Nuclear Segregation and the Growth of Clones of Spontaneous Mutants of Bacteria

Abstract

SUMMARY: An investigation of the rate of increase of spontaneous mutants and the subsequent increase in mutant proportion in a bacterial culture revealed discrepancies between the observed results and those expected on the assumption that mutant and parent grew with equal rates. These discrepancies could not be accounted for in terms of a selective difference between established mutants and their parents since, when the two were mixed together in reconstruction experiments, they fared equally well for hundreds of generations. Rather the discrepancies indicated a difference between parents and new mutants. The data were consistent with the hypothesis that the mutation occurred independently among one of four mutable units (nuclei) and that the mutant nucleus was 'dominant' over its sister non-mutant nuclei in the heterocaryon so formed. As a consequence, a delay of two generations ensued before the mutant unit segregated into the homocaryotic ancestor of a mutant clone. This process delayed the onset of an increase in mutant numbers after mutation. The accurate prediction of the pseudo-equilibrium level of mutants, based on verifiable assumptions of periodic selection, mutation and segregation lag, is added evidence for the occurrence of a two-generation delay before the increase of the mutant clone. This phenomenon, called segregation lag, is a source of error in the calculation of mutation rate by methods involving the numbers of mutants found in bacterial cultures. Furthermore, because bacteria may be multinucleate, the rate of mutation/bacterium/generation is not the same as the rate of mutation/mutable unit (nucleus)/generation.That a growing bacterium may contain more than one body consisting of deoxyribonucleic acid is well known. Witkin (1951) was the first, however, to offer direct evidence indicating that these bodies contain genetic material and are the counterparts of nuclei. Her evidence was threefold. First, the ratio of wholly mutant colonies to those containing mutant sectors was proportional to the ratio of uninucleate bacteria in the irradiated culture which gave rise to the colonies. This finding is consistent with the hypothesis that sectored colonies originate through segregation of the nuclear bodies during growth. Secondly, there was a proportionality between the frequency of induced mutants, whole and sectored, and the frequency of bacteria with more than one nuclear body. Finally, there was some correlation between the types of sectored colonies and the most frequent number of nuclear bodies/multinucleate cell; cultures with mostly binucleate bacteria gave rise predominantly to half sectors while cultures consisting for the most part of cells with four nuclei gave rise predominantly to quarter sectors. A more extensive correlation was not attempted because of the difficulty in determining nuclear numbers.This difficulty is even greater with bacteria that have been grown 0.n chemically defined media. Yet it is sometimes necessary to know the average number

Effect of Temperature on Natural Mutation in Escherichia coli

“…The nuclei were usually easy to count in all organisms in contrast to the preparations reported previously (Ryan & Wainwright, 1954). The average number of nuclei was 1.8 with an insignificant variation from 1.5 to 2-1, irrespective of stage or temperature.…”

“…The exponential results obtained indicated that there was a selection for the hf bacteria at 1 5 ' , a phenomenon not observed at 3 7 ' . The initial slope of the increase curve was, however, c. 9 x 10-9 which when compared with the value of 7 x found in the Poisson experiments, suggested the presence of 8 instead of 4 mutable units as had been found at 37' by the same scheme of comparison (Ryan & Wainwright, 1954). Therefore, when the mutation rates at other temperatures were divided by 4 and that at 15O by 8, indistinguishable mutation rates/ mutable unit/generation are obtained.…”

“…It cannot be investigated in the chemostat, probably for reasons of selection resulting at least in part from the fact that new mutants do not increase in number until they have become homocaryotic through division (Ryan & Wainwright, 1954; de Rothschild & Ryan, 1953). The basis of this segregation delay resides in the fact that h+ is a dominant condition.…”

Spontaneous Mutation in Spheroplasts of Escherichia colid

“…Yet proflavin, which induces auxotrophs, also does not revert the his-gene. Moreover, the possibility has not been excluded that the mutable unit under investigation is composed of RNA or protein even though it is located in a DNA-containing body (Ryan & Wainwright, 1954). Not enough evidence is as yet at hand to require rejection of the proposition that some natural mutations may result from copy-errors during the replication of the gene.…”

A Quantitative Measurement of the Human Somatic Mutation Rate

“…In microbial organisms, the rate of mutation can be measured by Luria-Delbrück fluctuation analysis (1) or in continuous cultures (2,3). By such methods, the rates of inactivating ( forward) mutations in lacI in Escherichia coli and CAN1 in Saccharomyces cerevisiae have been estimated to be 4 Â 10 À7 and 1 Â 10 À7 , respectively (4,5).…”