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A Revzin

Publications and source records attributed to A Revzin.

33 records · Page 2Linked to original sources
PubMed1980

Intracellular location of catabolite activator protein of Escherichia coli.

The catabolite activator protein was assayed in extracts from the minicell-producing Escherichia coli strain P678-54. The level of catabolite activator protein was found to be the same in both parent cells and purified minicells, regardless of whether the bacteria were grown on glucose (which leads to low intracellular cyclic adenosine monophosphate levels) or on glycerol-yeast extract or LB broth (which lead to high cyclic adenosine monophosphate concentrations in the cell). Thus, at any given time most catabolite activator protein molecules are found in the cytoplasm. The implications of this for the mechanism of catabolite activator protein action at catabolite-sensitive operons are discussed.

Bacterial Proteins↗
PubMed1977

Nonspecific DNA binding of genome-regulating proteins as a biological control mechanism: measurement of DNA-bound Escherichia coli lac repressor in vivo.

Binding of genome regulatory proteins to nonspecific DNA sites may play an important role in controlling the thermodynamics and kinetics of the interactions of these proteins with their specific target DNA sequences. An estimate of the fraction of Escherichia coli lac repressor molecules bound in vivo to the operator region and to nonoperator sites on the E. coli chromosome is derived by measurement of the distribution of repressor between a minicell-producing E. coli strain (P678-54) and the DNA-free minicells derived therefrom. Assuming the minicell cytoplasm to be representative of that of the parent E. coli cells, we find that less than 10% of the repressor tetramers of the average cell are free in solution; the remainder are presumed to be bound to the bacterial chromosome. The minimum in vivo value of the association constant for repressor to bulk nonoperator DNA (K(RD)) calculated from these results is about 10(3) M(-1), and analysis of the sources of error in the minicell experiment suggests that the actual in vivo value of K(RD) could be substantially greater. The value of K(RD), coupled with in vitro data on the ionic strength dependence of this parameter, can be used to estimate that the effective intracellular cation activity of E. coli is no greater than about 0.24 M (and probably no less than 0.17 M) in terms of sodium ion equivalents. The minicell distribution experiments also confirm that the association constant for the binding of inducer-repressor complex to bulk nonoperator DNA (K(RID)) is [unk] K(RD)in vivo. These results are used to calculate minimum in vivo values of K(RO) and K(RIO) (association constants for repressor and for inducer-repressor complex binding to operator) of about 10(12) M(-1) and about 10(9) M(-1), respectively. The results fit a quantitative model for operon regulation in which nonspecific DNA-repressor complexes play a key role in determining basal and constitutive levels of gene expression [von Hippel, P. H., Revzin, A., Gross, C. A. & Wang, A. C. (1974) Proc. Natl. Acad. Sci. USA 71, 4808-4812].

Bacterial Proteins↗
PubMed1977

Binding of E.coli lac repressor to non-operator DNA.

It is shown by melting profile analysis of lac repressor-DNA complexes that repressor binds tightly and preferentially (relative to single-stranded DNA) to double-stranded non-operator DNA. This binding stabilizes the DNA against melting and the repressor against thermal denaturation. Analysis of the extent of stabilization and the rate of dissociation of repressor from non-operator DNA as a function of sodium ion concentration shows, in confirmation of other studies,(3,4) that the binding constant (K(RD)) is very ionic strength dependent; K(RD) increases from approximately 10(6) M(-1) at approximately 0.1 M Na(+) to values in excess of 10(10) M(-1) at 0.002 M Na(+). Repressor bound to non-operator DNA is not further stabilized against thermal denaturation by inducer binding, indicating that the inducer and DNA binding sites probably represent separately stabilized local conformations. Transfer melting experiments are used to measure the rate of dissociation of repressor from operator DNA. These experiments show that most of the ionic strength dependence of the binding constant is in the dissociation process; the estimated dissociation rate constant decreases from greater than 10(-1) sec(-1) at [Na(+)] >/= 0.02 M to less than 10(-4) sec(-1) at [Na(+)] </= 0.002 M. Competition melting experiments are used to show that at 0.02 to 0.002 M Na(+) the affinity of lac repressor for various natural DNAs and synthetic double-stranded polynucleotides (including poly[d(m(6)A-T)], which carries a methyl group in the large groove) are approximately independent of base composition, except that the affinity of repressor for poly[d(A-T)] is approximately 2- to 3-fold greater than for the other DNAs tested. The affinity for single-stranded polynucleotides is atleast 50-fold less than for the doublehelical forms.

Bacterial Proteins↗
PubMed1976

Amphetamine, chlorpromazine and clonidine effects on self-stimulation in caudate or hypothalamus of the squirrel monkey.

In 2 separate groups of squirrel monkeys and within 3 animals low rates of intracranial self-stimulation (ICSS) elicited from caudate or lateral hypothalamic brain sites were increased by as much as 200% above control levels by amphetamine (0.5 mg/kg). Thresholds for responding were decreased by 50%. Increasing the drug dose from 2 to 10 mg/kg produced response inhibition at both brain sites. The duration of inhibitory action of amphetamine (2.0 mg/kg) on ICSS from the medial forebrain bundle (MFB) area of the lateral hypothalamus was 6 hr. At caudate sites ICSS did not occur until 48 hr had elapsed. A 10 mg/kg dose of amphetamine produced a duration of actine of 36 hr in the MFB and 84 hr in the caudate. Chlorpromazine (CPZ) doses of 0.5 and 1.0 mg/kg decreased caudate ICSS significantly more than lateral hypothalamic ICSS. At 1.0 mg/kg the duration of action of CPZ was 6 hr at lateral hypothalamic brain sites and 24 hr at caudate sites. At a 2.0 mg/kg CPZ dose the duration of action was 12 hr in the MFB and 36 hr in the caudate. A dose of 0.10 mg/kg of clonidine blocked high rates of MFB ICSS while within the same animal caudate ICSS was much less affected. Higher doses (0.25 mg/kg) sedated the animal and ICSS was equally inhibited at both sites. These findings, using ICSS as a behavioral measure, suggest that the effects of amphetamine and CPZ involve not only hypothalamic structures but more anterior telencephalic sites as well. The prolonged actions of amphetamine and CPZ on caudate ICSS suggest that drugs acting, in part, on dopamine containing neurons will interfere with certain caudate mediated behavior. Further, since hypothalamic but not caudate ICSS sites are more dose sensitive to drugs that selectively act on NE containing neurons, other amines in addition to NE may play a role in the support of ICSS.

Amphetamine↗
PubMed1974

Non-specific DNA binding of genome regulating proteins as a biological control mechanism: I. The lac operon: equilibrium aspects.

The regulatory system of the lactose operon has been "modeled" by a set of mass action equations and conservation constraints which describe the system at equilibrium. A "base-set" of values of binding constants and total component concentrations has been assembled from the available experimental data, and the simultaneous equations solved by computer procedures, to yield equilibrium concentrations of all the relevant molecular species. Considering the operator-repressor-inducer system alone, it is shown that the in vivo basal and induced (derepressed) levels of lac enzyme synthesis in both wild-type and certain mutant Escherichia coli can be accounted for only if binding of repressor and repressor-inducer complexes to non-specific DNA sites is included in the calculations as an integral component of the ovrall control system. A similar approach was applied to the RNA polymerase-promoter system to show that sigma factor may modulate the general level of transcription in the cell by "inducing" polymerase off non-specific DNA binding sites, thus making it available to promoters. Competitive and non-competitive models for the interaction of repressor and polymerase at the lac operon can, in principle, be distinguished by these computational procedures, though data sufficient to permit unambiguous differentiation between the models are not available at this time. However, for any competitive binding model the results show that repression in the entire (operator-repressor-RNA polymerase-lac promoter) system can occur only because non-specific binding of the regulatory proteins reduces the concentration of free polymerase, relative to that of repressor, to appropriate levels.

Bacterial Proteins↗
PubMedDate not supplied

Studies of DNA-protein interactions by gel electrophoresis.

The use of gel electrophoresis in studies of nucleic acid-protein (especially DNA-protein) interactions has yielded much qualitative and quantitative information about a variety of such systems. The reduction in mobility of complexes relative to free DNA allows isolation and characterization of the complexes as well as determination of thermodynamic and kinetic properties of the interactions. This article begins with a review of recent applications of the "gel retardation" assay, by way of introduction to experiments in two areas. In the first, a hypothesis is tested regarding whether a DNA molecule with sizable proteins bound very near to each end migrates through a polyacrylamide gel differently than does the corresponding complex having the proteins in the middle of the DNA fragment. The data show little mobility differences for these types of complexes, implying that both may move in a linear, "snakelike", manner through the gel. The experiments also provide results pertaining to questions of DNA bending caused by the binding of the E. coli catabolite activator protein (CAP) and RNA polymerase to the lactose promoter region. It appears that DNA bending by CAP at its wild type lac binding site is retained in complexes where RNA polymerase is bound simultaneously at the lac UV5 promoter.

Cloning, Molecular↗