Biomedical subjects
Publications and source records attributed to G Churchward.
We describe the mapping of several genetic loci involved in the replication of the pSC101 plasmid. These include the origin of replication and a short segment of DNA that encodes a pSC101 incompatibility function. This short segment lies within the origin region. Flanking the incompatibility segment are two loci, repA and repB, which are required for replication. The product of the repA locus is shown to be trans-acting.
We have constructed a plasmid cloning vector, pGB2, which is derived from the Escherichia coli plasmid pSC101. The plasmid, which specifies resistance to spectinomycin and streptomycin, contains unique restriction sites for the enzymes HindIII, PstI, SalI, BamHI, SmaI and EcoRI. pGB2 shows no sequence homology, as detected by DNA-DNA hybridization, to several widely used vectors such as pBR322, pUC8 and phage lambda L47.1. Amongst other applications, DNA fragments can be cloned into the plasmid and then radioactive plasmid DNA can be used as a probe to screen recombinant DNA libraries.
A derivative of pSC101, pLC709, was constructed by ligation of the HincII-A fragment of pSC101 to the mini-colEI plasmid pVH51 and to a DNA fragment encoding resistance to the antibiotics streptomycin and spectinomycin. Insertions of the transposon Tn1000 (gamma-delta) into the pSC101 replication region of pLC709 were isolated following cotransfer of the plasmid with the sex factor F. The sites of insertion of the transposon were determined by restriction enzyme analysis and the replication and incompatibility properties of the insertion plasmids and DNA fragments cloned from them were analysed. The insertion mutations defined a locus, inc, of approximately 200 base-pairs that is responsible for pSC101-specific incompatibility. Two mutations adjacent to this region inactivate pSC101 replication but can be complemented in trans by a wild-type pSC101 plasmid, and thus define a trans-acting replication function, rep. The inc locus is within a larger region of some 450 base-pairs that is essential for pSC101 replication and that includes the origin of replication. This 450 base-pair segment can replicate in the presence of a helper plasmid that supplies the rep function in trans.
The nucleotide sequence of 1100bp around the origin of replication of the pSC101 plasmid has been determined. This segment of DNA is capable of replication in the presence of a helper plasmid. The sequence data reveal similarities between pSC101 and several other replicons. The origin of replication contains three direct repeats of an 18bp sequence associated with a segment exceptionally rich in A-T base pairs. A promotor that probably directs transcription of a gene encoding an essential plasmid replication function is associated with a region of extensive potential secondary structure. The sequence presented here includes the sequence of the par region involved in partitioning of plasmids at cell division.
We have constructed a pBR322 plasmid derivative which expresses dnaA protein under the control of the E. coli lac UV5 promotor. Expression of the dnaA protein from the plasmid is inducible by isopropyl-beta-D-thiogalactoside. In a dnaA+ strain induction has no effect on the accumulation of DNA. In contrast, in a thermosensitive dnaA46 strain, induction, at either the permissive or the nonpermissive temperature, results in an immediate stimulation of DNA accumulation. We conclude that, while in a dnaA46 strain dnaA protein limits DNA replication, in a dnaA+ strain dnaA protein activity does not control the timing of replication initiation.
We have constructed two miniplasmids, derived from the resistance plasmid R100.1. In one of these plasmids 400 bp of R100.1 DNA have been replaced by DNA from the transposon Tn1000 (gamma-delta). This substitution removes the amino-terminal end of the repA2 coding sequence of R100.1 and results in an increased copy number of the plasmid carrying the substitution. The copy number of the substituted plasmid is reduced to normal levels in the presence of R100.1. The repA2 gene thus encodes a trans-acting repressor function involved in the control of plasmid replication.
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The effect of changing the DNA concentration on RNA synthesis, protein synthesis, and cell growth rate was studied in Escherichia coli B/r. The DNA concentration was varied by changing the replication velocity or by changing replication initiation in a thymine-requiring strain with a mutation in replication control. The results demonstrate that changes in DNA concentration (per mass) have no effect on the cell growth rate and the rates of synthesis (per mass) of stable RNA (rRNA, tRNA), bulk mRNA, or protein or on the concentration of RNA polymerase (total RNA polymerase per mass). Thus, transcription in E. coli is not limited by the concentration of DNA, but rather by the concentration of functional RNA polymerase in the cytoplasm. Changing the DNA concentration does, however, affect fully induced lac gene activity, here used as a model for constitutive gene expression. The magnitude of the effect of DNA concentration on lac gene activity depends on the distribution of replication forks over the chromosome, which is a function of the replication velocity. Analysis of these date reinforces the conclusion that transcription is limited by the concentration of functional RNA polymerase in the cytoplasm.
The initiation mass, defined as cell mass per origin of deoxyribonucleic acid replication (optical density units at 460 nm of culture/origins per milliliter of culture), reflects the intracellular concentration or activity of a hypothetical factor that controls initiation of chromosome replication in bacteria. In Escherichia coli B/r, the initiation mass was found to increase about twofold with increasing growth rate between 0.6 and 1.6 doublings per h; at higher growth rates it remained essentially constant (measured up to 2.4 doublings per h). A low-thymine-requiring (thyA deoB) derivative of E. coli B/r, strain TJK16, was found to have a 60 to 80% greater initiation mass than B/r which was independent of the replication velocity and not related to the thyA and deoB mutations. It is suggested that TJK16 had acquired, during its isolation, a mutation in a gene affecting the initiation of deoxyribonucleic acid replication. The initiation age was not altered by this mutation, but other parameters, including deoxyribonucleic acid concentration and cell size, were changed in comparison with the B/r parent, as expected from theoretical considerations.
The amounts of ribonucleic acid (RNA) polymerase (beta' subunits) and ribosomes (RNA), and the fraction of RNA polymerase actively engaged in transcription, were measured in Escherichia coli B/r as a function of growth rate. By an improved method of quantitating protein bands on electrophoresis gels, the systematic error and reproducibility of the RNA polymerase determination were estimated to be less than 15 and 6%, respectively. For a threefold increase in growth rate, the fractional synthesis of polymerase (relative to protein) increased 1.5-fold, whereas the fractional synthesis of ribosomal protein increased 2.2-fold. The decrease in the amount of RNA polymerase per ribosome with increasing growth rate is interpreted as an expression of the control of the transcriptional read-through from the genes for ribosomal protein, rplJ,L, to the adjacent genes for RNA polymerase subunits, rpoB,C. The number of active RNA polymerase molecules was determined from the synthesis rates of stable and messenger RNA and the known RNA chain growth rates. Comparison of active and total RNA polymerase indicates that the fraction of active enzyme increases from 20 to 30% in the range of growth rates between 0.6 and 2.0 doublings per hour. Possible causes for the inactive enzyme are discussed.
The syntheses of stable ribosomal ribonucleic acid (RNA) and transfer RNA in bacteria depend on the concentration and activity of RNA polymerase and on the fraction of active RNA polymerase synthesizing stable RNA. These parameters were measured in Escherichia coli B/r after a nutritional shift-up from succinate-minimal to glucose-amino acids medium and were found to change in complex patterns during a 1- to 2-h period after the shift-up before reaching a final steady-state level characteristic for the postshift growth medium. The combined effect of these changes was an immediate, one-step increase in the exponential rate of stable RNA synthesis and thus of ribosome synthesis. This suggests that the distribution of transcribing RNA polymerase over ribosomal and nonribosomal genes and the polymerase activity are continuously adjusted during postshift growth to some growth-limiting reaction whose rate increases exponentially. It is proposed that this reaction is the production of amino-acylated transfer RNA and that is exponentially increasing rate results in part from a gradually increasing concentration of aminoacyl transfer RNA synthetases after a shift-up. This idea was tested and is supported by a computer simulation of a nutritional shift-up.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
The theoretical relations between replication, initiation, termination, and deoxyribonucleic acid (DNA) accumulation were derived for experiments in which the length of the time required for the replication of the bacterial chromosome (C period) can be varied. This theory enables one to determine absolute values of the C period from kinetics of DNA accumulation after a "stepup" with thymine-requiring bacteria that are subjected to a sudden increase in the exogenous thymine concentration. Application of this method of data evaluation to an observed step-up experiment with a thy-derivative of Escherichia coli B/r (ATCC 12407) indicated that the theory describes the observed post-step accumulation of DNA accurately within experimental errors. It is also concluded that changes in the replication velocity (C) do not measurably affect the timing of initiation events in a culture.
The theory describing the effect of inhibition of initiation of rounds of deoxyribonucleic acid (DNA) replication on the accumulation of DNA is derived, and an analysis is presented which allows the determination of the time C taken to replicate the bacterial chromosome from the kinetic changes in the accumulation of DNA. This analysis is applied to experiments in which inhibition of initiation was achieved by inhibiting protein or protein and ribonucleic acid synthesis with chloramphenicol or rifampin. The results for both antibiotics are identical and indicate that there is a delay of 6 to 11 min in the effect of the antibiotics on initiation of rounds of replication. If this delay is taken into account, then the value of the C period estimated from such experiments agrees with values obtained by other methods, whereas by conventional data evaluation of such experiments the C period would be overestimated. In the low thymine-requiring derivative of Escherichia coli B/r ATCC 12407 used here, the C period was found to be between 38 and 41 min for cultures growing with a mass doubling time of 29 min in glucose-amino acids medium, supplemented with 20 micrograms of thymine/ml.
The time necessary to replicate the chromosome (C period) was measured in Escherichia coli B/r (ATCC 12407) and a low-thymine-requiring derivative of that strain. In the Thy- strain, C was measured as a function of growth rate and exogenous thymine concentration either from step-up or chloramphenicol experiments. In the Thy+ parental strain, C was measured only as a function of the growth rate and only by the chloramphenicol method. The C period was found to decrease with growth rate and, in the Thy- strain, the C period also decreased with increasing thymine concentration. It approached a value of approximately 37 min at high growth rates.