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13 p.-1 fig.-2 tab., A proliferating population of cells may be considered complex when its proliferative or growth fraction P is lower than 1 and/or when it is formed by subpopulations with different mean cycle times. The present paper shows that in such complex populations exponential growth is consistent with a steady-state distribution of cells. Obviously, when P = 1 then cell distribution is only a function of cell age. An analytical model has been developed to study complex populations including both quiescent fractions formed by cells with unreplicated genome (G0 cells) and cells with fully duplicated chromosomes (Q2 cells). The model also considers those quasi-quiescent cells in their last transit through G1 and S (Q1 and Qs cells) before becoming quiescent. In order to solve the difficulties of a direct analysis of the whole population, its kinetic parameters have been obtained by studying the negative exponential distribution of two subpopulations: one formed by the proliferating cells and another formed by the quasi-quiescent cells. Additionally, the model could be applied when quiescence is initiated at any other cycle phase different from G1 and G2, for instance, cells in the process of replicating their DNA or being at any other mitotic phases. The utility of the method was illustrated in populations which constitute the root meristems of both Allium cepa L. and Pisum sativum L. Three facts should be stressed: (1) the method seems to be rather powerful because it can be carried out from different sets of experimentally measured parameters; (2) the rate of division and, therefore, the population doubling time can be easily estimated by this method; and (3) it also allows the determination of the amount of cells that had become quiescent either before they had replicated their DNA (G0) or after having completed their replication (Q2), as well as those quasi-quiescent cells which are progressing throughout their last pre-replicative and replicative periods (thus Q1 and Qs, respectively). © 2002 Elsevier Science Ltd. All rights reserved., This work has been partially supported by the Direccio! n General de Ensen* anza Superior e Investigacio! n Cient!ıfica del Ministerio de Educacio! n y Cultura from Spain (Projects PB96-0909 and PB97-0909, by the CSIC-Universidad de Chile joint Project (99 CL-0009) and by the European Union (BIO6-CT96-0275). We thank Mr J. L. Marcilla and Ms M. Carrascosa for excellent technical assistance.

The distribution of cell surface area projection (cell size) has been measured at birth and at initiation of DNA synthesis in steady-state populations of Allium cepa root meristems. The conditional probability, P( I G 1 ) , that initiation occurs given that the event of being in g1 also occurs has been estimated from these data. p( I G 1 ) was found to increase when cells became larger. The distribution of G1 duration has been constructed from indicated cell size distributions. The absolute frequencies of G1 times showed a maximum in the zone of cells with short G1 periods; about 14% of cells appear to enter into S with G1 ∼- 1 h. These results suggest that the increase of p( I G 1 ) was due to cell enlargement and not to cell aging. By comparing the cell size distribution at initiation of S and at the end of this period, a drastic reduction of cell size variability during DNA replication was observed and both curves were seen as rather similar in shape although they obviously had different modal points. These observations support that there is a negative correlation between the initiation size and the duration of genome duplication, and that cells which initiate DNA synthesis with the same size have a similar replication time. From this hypothesis, a plot of S duration versus cell size at initiation of this period was constructed by comparing the distributions of cell size at start and end of replication; this plot was also consistent with the existence of a negative correlation between cell initiation size and S length.

7 p.-4 fig.-2 tab., The present work establishes a correlation between cell length and patterns of mitotic microtubular assemblies in Allium cepa L. root meristems. Binucleate cells were formed by a short caffeine treatment which aborted the formation of the phragmoplast during telophase. The largest binucleate cells (about 50 μm in length) behaved as two contiguous mononucleate cells in their next mitosis: they developed two preprophase bands (PPBs), one around each nucleus, where two spindles and two phragmoplasts were subsequently formed. On the other hand, the shortest binucleate cells (about 36 μm in length) formed a single PPB at the site of the aborted phragmoplast and, in the medium-sized cells (about 44 μm) in which the single PPB formed around the nucleus possessing the largest cytoplasmic environment, the two mitotic spindles and the new phragmoplasts moved to, or were assembled in the position of the phragmoplast that had been aborted one cycle earlier. Some rules derive from these observations. First of all, the aborted phragmoplast left a signal for microtubule positioning which was still operative one cycle later, in two-thirds of the bimitoses. Also, that formation of the PPB is dispensable. Moreover, its development does not always predict the future division plane, because of the presence of competing old signals which are stronger than those shed by the PPB in the same mitosis, but which fade away with distance. Finally, the positional signals were reinforced when the ratio of monomeric to fibrillar actin was increased by cytochalasin D during their shedding. When this drug was given simultaneously with caffeine, the frequency of bimitoses which, one cycle later, developed spindles and phragmoplasts in the positions of the old phragmoplast increased. On the other hand, those frequencies dropped in relation to control when the cytochalasin D treatment took place during bimitosis, indicating that at this time the treatment reinforced the signals produced in bimitosis itself.

Two cell size parameters, protein content and surface area projection, have been measured in proliferating cells of Allium cepa roots. Cells increase in size at birth and DNA synthesis termination when grown in the presence of hydroxyurea; under these conditions, G1 and G2 are reduced without affecting the growth rate. Opposite cell size changes at the same points are induced by growth in the presence of anisomycin under conditions in which G1 and G2 are extended, the former in higher proportion to the generation time and the later in less proportion. These results, while being consistent with cell size regulation of G1 and G2, suggest that the nature of G1 and G2 controls is different.

Fructose transport was not apparently affected in a number of Pseudomonas putida strains with deranged activity of a common glucose-gluconate uptake system, indicating the existence of an independent fructose uptake system. Fructose uptake by glucose-gluconate uptake mutants was induced by fructose and obeyed saturation kinetics (apparent Km equal 0.3 mM). The fructose uptake system serves to transport glucose in addition to fructose. The entry of fructose into P.putida cells appears to be mediated also by the glucose-gluconate uptake system, as shown by the ability to accumulate fructose of wild type cells grown on glucose, a substrate that induces the glucose-gluconate uptake system but not the fructose uptake system. In addition, fructose was found to be an inducer of the glucose-gluconate uptake system. The physiological significance of these observations is not clear because the fructose uptake system can provide the cell with a high enough internal concentration of fructose to support maximum growth rate on this hexose, as shown by following the growth course of a glucose-gluconate uptake mutants on fructose.

Previous studies with mutants of Salmonella typhimurium and Escherichia coli, which fail to grow on glucose-salts media unless such media are supplemented with intermediates of the tricarboxylic acid cycle or direct precursors thereof, have shown that the carboxylation of phosphoenolpyruvate (CH2=C.(COOH).O.PO3H2 + CO2 yields HOOC.CH2.CO.COOH + Pi) is necessarily involved in the maintenance of the tricarboxylic acid cycle: this reaction serves to replenish the cycle with C4-compounds, as intermediates of that cycle are revoved in the course of biosynthesis. It was difficult to explain why CoASAc and citrate synthase should be so much more efficient a trapping agent for labelled oxaloacetate than was malate dehydrogenase and NADH2, even when the extracts were supplemented with crystalline malate dehydrogenase: this observation suggested that CoASAc played a role in this system additional to its function as a trapping agent. This suggestion was confirmed with a mutant AB1623 of E. coli K-12.

The inducible enzymes responsible for the dissimilation of aromatic compounds through the β-ketoadipate pathway by Moraxella calcoacetica are subject to a high degree of coordinate control. The five enzymes which catalyze the conversion of protocatechuate to β-ketoadipyl-CoA are coordinately synthesized, the probable inducer being protocatechuate. In the conversion of catechol to β-ketoadipyl-CoA, there are two separate inductive events, though both are mediated by one metabolite-inducer, cis,cis-muconate. One is the induction of catechol oxygenase; the other is the coordinate induction of the four enzymes that catalyze conversion of cic,cis-muconate to β-ketoadipyl-CoA.

6 p.-1 fig.-2 tab., The rates of cell entrance into three cell cycle periods, G1, S and metaphase, have been measured in meristems of Allium cepa L. roots. The objective was to study, in a direct way, the question of cell age distribution in a tissue formed by proliferating cells with different average generation times. Entrance rates significantly changed from one period to another: the number of cells entering into G1 per unit time almost doubled the entry rate into metaphase. These data rules out a linear cell age distribution. The ratio of entry rates into different pairs of cell cycle periods was calculated, by assuming negative exponential cell age distribution, from the frequency of cell number between birth and the initiation of each period. Comparison of expected values with experimental ones strongly supports a negative exponential age distribution similar to that found in cell cultures with a single cell cycle average time., This work was supported by a grant from the CICYT (Spain). M.I. Gimenez-Abian is the recipient of a postdoctoral fellowship from the CSIC. Secretarial and technical work carried out by respectively, Ms. M.O. Partearroyo and Ms. M. Carrascosa, is acknowledged.

Meristematic cells from Allium cepa L roots can attain a steady-state of growth at both 15 and 25 °C in the presence of drugs, hydroxyurea and 5-amino-uracil, which reduce the rate of DNA synthesis. These drugs, at used concentrations, significantly lengthen the S period without altering the cell growth rate, as indicated by the maintenance of the generation time. It has been observed that steady-state populations respond to a gradual increase in S by a reduction of G2 until a minimum value; with larger lengthening of S, both G1 and G2 are reduced. Natural synchronous populations have been used to study cell cycle parameters during transition from the physiological steady-state to the new one created by the presence of the drug. G2 (but not G1) is reduced during transition even in the presence of maximum drug concentrations that do not alter the cell growth rate. Both the S period and the division time are lengthened during transition. These observations support the concept that certain fractions of G1 and G2 are expendable, because they have no role in the DNA-division sequence of cell cycle events. We conclude that cell size regulates the length of these fractions by means of a negative correlation.

The variability of (1) surface area projection (size) at which cells terminate DNA replication, (2) the area at which they initiate mitosis, (3) the area at which they divide, (4) the duration of G2, and (5) the duration of G2 plus mitosis (in fact, prophase + metaphase + anaphase) has been estimated in steady-state cell populations of Allium cepa root meristems. The coefficient of variation of cell area at termination of DNA synthesis was found to be 14% while the coefficient of variation of cell area at mitosis initiation was 13%. As there is also a substantial variability of G2 (the coefficient of variation was estimated to be 38%), the combination of these data indicates that cell size regulation of G2 contributes to maintaining cell size variability (and therefore DNA concentration) within certain limits. Mitosis also varies but less than G2 (the coefficient of variation of G2 + mitosis was found to be 31%). As the coefficient of variation of cell area at division (14%) is hardly larger than the coefficient of variation of cell area at initiation of mitosis, it can be suggested that coordination between cell size and mitosis duration helps to avoid a significant increase in the variability of cell size at the end of the division cycle.