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Biomedical subjects

A Newton

Publications and source records attributed to A Newton.

At least 91 records · Page 5Linked to original sources
PubMed1982

Isolation and expression of cloned hook protein gene from Caulobacter crescentus.

Previous genetic analysis of Caulobacter crescentus showed that the periodic synthesis of hook protein, flagellin A, and flagellin B, the major flagellar subunits, is coupled in some way to chromosome replication. To examine the regulation of flagellar gene expression at the molecular level, we isolated the gene that codes for the 72,000-dalton hook protein. A specific 125I-labeled anti-hook protein IgG was used to screen a hybrid lambdaL47.1 bank of 4,500 clones and to compare peptide maps of the cloned gene product with purified hook protein. Restriction analysis of DNA from the positive lambda clones and plasmid subclones showed that the structural gene for the hook protein is contained on a 2.3-kilobase (kb) BamHI fragment. The direction of transcription was established by demonstrating the inducibility of hook protein gene in strains with the 2.3-kb fragment fused to the Escherichia coli lipoprotein gene-lactose gene promoter-operator region of pIN-II. Preliminary genomic analysis showed that the hook gene occupies a single location on the C. crescentus chromosome. These results suggest that the periodic expression of the hook protein gene in the cell cycle does not involve a major or persistent rearrangement of the 2.3-kb coding sequence during the cell cycle.

Bacteria↗
PubMed1982

Localization of surface structures during procaryotic differentiation: role of cell division in Caulobacter crescentus.

Asymmetric cell division in Caulobacter crescentus produces two cell types, a stalked cell and a new swarmer cell, with characteristics surface structures. We have examined the role of the cell cycle in the differentiation of these two cells using adsorption of bacteriophage phi LC72, the assembly of the polar flagellum, and stalk formation as assays for changes in surface morphology. Previous studies of this aquatic bacterium [17,25] have suggested that the replicating chromosome acts as a "clock' in timing the formation of the flagellar filament at one pole of the new swarmer cell. the analysis of conditional cell cycle mutants presented here extends these results by showing that DNA synthesis is also required for adsorption of phage phi LC72 and, more importantly, they also suggest that a late cell division step is involved in determining the spatial pattern in which the phage receptors and flagella are assembled. We propose that this cell division step is required for formation of "organizational' centers which direct the assembly of surface structures at the new cell poles, and for the polarity reversal in assembly that accompanies swarmer cell to stalked cell development.

Bacterial Physiological Phenomena↗
PubMed1982

Inhibition of herpes simplex virus multiplication by activated macrophages: a role for arginase?

Proteose-peptone-activated mouse macrophages can prevent productive infection by herpes simplex virus in neighboring cells in vitro whether or not those cells belong to the same animal species. The effect does not require contact between the macrophages and the infected cells, may be prevented by adding extra arginine to the medium, and may be reversed when extra arginine is added 24 h after the macrophages. Arginase activity was found both intracellularly and released from the macrophages. The extracellular enzyme is quite stable; 64% activity was found after 48 h of incubation at 37 degrees C in tissue culture medium. No evidence was found that the inefficiency of virus replication in macrophages was due to self-starvation by arginase. As might be predicted macrophages can, by the same mechanism, limit productive infection by vaccinia virus.

Animals↗
PubMed1982

Circular organization of the DNA synthetic pathway in Caulobacter crescentus.

Genetic analysis of the cell cycle of Caulobacter crescentus has identified a DNA synthetic pathway and a cell division pathway (M. A. Osley and A. Newton, J. Mol. Biol. 138:109-128, 1980). The results presented here show that in double-shift experiments the function of the PC2076 gene product, which is required for the initiation of DNA synthesis, depends on completion of a late stage of chromosome replication in the previous cell cycle. These findings suggest a circular organization of steps in the DNA synthetic pathway in C. crescentus.

Bacteria↗
PubMed1979

Purification and characterization of a polyhook protein from Caulobacter crescentus.

A polyhook-producing strain of Caulobacter crescentus was isolated, and the polyhook protein was purified. The antigenicity and morphology of the polyhook structure are similar to the wild-type hook except that the mutant strain produces a hook structure at least 10-fold the length of wild-type hooks (1.0 versus 0.1 micrometers). The molecular weight of the polyhook protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 72,000, and the protein has a pI of approximately 6.1. Antibodies prepared against the polyhook protein were used to show that this protein is antigenically distinct from the Caulobacter flagellins. Amino acid analysis of the polyhook protein revealed compositional similarities to other gram-negative, bacterial hook proteins.

Amino Acids↗
PubMed1978

Polyadenylic acid synthesis activity of purified DNA-dependent RNA polymerase from Caulobacter.

Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulobacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell extracts, DEAE-cellulose column, DNA-affinity chromatography, and low salt glycerol gradient centrifugation. Holoenzyme can be resolved into core and sigma subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure sigma factor.

Bacteria↗
PubMed1978

Characterization of unstable poly (A)-RNA in Caulobacter crescentus.

A significant amount of poly(A)-RNA in Caulobacter crescentus is located on polysomes and the size distribution of this polysomal poly(A)-RNA is small compared to the total pulse-labeled RNA in these cells. These observations suggest that the poly (A)-RNA represents a subset of small messenger RNA molecules. Poly (A)-RNA, and presumably the poly (A) portion of these molecules is extremely unstable: as assayed by binding to oligo (dT)-cellulose the poly (A)-RNA turns over with a chemical half-life of 15--20 s compared to a half-life of approx. 2 min for total cellular messenger RNA. The presence of adenosine in hydrolysates of poly(A) tracts showed that these sequences are located at the 3'-OH end of the RNA. The ratios of AMP/adenosine in the samples confirmed that the length of the A-tracts is approx. 13-17 nucleotides (Ohta, N., Sanders, M. and Newton, A. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2343--2346)

Bacteria↗
PubMed1978

Regulation of cell cycle events in asymmetrically dividing cells: functions required for DNA initiation and chain elongation in Caulobacter crescentus.

To study the regulation of cell cycle events after asymmetric cell division in Caulobacter crescentus, we have identified functions that are required for DNA synthesis in the stalked cell produced at division and in the new stalked cell that develops from the swarmer cell 60 min after division. The initiation of DNA synthesis in the two progeny cells is dependent upon at least two common functions. One of these is a requirement for protein synthesis and the other is a gene product identified in a temperature-sensitive cell cycle mutant. DNA chain elongation requires a third common function. The characteristic pattern of DNA synthesis in C. crescentus appears to be controlled in part by the expression of these functions in the two stalked cells at different times after cell division. The age distribution for Caulobacter cells in an exponential population has been calculated (Appendix by Robert Tax) and used to analyze some of the results.

Bacteria↗
PubMed1977

Regulation of flagellin synthesis in the cell cycle of caulobacter: dependence on DNA replication.

Synthesis of the two filament proteins (flagellin A and flagellin B) of the Caulobacter creascentus flagellum was measured during the cell cycle. Synchronous cells were pulse-labeled with 36S-methionine, and flagellin proteins were isolated from crude extracts by radioimmune precipitation. The results showed that both proteins are maximally induced during the G2 period and that their induction requires de novo transcription. Flagellin A, however, continues to be made in the progeny swarmer cells after flagellin B synthesis has stopped. This discoordination in flagellin A and B synthesis and the relative abundance of the two proteins may result in part from the longer functional half-life of the flagellin A messenger RNA. Analysis of temperature-sensitive DNA chain elongation mutants suggests that the periodicity of flagellin A and B synthesis in the cell cycle is controlled by a late cell cycle event, presumably the completion of chromosome replication.

Bacteria↗
PubMed1977

Mutational analysis of developmental control in Caulobacter crescentus.

The relationship between the cell cycle and control of development has been studied by a genetic analysis of Caulobacter crescentus. The behavior of conditional cell division mutants showed that cell cycle events, such as DNA replication and cell division, are organized into a dependent pathway(s), i.e., later steps cannot preceed until earlier ones are completed. The ability of these strains to develop normally under nonpermissive conditions suggested that flagellin synthesis and stalk formation are dependent on the completion of different cell-cycle steps: the periodicity of flagellin synthesis is achieved by coupling it to either DNA chain elongation or completion, and stalk formation is dependent on a later step in the cell-division pathway. These developmental events are not themselves required for cell division, however.

Bacteria↗
PubMed1977

Reconstitution and purification of flagellar filaments from Caulobacter crescentus.

Filaments from isolated flagella of Caulobacter crescentus have been purified by successive dissociation and reconstitution. After the second and third reconstitutions from subunits in 0.8 M sodium citrate, filament preparations contained only two proteins, flagellin A (26,000 daltons) and flagellin B (28,000 daltons). There was some enrichment for flagellin A during reconstitution by this procedure, since isolated flagella contained flagellin A and flagellin B in a ratio of approximately 3.8:1 and filaments after the third reconstitution contained the two proteins in a ratio of 5.0:1.

Bacteria↗
PubMed1976

Requirement of a cell division step for stalk formation in Caulobacter crescentus.

Penicillin G at low concentrations blocked cell division in Caulobacter crescentus without inhibiting cell growth. The long filamentous cells formed after two to three generations under these conditions had a stalk at one pole and usually one or more flagella at the opposite pole. The failure of the filaments to form a second stalk at the flagellated pole indicates that stalk formation was dependent upon completion of a step that was also required for cell division. Two observations support this conclusion. (i) Penicillin did not stop the normal development of synchronous swarmer cells into stalked initiation and stalk elongation. (ii) When the action of penicillin was reversed by the addition of penicillinase to cultures of filaments, stalks were not formed at the nonstalked pole until after cell division had occurred; thus the normal order of development events was maintained: cell division leads to stalk formation. These results are consistent with a model in which the organization of the developmental program for stalk formation occurs before cell division as a consequence of steps that branch from the cell division pathway.

Bacteria↗
PubMed1975

Poly(adenylic acid) sequences in the RNA of Caulobacter crescenus.

Poly(adenylic acid) sequences have been isolated from the Gram-negative bacterium Caulobacter crescentus. Most of these A-rich tracts are associated with large RNA molecules that constitute an important fraction of the unstable RNA in these bacteria, and, as estimated by poly(U) filter binding, they are not present in the 16S or 23S ribosomal RNA. Preliminary estimates of size from polyacrylamide gel electrophoresis suggest that the majority of the A-rich tracts ranges from 15 to approximately 50 residues in length.

Adenine Nucleotides↗