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DNA-membrane attachment in cultures of E. coli B/r is found to depend on the pattern of DNA synthesis, (a) When the number of replication points increases, the fraction of the DNA that is membrane bound also increases. The relationship between the amount of DNA-membrane attachment and the number of replication points is most consistent with attachment of all replication points and attachment of at least one other chromosomal site; it is inconsistent with attachment of the replication points only, the chromosomal origin only, or the chromosomal terminus only. (b) When repair DNA synthesis is stimulated by irradiation with ultraviolet light, the DNA-membrane attachment remains unchanged.

Several newly isolated temperature-sensitive dnaE mutants of Escherichia coli exhibit powerful mutagenic action at permissive temperatures. Mutation rates for the two most active mutants were assayed at four different temperatures and compared to wild-type behavior. Temperature-resistant revertants of the original temperature-sensitive dnaE mutants exhibited lower, nearly normal, mutation rates, but no antimutator strains were found.

The potassium, calcium, magnesium, and zinc ion content of cells in exponential and synchronously growing cultures of Escherichia coli B/r was determined with an X-ray fluorescence spectrometer and an atomic absorption spectrophotometer. Cellular potassium, calcium, and magnesium content increased smoothly during the cell cycle, but cellular zinc showed a steplike increase about 10 to 15 min after cell division in a culture having a doubling time of 47 min. The possible role of cellular zinc in the control of cell division is discussed.

Ten cold-sensitive mutants defective in deoxyribonucleic acid (DNA) synthesis at 20 C have been identified among 218 cold-sensitive mutants isolated from a mutagenized population of Escherichia coli K-12. Four of the ten mutant alleles, dna-339 dna-340, dna-341, and dna-342, cotransduce with serB(+) and hence may be dnaC mutants. Two of these, dna-340 and dna-341, are recessive to their wild-type allele. The gene product of their wild-type allele is trans acting. Complementation tests have demonstrated that dna-340 and dna-341 are in the same cistron. The mapping of the remaining six mutations is in progress. In an attempt to determine whether LW4 and LW21 were initiator mutants, cultures of these strains were starved of an essential amino acid at 37 C and then incubated at 15 C with the essential amino acid. The amount of DNA synthesis observed under these circumstances was insignificant. These data are consistent with the idea that LW4 and LW21 are initiator mutants. However, attempts to integratively suppress LW4 and LW21 with F' factors were unsuccessful. To resolve the question of whether or not LW4 and LW21 are initiator mutants, more specific tests and criteria are required. Cultures of LW4 and LW21 were toluene treated and used to measure in vitro DNA synthesis. If the cells were incubated either at 15 or 20 C before toluene treatment, they were capable of markedly less DNA synthesis than if preincubation had not occurred. The amount of in vitro DNA synthesis is directly proportional to the amount of DNA synthesis occurring during preincubation in vivo; i.e., more DNA synthesis is observed at 20 than at 15 C. The fact that the cold-sensitive mutants are unable to synthesize DNA when supplied with deoxyribonucleoside triphosphates, DNA precursors, is evidence they are not defective in precursor synthesis.

Four cold-sensitive mutants of Escherichia coli have been isolated which show a reduced ability to synthesize deoxyribonucleic acid at low temperature. The mutants also have a reduced ability to incorporate nucleoside triphosphates into deoxyribonucleic acid at low temperature in cell preparations made permeable with toluene. All four mutations are located at or near the dnaA locus on the E. coli genetic map. They are recessive to the wild-type allele and two of them can be integratively suppressed by F episomes.

A zonal centrifugation technique that can select the smallest newborn cells in an exponentially growing culture of Escherichia coli B/r is described.

A mutant of Escherichia coli (sof) which was previously shown to have increased recombination frequency, to produce abnormally short "Okazaki fragments," and to be deficient in deoxyuridine triphosphatase has now been found also to possess mutator activity for several genes; point mutation rates and deletion rates are affected. The mutational stimulation effects are consistent with the hypothesis that incorporation of uracil into DNA is directly or indirectly responsible for the observed mutator activity.

We have screened about 1.4 million colonies of Escherichia coli K-12 for their ability to grow on nutrient agar at 30 degrees and 41 degrees. Among the 2266 temperature-sensitive mutants found, 110 were defective in DNA synthesis but not in protein synthesis at 41 degrees. Three of these dna mutations mapped at two previously undescribed loci on the E. coli genetic map and may represent new genes involved in DNA replication in E. coli. The mutant isolation was aided by novel automatic machinery that inoculated agar-filled petri dishes with mutagenized E. coli cells laid down in square arrays of evenly spaced rows and columns on the agar. Time-lapse photographs taken before and after a temperature shift were used to find colonies of temperature-sensitive mutants. These mutations were mapped by interrupted conjugation and viral cotransduction methods, and the mutants were divided into three classes according to the kinetics of DNA synthesis at the restricted temperature. Some of the mutants exhibited mutator activity at partially restrictive temperatures. It is argued that some genes involved in DNA synthesis remain to be discovered.

Synchronized cultures of rapidly growing E. coli B/r cells were starved for a required amino acid at various cell ages to allow ongoing chromosome replication to be completed without initiation of new replication cycles. It has been found that when such synchronized cultures are exposed to the mutagen nitrosoguanidine, genetic markers just in the process of replication show a markedly higher mutation rate than markers elsewhere on the chromosome. The number of growing points on the chromosome at each cell age can then be determined by observing the nitrosoguanidine-induced mutation rates for specific genetic markers on the genome. These experiments indicate that there exist multiple growing points on the genome for about ten minutes during the life cycle when the cells are growing with a doubling time of 28 minutes.

Nalidixic acid has been used to interrupt DNA synthesis in exponentially growing cultures of E. coli B/r. Within three minutes after removal of the nalidixic acid block, premature rounds of chromosome replication are initiated at the normal origin of vegetative DNA synthesis. The new growing forks traverse the chromosome in about 40 minutes, the normal replication time. These rounds continue synchronously into the second generation after removal of the nalidixic acid. These phenomena are similar in some respect to the premature initiation of chromosomal replication observed after thymine starvation or after release of thermo-sensitive DNA synthesis mutants from the restrictive temperature that prevents DNA synthesis.

Two processes that are required for the initiation of a new round of bacterial DNA synthesis and which are blocked by low and high concentrations of chloramphenicol, respectively, have been examined in order to determine the cell age at which each of these processes occurs as a function of growth rate. The timing of these steps in the life cycle was determined by observing changes in the variation with cell age of the rate of DNA synthesis during growth in medium containing chloramphenicol of the desired concentration. For cell-doubling times ranging from 27 to 49 minutes, it was found that the step which is resistant to low concentrations of chloramphenicol occurs at the cell age at which a new round of DNA synthesis is initiated. The chloramphenicol-sensitive step was found to occur about 21 minutes before the start of rounds, independent of growth rate for doubling times between 27 and 49 minutes. For doubling of times 41 minutes or less, the chloramphenicol-sensitive processes occurred within five minutes after cell division.

Replication of the chromosome of E. coli B/r has been studied by measuring the change with cell age of the rates of reversion to wild type of auxotrophic markers induced by nitrosoguanidine. Synchronous populations of bacteria were obtained by the procedure of Helmstetter and Cummings. The origin of DNA synthesis was determined to occur at 49 minutes +/- 5 minutes on the genetic map, and the direction of synthesis was found to be clockwise.

The rates of cell growth and of DNA synthesis have been measured in synchronized cultures of Escherichia coli B/r growing with three different doubling times, from 27 to 80 min. Cell volume was found to increase linearly over the cell cycle, the rate of increase doubling at a precise cell age for each growth rate. The time at which the rate of cell growth doubled was also found to be the time at which new rounds of DNA synthesis were initiated. However, it was found that these two events could be separated in time by inhibiting DNA synthesis for a short period by the addition of nalidixic acid. It is concluded that neither event is the direct trigger for the other.

The pattern of new growing forks in the Escherichia coli genome after a period of inhibition of DNA synthesis has been examined. Synchronous cultures of E. coli B/r were treated with nalidixic acid to stop DNA synthesis for various lengths of time. After removal of the nalidixic acid the location of active growing forks was determined by measuring the reversion rate of a histidine requirement induced by the "growing-point mutagen," nitrosoguanidine. It was found that the ability to initiate a new round of DNA synthesis upon release from the nalidixic acid block was attained at the cell age at which a new round of replication would have been initiated had DNA synthesis not been blocked. However, if the nalidixic acid was removed before the normal initiation age, the initiation of the next round of synthesis was delayed past the normal time, which indicates that initiation of new rounds is not completely independent of DNA synthesis.

Nalidixic acid inhibits DNA synthesis in toluene-treated E. coli, strain B/r, as it is well known to do in vivo. Both semiconservative and repair syntheses are affected, though to different degrees. Density-transfer experiments indicate that chromosomal replication is reinitiated when nalidixic acid is removed from toluene-treated cells after exposure to the acid for one generation in vivo. For cells in vivo or after toluene-treatment, reinitiation is not seen in asynchronous cultures exposed briefly to nalidixic acid or in cells prevented from synthesizing proteins during their exposure to the acid. Reinitiation occurs at the chromosomal origin but, unlike the effect seen in vivo, replication at the old site persists.