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K F Jensen

Publications and source records attributed to K F Jensen.

At least 91 records · Page 5Linked to original sources
PubMed1985

Role of translation in the UTP-modulated attenuation at the pyrBI operon of Escherichia coli.

A 273 bp DNA fragment containing the attenuator of the pyrBI operon was inserted into a synthetic cloning site early in the lacZ gene on a plasmid. By this operation the first few codons of lacZ were joined through a linker to the last 39 codons of the open reading frame for the putative pyrB leader peptide. In addition a gene fusion encoding a hybrid protein with beta-galactosidase activity was formed between the pyrB start and the rest of lacZ. This gene fusion is expressed from the lac promoter and the transcript is subject to facultative termination at the pyrBI attenuator. Different variants of the lacZ start were used that either contained a stop codon or directed the translation toward the attenuator in any of the alternative reading frames. The following results were obtained. No significant read-through of transcription over the pyrB attenuator was seen when the leader translation ended 49 nucleotide residues, or more, upstream of the attenuator symmetry, but a UTP-modulated attenuation was established if the leader translation was allowed to proceed across the attenuator as for the putative leader peptide or in a frame-shifted version. The regulation, however, was not as great as for the native pyrB gene. This is probably because the substitution of the normal start of the leader peptide by the start of lacZ alters the coupling between transcription and translation and thereby the attenuation frequency. It cannot, however, be ruled out that the pyrBI operon is regulated at the promoters in addition to the control by attenuation.

Aspartate Carbamoyltransferase↗
PubMed1984

Mechanism of UTP-modulated attenuation at the pyrE gene of Escherichia coli: an example of operon polarity control through the coupling of translation to transcription.

The pyrE gene of Escherichia coli is part of an operon where it is preceded by an unknown gene (orfE) that ends 8 bp before the start of the symmetry of the UTP-modulated pyrE attenuator. On a plasmid we have inserted this attenuator region in a synthetic cloning site early in lacZ. The resulting structure contains the lac promoter-operator, the first few codons of lacZ, 42 bp of DNA from the orfE end, the pyrE attenuator, and an in-frame fusion pyrE-lacZ+. The synthetic cloning sites have been used to vary the length and reading frame of the translation that begins at the lacZ start and proceeds towards the attenuator. The effects of these variations on pyrE attenuation were determined by monitoring the synthesis of beta-galactosidase from the pyrE-lacZ hybrid gene in cells grown with either low or high pools of UTP. Thus, a very low level of pyrE expression was observed, regardless of UTP pool size, when the translation from the lacZ start ended 31 or 62 nucleotide residues upstream to the pyrE attenuator symmetry, but a proper UTP controlled attenuation could be established if this translation ended only 8 bp before the symmetry region of the attenuator (as the native orfE gene) or 10 bp after this structure. However, a single 'leader peptide' read from only frequently used codons gave a high level of pyrE expression both at high and low UTP pools. These observations indicate that the coupling between transcription and translation determines the degree of mRNA chain terminations at the pyrE attenuator.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1984

Studies on the structure and expression of Escherichia coli pyrC, pyrD, and pyrF using the cloned genes.

The Escherichia coli pyrC, pyrD and pyrF genes were cloned on multicopy plasmids derived from pBR322 and analysed by means of restriction endonucleases. It was found that the pyrC gene is destroyed by cutting with the restriction endonuclease BamHI, that the entire pyrD gene can be isolated on a 1300-base pairs DNA fragment generated by EcoRI cleavage and that cutting with EcoRI removes the promotor and probably also the translational start site from the pyrF gene. More details on the restriction maps are presented. Further, it was found that the presence of a pyr gene in multiple copies on a plasmid does not significantly interfere with the activity of the chromosomal pyr genes. Using the 'minicell' technique, the polypeptides encoded by the three cloned pyr genes were identified. The relative molecular masses for the pyrC-encoded and pyrD-encoded polypeptides are 38 000-40 000 and 36 000-38 000, respectively. Thus in their native form, dihydroorotase and dihydroorotate oxidase appear to be dimeric proteins. The 'minicell' experiments positively identified a protein chain of Mr 23 000-24 000 as being a subunit of OMP decarboxylase encoded by pyrF. Moreover, the coding frame for this polypeptide seems to be expressed as the first gene in the operon with the coding frame for another protein chain of Mr 13 000-14 000. Since, however, the native OMP decarboxylase during sedimentation and gel filtration behaves as a protein of Mr 45 000 +/- 4000, this latter polypeptide (Mr 13 000-14 000) is hardly a component of the enzyme. Pyr-lac+ operon fusions were constructed by the Mu d1 procedure. By integrating an F'lac episome into the lac part of the fusions and determining the direction of chromosomal transfer from the resultant Hfr strains, the direction of pyrC transcription was found to be counter-clockwise, while pyrD and pyrF were found to be transcribed in a clockwise direction.

Base Composition↗
PubMed1984

Structure of the Escherichia coli pyrE operon and control of pyrE expression by a UTP modulated intercistronic attentuation.

Protein synthesis in 'minicells' showed that the DNA immediately preceding the pyrE gene of Escherichia coli directs the formation of considerable amounts of a polypeptide (mol. wt. approximately 30 000) of unknown function. The nucleotide sequence of this DNA revealed the existence of an open reading frame (ORF) of 238 codons that ends 68 nucleotide residues upstream to the structure start of pyrE, just prior to the GC-rich symmetry region of a sequence with features characteristic of a rho-independent transcription terminator. Deletion of the start of this 238 codons long ORF gene resulted in a dramatic fall in the level of pyrE expression, indicating that the two genes (ORF and pyrE) constitute an operon. S1-nuclease digestion of RNA-DNA hybrids revealed that both genes are transcribed from two promoters (P1 and P2) located in front of the ORF start. Furthermore, when the RNA used in these experiments was prepared from cells with different levels of pyrE expression, created by manipulations in their pyrimidine nucleotide supply, the frequency of transcription initiations at P1 and P2 was found to be constitutive, while only a pyrimidine regulated fraction of the mRNA chains reached into the pyrE gene. In vitro transcription of isolated DNA fragments showed that the mRNA chains are terminated between the ORF gene and pyrE. From these observations we conclude that pyrE expression is controlled by a UTP modulated intercistronic attentuation.

Bacterial Proteins↗
PubMed1984

Subcortical projections from ectopic neocortical neurons.

There is a high degree of specificity in the efferent connections of the cerebral cortex. In the rodent neocortex, the characteristic band of corticospinal neurons within layer V is present at birth even though changes still occur in the areal distribution of these neurons. Disruption of neocortical development with ionizing radiation before, during, or after the production of neurons destined for layer V results in abnormally located corticospinal neurons. One abnormal location in which corticospinal neurons are found is in ectopic cell clusters beneath the cortical white matter bordering the dorso-medial aspect of the lateral ventricle. Corticospinal neurons only occur in these periventricular ectopias in adult rats irradiated on or before embryonic day 17. A second abnormal location of corticospinal neurons is between layer V and the pial surface. These scattered supragranular corticospinal neurons occur in all adult animals irradiated on embryonic days 16, 17, 18, or 19. The fact that neurons having an unusual position project to a subcortical target appropriate for one neocortical sublayer indicates that neither migratory path nor final position is essential to specifying a subcortical target. In addition, the fact that labeled corticospinal neurons are located in periventricular ectopias only when irradiation occurs on or before embryonic day 17 suggests that the initial projections of corticospinal neurons are determined early in their individual ontogeny prior to migration.

Animals↗
PubMed1983

Phosphorolytic activity of Escherichia coli glycyl-tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P-NMR spectroscopy and thin-layer chromatography.

The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethyleneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses of adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')triphospho(5')adenosine (Ap3A), also catalyzes the formation of ADP from inorganic phosphate and the enzyme-bound glycyl adenylate. Accordingly it was shown that E. coli glycyl-tRNA synthetase, in the presence of inorganic phosphate, glycine, and Mg2+ ions, catalyzes the synthesis of ADP from three different substrates which all lead to enzyme-bound glycyl adenylate, that is, ATP, adenosine 5'-[beta, gamma-methylene]triphosphate and Ap4A. It was furthermore demonstrated that the only pathway by which a synthetase-catalyzed degradation of Ap4A can occur is through the reaction between inorganic phosphate and the enzyme-bound glycyl adenylate, synthesized from Ap4A. Likewise a 20-fold increase of the phosphorolytic activity of the investigated synthetase was observed when Mg2+ was replaced by Mn2+. Besides establishing the phosphorolytic activity of the applied enzyme, the study also showed that the preparation catalyzes a glycine-independent transfer of the gamma-phosphate group from ATP to nucleoside 5'-diphosphates. The importance of the observed reaction between inorganic phosphate and enzyme-bound aminoacyl adenylate in relation to the remaining catalytic activities of aminoacyl-tRNA synthetases is discussed, as well as the biological significance of the reaction.

Adenine Nucleotides↗
PubMed1983

Nucleotide sequence of the Escherichia coli pyrE gene and of the DNA in front of the protein-coding region.

Orotate phosphoribosyltransferase (EC 2.4.2.10) was purified to electrophoretic homogeneity from a strain of Escherichia coli containing the pyrE gene cloned on a multicopy plasmid. The relative molecular masses (Mr) of the native enzyme and its subunit were estimated by means of gel filtration and electrophoresis in the presence of dodecyl sulfate. The amino acid sequences at the N and C termini, as well as the amino acid composition, were determined. The nucleotide sequence of the structural pyrE gene, including 394 nucleotide residues preceding the beginning of the coding frame, was also established. From the results the following conclusions may be drawn. Orotate phosphoribosyltransferase is a dimeric protein with subunits of Mr 23 326 consisting of 211 amino acid residues. The pyrE gene is transcribed in a counter-clock wise direction from the E. coli chromosome as an mRNA with a considerable leader segment in front of the protein-coding region. This leader contains a structure with features characteristic for a (translated?) rho-independent transcriptional terminator, which is preceded by a cluster of uridylate residues. This indicates that the frequency of pyrE transcription is regulated by an RNA polymerase (UTP) modulated attenuation.

Bacterial Proteins↗
PubMed1983

Role of hypoxanthine and guanine in regulation of Salmonella typhimurium pur gene expression.

Data are presented which indicate that the repression of pur gene expression seen after the addition of preformed purines to cultures of Salmonella typhimurium is the consequence of the presence or the formation of the purine bases, hypoxanthine and guanine. This conclusion is based on the following observations. First, it was impossible to find a correlation between the size of any individual purine nucleotide pool and the level of the first four enzymes in the de novo biosynthetic pathway. Second, adenine plus guanosine served as a perfect source of purine nucleotides, but their presence caused no repression of pur gene expression if the cells lacked purine nucleoside phosphorylase activity. This enzyme is needed to convert adenine and guanosine to hypoxanthine and guanine, but not for their conversion to nucleotides. Third, addition of guanine to a strain lacking guanine phosphoribosyltransferase (gpt) resulted in a repression of the level of the purine de novo biosynthetic enzymes, a reduction of the growth rate, and a fall in the pools of ATP and GTP. Addition of hypoxanthine to a strain lacking hypoxanthine phosphoribosyltransferase (hpt) had a similar, although weaker, effect. If the cells lacked both hypoxanthine and guanine phosphoribosyltransferases (hpt gpt), their basal level of the purine de novo biosynthetic enzymes was repressed in minimal medium. Such cells grow slower than wild-type cells and excrete purines, probably due to the inability to salvage endogenously formed hypoxanthine and guanine.

Adenosine Triphosphate↗
PubMed1982

The contribution of late-generated neurons to the callosal projection in rat: a study with prenatal x-irradiation.

Studies utilizing horseradish peroxidase tracing methods have suggested that there are species differences in the relative contribution of the different neocortical layers to the callosal projection. The present investigation utilized x-irradiation at different gestational ages to eliminate the late-generated neurons in the rat neocortex. The caudorostral gradient of reduction in the neuronal population of the supragranular layers is closely correlated with the gradient of reduction in the size of the corpus callosum. Furthermore, the callosal projection is absent in anteroposterior cortical segments in which the development of the supragranular layers was prevented without a reduction of the number of neurons in the infragranular layers of the neocortex. These results indicate that late-generated neurons residing primarily in the supragranular layers are essential for the formation of the corpus callosum.

Animals↗
PubMed1982

RNA polymerase involvement in the regulation of expression of Salmonella typhimurium pyr genes. Isolation and characterization of a fluorouracil-resistant mutant with high, constitutive expression of the pyrB and pyrE genes due to a mutation in rpoBC.

A mutant of Salmonella typhimurium with a defect in the regulation of pyr-gene expression was obtained during a selection for mutants resistant to a combination of the two pyrimidine analogs, 5-fluorouracil and 5-fluorouridine. The mutant possesses 4-fold elevated pools of the pyrimidine nucleoside triphosphatases, UTP and CTP. The specific activities of aspartate transcarbamylase and orotate phosphoribosyltransferase are 40-fold and 7-fold higher in the mutant than in the parent strain when grown in minimal media. Furthermore, the synthesis of the two enzymes in the mutant is not repressed following addition of exogenous pyrimidines. The levels of carbamoylphosphate synthase and orotidine 5'-monophosphate decarboxylase are approximately 3-fold enhanced, while the activities of dihydroorotase and dihydroorotate oxidase appear largely unaffected by the mutation. The mutation responsible for these effects was shown to map between two known point mutations in the rpoBC gene cluster encoding the beta and beta' subunits of RNA polymerase. These observations indicate a regulatory function of RNA polymerase in the control of pyr-gene expression in S. typhimurium.

Aspartate Carbamoyltransferase↗
PubMed1982

Salmonella typhimurium mutants with altered expression of the pyrA gene due to changes in RNA polymerase.

Rifampicin-resistant mutants of Salmonella typhimurium were isolated and tested for pleiotropic defects in the regulation of pyr gene expression. Seven per cent of all the Rifr mutants were inhibited in growth by addition of uracil (uracil-sensitive). The uracil-sensitive phenotype ( UraS ) was reversed by arginine or citrulline, but not by ornithine, and it was suppressed by mutations in either argR or pyrH , which causes increased expression of pyrA . It was shown that the basal levels of carbamoylphosphate synthase (the pyrA gene product) was reduced to approximately 60% in the mutants, and that addition of arginine and/or uracil to the growth medium caused hyperrepression of pyrA expression. The expression of other genes of the arginine and pyrimidine biosynthetic pathways was not affected significantly in the mutants. The mutations were located in the rpoB gene coding for the beta-subunit of RNA polymerase, suggesting a regulatory function of RNA polymerase in the control of pyrA expression.

Carbamoyl-Phosphate Synthase (Ammonia)↗
PubMed1979

Apparent involvement of purines in the control of expression of Salmonella typhimurium pyr genes: analysis of a leaky guaB mutant resistant to pyrimidine analogs.

A leaky guaB mutant of Salmonella typhimurium LT-2 was obtained during a selection for mutants resistant to a combination of the two pyrimidine analogs, 5-fluorouracil and 5-fluorouridine. In the absence of exogenous guanine compounds, the growth rate of this mutant is limited by the endogenous supply of guanine nucleotides due to a defective inosine 5'-monophosphate dehydrogenase. Under these conditions the guanosine 5'-triphosphate pool is about 20% of normal, the cytidine 5'-triphosphate pool is reduced to below 60%, and the uridine 5'-triphosphate pool is slightly elevated. Simultaneously, levels of the pyrimidine biosynthetic enzymes are abnormal: aspartate transcarbamylase, orotate phosphoribosyltransferase, and orotidylic acid decarboxylase levels are increased 4-, 11-, and 3-fold, respectively. Levels of dihydroorotase and dihydroorotate dehydrogenase are decreased to 10 and 20%, respectively. The pyrimidine metabolism of the guaB mutant is restored completely by addition of guanine (or xanthine) to the growth medium. The data indicate purine nucleotide involvement in the regulation of expression of the pyr genes of S. typhimurium.

Drug Resistance, Microbial↗
PubMed1978

Two purine nucleoside phosphorylases in Bacillus subtilis. Purification and some properties of the adenosine-specific phosphorylase.

Two purine nucleoside phosphorylases (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) were purified from vegetative Bacillus subtilis cells. One enzyme, inosine-guanosine phosphorylase, showed great similarity to the homologous enzyme of Bacillus cereus. It appeared to be a tetramer of molecular weight 95 000. The other enzyme, adenosine phosphorylase, was specific for adenosine and deoxyadenosine. The molecular weight of the native enzyme was 153 000 +/- 10% and the molecular weight of the subunits was 25 500 +/- 5%. This indicates a hexameric structure. The adenosine phosphorylase was inactivated by 10(-3) M p-chloromercuribenzoate and protected against this inactivation by phosphate, adenosine and ribose 1-phosphate.

Adenosine↗
PubMed1978

Detergent-inhibited, heat-labile nucleoside triphosphatase in cores of avian myeloblastosis virus.

Endogenous DNA synthesis was studied in isolated core particles of avian myeloblastosis virus. It was found that cores contained an enzymatic activity which rapidly converted the added nucleoside triphosphates to diphosphates (but not further) at 0 degrees C, thus inhibiting DNA synthesis. This triphosphatase probably originates from the viral membranes. In the cores the enzyme is completely inactivated by low concentrations (0.02%) of Nonident P-40. Also, the enzyme is very thermolabile and denatures rapidly at 38 degrees C.

Adenosine Triphosphate↗