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At least 703 records · Page 39Linked to original sources
PubMed1990

[Construction of recombinant genes coding for products containing the fragment of Staphylococcus aureus protein A and the fragment of Pseudomonas aeruginosa exotoxin A].

The genes coding for Pseudomonas aeruginosa exotoxin-A and Staphylococcus aureus A-protein have been cloned. Different fragments of the genes were subcloned on the plasmid vestors. The plasmids pAPA4 and pAPA42 have been constructed. The plasmids are coding for the hybrid protein which consists of the immunoglobulin binding A-protein domain at its NH2-terminus and the catalytically active fragment of exotoxin-A at its COON-terminus. The hybrid gene is expressed in Escherichia coli cells under the control of lambda cro-gene expression elements (pAPA42) or lac-operon expression elements (pAPA4). The latter proved to be most productive.

ADP Ribose Transferases↗
PubMed1989

[Structure of the recombination zone during abnormal excision of the transducing bacteriophage lambda plac9].

Investigating molecular mechanism of illegitimate recombinations in prokaryote we study transducing bacteriophages of the lambda lac series. We have carried out physical mapping of bacteriophage lambda plac9 DNA and, by comparing the obtained results with the data on the structure of lambda DNA and lac operon of E. coli, located the phage-bacterial junction corresponding to the lambda-lac9 abnormal excision and elucidated the nucleotide sequence around the junction. It led to the primary structure of phage and bacterial segments in the lysogenic bacterium which took part in the recombinational act leading to the abnormal excision and lambda lac9 formation. Structural homology of the partners in the lambda plac9 excision proved to be lower than in case of the earlier studied lambda plac5 and lambda plac10 whose excision proceeded regioselectively. Various aspects of the crossover area, including the crossover point's probable position and enzymic systems participating in the abnormal excision, are discussed.

Bacteriophage lambda↗
PubMed1989

[Mutants of Escherichia coli K-12 with altered excision efficiency of transposons Tn5 and Tn9].

HFETn5, HFETn9 and LFETn9 mutants of Escherichia coli K-12 have been isolated. The frequency of Tn5 precise excision from the chromosomal lac operon is increased 3-660-fold in nine HFETn5 mutants. The majority of these mutations have no influence on the efficiency of precise excision of transposon Tn9, though hfeTn5-04 and hfeTn5-06 mutations decrease excision efficiency 2-13-fold. The Tn9 transposon is excised in HFETn9 mutant about 20-fold more efficiently than in the wild type strain. This mutation does not stimulate excision of Tn5 and Tn10. LfeTn9 mutation decreases excision frequency of Tn9 11-17-fold, but has no effect on Tn5 excision and increases that of Tn10 about 20-fold. The differences in genetic control and mechanisms of excision of the transposons with long and short inverted repeats are discussed.

DNA Transposable Elements↗
PubMed1988

[SOS-induction of the RP4 plasmid tet-determinant].

Induction of the tetracycline-resistance genes function by the inducer of the DNA-repair and mutability SOS-system, UV-light, has been tested. Activity of the tet-genes residing on the plasmid RP4 in Escherichia coli cells has been shown to be inducible by the low doses of tetracycline as well as by the mutagenic doses of UV-light. The induction was quantitatively registered by measuring the activity of beta-galactosidase of bacteriophage Mud1 (Ap, Lac) inserted into the tet-genes of the plasmid RP4. The bacteriophage integration inactivates the tet-genes function of the constructed plasmid fusing the lac-operon to a promoter of inactivated genes. Precise excision of bacteriophage restores the activity of the tet-genes proving together with the plasmid DNA-restriction analysis the fusion of tet-promoter with Iac-operon. The tet-genes of RP4 are concluded to be a part of the SOS-regulon, a set of genes inducible by the conditions harming the bacterial cell. Preliminary data on the mutagenic activity of tetracycline obtained in the bacterial test-system of mutagens are discussed.

DNA Repair↗
PubMed1986

[Isolation and properties of recombinant DNA from a plasmid and filamentous phage].

In the course of studying extrachromosomal DNA with composite replicons, a hybrid has been constructed by the in vitro recombination of the filamentous phage M13mp2 DNA (RF) and plasmid pUR222 (ApR). Both parental DNAs contain a fragment of lac-operon (ca. 800 bp), which includes the distal end of lacI gene, lacPO segments, and the lacZ gene proximal region coding for 145 N-terminal amino acid residues of beta-galactosidase and thus providing for alpha-complementation, the effect being cancelled with a polynucleotide insertion at the unique EcoRI site in the lacZ gene segment. E. coli BMH71-18 cells were transformed with the ligated mixture of EcoRI restricts of both DNAs. A phage-like nucleoprotein was isolated from colourless plaques (on the Xgal- and IPTG-supplemented medium); its deproteinization yielded a DNA which contains the ApR-determinant and, according to PAGE, structurally specific staining, restriction analysis, sequencing by the Sanger procedure, and electron microscopy data, is a linear double-stranded molecule comprising the phage and plasmid genomes in an equimolar ratio. Since the hybrid DNA does not display the alpha-complementation effect, both bacterial inserts are in the opposite orientation. Transformation of both phage (F+) and plasmid (F-) hosts with the hybrid DNA led to cultures which, after precipitation of the nucleoprotein from the extracellular medium and deproteinization, afforded the same composite DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Mapping↗
PubMed1988

Expression of rat and mouse cathepsin B precursors in Escherichia coli.

Vectors for the expression of mouse preprocathepsin B and rat procathepsin B, under the control of a modified lac operon regulatory region, in plasmid pKK233-2 were constructed. In rapidly growing cells induction of preprocathepsin B was cytotoxic. Immune precipitable cathepsin B, compatible in size with that expected for the unglycosylated proenzyme, was produced in both constructs. However, pepsin digestion of extracts did not produce any cathepsin B activity, indicating either that the recombinant forms are not correctly folded or were produced at such low levels that activity could not be detected.

Animals↗
PubMed1985

Amplified expression of streptomyces endo-beta-N-acetylglucosaminidase H in Escherichia coli and characterization of the enzyme product.

The endo-beta-N-acetylglucosaminidase H (Endo H) gene from Streptomyces plicatus has been cloned into the Escherichia coli plasmid pKC30 (Shimatake, H., and Rosenberg, M. (1981) Nature 272, 128-132), thus placing expression of this gene under control of the strong lambda promoter pL. The construction, pKCE3, which includes a properly positioned E. coli ribosome binding site from the lac operon (Robbins, P.W., Trimble, R. B., Wirth, D.F., Hering, C., Maley, F. Maley, G. F., Das, R., Gibson, B.W., and Biemann, K. (1984) J. Biol. Chem. 259, 7577-7583), was used to transform an E. coli strain lysogenic for a lambda prophage containing a temperature-sensitive repressor. By shifting cultures of pKCE3 lysogens to 42 degrees C, the production of Endo H commenced and was linear for about 1 h. Enzyme yields were amplified 150-fold above those obtained from comparable cultures of S. plicatus and represented 3 to 4% of total cellular protein, which enabled purification of Endo H to homogeneity by a rapid fourstep procedure. Although most of the cloned Endo H was secreted into the periplasmic space by E. coli, its 4 kDa leader sequence peptide (Robbins et al. (1984] was only partially removed during processing. As a result the purified pKCE3 Endo H was a heterogeneous population of molecules with an average molecular mass of 31 kDa compared to the 28.9 kDa fully processed product normally secreted by S. plicatus. Despite the residual approximately 2 kDa of leader sequence on the cloned pKCE3 product, there were no detectable differences in either the substrate specificity or the stability characteristics of the enzyme purified from E. coli or from S. plicatus. Of particular value for studies on glycoproteins was the finding that the genetically engineered Endo H was completely free of proteolytic contaminants.

Acetylglucosaminidase↗
PubMed1984

Nucleotide sequence and expression of the Escherichia coli dapB gene.

The Escherichia coli dapB gene encodes dihydrodipicolinate reductase. This enzyme is part of the diaminopimelate-lysine pathway, and its synthesis is repressed by lysine. The dapB gene was cloned into pBR322 from a transducing lambda bacteriophage, its complete nucleotide sequence established, and the transcriptional start localized. The DNA sequence predicts that the dapB gene codes for a 273-amino acid polypeptide, Mr 28,798. No attenuation-type sequence can be found between the mRNA start and the coding sequence. The dapB promoter signals appear to be weak as compared to RNA polymerase consensus sequences. Nevertheless an efficient in vivo synthesis of beta-galactosidase was obtained when the lac operon was inserted in vitro in the dapB gene, downstream of the dapB regulatory signals. Further studies were performed on an in-frame gene fusion constructed in vitro between the dapB and the lacZ genes. They indicated that repression by lysine is exerted on a DNA region restricted to a 153-base pair fragment with only 102 nontranscribed nucleotides. Finally, dapB gene expression showed a gene dosage effect which suggests that it is not controlled by an element present in limiting amounts in the cell.

Amino Acid Sequence↗
PubMed1983

Tandem promoters in the gene for ribosomal protein S20.

Examination of the nucleotide sequence of the gene for ribosomal protein S20 (rpsT) of Escherichia coli suggested the presence of two promoters ("sites 1 and 2") separated by 90 base pairs (Mackie, G. A. (1981) J. Biol. Chem. 256, 8177-8182). We have investigated the properties of purified or cloned DNA fragments containing one or other or both these sites for their ability to promote transcription in vivo and in vitro. In reactions in vitro containing DNA and purified RNA polymerase as the sole macromolecular components, both sites 1 and 2 act as promoters directing the synthesis of "runoff" transcripts. The 5' termini of such transcripts have been determined by direct sequencing or by identification of the 5' terminal nucleoside 5'-triphosphate, 3'-monophosphate. In site 1, the major transcript initiates with GTP at residue 141 in the DNA sequence. A minor start occurs at residue 142 and uses CTP as the initiating nucleotide. In site 2, the major transcript (approximately 55% of all initiations in site 2) initiates with CTP at residue 232 while minor transcripts, each comprising approximately 20% of the total, initiate at residues 231 and 233 with GTP and CTP, respectively. In four methods of assay which reflect to varying extents the usage of promoters in vivo, site 1 is responsible for 10-30% of the total transcription of the gene for S20 and site 2 the remainder. Sites 1 and 2 appear to act independently and additively in assays based on the rate of synthesis of S20 in a system for coupled transcription and translation. Together, the two promoters for S20 are from 10-25-fold more active than the fully induced lac operon promoter.

Base Sequence↗
PubMedDate not supplied

[Catabolyte repression of Escherichia coli K12 mutants with defects in different systems of glucose transport].

The phenomenon of glucose catabolite repression was studied in E. coli mutants inable to transport this carbohydrate. The pts 1, H mutant P34 was much less sensitive to the repressive effect of glucose on beta-galactosidase synthesis than the parent type. The 1103 mutant devoid of enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) behaves in the same way as P34 mutant after addition of glucose to casamino acid mineral medium. However, in minimal medium with succinate as the sole source of carbon, cells of the 1103 mutant show enhanced sensibility to transient glucose repression. The effect of hypersensibility disappears when the lac I mutation leading to constitutive the beta-galactosidase synthesis is introduced in 1103 mutant. It is shown that the enhanced sensibility of beta-galactosidase synthesis to glucose transient repression in 1103 mutant is an effect of the aburpt decrease in its growth rate in the presence of succinate and most probably this decrease leads to "inducer exclusion" of the lac operon. It is also shown that if one introduces the P34 mutation in strain JD3 devoid of one of the enzymes II for glucose (and due to this resistant to glucose catabolite respression) then the level of resistance in double mutant does not increase in spite of considerable supression of 14C glucose accumulation. In connection with this the role is discussed of separate components of the E. coli K 12 glucose transport system in realization of the phenomenon of catabolite repression.

Biological Transport, Active↗
PubMed1993

Plasmid rescue from transgenic mouse DNA using LacI repressor protein conjugated to magnetic beads.

A method for the efficient rescue of lac operator containing plasmids from transgenic mouse genomic DNA is described. The method is based on the high affinity of the LacI repressor protein for the lac operator sequence. Using the LacI repressor protein conjugated to magnetic beads, more than 95% of plasmid sequences could be purified from restriction enzyme digested genomic DNA. After circularization, the plasmids were introduced into Escherichia coli by means of electroporation. Since the plasmid was cloned into a bacteriophage lambda vector, the efficiency of plasmid rescue could easily be compared with in vitro packaging. Our results indicate that plasmid rescue is about 25 times more efficient. Application of this method should be especially useful with transgenic mouse models harboring LacZ plasmid shuttle vectors for studying spontaneous or induced mutations in vivo.

Animals↗
PubMed1996

High resolution mapping of E.coli transcription elongation complex in situ reveals protein interactions with the non-transcribed strand.

We have used chemical probes and UV light to perform a high resolution mapping of an Escherichia coli transcription elongation complex that was arrested in vivo by a protein readblock at a position distal to the promoter. The in situ probing data provide a precise picture of a constrained ternary complex in which the front edge of the polymerase is located at <6 bp from the catalytic center. Furthermore, our analyses reveal protein contacts with the non-transcribed strand within the arrested ternary complex. Thus, these results contribute substantially to the emerging view of a flexible transcription elongation complex in which the non-transcribed strand is an important regulatory element.

Acetaldehyde↗
PubMed1990

Two different mechanisms for urea action at the LAC and TNA operons in Escherichia coli.

Urea, at concentrations which do not interfere with bacterial growth, specifically inhibits the expression of catabolite sensitive operons. To search for the target and the mechanism of urea action we measured lactose (lac) and tryptophanase (tna) specific mRNA synthesis in vivo and in vitro. We show that urea acts by two different mechanisms at these two catabolite sensitive operons, resembling the manner in which catabolite repression regulates lac and tna. At the lac promoter, urea abolishes transcription initiation or blocks an early step in mRNA elongation without interfering with the binding of RNA polymerase and catabolite gene activator protein (CAP). At the tna promoter, urea does not abolish transcription initiation but could interfere with tnaC translation.

Cyclic AMP Receptor Protein↗
PubMed1984

Molecular cloning and sequence analysis of trp-lac fusion deletions.

DNA fragments containing deletions that fuse the trp operon to the lac operon were cloned and the end-points of the fusions were determined. The results from DNA sequence analysis correlated well with those from genetic, biochemical and physiological studies previously reported. The sequence data from this study, in combination with the known properties of these fusion strains, provided information on: (1) the precise lac operon distal boundary of the lac operator; (2) the nature of the trp operon terminator; and (3) the messenger RNA sequences that result in inhibition of lacZ translation initiation in trp-lac fused mRNAs.

Amino Acid Sequence↗
PubMed1980

Fusion of the promoter region of rRNA operon rrnB to lac Z gene.

A Lambda phage was constructed in which the structural gene for beta galactosidase is fused to a DNA segment carrying the ribosomal promoter rrnB of E. coli. In this hybrid operon beta galactosidase synthesis in vitro is repressed by ppGpp. Repression of beta galactosidase synthesis by cAMP is reported.

Bacteriophage lambda↗
PubMed1985

Identification of the merR gene of R100 by using mer-lac gene and operon fusions.

Transcriptional (operon) and translational (gene) fusions between the R100 merR gene and lacZ were constructed in vitro in a pBR322 plasmid carrying the mer genes derived from plasmid R100. The translational fusions were oriented in the opposite direction to and divergently from the merTCAD genes. This shows that the reading frame previously thought to be merR was incorrect. Expression of the gene fusion was repressed in trans by a compatible plasmid carrying the R100 merR+ gene, as was a similarly oriented transcriptional fusion. In contrast, expression of beta-galactosidase by the lac fragment located at the same site but in the opposite orientation was at a lower level and was not repressed by merR+.

DNA Restriction Enzymes↗
PubMed1980

Lactose operon transcription from wild-type and L8-UV5 lac promoters in Escherichia coli treated with chloramphenicol.

In cells treated with chloramphenicol and the inducer isopropyl-beta-D-thiogalacto-pyranoside, messenger ribonucleic acid transcription from the wild-type lac promoter was not detected. Transcription occurred from the mutant UV5-L8 promoter. The transcripts were of variable length; some included the whole Z gene. No major site of transcription arrest within the Z gene was apparent.

Chloramphenicol↗