Concurrent DNA Research Articles

Characterization of Simian Virus 40 tsA 58 Transcriptional Intermediates at Restrictive Temperatures: Relationship Between DNA Replication and Transcription

When nuclei from simian virus 40 (SV40)-infected cells are lysed with Sarkosyl and the chromatin is pelleted, the supernatant fluid contains a nucleoprotein complex capable of synthesizing viral RNA (Laub and Aloni, Virology 75:346-354, 1976; Gariglio and Mousset, FEBS Lett. 56:149-155, 1975). The level of activity of the RNA polymerase in the complex increased during infection in parallel with the amount of viral DNA that had been synthesized. If cells infected at 33 degrees C with the SV40 mutant tsA 58 were shifted to the nonpermissive temperature of 40 degrees C at any time between 18 and 48 h postinfection, no viral DNA replication was detected after 45 min and no new rounds of synthesis were initiated after 20 to 30 min. However, after this shift, polymerase activity associated with the nucleo-protein complex did continue to increase for 5 h, at which time it reached a plateau. There was an increase of RNA synthesized from both the early (E) and late (L) SV40 DNA strands, and there was a threefold increase in the ratio of early-to-late RNA species after the shift. In comparable experiments with cells infected with wild-type virions, no increase in polymerase activity occurred because of the temperature change alone. At 33 degrees C, the relative amount of RNA transcribed from the wild-type E-strand was less than tsA 58 at 33 degrees C and did not increase after a shift to 40 degrees C. The tsA 58 transcriptional complexes extracted from cells grown at 33 degrees C sedimented heterogeneously in sucrose gradients, with a peak near 26S. There were no detectable alterations in the sedimentation properties of the complexes when tsA 58-infected cells were shifted to 40 degrees C for 2 h. We conclude that continued synthesis of viral DNA is not an obligatory prerequisite for maintenance of late viral transcription nor is the sedimentation of the transcriptional complex at 26S related to actively replicating DNA molecules serving as templates for transcription. Further, an increase in late transcription can occur under conditions where reinitiation of viral DNA synthesis is prevented. The increase in the synthesis of early and late RNA at the restrictive temperature without concurrent DNA synthesis is discussed in relationship to the function of the A gene product.

Excision of thymine dimers by proteolytic and [formula omitted] fragments of [formula omitted] DNA polymerase I

Excision of thymine dimers from specifically incised ultraviolet irradiated DNA by E. coli DNA polymerase I is stimulated by concurrent DNA synthesis. The 36,000 molecular-weight “small fragment” obtained by limited proteolysis of DNA polymerase I, which retains only the 5′ → 3′ exonuclease activity, also excises thymine dimers, but at one-tenth the rate of the intact enzyme. However, the rate of excision is increased by addition of the “large” 76,000-molecular weight fragment. With the further addition of the 4 deoxynucleoside triphosphates, permitting DNA synthesis to occur, excision approaches rates observed with the intact enzyme. The same result was obtained with a fragment of DNA polymerase I with 5′ → 3′ exonuclease activity that is present uniquely in polymerase I amber mutants.

T4 DNA replication and viral gene expression

The normal dependence of “late” T4 gene expression on concurrent viral DNA replication is circumvented in cells infected with a triple mutant in which viral DNA polymerase, DNA ligase, and the exonuclease functions of genes 46 or 47 are defective. Acrylamide gel electrophoresis of labeled proteins from infected cells has made possible an extension of the analysis of replication-uncoupled T4 protein synthesis. We find a number of late T4 proteins synthesized: the products of genes 34, 37, 18, 23 and 24. Processing of the gene 23 product, normally headassembly dependent, occurs, but with considerably diminished efficiency compared to wild-type infection. Late T4 protein synthesis in replication-uncoupled infection retains a requirement for T4 gene 33 and gene 55 function. Finally, a number of “early” T4 gene products, normally shut off late in wildtype infection, continue to be synthesized late in replication-uncoupled infection, concurrently with the late proteins.

Patterns of Ribonucleic Acid Synthesis in T5-infected Escherichia coli: IV. EXAMINATION OF THE ROLE OF DEOXYRIBONUCLEIC ACID REPLICATION

The role of DNA replication in the synthesis of RNA and proteins during the period of the infective cycle of T5-infected Escherichia coli F has been examined. Inhibition of DNA synthesis was achieved by addition of chloramphenicol to T5-infected cultures or by use of a mutant of T5 (T5 am I), which is unable to cause the synthesis of an active DNA polymerase upon infection of the nonpermissive host, E. coli F. The synthesis of late or Class III RNA and lysozyme, a presumed late protein, was measured. In the absence of DNA synthesis a small amount of late or Class III RNA species is formed by 9 to 12 min after infection, but much greater quantities are produced under conditions allowing concurrent DNA replication. The synthesis of T5 lysozyme occurs normally in both the permissive and nonpermissive hosts.

A Mutant in the Initiation of DNA Synthesis in Salmonella typhimurium

SUMMARY: A mutant of Salmonella typhimurium was isolated which showed temperature-sensitive synthesis of DNA. The mutant (11 G) increased in cell mass at 38° without concurrent DNA synthesis, resulting in loss of viability and the production of long filaments. DNA synthesis at 38° continues for approximately 40 min. at a gradually decreasing rate giving an increase of DNA in Casamino acid minimal medium of about 55% over that present at the time of the shift. This residual DNA synthesis is not reduced by increasing the temperature to 42°, but can be increased by enriching of the medium in which the organisms are grown before the shift. The effect of high temperature on DNA synthesis in 11 G mimics the effect of chloramphenicol, which is known to allow the completion of rounds of replication of DNA without allowing new initiations. The lesion is essentially irreversible and is not corrected by increasing the osmotic pressure of the growth medium. Phage P22 can develop normally in 11 G if added at the time of the shift or 30 min. later. Even if the phage is added 3½ h. after the shift substantial multiplication occurs. Cell division continues for several hours after the temperature shift, resulting in the production of DNA-less cells as reported in a separate publication.

The synchronization of oral development without cell division in Tetrahymena pyriformis GL-C.

AbstractCells of T. pyriformis strain GL‐C synchronized in an amino‐acid‐free medium underwent synchronous oral replacement in place of cell division. This involved development of an oral primordium at the anterior end of the cell, with a concomitant rounding of the cell. The timing of this development was very similar to that of primordium development during synchronous division. In some experiments a second synchronous oral replacement was observed about 90 minutes after the first.Synchronization of oral replacement was similar to that of cell division. Prior to the heat shocks cells maintained in amino‐acid‐free medium were undergoing oral replacement asynchronously (with no concurrent macronuclear DNA synthesis). During the synchronizing treatment, development was initiated in the inter‐shock intervals but was arrested by the 34° heat shocks. As a result, cells became progressively accumulated in the earliest stage of oral replacement development. This accumulation did not take place if a single continuous heat treatment was given, or if cells were maintained in 2,4‐dinitrophenol, actinomycin D, or cycloheximide during the periodic heat shock treatment.The synchronous oral replacement which took place after the end of the synchronizing treatment (EST) could be blocked completely by actinomycin D, cycloheximide, and puromycin, and partially by p‐fluorophenylalanine. Exposure to cycloheximide (1 μg/ml) during stage 1 of stomatogenesis (10–30 minutes after EST) resulted in arrest of development, exposure during stages 3 and 4 (45 minutes) brought about primordium resorption, while exposure in stage 5 (60 minutes) permitted completion of development, with subsequent partial resorption. These responses were identical to those of synchronized dividing cells treated at comparable stages.

Synthesis of chromosomal proteins during the maturation of chicken erythrocytes

During their final maturation, nondividing chicken erythrocytes undergo heterochromatinization and decline of RNA synthesis. Upon incubation of the maturing erythrocytes in vitro with [ 14C]leucine, the chromosome-bound histone and nonhistone proteins become highly labeled. Chromatographic analysis of acid-extractable chromosomal proteins revealed that these cells synthesize the arginine-rich but not the lysine-rich or slightly lysine-rich histones. Tryptic peptide map analysis of the radioactive proteins showed that the labeled amino acid was incorporated into the chromosome-specific polypeptide chains. The results demonstrate that the synthesis of chromosomal nonhistone proteins and of arginine-rich histones can proceed without concurrent DNA synthesis. We propose that the chromosomal proteins which are synthesized during erythrocyte maturation may be involved in heterochromatinization and associated gene repression.

The relationship between histone and DNA synthesis in HeLa cells

The relationship between histone and DNA synthesis in HeLa cells has been investigated. Radioactive amino acids were incorporated in histones using synchronized as well as logarithmic cultures treated with inhibitors of the DNA synthesis. It is demonstrated that only a part of the synthesis of all histone fractions in HeLa cells is dependent on concurrent DNA synthesis, whereas all histones are synthesized in varying degrees even in the absence of DNA synthesis, i.e. during the G 1 phase. Different histone fractions of HeLa cells show differences in their degree of dependence on DNA synthesis. On the basis of previously published studies of other organisms, we suggest that the relationship between the synthesis of histones and DNA may be governed by different mechanisms in cells belonging to different physiological and developmental stages.