scholarly journals Chimeric rpoC'- lacZ' gene of the recombinant pUC19 plasmid reserving the β-galactosidase activity in Escherichia coli cells

1987 ◽  
Vol 3 (5) ◽  
pp. 276-279 ◽  
Author(s):  
E. B. Paton ◽  
A. N. Zhyvoloup
Keyword(s):  
Escherichia Coli ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
Escherichia Coli Cells ◽  
Beta Galactosidase

Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica

1990 ◽  
Vol 10 (9) ◽  
pp. 4795-4806
Author(s):  
J W Xuan ◽  
P Fournier ◽  
N Declerck ◽  
M Chasles ◽  
C Gaillardin
Keyword(s):  
Yarrowia Lipolytica ◽  
Stop Codon ◽  
Open Reading Frames ◽  
Yeast Cells ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
Phenotypic Effect ◽  
Frame Fusion ◽  
Beta Galactosidase ◽  
Reading Frames

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

Eight UCA codons differentially affect the expression of the lacZ gene in the divE42 mutant of Escherichia coli

2000 ◽  
Vol 46 (6) ◽  
pp. 577-583 ◽  
Author(s):  
Takashi Kubo ◽  
Toshiko Aiso ◽  
Reiko Ohki
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Temperature Sensitive ◽  
Lacz Gene ◽  
Permissive Temperature ◽  
Wild Type ◽  
Double Mutation ◽  
Mutant Genes ◽  
Beta Galactosidase ◽  
Serine Codons

In the divE mutant, which has a temperature-sensitive mutation in the tRNA1Ser gene, the synthesis of beta-galactosidase is dramatically decreased at the non-permissive temperature. In Escherichia coli, the UCA codon is only recognized by tRNA1Ser. Several genes containing UCA codons are normally expressed at 42°C in the divE mutant. Therefore, it is unlikely that the defect is due to the general translational deficiency of the mutant tRNA1Ser. In this study, we constructed mutant lacZ genes, in which one or several UCA codons at eight positions were replaced with other serine codons such as UCU or UCC, and we examined the expression of these mutant genes in the divE mutant. We found that a single UCA codon at position 6 or 462 was sufficient to cause the same level of reduced beta-galactosidase synthesis as that of the wild-type lacZ gene, and that the defect in beta-galactosidase synthesis was accompanied by a low level of lacZ mRNA. It was also found that introduction of an rne-1 pnp-7 double mutation restored the expression of mutant lacZ genes with only UCA codons at position 6 or 462. A polarity suppressor mutation in the rho gene had no effect on the defect in lacZ gene expression in the divE mutant. We propose a model to explain these results.Key words: divE gene, tRNA1Ser, lacZ gene expression, UCA codon.

scholarly journals Kinetics and mechanism of the bactericidal action of human neutrophils against Escherichia coli

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 635-641
Author(s):  
MN Hamers ◽  
AA Bot ◽  
RS Weening ◽  
HJ Sips ◽  
D Roos
Keyword(s):  
Escherichia Coli ◽  
Computer Program ◽  
Rate Constants ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Human Neutrophils ◽  
Galactosidase Activity ◽  
E Coli ◽  
Bacterial Proteins ◽  
Beta Galactosidase

A mutant strain of Escherichia coli (E. coli ML-35) was used to follow the kinetics of phagocytosis, perforation of the bacterial cell envelope, and inactivation of bacterial proteins by human neutrophils. This particular E. coli mutant strain has no lactose permease, but constitutively forms the cytoplasmic enzyme beta-galactosidase. This implies that the artificial substrate ortho-nitrophenyl-beta-D- galactopyranoside cannot reach the beta-galactosidase unless the bacterial cell envelope has been perforated. Thus, the integrity of the E. coli envelope can be measured simply by the activity of beta- galactosidase with this substrate. Indeed, ingestion of E. coli ML-35 by human neutrophils was followed by perforation of the bacteria (increase in beta-galactosidase activity). Subsequently, the beta- galactosidase activity decreased due to inactivation of the enzyme. With a simple mathematical model and a curve-fitting computer program, we have determined the first-order rate constants for phagocytosis, perforation, and beta-galactosidase inactivation. With 32 normal donors, we found an interdonor variation in these rate constants of 20% to 30% (SD) and an assay variance of 5%. The perforation process closely correlated with the loss of colony-forming capacity of the bacteria. This new assay measures phagocytosis and killing in a fast, simple, and accurate way; it is not hindered by extracellular bacteria. Moreover, this method also measures the postkilling event of inactivation of a bacterial protein, which permits a better detection of neutrophils deficient in this function. The assay can also be used for screening neutrophil functions without the use of a computer program. A simple calculation suffices to detect neutrophil abnormalities. Neutrophils from patients with chronic granulomatous disease (CGD) showed an impaired rate of perforation and thus also of inactivation. Neutrophils from myeloperoxidase-deficient patients or from a patient with the Chediak-Higashi syndrome only showed a retarded inactivation of beta-galactosidase, but normal ingestion and perforation. The role of myeloperoxidase in the killing process is discussed. Although myeloperoxidase does not seem to be a prerequisite for perforation, it probably plays a role in bacterial destruction by normal cells, because the inactivation of bacterial proteins seems strictly myeloperoxidase dependent.

Identification of the upstream activating sequence of MAL and the binding sites for the MAL63 activator of Saccharomyces cerevisiae

1990 ◽  
Vol 10 (7) ◽  
pp. 3797-3800
Author(s):  
B F Ni ◽  
R B Needleman
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
Lacz Gene ◽  
Maltose Permease ◽  
Upstream Activating Sequence ◽  
The Subject ◽  
Dna Fragment ◽  
Mal Genes ◽  
Beta Galactosidase

Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4, and MAL6. Each of these loci consists of a complex of genes involved in maltose metabolism; the complex includes maltase, a maltose permease, and an activator of these genes. At the MAL6 locus, the activator is encoded by the MAL63 gene. While the MAL6 locus has been the subject of numerous studies, the binding sites of the MAL63 activator have not been determined. In this study, we used Escherichia coli extracts containing the MAL63 protein to define the binding sites of the MAL63 protein in the divergently transcribed MAL61-62 promotor. When a DNA fragment containing these sites was placed upstream of a CYC1-lacZ gene, maltose induced beta-galactosidase. These sites therefore constitute an upstream activating sequence for the MAL genes.

Regulation of HIS4-lacZ fusions in Saccharomyces cerevisiae

1982 ◽  
Vol 2 (10) ◽  
pp. 1212-1219
Author(s):  
S J Silverman ◽  
M Rose ◽  
D Botstein ◽  
G R Fink
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Base Pair ◽  
Yeast Genome ◽  
Galactosidase Activity ◽  
Lacz Fusions ◽  
Translation Initiation Codon ◽  
Beta Galactosidase ◽  
His4 Gene

The beginning of the Saccharomyces cerevisiae HIS4 gene has been fused to the structural gene for Escherichia coli beta-galactosidase. This construction, which contains HIS4 DNA from -732 to +30 relative to the translation initiation codon, has been integrated into the yeast genome at two chromosomal locations, HIS4 and URA3. At both locations, this 762-base-pair stretch of DNA is sufficient for initiating expression of beta-galactosidase activity in S. cerevisiae and confers upon this activity the regulatory response normally found for HIS4.

scholarly journals Arginine restriction induced by delta-N-(phosphonacetyl)-L-ornithine signals increased expression of HIS3, TRP5, CPA1, and CPA2 in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (11) ◽  
pp. 4882-4888 ◽  
Author(s):  
D M Kinney ◽  
C J Lusty
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Translational Control ◽  
Mrna Translation ◽  
Single Amino Acid ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
General Amino Acid Control ◽  
Acid Control ◽  
Beta Galactosidase

delta-N-(Phosphonacetyl)-L-ornithine (PALO), a transition state analog inhibitor of ornithine transcarbamylase, induced arginine limitation in vivo in Saccharomyces cerevisiae. Arginine restriction caused increased expression of HIS3 and TRP5, measured by the beta-galactosidase activity in strains carrying chromosomally integrated fusions of the promoter regions of each gene with the lacZ gene of Escherichia coli. The increase in beta-galactosidase activity induced by PALO was reversed by the addition of arginine and was dependent on GCN4 protein. These results indicate that PALO, like 3-amino-1,2,4-triazole DL-5-methyltryptophan, can be used to study the effect of limitation of a single amino acid, arginine, on the expression of genes under the general amino acid control regulatory system. Arginine deprivation imposed by PALO also caused increased expression of CPA1 and CPA2, coding respectively for the small and large subunits of arginine-specific carbamyl-phosphate synthetase. The observed increase was GCN4 dependent and was genetically separable from arginine-specific repression of CPA1 mRNA translation. The 5'-flanking regions of CPA1 (reported previously) and CPA2 determined in this study each contained at least two copies of the sequence TGACTC, shown to bind GCN4 protein. The beta-galactosidase activities expressed from CPA1- and CPA2-lacZ fusions integrated into the nuclear DNA of gcn4 mutant strains were five to six times less than in the wild type, when both strains were grown under depressed conditions. The gcn4 mutation reduced basal expression of both CPA1 and CPA2. The addition of arginine to strains containing the CPA1-lacZ fusion further reduced beta-galactosidase activity of the gcn4 mutant, indicating independent regulation of the CPA1 gene by the general amino acid control and by arginine-specific repression. In strains overproducing GCN4 protein, the translational control completely overrode transcriptional activation of CPA1 by general amino acid control.

scholarly journals Escherichia coli ribonucleotide reductase expression is cell cycle regulated.

1992 ◽  
Vol 3 (10) ◽  
pp. 1095-1105 ◽  
Author(s):  
L Sun ◽  
J A Fuchs
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Ribonucleotide Reductase ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Blot Hybridization ◽  
Regulatory Region ◽  
Lacz Gene ◽  
Dot Blot ◽  
Beta Galactosidase

The expression of the genes encoding ribonucleotide reductase in Escherichia coli was investigated in cultures synchronized by obtaining the smallest cells in a population after sucrose gradient centrifugation. Specific activity of ribonucleotide reductase and DNA initiation were found to increase in parallel, periodically as a function of the cell cycle. The expression of nrd was also determined in cells synchronized by periodic repeated doubling in a phosphate limited medium. Antibodies directed against the B2 subunit of ribonucleotide reductase were raised in a rabbit and purified. Immunoprecipitation of the B2 subunit and RNA-DNA dot blot hybridization assays were developed and employed to determine the expression of ribonucleotide reductase translational and transcriptional products during the cell cycle. Both of nrd-mRNA and B2 subunit expression were found to increase each generation at approximately the same time DNA synthesis was initiated and then to decrease back to the basal level shortly after DNA initiation. These results provided evidence of cell cycle dependent regulation of ribonucleotide reductase in E. coli. When the upstream regulatory region of nrd was fused to a promoterless lacZ gene on a single copy plasmid, lac-mRNA and beta-galactosidase were found to be synthesized in parallel to nrd expression from the chromosomal operon. When nrd sequences surrounding the promoter were removed from this construct, lac-mRNA and beta-galactosidase synthesis were no longer cell cycle regulated.

Protective Effect of Glycine Betaine on Survival of Escherichia coli Cells in Marine Environments

1991 ◽  
Vol 24 (2) ◽  
pp. 129-132 ◽  
Author(s):  
M. J. Gauthier ◽  
G. N. Flatau ◽  
V. A. Breittmayer
Keyword(s):  
Escherichia Coli ◽  
Glycine Betaine ◽  
Previous History ◽  
Organic Carbon Content ◽  
Lacz Gene ◽  
Genetic Changes ◽  
Escherichia Coli Cells ◽  
Expression Of Genes ◽  
Adaptive Processes ◽  
History Of

Under marine conditions, Escherichia coli cells develop extensive structural, metabolic and (to some extent) genetic changes which can be considered as adaptive processes rather than the expression of an irreversible decay. Such adaptation strongly depends on the previous history of growth of the cells and on whether they can regulate internal osmotic pressure when entering the sea. All known osmoregulation mechanisms can help the cells to survive at high osmolarity, but their efficiency depends both on expression of genes responsible for osmoregulation and normal functioning of osmoregulatory mechanisms under marine conditions. The paper presents some experimental evidence on the influence of osmoregulation processes on the survival of E. coli cells in seawater and sediments. This was investigated using strains harboring proP- and proll-lacZ gene or operon fusions. It is shown that the uptake of glycine betaine (GB) and the expression of both proP and proU were weak in seawater. In marine sediments, proP expression was weak, and GB uptake and proU expression were variable, probably depending on the availability of nutrients. In sediment with a high total organic carbon content, GB uptake was very high and proU expression enhanced; cells previously incubated in this sediment showed a higher resistance to decay in seawater. These results confirm that marine deposits can act as reservoirs for enteric pathogens and may give rise to resistant cells.

scholarly journals Kinetics and mechanism of the bactericidal action of human neutrophils against Escherichia coli

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 635-641 ◽  
Author(s):  
MN Hamers ◽  
AA Bot ◽  
RS Weening ◽  
HJ Sips ◽  
D Roos
Keyword(s):  
Escherichia Coli ◽  
Computer Program ◽  
Rate Constants ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Human Neutrophils ◽  
Galactosidase Activity ◽  
E Coli ◽  
Bacterial Proteins ◽  
Beta Galactosidase

Abstract A mutant strain of Escherichia coli (E. coli ML-35) was used to follow the kinetics of phagocytosis, perforation of the bacterial cell envelope, and inactivation of bacterial proteins by human neutrophils. This particular E. coli mutant strain has no lactose permease, but constitutively forms the cytoplasmic enzyme beta-galactosidase. This implies that the artificial substrate ortho-nitrophenyl-beta-D- galactopyranoside cannot reach the beta-galactosidase unless the bacterial cell envelope has been perforated. Thus, the integrity of the E. coli envelope can be measured simply by the activity of beta- galactosidase with this substrate. Indeed, ingestion of E. coli ML-35 by human neutrophils was followed by perforation of the bacteria (increase in beta-galactosidase activity). Subsequently, the beta- galactosidase activity decreased due to inactivation of the enzyme. With a simple mathematical model and a curve-fitting computer program, we have determined the first-order rate constants for phagocytosis, perforation, and beta-galactosidase inactivation. With 32 normal donors, we found an interdonor variation in these rate constants of 20% to 30% (SD) and an assay variance of 5%. The perforation process closely correlated with the loss of colony-forming capacity of the bacteria. This new assay measures phagocytosis and killing in a fast, simple, and accurate way; it is not hindered by extracellular bacteria. Moreover, this method also measures the postkilling event of inactivation of a bacterial protein, which permits a better detection of neutrophils deficient in this function. The assay can also be used for screening neutrophil functions without the use of a computer program. A simple calculation suffices to detect neutrophil abnormalities. Neutrophils from patients with chronic granulomatous disease (CGD) showed an impaired rate of perforation and thus also of inactivation. Neutrophils from myeloperoxidase-deficient patients or from a patient with the Chediak-Higashi syndrome only showed a retarded inactivation of beta-galactosidase, but normal ingestion and perforation. The role of myeloperoxidase in the killing process is discussed. Although myeloperoxidase does not seem to be a prerequisite for perforation, it probably plays a role in bacterial destruction by normal cells, because the inactivation of bacterial proteins seems strictly myeloperoxidase dependent.

scholarly journals Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica.

1990 ◽  
Vol 10 (9) ◽  
pp. 4795-4806 ◽  
Author(s):  
J W Xuan ◽  
P Fournier ◽  
N Declerck ◽  
M Chasles ◽  
C Gaillardin
Keyword(s):  
Yarrowia Lipolytica ◽  
Stop Codon ◽  
Open Reading Frames ◽  
Yeast Cells ◽  
Lacz Gene ◽  
Galactosidase Activity ◽  
Phenotypic Effect ◽  
Frame Fusion ◽  
Beta Galactosidase ◽  
Reading Frames

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

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