Charles E. Helmstetter

Correlation of gene transcription with the time of initiation of chromosome replication in Escherichia coli

Transcriptional levels of the Escherichia coli mioC and gidA genes, which flank the chromosomal origin of replication (oriC) and the dnaA gene, were correlated with the time of initiation of chromosome replication. The transcripts were measured either in dnaC2(ts) mutants that had been aligned for initiation of chromosome replication by a temperature shift or in synchronous cultures of cells obtained using the baby machine technique. In both types of experiments, mioC transcription was inhibited prior to initiation of chromosome replication and resumed several minutes after initiation. Conversely, gidA and dnaA transcription were both inhibited after initiation of replication, coincident with the period of hemimethylation of oriC DNA. It is proposed that mioC transcription prevents initiation of chromosome replication, and must terminate before replication can begin. It is further proposed that the eclipse period between rounds of replication, i.e. the minimum interval between successive initiations, encompasses the time required to methylate GATC sequences in newly replicated oriC plus the time required to terminate mioC transcription. Conversely, the active transcription of gidA and dnaA prior to initiation is consistent with their positive effects on initiation, and their shutdown after initiation could serve to limit premature reinitiation.

DNA sequestration and transcription in the oriC region of Escherichia coli

In Escherichia coli, gidA and dnaA transcription are shut off transiently after initiation of chromosome replication, while mioC transcription is shut off before initiation. The possible involvements of seqA and dam in these transcriptional periodicities were evaluated by examining transcription of the genes in seqA and dam mutants of E. coli PC2 dnaC2(ts) aligned for initiation of chromosome replication. In both seqA− and dam− cells, gidA and dnaA continued to be transcribed after initiation, whereas the inhibition of mioC transcription before initiation was unaltered. After initiation, transcripts from mioC that traversed oriC reappeared more slowly in seqA+dam+ cells than in seqA− or dam− cells, but before the release of oriC from sequestration. Apparently, initiation of transcription at a promoter can be completely prevented by sequestration, but established transcription forks can traverse sequestered DNA. These findings, plus analyses of transcript levels in steady‐state cultures, support the idea that initiation capacity in seqA mutants is elevated because of the combined influences of increased durations of expression of both gidA and dnaA during the division cycle and defective sequestration at oriC. Accordingly, a proposal for the sequence of events during the interinitiation interval in E. coli is presented based on the evident coupling of transcription to replication.

Synchrony in human, mouse and bacterial cell cultures--a comparison

Growth characteristics of synchronous human MOLT-4, human U-937 and mouse L1210 cultures produced with a new minimally-disturbing technology were compared to each other and to synchronous Escherichia coli B/r. Based on measurements of cell concentrations during synchronous growth, synchrony persisted in similar fashion for all cells. Cell size and DNA distributions in the mammalian cultures also progressed synchronously and reproducibly for multiple cell cycles. The results demonstrate that unambiguous multi-cycle synchrony, critical for verifying the absence of significant growth imbalances induced by the synchronization procedure, is feasible with these cell lines, and possibly others.

Cyclin mRNA stability does not vary during the cell cycle.

The cyclins are tightly regulated elements governing eukaryotic cell cycle progression by means of sequential activation-inactivation of cyclin-dependent kinases. In one manifestation of this regulation, the mRNA levels of several cyclin genes oscillate during the cycle in mammalian cells. Such cycle-dependent fluctuations in transcript levels could result from changes not only in rates of transcription, but also in mRNA stability. Here we used a new, minimally-disturbing method for producing multi-cycle synchronous growth of human MOLT-4 cells, in combination with quantitative real-time RT-PCR, to compare cell cycle-dependent transcript levels and half-lives of cyclin A2, B1, D3, E and PCNA mRNAs. While all mRNA levels except cyclin D3 varied in the cycle, there were no apparent variations in message half-lives. This differs from several previous reports of dramatic fluctuations in the stabilities of cyclin mRNAs, and infers that fluctuations in cyclin mRNA transcript levels during the MOLT-4 cell cycle are not due to variations in half-lives. The discrepancy in mRNA stability determinations could be due to differences in cell types or synchronization methods, but our findings may be representative of mRNA processing in the cycle of cells in unstressed steady-state growth.

Dimensional rearrangement of Escherichia coli B/r cells during a nutritional shift-down

In a search for the mechanism underlying dimensional changes in bacteria, the glucose analogue methyl alpha-D-glucoside was used to effect a rapid reduction in the mass growth rate of Escherichia coli by competitively inhibiting glucose uptake, a so-called nutritional shift-down. The new steady-state cell mass and volume were reached after 1 h, during which the rate of cell division was maintained; rearrangement of the linear dimensions (cell length, diameter), however, required an additional 2 h and caused an undershoot in cell length, consistent with the view that E. coli is slow to modify its diameter. The results are compared with the overshoot in cell length that occurs following nutritional shift-up.

Cell-cycle research with synchronous cultures: an evaluation.

The baby-machine system, which produces new-born Escherichia coli cells from cultures immobilized on a membrane, was developed many years ago in an attempt to attain optimal synchrony with minimal disturbance of steady-state growth. In the present article, we put forward a model to describe the behaviour of cells produced by this method, and provide quantitative evaluation of the parameters involved, at each of four different growth rates. Considering the high level of selection achievable with this technique and the natural dispersion in interdivision times, we believe that the output of the baby machine is probably close to optimal in terms of both quality and persistence of synchrony. We show that considerable information on events in the cell cycle can be obtained from populations with age distributions very much broader than those achieved with the baby machine and differing only modestly from steady state. The data presented here, together with the long and fruitful history of findings employing the baby-machine technique, suggest that minimisation of stress on cells is the single most important factor for successful cell-cycle analysis.

F plasmid replication and the division cycle of Escherichia coli B/r

Abstract A terminal stage in the duplication of many bacterial plasmids involves the transient formation of catenated molecules containing two interlocked monomeric plasmid units. This property of plasmid replication was exploited to examine the relationship between F replication and the division cycle of Escherichia coli B/r cells growing in undisturbed, exponential-phase cultures. Various cultures of F′lac- or FKmr-containing cells were briefly exposed to [3H]thymidine, and then the transfer of radioactivity into, and out of, a catenated dimer consisting of two closed circular monomers was measured during a chase period. The fraction of plasmid molecules present in this dimer form was determined by separating cellular DNA in alkaline sucrose gradients. In addition, plasmid replication was studied in synchronously growing cultures by measuring both [3H]thymidine incorporation into covalently closed circular DNA and β-galactosidase inducibility. The results suggest that replication of F plasmids can take place throughout the cell division cycle, with the probability of replication increasing toward the end of the cycle. The presence of DNA homologous to the chromosome on the F′lac did not alter the replication pattern of the plasmid during the division cycle.

A ten-year search for synchronous cells: obstacles, solutions, and practical applications

My effort to use synchronously dividing cultures to examine the Escherichia coli cell cycle involved a 10-year struggle with failure after failure punctuated by a few gratifying successes, especially at the end. In this essay, I recount my personal journey in this obsessive experimental pursuit. That narrative is followed by a description of a simplified version of the “baby machine,” a technique that was developed to obtain minimally disturbed, synchronously growing E. coli cells. Subsequent studies with this methodology led to an understanding of the basic properties of the relationship between chromosome replication and cell division. Accordingly, I end this reminiscence with a simple, fool-proof graphical strategy for deducing the pattern of chromosome replication during the division cycle of cells growing at any rate.

Characterization of cell-cycle-specific events in synchronous cultures of Escherichia coli: a theoretical evaluation.

Synchronous growth studies are often used to assess the presence, timing and duration of periodic phenomena in the bacterial cell cycle. In an effort to evaluate the quality and quantity of information on cycle-specific events that can reasonably be expected from such inquiries, a model was constructed of a synchronous culture of Escherichia coli cells as would be derived from a growing population immobilized on a surface, and applied to the case of one stable and one unstable cellular component. The results indicated that, while the presence of cycle-specific events may be easily detectable, their timing and duration are very difficult to establish in synchronous growth experiments. Furthermore, differences in timing can be misconstrued as differences in duration, and vice versa, when interpretations are based on the qualitative analysis of the data.

Technology for cell cycle research with unstressed steady-state cultures

A culture system for performing cell cycle analyses on cells in undisturbed steady-state populations was designed and tested. In this system, newborn cells are shed continuously from an immobilized, perfused culture rotating about the horizontal axis. As a result of this arrangement, the number of newborn cells released into the effluent medium each generation is identical to the number of cells residing in the immobilized population, indicating that one of the two new daughter cells is shed at each cell division. Thus, the immobilized cells constitute a continuous, steady-state culture because the concentrations, locations and microenvironments of the cells in the culture vessel do not vary with time. In tests with mouse L1210 lymphocytic leukemia cells, about 108 newborn cells were produced per day. This new culture system enables a multiplicity of cell cycle analyses on large numbers of cells assured to be from populations in steady-state growth.