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Biomedical subjects

F G Hansen

Publications and source records attributed to F G Hansen.

At least 37 records · Page 2Linked to original sources
PubMed1991

Initiator (DnaA) protein concentration as a function of growth rate in Escherichia coli and Salmonella typhimurium.

The DnaA protein concentration was determined in five different Escherichia coli strains and in Salmonella typhimurium LT2 growing at different growth rates. The DnaA protein concentration was found to be invariant over a wide range of growth rates in the four E. coli K-12 strains and in S. typhimurium. In E. coli B/r the DnaA protein concentration was generally higher than in the K-12 strains, and it increased with decreasing growth rates. For all the strains, there appears to be a correlation between the DnaA protein concentration and the initiation mass. This supports the concept of the concentration of DnaA protein setting the initiation mass and, thus, that the DnaA protein is a key molecule in the regulation of initiation of chromosome replication in members of the family Enterobacteriaceae.

Bacterial Proteins↗
PubMed1991

A missense mutation in the rpoC gene affects chromosomal replication control in Escherichia coli.

An RNA polymerase mutant with a single-base-pair change in the rpoC gene affects chromosome initiation control. The mutation, which is recessive, is a G to A transition leading to the substitution of aspartate for glycine at amino acid residue 1033 in the RNA polymerase beta' subunit. The chromosome copy number is increased twofold in the mutant at semipermissive growth temperatures (39 degrees C). In a delta oriC strain, in which chromosome initiation is governed by an F replicon, chromosome copy number is not affected. Plasmid pBR322 copy number is also increased in the mutant at 39 degrees C. The mutation causes a more than fivefold increased expression of the dnaA gene at 39 degrees C. It is conceivable that it is this high DnaA concentration which causes the high chromosome copy number and that the mutant RNA polymerase beta' subunit exerts its effect by altering the expression of the dnaA gene. However, other factors must be affected as well to explain why the RNA polymerase mutant can grow in a balanced fashion with a high chromosome concentration. This is in contrast to wild-type cells, which exhibit higher origin concentrations when DnaA protein is overproduced, but in which the overall DNA concentration is only moderately affected.

Aspartic Acid↗
PubMed1989

The DnaA protein determines the initiation mass of Escherichia coli K-12.

DNA replication was studied in a dnaA(Ts) strain containing a plasmid with the dnaA+ gene under plac control. At 42 degrees C, initiation of DNA replication was totally dependent upon the gratuitous inducer isopropyl beta-D-thiogalactopyranoside (IPTG). Flow cytometric measurements showed that at 13% induction of the lac promoter the growth rate, cell size, DNA content, and timing of initiation of DNA replication were indistinguishable from those observed in a wild-type control cell. Higher levels of induction resulted in initiations earlier in the cell cycle and a corresponding increase in the time from initiation to termination. We conclude that the concentration of DnaA protein determines the time of initiation and thereby the initiation mass. With an induction level equal to or above 13%, the synchrony of multiple initiations within one cell was close to that found in a wild-type control cell, showing that a cyclic variation in DnaA content is not necessary for a high degree of synchrony.

Bacterial Proteins↗
PubMed1989

Cloning and characterization of the Escherichia coli phosphoglycerate kinase (pgk) gene.

The pgk gene of Escherichia coli coding for the phosphoglycerate kinase was subcloned from the Carbon and Clarke collection plasmid pLC33-5. The position and direction of transcription of the pgk gene was determined by Tn5 insertion mutagenesis. Analysis of proteins encoded from these plasmids showed that the pgk gene product is a 40-kDa protein, and that the gene is transcribed from two promoters, one immediately in front of the gene and one in front of an upstream gene coding for a 38-kDa polypeptide of unknown function. The position of the Pgk protein on two-dimensional O'Farrel gels was identified, and from this we conclude that it is one of the proteins induced by anaerobiosis [Smith and Neidhardt, J. Bacteriol. 154 (1987) 336-343]. The pgk gene was also found to show growth phase regulation; the synthesis of Pgk protein was induced more than ten-fold during transition from the exponential to the stationary growth phase.

Anaerobiosis↗
PubMed1989

In vivo studies of DnaA binding to the origin of replication of Escherichia coli.

We have shown that DnaA, a protein required for initiation of DNA replication in Escherichia coli, binds to three of four DnaA binding sequences in the replicative origin oriC (boxes R1, R2 and R4). Protein-oriC DNA interactions in minichromosomes carried by wild-type and dnaA mutant strains were demonstrated by in vivo footprinting using dimethylsulfate treatment of intact cells. The same characteristic enhancement/protection pattern was seen in wild-type minichromosomes or mutants defective in oriC function but carrying the four DnaA boxes. Minichromosomes in dnaA (Ts) mutants showed no protein binding at non-permissive temperatures and reduced binding even at permissive temperatures. In vivo footprints of the wild-type strain were identical to those obtained in vitro using purified DnaA proteins and oriC DNA. Transcription into oriC affected the binding of DnaA protein to the DnaA boxes. These findings suggest that the protein causing the in vivo footprints at oriC is DnaA.

Bacterial Proteins↗
PubMed1989

Isolation, characterization, and nucleotide sequence of appY, a regulatory gene for growth-phase-dependent gene expression in Escherichia coli.

A plasmid carrying a regulator gene, designated appY, was found in the screening of an Escherichia coli gene library for clones overproducing AppA, an acid phosphatase which is induced as a culture approaches the stationary phase. In cells containing multicopy plasmids carrying the appY gene, the expression of the chromosomal appY gene was stimulated 10- to 40-fold in the stationary phase and more than 100-fold during exponential growth. The appA plasmid also changed the rate of synthesis of more than 30 other proteins in a growth-phase-dependent way. The appY gene was mapped to 13 min on the E. coli genetic map. The position of the appY gene on the 4.9-kilobase HindIII fragment of the original clone was located by Tn5 mutagenesis and deletion analysis, and the nucleotide sequence of a 1.9-kilobase region containing the gene was determined. The appY gene product was identified as a weakly expressed 243-amino-acid polypeptide which contains a stretch of 20 amino acids showing very good similarity to the conserved DNA-binding domain of repressors and transcriptional activators.

Amino Acid Sequence↗
PubMed1988

Coordination of chromosome replication initiation in Escherichia coli: effects of different dnaA alleles.

The synchrony of initiation of chromosomal replication in single cells was determined in ten different dnaA(Ts) mutants. After inhibiting the initiation of replication but allowing initiated rounds of replication to terminate, we measured the number of fully replicated chromosomes per individual cell by flow cytometry. Synchronous initiation at the several independent origins (oriC) in single rapidly growing cells would give 2'' (n = 0,1,2,3,...) chromosomes per cell, whereas asynchronous initiation was indicated by the presence of a different number of chromosomes. Mutations mapping in the central part of the dnaA gene (dnaA5, dnaA46, dnaA601, dnaA602, and dnaA604) lead to a high degree of asynchrony (class I mutants), whereas mutations mapping in either of the distal parts of the gene (dnaA508, dnaA167, dnaA203, and dnaA204) yielded a low degree of asynchrony at the permissive temperature (class 2 mutants). The dnaA205 mutant exhibited an intermediate degree of asynchrony. Mutants dnaA203 and dnaA204 (promoter distal) differed from the other class 2 mutants (dnaA167, dnaA508; promoter proximal) in that asynchrony increased no more than twofold between 25 and 37 degrees C compared with the more-than-fourfold increase in the latter. The high degree of asynchrony in class 1 mutants was independent of temperature and was not due to insufficient functional DnaA protein, because overproduction of DnaA46 protein did not decrease the asynchrony. The data demonstrate that the DnaA protein has functions in addition to acting positively in the initiation process and negatively as its own repressor, namely in coordinating initiations at all oriC sites within a single cell.

Alleles↗
PubMed1988

Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein.

Plasmids carrying the intact Bacillus subtilis dnaA-like gene and two reciprocal hybrids between the B. subtilis and Escherichia coli dnaA genes were constructed. None of the plasmids could transform wild-type E. coli cells unless the cells contained surplus E. coli DnaA protein (DnaAEc). A dnaA (Ts) strain integratively suppressed by the plasmid R1 origin could be transformed by plasmids carrying either the B. subtilis gene (dnaABs) or a hybrid gene containing the amino terminus of the E. coli gene and the carboxyl terminus of the B. subtilis gene (dnaAEc/Bs). In cells with surplus E. coli DnaA protein, expression of the E. coli dnaA gene was derepressed by the B. subtilis DnaA protein and by the hybrid DnaAEc/Bs protein, whereas it was strongly repressed by the reciprocal hybrid protein DnaABs/Ec. The plasmids carrying the different dnaA genes probably all interfere with initiation of chromosome replication in E. coli by decreasing the E. coli DnaA protein concentration to a limiting level. The DnaABs and the DnaAEc/Bs proteins effect this decrease possibly by forming inactive oligomeric proteins, while the DnaABs/Ec protein may decrease dnaAEc gene expression.

Bacillus subtilis↗
PubMed1987

Titration of DnaA protein by oriC DnaA-boxes increases dnaA gene expression in Escherichia coli.

Binding of the DnaA protein to its binding sites, the DnaA-boxes (TTATCCACA), was measured by a simple physiological approach. The presence of extra DnaA-boxes in growing cells leads to a derepression of dnaA gene expression, measured as beta-galactosidase activity of a dnaA-lacZ fusion polypeptide. Different DnaA-boxes caused different degrees of derepression indicating that the DnaA protein requires sequences in addition to the DnaA-box for efficient binding. The DnaA-boxes in oriC might act cooperatively in binding of the DnaA protein. The derepressed levels of DnaA protein obtained in a strain carrying an oriC+-pBR322 chimera were very high and sufficient to activate oriC on the chimeric plasmid, which was maintained at a copy number more than three times that of pBR322.

Bacterial Proteins↗
PubMed1987

Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli.

Increased synthesis of DnaA protein, obtained with plasmids carrying the dnaA gene controlled by the heat inducible lambda pL promoter, stimulated initiation of replication from oriC about threefold. The overinitiation was determined both as an increase in copy number of a minichromosome and as an increase in chromosomal gene dosage of oriC proximal DNA. The additional replication forks which were initiated on the chromosome did not lead to an overall increase in DNA content. DNA/DNA hybridization showed an amplification encompassing less than a few hundred kilobases on each side of oriC. Kinetic studies showed that the overinitiation occurred very rapidly after the induction, and that the initiation frequency then decreased to a near normal frequency per oriC. The results indicate that the DnaA protein is one important factor in regulation of initiation of DNA replication from oriC.

Bacterial Proteins↗
PubMed1987

Comparison of dnaA nucleotide sequences of Escherichia coli, Salmonella typhimurium, and Serratia marcescens.

The dnaA genes of Salmonella typhimurium and Serratia marcescens, which complemented the temperature-sensitive dnaA46 mutation of Escherichia coli, were cloned and sequenced. They were very homologous to the dnaA gene of E. coli. The 63 N-terminal amino acids and the 333 C-terminal amino acids of the corresponding DnaA proteins were identical. The region in between, corresponding to 71 amino acids in E. coli, exhibited a number of changes. This variable region coincided with a nonhomologous region found in the comparison of E. coli dnaA and Bacillus subtilis "dnaA" genes. The regions upstream of the genes were also homologous. The ribosome-binding area, one of the promoters, the DnaA protein-binding site, and many GATC sites (Dam methyltransferase-recognition sequence) were conserved in these three enteric bacteria.

Amino Acid Sequence↗
PubMed1985

Conservation of genes and their organization in the chromosomal replication origin region of Bacillus subtilis and Escherichia coli.

The organization of six open reading frames which were deduced from the nucleotide sequence of some 10 kb from the replication origin region of Bacillus subtilis resembles the organization of the genes in the rnpA-dnaA-gyrB region of the Escherichia coli chromosome. Based on the detection of homology with the E. coli genes the open reading frames were found to represent the Bacillus 'rnpA', 'rpmH', 'dnaA', 'dnaN', recF and gyrB genes. Only the latter two have also been defined by genetic analysis. Two regulatory regions containing nine and four copies of a repeating sequence, DnaA-box, which is identical with the DnaA protein-binding sequence repeated four times in the E. coli origin of replication, flank the 'dnaA' gene of B. subtilis. One or both of them are proposed to function as origins in the initiation of chromosomal replication. Transcription of the 'dnaA' gene of Bacillus starts in one of these regions and appears to be coupled to initiation of chromosomal replication. We propose that the conserved gene organization in the 'dnaA'-'gyrB' region of B. subtilis is representative of the replication origin region of a primordial replicon. The oriC sequence of E. coli has either been translocated to its present location 44 kb away from the primordial origin or has independently evolved there.

Amino Acid Sequence↗
PubMed1985

Autoregulation of the dnaA gene of Escherichia coli K12.

Regulation of the dnaA gene, which codes for an essential factor for the initiation of replication from the chromosomal origin, was studied in vivo using transcriptional and translational gene fusions. We found that the dnaA gene was autoregulated over a 30-fold range by the activity of dnaA protein. Expression from the dnaA promoter region of a dnaA"lacZ fusion was inhibited up to sevenfold by surplus dnaA protein and was stimulated up to fivefold upon thermoinactivation of the mutant protein in five different dnaA(Ts) strains. The autoregulation was found to be exerted at transcription from the major dnaA promoter and was eliminated by deletion of sequences around position -65 of this promoter where a 9-bp sequence, which is also found four times in the chromosomal origin, is located.

Bacterial Proteins↗
PubMed1985

Physical mapping and nucleotide sequence of the rnpA gene that encodes the protein component of ribonuclease P in Escherichia coli.

The rnpA gene, coding for the protein component of ribonuclease P (RNase P), was allocated to the dnaA region at 83 min of the E. coli K-12 map. This was accomplished through analysis of recombinant pBR322 plasmids, some of which complemented the temperature sensitivity of a strain carrying the rnpA 49 allele and restored the RNA processing activity. Although the temperature sensitivity of a strain carrying the rnp-241 allele could not be complemented by the rnpA+ plasmid, the RNA-processing activity was restored, suggesting that the rnp-241 mutation is allelic with rnpA 49. In this analysis we also found two genes coding for proteins (60 and 50 kDal) of unknown function. The order of the genes located in this region is in the clockwise orientation: rpmH (5.4 kDal; ribosomal protein L34), rnpA (14 kDal; protein component of RNase P), a gene for a 60-kDal protein (inner membrane protein), a gene for a 50-kDal protein, and tnaA. All these genes are expressed in the clockwise orientation. From the DNA sequence of the rnpA gene region a very basic polypeptide with an Mr of 13773 could be deduced. We conclude that this polypeptide is the rnpA gene product, and is the protein component of RNase P. Comparison with previously published data on the transcription of rpmH suggests that the rnpA gene is the second gene in the rpmH operon.

Amino Acid Sequence↗
PubMed1984

The promoters of the atp operon of Escherichia coli K12.

The nucleotide sequence has been determined of a 900 bp segment of chromosomal DNA located between 2.6 and 3.5 kb left of the origin of replication, oriC. This segment, which overlaps with the known sequence of the atp operon coding for the eight subunits of the Escherichia coli K12 ATP synthase, contains two coding sequences with the same polarity (counterclockwise) as the atp genes: One of these, designated atpI, which codes for the N-terminal part of a 14 kD polypeptide, is located in front (upstream) of the atpB gene (the first structural gene in the atp operon), the other one codes for the C-terminal part of the gidB gene. The 606 bp segment located between the gidB and the atpI genes contains no coding sequences. By employing the nuclease S1 mapping technique, we have determined a promoter, designated atpIp, for the atp operon located in front of the atpI gene; two additional, weak transcription starts were located within the atpI gene. No transcription start sites were detected up to 1,000 bp upstream of the atpIp promoter, neither were any transcription start sites detected within the cluster of the eight structural atp genes. The atp operon transcription terminates at a site approximately 50 bp downstream from the atpC gene.

Amino Acid Sequence↗
PubMed1984

Fine structure genetic map and complementation analysis of mutations in the dnaA gene of Escherichia coli.

A fine structure genetic map of several mutations in the dnaA gene of Escherichia coli was constructed by the use of recombinant lambda and M13 phages. The dnaA508 mutation was found to be the mutation most proximal to the promoter, while the dnaA203 mutation was found to be the most distal one. The order of mutations established in this analysis was: dnaA508, dnaA167, (dnaA5, dnaA46, dnaA211), dnaA205, dnaA204, dnaA203. The mutations dnaA601, dnaA602, dnaA603, dnaA604 and dnaA606 were found to map very close to each other and close to dnaA205 in the middle third of the dnaA gene. In analysing the dominance relationship all 13 dnaA mutations were found to be recessive to the wild type. Characteristic phenotypes of the dnaA(Ts) mutants, like reversibility of the temperature inactivation of the dnaA protein, cold sensitivity of haploid or of merodiploid strains and suppressibility by rpoB mutations, are found to correlate with clusters of mutations within the gene.

Bacterial Proteins↗
PubMed1983

Conditional change of DNA replication control in an RNA polymerase mutant of Escherichia coli.

A temperature-sensitive mutant of Escherichia coli with a temperature-dependent change in the control of initiation of DNA replication was isolated. The phenotype of the mutant was dependent on a mutation in the RNA polymerase gene rpoC. In vitro RNA polymerase activity was temperature sensitive. The mutant grew and synthesized DNA at 30 degrees C as did the wild type. After a shift to 39 degrees C, a temperature still permissive for growth, the mutant increased its origin concentration more than twofold. After a shift from 39 to 30 degrees C, initiation of DNA replication was inhibited until the normal origin concentration was reestablished.

DNA Replication↗