natural bioantimutagens and their mechanisms of action with bacterial assay-system.

Escherichia coli K12 assay-system is designed in order to detect bioantimutagens, agents preventing mutagenesis by modulation of DNA repair and replication. The assay is composed of four tests aimed at the detection of inhibition of spontaneous and induced mutations (Tests A and B) and at the estimation whether the anti-mutagenic agent acts by increasing the fidelity of DNA replication (Test B), by inhibition of SOS error prone repair (Test C), or by favoring error-free recombinational repair (Test D). In Test A, repair proficient strain and its uvrA counterpart are used for detection of spontaneous and UV-induced mutations, while in Test B mismatch repair deficient strains (mutH, mutS, mutL and uvrD) are used for amplified detection of spontaneous mutations caused by replication errors. In Test C, repair proficient strain carrying sfiA::lacZ fusion is used for measuring the level of SOS induction by monitoring the level of beta-galactosidase. In Test D, the strains carrying different recA alleles (recA+, recA730 and DeltarecA) are used for measuring intrachromosomal recombination between nonoverlapping deletions in duplicated lac operon, by monitoring Lac+ recombinants. The assay-system is validated with model bioantimutagens and used for detection of anti-mutagenic potential of different terpenoid fractions from sage (Salvia officinalis L.). Extract E1/3 of cultivated sage, distinguished from others by its high content of monoterpenoid camphor, reduces UV-induced mutagenesis in Test A, while it has no effect in Tests B and C. In Test D, it enhances intrachromosomal recombination in untreated and UV-irradiated recA+ and recA730 strains. The results suggest that the protective effect is due to stimulation of recombinational repair, similarly to coumarin. We speculate that monoterpenoids from sage enhance genetic recombination by intervening in a formation of RecA-DNA complex and channeling it into recombination reaction.

Antimutagenic Agents↗

PubMed2006

Modeling the Lac repressor-operator assembly: the influence of DNA looping on Lac repressor conformation.

Repression of transcription of the Escherichia coli Lac operon by the Lac repressor (LacR) is accompanied by the simultaneous binding of LacR to two operators and the formation of a DNA loop. A recently developed theory of sequence-dependent DNA elasticity enables one to relate the fine structure of the LacR-DNA complex to a wide range of heretofore-unconnected experimental observations. Here, that theory is used to calculate the configuration and free energy of the DNA loop as a function of its length and base-pair sequence, its linking number, and the end conditions imposed by the LacR tetramer. The tetramer can assume two types of conformations. Whereas a rigid V-shaped structure is observed in the crystal, EM images show extended forms in which two dimer subunits are flexibly joined. Upon comparing our computed loop configurations with published experimental observations of permanganate sensitivities, DNase I cutting patterns, and loop stabilities, we conclude that linear DNA segments of short-to-medium chain length (50-180 bp) give rise to loops with the extended form of LacR and that loops formed within negatively supercoiled plasmids induce the V-shaped structure.

Bacterial Proteins↗

PubMed1999

Functional and biochemical characterization of Escherichia coli sugar efflux transporters.

A family of bacterial transporters, the SET (sugar efflux transporter) family, has been recently reported (Liu, J. Y., Miller, P. F., Gosink, M., and Olson, E. R. (1999) Mol. Microbiol. 31, 1845-1851). In this study, the biochemical and cell biological properties of the three Escherichia coli members (SetA, SetB, and SetC) of the family are characterized. We show that both SetA and SetB can transport lactose and glucose. In addition, SetA has broad substrate specificity, with preferences for glucosides or galactosides with alkyl or aryl substituents. Consistent with the observed in vitro substrate specificities, strains that hyperexpress SetA or SetB are desensitized to lactose analogues as measured by induction of the lac operon. In addition, strains that hyperexpress SetA are resistant to the growth inhibitory sugar analogue o-nitrophenyl-beta-D-thiogalactoside. Strains disrupted for any one or all of the set genes are viable and show no defects in lactose utilization nor increased sensitivity to inducers of the lac operon and nonmetabolizable sugar analogues. The data suggest that the set genes are either poorly expressed under normal laboratory growth conditions or are redundant with other cellular gene products.

Biological Transport↗

PubMed2002

Intracellular cyclic AMP concentration is decreased in Salmonella typhimurium fur mutants.

It is known that the Fur protein negatively regulates iron-uptake systems in different bacterial species, including Salmonella typhimurium. In this study it has been shown that the intracellular concentration of cyclic AMP (cAMP) is lower in a knockout S. typhimurium fur mutant than in the wild-type strain. According to this, the expression of two cAMP-regulated genes, such as pepE (encoding an alpha-aspartyl dipeptidase) and the Escherichia coli lac operon, is decreased in S. typhimurium fur cells in comparison with wild-type cells. Introduction of an additional mutation in cpdA, encoding a cyclic 3',5'-cAMP phosphodiesterase, recovers wild-type intracellular cAMP concentration in the S. typhimurium fur mutant. Likewise, expression of pepE and the E. coli lac operon was the same in the S. typhimurium fur cpdA double mutant and the wild-type strain. Moreover, these results also demonstrate that the S. typhimurium Fur protein positively regulates the expression of the flhD master operon governing the flagellar regulon. This positive control must be mediated by binding of the S. typhimurium Fur protein to the flhD promoter as indicated by the fact that this promoter tests positive in a Fur titration assay.

Aspartic Acid Endopeptidases↗

PubMed1988

Genetic determinants for catabolite induction of antibiotic biosynthesis in Pseudomonas fluorescens HV37a.

Antibiotic biosynthesis is regulated by glucose in Pseudomonas fluorescens HV37a. Fusions between antibiotic biosynthetic operons (afu operons) and the Escherichia coli lac operon were isolated to evaluate the genetic determinants for the regulation of antibiotic biosynthesis. Four afu transcriptional units were defined, afuE, afuR, afuAB, and afuP. The afuE and afuR transcripts were promoted divergently at one locus and were catabolite induced, by 250-fold and 5-fold, respectively; the afuAB and afuP transcriptional units were not linked to the others and were not catabolite induced. Thus, regulation of afuE and afuR operon transcription is apparently the mechanism whereby glucose regulates antibiotic biosynthesis. Catabolite induction of the afuE and afuR transcriptional unit was dependent on the products of the afuA, afuB, and afuP genes. Expression of the afuE transcriptional unit was altered quantitatively in afuE mutants. Apparently the afuE transcriptional unit is regulated, at least in part, by its own gene products. Under inducing conditions, expression of the afuE, afuR, and afuP transcriptional units increased rapidly during a 6-h period.

Antifungal Agents↗

PubMed1984

Use of a cloned bacteriophage gene to disrupt bacteria.

A plasmid, pUH51, was constructed, which contains the lysis gene E of bacteriophage phi X174, subjected to the regulatory region of the lac operon, as well as the lac repressor gene. This plasmid can readily replicate in any strain of E. coli and mediates lysis of the bacteria after induction of the cloned phi X174 gene E. Taking advantage of these properties, plasmid pUH51 was used as a tool for gentle disruption of E. coli. At cell concentrations below 5 X 10(10)/ml, the efficiency of this method, as measured by release of beta-galactosidase from the cells, exceeded the efficiency of conventional methods for cell breakage.

Alleles↗

PubMed1989

Insertional mutagenesis using a synthetic lac operator.

We have developed a novel cassette for generating insertion mutants in multi-copy bacterial plasmids. The cassette consists of synthetic oligodeoxyribonucleotides (oligos) which form a DNA duplex following reconstitution in vitro, due to sequence complementarity. It contains a 21-bp segment of the lac operator (lacZo), to provide a readily detectable phenotypic marker. Bacterial colonies harboring plasmids with insertions of this cassette are blue due to constitutive expression of the lac operon resulting from titration of lac repressor molecules by plasmid-borne lacZo sequences. Synthetic oligos containing a desire sequence may be added to the cassette by complementary ends for targeted insertion into plasmids. Sequencing of the resulting insertion mutants is facilitated by using oligos within the cassette as primers for bidirectional sequencing. This allows a complete characterization of each insertion in terms of location, structure of flanking sequences, and orientation of the inserted oligo. We have used this system to construct a series of mutants in early region 1a genes of human adenovirus type 5. For this purpose we designed a cassette which had all three possible translational reading frames open when inserted in one orientation, and all reading frames closed in the other orientation. The cassette also had BamHI restriction sites at each end which could be used to 'collapse' mutants, reducing the size of each insert to 6 bp.

Base Sequence↗

PubMed1985

Effects of heterologous ribosomal binding sites on the transcription and translation of the lacZ gene of Escherichia coli.

A vector (pKL203) was constructed which contains the promoter-operator region of the lacZ gene and the major part of the coding sequence of the lac operon. The lacZ translation initiation signals [Shine-Dalgarno (SD) sequence and AUG codon] were deleted, and in their place a synthetic linker sequence was inserted, providing single restriction sites for SmaI and BamHI. With this vector constructions were made in which initiation signals of other prokaryotic genes (phage MS2 maturation protein, phage Q beta A2 gene and tufB gene) were fused to the lacZ gene, giving rise to various fusion proteins. The introduction of N-terminal amino acids (aa) in beta-galactosidase (beta-gal) which differ from the wild-type aa invariably leads to an enzyme with a strongly reduced thermostability as compared to the wild-type enzyme. Therefore an immunoprecipitation method was used to measure the amount of fusion protein. It was found that these amounts varied strongly from one construction to another. Concomitant determinations of the amounts of lac-operon-specific mRNA showed an unexpectedly large variation among the clones. No strict correlation could be found between the level of lac mRNA and beta-gal production. Per molecule of lac mRNA, translation appears to be most efficient when the homologous lacZ initiation signal is present.

Binding Sites↗

PubMed1984

A genetic switch in vitro: DNA inversion by Gin protein of phage Mu.

Inversion of the G segment in the DNA of Escherichia coli phage Mu depends on the Mu Gin protein and alters the host range of the phage. The frequency of the inversion reaction is low both in the lysogenic state and during lytic growth. A sensitive assay was developed to detect low levels of G inversion: the E. coli lac operon was inserted within the invertible G segment in such a way that the lac operon was expressed only by G(-) clones. As a result Gin-catalyzed inversion from G(+) to G(-) can be monitored as a lactose-negative to lactose-utilizing switch. Using a crude extract from a Gin-overproducing strain and this assay plasmid, we could detect a low level of G inversion in vitro (1% in 30 min). The reaction depends on Mg2+ and a supercoiled substrate. Under optimized reaction conditions over 15% of the plasmids had the G segment inverted after incubation with Gin in vitro. The inversion was then visualized by agarose gel analysis of plasmid DNA digested by restriction endonucleases. The Gin protein retains its catalytic properties upon partial purification. The mechanism of this genetic switch can now be studied in vitro.

Bacteriophage mu↗

PubMed2003

LacSwitch II regulation of connexin43 cDNA expression enables gap-junction single-channel analysis.

Metabolic and electrical coupling through gap junction channels is implicated in cell differentiation, tissue homeostasis, and electrotonic propagation of signals in excitable tissues. The characterization of gating properties of these channels requires electrophysiological recordings at both single- and multiple-channel levels. Hence, a system that is able to control connexin expression by external means would provide a useful tool. To regulate the expression of connexins in cells, plasmids encoding a transactivator and/or a lac-operon IPTG response-dependent Cx43 target gene were transfected into communication-deficient N2a neuroblastoma cells. Immunoblotting, dye coupling, and electrophysiological methods revealed that expression of Cx43 in selected clones could be tightly regulated. After 15-20 h of acute induction with IPTG, cell-to-cell communication reached its peak with junctional conductances of 15-30 nS. Chronic induction at specific doses of IPTG produced constant, controlled levels of Cx43 expression, which were reflected by predictable junctional coupling levels. These conditions allowed prolonged recordings from either lowly or highly coupled cells, making lac operon an ideal regulatory system for channel gating studies at a single-channel level.

Animals↗

PubMed1990

The cyanase operon and cyanate metabolism.

Cyanase is an inducible enzyme in E. coli that catalyzes bicarbonate-dependent decomposition of cyanate. It is encoded as part of an operon we have named the cyn operon, which includes three genes in the following order: cynT (cyanate permease), cynS (cyanase), and cynX (protein of unknown function). The direction of transcription is opposite to that of the lac operon, and the 3'-end of the cyn operon overlaps the 3'-end of the lac operon by 98 nucleotides. The gene cynR (regulatory protein) is located upstream from the cyn operon, and its transcription is opposite that of the cyn operon. The genes of the cyn operon and the cynR gene have been cloned, sequenced and over-expressed. Cyanate at concentrations of about 1 mM is toxic to strains of E. coli lacking the cyanase gene, but strains in which the inducible gene for cyanase is present can grow on cyanate as the sole source of nitrogen at concentrations as high as 20 mM. The presence of cyanase itself is not sufficient to overcome cyanate toxicity--the permease must also be present. Strains lacking the cyanase gene, but having a functional permease gene, are extremely sensitive to cyanate. Uptake of cyanate involves the product of the permease gene in an energy-dependent process. It appears that the cyn operon has evolved to function in detoxification/decomposition of cyanate arising from both intra- and extracellular sources.

Aminohydrolases↗

PubMed1984

Location and analysis of nucleotide sequences at one end of a putative lac transposon in the Escherichia coli chromosome.

A segment of Escherichia coli DNA that contained a discontinuity of homology with Salmonella typhimurium DNA was isolated. The segment, 1,430 base pairs long, was derived from one end of the lac "loop," a region of about 12 kilobase pairs of E. coli DNA, including the lac operon which has no detectable homology with S. typhimurium DNA (K. Lampel and M. Riley, Mol. Gen. Genet. 186:82-86, 1982). The nucleotide sequence of the 1,430-base-pair segment of DNA was determined. The location of the junction of discontinuity of homology within the segment was established by hybridization experiments. Nucleotide sequences at or near the junction were determined to be similar to sequences that are involved in site-specific inversion in S. typhimurium, E. coli, phage P1, and phage Mu. Similar sequences are also present within the terminal inverted repeat sequences of transposon Tn5 and at the V-D-J joining sequences of eucaryotic immunoglobulin genes. Therefore, the lac operon, together with flanking DNA, may have been inserted into the E. coli chromosome at one time via a site-specific recombination event. Rearrangement events of this kind undoubtedly have played a significant role in the evolutionary divergence of chromosomal DNAs.

Base Composition↗

PubMed1991

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.

Blotting, Southern↗