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Biomedical subjects

K F Jensen

Publications and source records attributed to K F Jensen.

At least 73 records · Page 4Linked to original sources
PubMed1990

Metabolic growth rate control in Escherichia coli may be a consequence of subsaturation of the macromolecular biosynthetic apparatus with substrates and catalytic components.

In this paper, the Escherichia coli cell is considered as a system designed for rapid growth, but limited by the medium. We propose that this very design causes the cell to become subsaturated with precursors and catalytic components at all levels of macromolecular biosynthesis and leads to a molecular sharing economy at a high level of competition inside the cell. Thus, the promoters compete with each other in the binding of a limited amount of free RNA polymerase and the ribosome binding sites on the mRNA chains compete with each other for the free ribosomes. The macromolecular chain elongation reactions sequester a considerable proportion of the total amount of RNA polymerase and ribosomes in the cells. We propose that the degree of subsaturation of the macromolecular biosynthetic apparatus renders a variable fraction of RNA polymerase and ribosomes unavailable for the initiation of new chain synthesis and that this, at least in part, determines the composition of the cell as a function of the growth rate. Thus, at rapid growth, the high speed of the elongation reactions enables the cell to increase the concentrations of free RNA polymerase and ribosomes for initiation purposes. Furthermore, it is proposed that the speed of RNA polymerase movement is adjusted to the performance speed of the ribosomes. Mechanistically, this adjustment of the coupling between transcription and translation involves transcriptional pause sites along the RNA chains, the adjustment of the saturation level of RNA polymerase with the nucleoside triphosphate substrates, and the concentration of ppGpp, which is known to inhibit RNA chain elongation. This model is able to explain the stringent response and the control of stable RNA and of ribosome synthesis in steady states and in shifts, as well as the rate of overall protein synthesis as a function of the growth rate.

Bacterial Proteins↗
PubMed1989

Ca2+/calmodulin-dependent protein kinase enriched in cerebellar granule cells. Identification of a novel neuronal calmodulin-dependent protein kinase.

Ca2+-sensitive protein kinases are thought to play a pivotal role in Ca2+-mediated neuronal communication. We describe here the cloning, purification, and characterization of a major Ca2+/calmodulin-dependent, brain-specific protein kinase which is particularly enriched in cerebellar granule cells. The enzyme is comprised of Mr 65,000 and 67,000 polypeptides which copurify to homogeneity and phosphorylate synapsin I. The protein kinase is coded for by two poly(A+) RNAs of 2.0 and 3.5 kilobases which probably derive from a single gene. Two cDNA inserts, one of 198 base pairs and one of 1225 base pairs, contain a total of 677 base pairs of the protein coding sequence which includes sequences homologous to other calmodulin-dependent protein kinases including part of the calmodulin-binding domain. The surprising presence of extended sequences which are enriched in glutamate residues may influence the subcellular distribution of this kinase. Immunohistochemical localization with an affinity-purified antibody reveals that whereas the enzyme is expressed in several neuronal subpopulations, it is exceptionally enriched in the granule cells of the cerebellum. The relevance of the biochemical, molecular, and histologic properties of this enzyme is discussed in the context of neuronal Ca2+ signaling.

Amino Acid Sequence↗
PubMed1989

Regulation of Salmonella typhimurium pyr gene expression: effect of changing both purine and pyrimidine nucleotide pools.

The synthesis of the pyrimidine biosynthetic enzymes is repressed by the pyrimidine nucleotide end-products of the pathway. However, purine nucleotides also play a role. In this study, I have measured expression of the pyr genes (pyrA-E) in Salmonella typhimurium strains harbouring mutations that permit manipulation of the intracellular pools of both pyrimidine and purine nucleotides. The results identify the effectory purine compound as being a guanine nucleotide; it is probably GTP, but it may be GDP or GMP. The synthesis of carbamoylphosphate synthase, encoded by pyrA, and particularly dihydroorotase, encoded by pyrC, and dihydroorotate dehydrogenase, encoded by pyrD, is stimulated by the guanine nucleotide, while the synthesis of aspartate transcarbamoylase, encoded by pyrBI, and orotate phosphoribosyltransferase, encoded by pyrE, is inhibited by guanine nucleotides. The regulatory pattern of each pyr gene is discussed in relation to present knowledge on gene structure and regulatory mechanism.

Gene Expression Regulation, Bacterial↗
PubMed1989

Molecular and mutational analysis of three genes preceding pyrE on the Escherichia coli chromosome.

The nucleotide sequence of two kilobase pairs (kb) 5' to the orfE-pyrE operon has been determined. The sequence revealed two open reading frames, orfX and orfY, consisting of 286 and 274 codons, respectively, and having a transcriptional orientation opposite that of the orfE-pyrE operon. Analysis of transcription initiations showed that the promoters of orfE and orfX constitute a pair of divergent promoters with overlapping -35 regions and that orfY is transcribed from an independent promoter. Translational analysis indicated that the orfs are expressed in Escherichia coli. The orfE, orfX, and orfY genes were inactivated on the bacterial chromosome by deletion-insertion mutagenesis using a kanamycin resistance cassette. The mutants were all viable. However, the orfE deletion caused a dramatic reduction in the level of pyrE expression and a partial pyrimidine requirement, because this mutation prevented transcription of pyrE. the orfE protein seemed without significance for pyr-gene expression in E. coli, and the mutations in orfX and orfY were without detectable phenotypes.

Amino Acid Sequence↗
PubMed1989

Translation rates of individual codons are not correlated with tRNA abundances or with frequencies of utilization in Escherichia coli.

We analyzed 12 individual codons, which differed widely with respect to the frequency of use in Escherichia coli and the abundance of the corresponding tRNAs, for their influence on the coupling between transcription and translation. This was probed by determining the effects of codon substitutions in the leader peptide gene on transcription past the pyrE attenuator, as described previously by Bonekamp et al. (F. Bonekamp, H. D. Andersen, T. Christensen, and K. F. Jensen, Nucleic Acids Res. 13:4113-4123, 1985). In principle, the results revealed that either RNA polymerase or the (leading) ribosomes pass the different codon strings at different rates. However, under the assumption that the rate of transcription elongation is unaffected by the sequence changes, the results may be interpreted as indicating that different codons are translated at different rates and that these rates do not generally reflect the concentrations of the corresponding tRNAs or the frequencies with which the codons are used in E. coli. Moreover, it seems that codon synonyms that are served by the same isoaccepting tRNA species can deviate as much from each other in translational behavior as synonymous codons that are served by isoacceptors present in the cell in widely different amounts can.

Base Sequence↗
PubMed1988

A specific and efficient photoreaction between E. coli RNA polymerase and T+1 in the lacUV5 or deoP1 promoter.

Upon irradiation of the RNA polymerase-lacUV5 or deoP1 promoter complex with short wavelength ultraviolet light (lambda less than or equal to 300 nm) the polymerase is covalently crosslinked at an efficiency of greater than 10% to the first transcribed base of the template DNA strand when this is a thymine. The temperature dependence of this RNA polymerase-T+1 photoreaction strongly indicates a relation to the formation of the open complex. It is suggested that open complex formation is preceded or accompanied by a specific contact between the RNA polymerase and the first transcribed base of the DNA template.

Cross-Linking Reagents↗
PubMed1988

Hyper-regulation of pyr gene expression in Escherichia coli cells with slow ribosomes. Evidence for RNA polymerase pausing in vivo?

UTP-modulated attenuation of transcription is involved in regulating the synthesis of pyrimidine nucleotides in Escherichia coli. Thus, expression of two genes, pyrBI and pyrE, was shown to be under this type of control. The genes encode the two subunits of aspartate transcarbamylase and orotate phosphoribosyltransferase respectively. The levels of these enzymes are inversely correlated with the intracellular concentration of UTP. Modulation of attenuation seems to be a consequence of the effect of UTP concentration on the mRNA chain growth rate. Reducing the UTP pool retards RNA polymerase movement. Mechanistically this will couple the ribosomes translating a leader peptide gene more tightly to the elongating RNA polymerase. The ribosomes will then be more prone to prevent the folding of the mRNA chains into terminating hairpin structures when RNA polymerase is at the attenuator and has to decide whether transcription should terminate or continue into the structural genes. This paper described a study of pyrBI and pyrE gene regulation in cells where the ribosomes move slowly as a result of mutation in rpsL. It appears that expression of the two genes is hyper-regulated by the UTP pool in this type of cells. Furthermore, the attenuator model can only account for the results if it is assumed that UTP-concentration-dependent pausing of transcription occurs in vivo in the two pyr gene leaders such that RNA polymerase waits for the coupled ribosomes before transcribing into the attenuator regions.

Aspartate Carbamoyltransferase↗
PubMed1988

The AGG codon is translated slowly in E. coli even at very low expression levels.

Data are presented which indicate that AGG codons for arginine are translated significantly more slowly than the CGU codons for the same amino acid even when their expression level from the probe is very low. The two types of codons were inserted (three in tandem) on a multicopy plasmid in an artificial leader peptide gene in front of the pyrE attenuator where the frequency of transcription termination is regulated by the degree of coupling between transcription and translation. Transcription of the operon is initiated from the lac-promoter dependent on the concentration of the lac-operon inducer IPTG. At all induction levels it was found that the frequency of transcription past the pyrE attenuator was approximately nine times lower when the AGG codons were present in the leader than with CGT codons present. This shows that AGG codons decouple translation from transcription in the pyrE attenuator region even when the concentration of this codon is not increased significantly relative to that in the unperturbed wild type strain. Thus the results indicate that AGG codons are always slowly translated in Escherichia coli.

Adenine↗
PubMed1987

Effect of UTP and GTP pools on attenuation at the pyrE gene of Escherichia coli.

We have used the galK gene, minus its promoter, to quantitate transcription of the orfE--pyrE operon of Escherichia coli in front of and after the intercistronic attenuator. Expression of the hybrid genes was studied in a bacterium with mutations that permit changes in the UTP and GTP pools during exponential growth. It was found that the greater part of pyrE gene regulation by the nucleotides takes place at the intercistronic attenuator and that promoter control contributes only little, ca. twofold. When pools of both UTP and GTP were high only 5%-6% of the mRNA chains were continued into the pyrE gene. However, when the UTP pool was reduced (from 1.3 to 0.2 mumol/g dry weight) nearly 100% of transcription passed the attenuator. Likewise, a reduction in the GTP pool (from 3.2 to 0.8 mumol/g dry weight) resulted in 25%-30% escape of attenuation. Regulation by attenuation disappeared when a premature stop-codon was introduced near the end of orfE such that translational coupling to transcription was prevented in the attenuator area. Therefore, we attribute the modulation of attenuation to nucleotide-induced variations in the kinetics of mRNA chain elongation. In support for this it was found that an RNA polymerase mutant with reduced RNA chain growth rate transcribed past the pyrE attenuator at a high frequency in the presence of a high UTP pool, but only when coupling of translation to transcription was allowed at the end of orfE.

DNA, Recombinant↗
PubMed1987

Focal lesions of visual cortex--effects on visual evoked potentials in rats.

Focal lesions were placed in the visual cortex of Long-Evans hooded rats, immediately below skull screw recording electrodes. Lesions were produced by heat, and extended an average depth of about 0.9 mm below the cortical surface. Evoked potentials recorded from the electrode overlying the cortical lesion were compared with simultaneously recorded potentials from a contralateral homotopic site. The effects of the lesion were selective. Flash-evoked potential peaks P1, P2, and N2 were depressed by the lesion, and peaks N1 and P3 were augmented; peak N3 was unaffected. Pattern reversal evoked potential peak N3 was depressed by the lesion, and peaks N1 and P2 were made more distinct. The results emphasized that different peaks have different generators, and suggest in particular that flash-evoked potential peaks P1 and N2, and peak N3 of the pattern reversal-evoked potential require the superficial layers of the cortex.

Animals↗
PubMed1987

Isolation of Escherichia coli rpoB mutants resistant to killing by lambda cII protein and altered in pyrE gene attenuation.

Escherichia coli mutants simultaneously resistant to rifampin and to the lethal effects of bacteriophage lambda cII protein were isolated. The sck mutant strains carry alterations in rpoB that allow them to survive cII killing (thus the name sck), but that do not impair either the expression of cII or the activation by cII of the lambda promoters pE and pI. The sck-1, sck-2, and sck-3 mutations modify transcription termination. The growth of lambda, but not of the N-independent lambda variant, lambda nin-5, is hindered by these mutations, which act either alone or in concert with the bacterial nusA1 mutation. In contrast to their effect on lambda growth, the three mutations reduce transcription termination in bacterial operons. The E. coli pyrE gene, which is normally regulated by attenuation, is expressed constitutively in the mutant strains. The sck mutations appear to prevent pyrE attenuation by slowing the rate of transcriptional elongation of the pyrE leader sequence. The sck-6 mutation, unlike the other sck mutations, neither increases pyrE expression nor inhibits the ability of lambda to suppress transcription termination. Instead, the sck-6 mutation blocks the growth of the lambda variants lambda nin-5 and lambda red-3.

Bacteriophage lambda↗
PubMed1987

Terminal arbors of axons projecting to the somatosensory cortex of the adult rat. I. The normal morphology of specific thalamocortical afferents.

A characteristic feature of the rat somatosensory neocortex is a discrete topographic representation of the facial whiskers. Afferent fibers projecting to this vibrissae representation were "bulk-labeled" by injecting horseradish peroxidase into the white matter. Terminal arbors with the morphological characteristics of Lorente de No's (1949) "specific" thalamocortical afferents were then reconstructed through serial sections. These terminal arbors, characterized by the discrete organization of their dense plexus in layer IV, have a laminar distribution of boutons that parallels the laminar pattern of terminal degeneration resulting from lesions of the ventral posterior nucleus of the thalamus. The regional distribution of different-sized arbors corresponds to the distribution of vibrissae-related clusters of different sizes. Larger arbors were found in the posteromedial region corresponding to the mystacial vibrissae representation, while smaller arbors were found in the anterolateral region corresponding to the representation of the anterior sinus hairs. Terminal arbors were also reconstructed from sections stained simultaneously to demonstrate the pattern of vibrissae-related clusters. The greatest concentration of boutons on these axons occurred within a single vibrissae-related cluster. Furthermore, when 2 fibers terminated within a single cluster, their terminal arbors appeared to be largely coextensive. The morphology, size, and distribution of these terminal arbors support the hypothesis that the layer IV plexus of a single specific thalamocortical afferent tends to fill a vibrissae-related cluster. Thus, the organization of specific thalamocortical afferents may be responsible for clustered organization within the somatotopic map of the rodent neocortex.

Animals↗
PubMed1987

Terminal arbors of axons projecting to the somatosensory cortex of the adult rat. II. The altered morphology of thalamocortical afferents following neonatal infraorbital nerve cut.

The organization of the whisker representation within the neocortex of the rat is dependent on an intact periphery during development. To further investigate how alterations in this cortical map arise we examined the organization of thalamocortical afferents to the whisker representation in adult animals in which the infraorbital branch of the trigeminal nerve was cut on the day of birth. The disrupted pattern of thalamocortical projections to the vibrissae representation was apparent in the abnormal pattern of the anterograde transport of horseradish peroxidase from the thalamus, as well as in the abnormal pattern of succinate dehydrogenase activity. To determine the morphology of individual thalamocortical axons associated with this disrupted pattern, terminal arbors were "bulk-labeled" by injections of horseradish peroxidase into the white matter beneath the somatosensory cortex. Terminal arbors were identified by their laminar distribution of boutons corresponding to the specific thalamocortical afferent. The medial to lateral extent of these terminal arbors varied dramatically, from 350 to 1500 microns. In addition, terminal arbors innervating the same local area of cortex appeared to have varying degrees of overlap. Thus, the disruption of the neocortical vibrissae representation appears to involve the abnormal arborization of individual thalamocortical afferents. This finding supports the hypothesis that the fine-grain organization of the somatotopic map is dependent on the morphology and organization of individual thalamocortical arbors, which, in turn, are dependent on the periphery during development.

Animals↗
PubMed1986

Comparison of yellow poplar growth models on the basis of derived growth analysis variables.

Quadratic and cubic polynomials, and Gompertz and Richards asymptotic models were fitted to yellow poplar growth data. These data included height, leaf area, leaf weight and new shoot height for 23 weeks. Seven growth analysis variables were estimated from each function. The Gompertz and Richards models fitted the data best and provided the most accurate derived variables. However, the Richards model was more complex to fit.

Journal Article↗
PubMed1986

Association of RNA polymerase having increased Km for ATP and UTP with hyperexpression of the pyrB and pyrE genes of Salmonella typhimurium.

We investigated the transcription kinetics of RNA polymerase from an rpoBC mutant of Salmonella typhimurium which showed highly elevated, constitutive expression of the pyrB and pyrE genes as well as an increased cellular pool of UTP. When bacterial cultures containing an F' lac+ episome were induced for lac operon expression, the first active molecules of beta-galactosidase were formed with a delay of 73 +/- 3 s in rpo+ cells. The corresponding time was 104 to 125 s for cells carrying the rpoBC allele, indicating that this mutation causes a reduced RNA chain growth rate. In vitro the purified mutant RNA polymerase elongated transcripts of both T7 DNA and synthetic templates more slowly than the parental enzyme at a given concentration of nucleoside triphosphates. This defect was found to result from four- to sixfold-higher Km values for the saturation of the elongation site by ATP and UTP. The saturation kinetics of the RNA chain initiation step also seemed to be affected. The maximal elongation rate and Km for GTP and CTP were less influenced by the rpoBC mutation. Open complex formation at the promoters of T7 DNA and termination of the 7,100-nucleotide transcript showed no significant difference between the parental and mutant enzymes. Together with the phenotype of the rpoBC mutant, these results indicate that expression of pyrB and pyrE is regulated by the mRNA chain growth rate, which is controlled by the cellular UTP pool. The rate of gene expression is high when the saturation of RNA polymerase with UTP is low and vice versa.

Adenosine Triphosphate↗
PubMed1985

Nucleotide sequence of the pyrD gene of Escherichia coli and characterization of the flavoprotein dihydroorotate dehydrogenase.

Dihydroorotate dehydrogenase (EC 1.3.3.1) was purified to near electrophoretic homogeneity from the membranes of a strain of Escherichia coli carrying the pyrD gene on a multicopy plasmid. The preparation had a specific activity of 120 mumol min-1 mg-1 and contained flavin mononucleotide (FMN) in amounts stoichiometric to the dihydroorotate dehydrogenase subunit (Mr = 37000). The flavin group was reduced when dihydroorotate was added in the absence of electron acceptors. The complete sequence of 1357 base pairs of an EcoRI-EcoRI DNA fragment containing the pyrD gene was established. Dihydroorotate dehydrogenase is encoded by a 336-triplets open reading frame. The molecular mass (Mr = 36732), the amino acid composition and the N-terminal sequence of the predicted polypeptide agree well with the data obtained by analysis of the purified protein. A region of the amino acid sequence (residues 292-303, i.e. Ile-Ile-Gly-Val-Gly-Gly-Ile-Asp-Ser-Val-Ile-Ala) shows distinct homology to the cofactor binding site of other flavoproteins. No hydrophobic regions large enough to span the cytoplasmic membrane were observed. By the S1-nuclease technique an mRNA start was mapped 34 +/- 2 nucleotide residues upstream of the beginning of the coding frame of pyrD. The leader region contains no similarity to the attenuators of the pyrB and pyrE genes of E. coli.

Amino Acids↗
PubMed1985

Codon-defined ribosomal pausing in Escherichia coli detected by using the pyrE attenuator to probe the coupling between transcription and translation.

This communication describes an assay for the relative translation efficiency of individual codons which makes use of the pyrE attenuator to probe the coupling between transcription and translation at the end of an artificial leader peptide. By cloning of short synthetic DNA fragments the codons to be tested were placed in the middle of the leader peptide and the downstream transcription of a pyrE"lacZ gene was monitored by measuring beta-galactosidase activity. The substitution, one by one, of three AGG codons for arginine with three CGT codons for the same amino acid residue was found to cause a two fold increase per codon of transcription over the pyrE attenuator, such that an eight fold higher frequency of pyrE expression was seen when all three AGG codons were replaced by CGT codons. No such effect of codon composition was observed, when the cells were grown with a low UTP pool which causes a reduction of the mRNA chain growth rate.

Base Sequence↗