X-ray-induced perturbations in the replication of the bacterial chromosome

Billen, Daniel; Hewitt, Roger R.; Jorgensen, George N.

doi:10.1016/0005-2787(65)90137-1

Cited by 36 publications

(17 citation statements)

References 16 publications

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“…2 a shows the profile of DNA from lysates that had been sheared by six strokes in a Potter homogenizer (Potter Instrument Co., Inc., Plainview, N.Y.). Such treatment of bacterial lysates results in DNA fragments with an average mol wt of at least I 5< 107 daltons (2). In this case, the parental (1'C-labeled) DNA banded at a density of about 1.700 g/cm' as expected for mammalian DNA.…”

Section: Resultsmentioning

confidence: 86%

DNA replication in Chinese hamster ovary cells made permeable to nucleotides by tween-80 treatment.

Billen¹,

Olson²

1976

The Journal of Cell Biology

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We have developed a method for permeabilizing CHO cells to nucleotides under conditions which allow most cells to remain viable. Permeabilized cells can carry out ATP-dependent, semiconservative synthesis of DNA. The data are consistent with the continuation of DNA synthesis in those cells in S phase at the time of treatment, possibly limited to completion of replicon synthesis without new initiations.

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Section: Resultsmentioning

confidence: 86%

DNA replication in Chinese hamster ovary cells made permeable to nucleotides by tween-80 treatment.

Billen¹,

Olson²

1976

The Journal of Cell Biology

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“…The method for studying replication of the chromosome during various growth conditions involves selective radioisotopic and density labeling of cellular DNA. The details of the method used have been previously described (6).…”

Section: Methodsmentioning

confidence: 99%

Influence of Starvation for Methionine and Other Amino Acids on Subsequent Bacterial Deoxyribonucleic Acid Replication

Billen

Hewitt

1966

J Bacteriol

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Billen, Daniel (University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Tex.), and Roger Hewitt . Influence of starvation for methionine and other amino acids on subsequent bacterial deoxyribonucleic acid replication. J. Bacteriol. 92: 609–617. 1966.—A study has been made of the subsequent replicative fate of deoxyribonucleic acid (DNA) synthesized during amino acid starvation by several multiauxotrophic strains of Escherichia coli . Using radioisotopic and density labels and a procedure whereby total cellular DNA is analyzed, we have confirmed and extended a recent report that the DNA made during amino acid starvation behaves anomalously during subsequent DNA replication. When 5-bromouracil (BU) serves as the density lable, 40% or more of the DNA synthesized during starvation will subsequently fail to replicate during three cell generations. Selective amino acid effects were noted. In two methionine-requiring bacteria, methionine deprivation appeared to be of singular importance in influencing the subsequent replicative fate of the DNA made in its absence. When a non-BU density label (N 15 , C 13 ) was utilized, the effects of amino acid starvation were less obvious. Although the DNA synthesized during complete amino acid starvation in a methionine-requiring E. coli was subsequently more slowly replicated, most of the DNA was finally duplicated during three generations of growth. If methionine was present during starvation for other required amino acids, the subsequent replication rate of the DNA synthesized during this time was more nearly normal, and complete replication was observed. The results have been interpreted as indicating that DNA synthesized during amino acid starvation, and especially during methionine starvation, is somehow altered, and that BU substitution for thymine may interfere with the restoration of such DNA to its replicative state.

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“…All irradiations were carried out at room temperature in minimal salts. The details for X-ray and UV exposure were as described earlier (7).Determination of per cent of radioisotopically labeled deoxyribonucleic acid (DNA) replicated. Details of the procedure for determining per cent of radioisotopically labeled DNA, and the rationale of the method, were 1169 on August 1, 2020 by guest…”

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confidence: 99%

“…All irradiations were carried out at room temperature in minimal salts. The details for X-ray and UV exposure were as described earlier (7).…”

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confidence: 99%

“…An alteration in the expected sequence of chromosome replication in Escherichia coli occurs after exposure to ultraviolet light (UV; reference 13) or X-rays (7). The reorientation of the replication sequence is not an artifact of the methodology (14) or a consequence of repair synthesis (8), but results from a semiconservative synthesis starting at a region differing from that occupied by the growth point at the time of irradiation.…”

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confidence: 99%

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Replication of the Bacterial Chromosome: Location of New Initiation Sites After Irradiation

Billen

1969

J Bacteriol

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New loci of replication along the bacterial chromosome are observed after irradiation of Escherichia coli. It was conjectured that, after X-irradiation, the new initiation site was random with respect to the fixed-origin, whereas, after ultraviolet light exposure, it was selective and appeared to be from the fixed-origin. Evidence presented here shows that, after X-irradiation of E. coli, the new initiation site(s) for the onset of deoxyribonucleic acid replication is induced at chromosomal regions not restricted to the fixed-origin. After ultraviolet light exposure, the new initiation site is preferentially from the fixed-origin. In these studies amino acid starvation was used to synchronize chromosome replication and to allow for differential radioisotopic labeling of the chromosomal origin and terminus. To facilitate interpretation, growing cells actively replicating their chromosome were compared with cells lacking growth points at the time of irradiation. The role of these new replication sites in the observed kinetics of deoxyribonucleic acid replication following X-ray or ultraviolet light exposure is discussed.An alteration in the expected sequence of chromosome replication in Escherichia coli occurs after exposure to ultraviolet light (UV; reference 13) or X-rays (7). The reorientation of the replication sequence is not an artifact of the methodology (14) or a consequence of repair synthesis (8), but results from a semiconservative synthesis starting at a region differing from that occupied by the growth point at the time of irradiation.The present studies were designed to determine whether the new initiation sites were at the fixedorigin of cycles of replication or elsewhere on the chromosome. Advantage was taken of the amino acid deprivation method which allows synchronization of replication starting from the vegetative origin (fixed-origin) of the bacterial chromosome (1,9,21,23). The sequence of replication of radioisotopically marked origin and terminus was then followed by density-transfer.The resulting data show that, in the immediate round of replication following X-irradiation, the new initiation sites are non-origin in location, whereas, after UV they are predominantly from the origin.MATERIALS AND METHODS Bacterial growth. The multiauxotrophic E. coli 15T-(555-7) requiring thymine, arginine, methionine, and tryptophan was used. The cells were grown with forced aeration at 37 C in a minimal salts medium (3) supplemented with 20 ,ug of arginine, 30 /g of methionine, 14 Mug of tryptophan, and 20 MAg of thymine per ml.Harvesting of cells. Cells were harvested and washed on membrane filters (Millipore Filter Corp., Bedford, Mass.) at ambient temperature to reduce trauma. Care was exercised to avoid drying the cells on the filter. The washing consisted of adding an equal volume of minimal salts solution followed by filtration. The sequence was repeated three times. The total time required for washing was less than 5 min.There was no observable deleterious effect on cell growth or macromolecular synt...

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X-ray-induced perturbations in the replication of the bacterial chromosome

Cited by 36 publications

References 16 publications

DNA replication in Chinese hamster ovary cells made permeable to nucleotides by tween-80 treatment.

DNA replication in Chinese hamster ovary cells made permeable to nucleotides by tween-80 treatment.

Influence of Starvation for Methionine and Other Amino Acids on Subsequent Bacterial Deoxyribonucleic Acid Replication

Replication of the Bacterial Chromosome: Location of New Initiation Sites After Irradiation

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