Volume growth of daughter and parent cells during the cell cycle of Saccharomyces cerevisiae a/alpha as determined by image cytometry.

The pattern of volume growth of Saccharomyces cerevisiae a/alpha was determined by image cytometry for daughter cells and consecutive cycles of parent cells. An image analysis program was specially developed to measure separately the volume of bud and mother cell parts and to quantify the number of bud scars on each parent cell. All volumetric data and cell attributes (budding state, number of scars) were stored in such a way that separate volume distributions of cells or cell parts with any combination of properties--for instance, buds present on mothers with two scars or cells without scars (i.e., daughter cells) and without buds--could be obtained. By a new method called intersection analysis, the average volumes of daughter and parent cells at birth and at division could be determined for a steady-state population. These volumes compared well with those directly measured from cells synchronized by centrifugal elutriation. During synchronous growth of daughter cells, the pattern of volume increase appeared to be largely exponential. However, after bud emergence, larger volumes than those predicted by a continuous exponential increase were obtained, which confirms the reported decrease in buoyant density. The cycle times calculated from the steady-state population by applying the age distribution equation deviated from those directly obtained from the synchronized culture, probably because of inadequate scoring of bud scars. Therefore, for the construction of a volume-time diagram, we used volume measurements obtained from the steady-state population and cycle times obtained from the synchronized population. The diagram shows that after bud emergence, mother cell parts continue to grow at a smaller rate, increasing about 10% in volume during the budding period. Second-generation daughter cells, ie., cells born from parents left with two scars, were significantly smaller than first-generation daughter cells. Second- and third-generation parent cells showed a decreased volume growth rate and a shorter budding period than that of daughter cells.     

Fractionation of a population ofSaccharomyces cerevisiae yeasts by centrifugation in a dextran gradient

A method of the fractionation of aSaccharomyces cerevisiae yeast population in dextran gradients is described. The elaboration of this method was based on the finding of a correlation between the size of individual cells and the number of bud scars on their surface and rapid indication of the scars by fluorescence microscopy. The basic conditions for fractionation (determined experimentally) were as follows: 2 ml. yeast suspension (100 mg. dry weight) was applied to the surface of a continuous dextran gradient of 9–16% concentration and was centrifuged at a relative centrifugal force of 200 G for 15 minutes. In fractionation of a whole population, the best fractionation was obtained in a linear gradient. Repeated separation of fractions obtained by centrifugation in a linear gradient in a concave gradient further separated cells without bud scars and accumulated cells with five scars and over. Three fractions were obtained by this technique. The first contained 90–98% cells without bud scars, the second 55–65% cells with 1–4 bud scars and the third 50% cells with five bud scars and over.     

A STUDY OF THE RELIABILITY OF SYNCHRONY IN THE DEVELOPMENT OF POLLEN MOTHER CELLS OF LILIUM LONGIFLORUM AT THE FIRST MEIOTIC PROPHASE

Lilium longiflorum anthers have been used by a number of investigators as a source of supposedly synchronous cells for studying genetic, chromosomal and molecular events of meiotic prophase I. Because of questions raised by the literature as to the reliability of such synchrony, new baseline data were obtained for L. longiflorum ‘Croft’ in preparation for experiments requiring homogeneity or synchrony. Homogeneity was found to be reliable at the same level of locules of the same anther and of anthers from the same bud up to the onset of diplotene. Synchrony deteriorates rapidly from diplotene on. Less than half the anthers examined had PMC's at the same stage from apex to base. Hence experiments requiring complete homogeneity within anthers would call for rigorous monitoring of each anther included in the sample. Synchrony between buds of the same length was insufficient to allow bud length to be used as a reliable index of meiotic stage.     

Apoptosis at inflection point in liquid culture of budding yeasts

Budding yeasts are highly suitable for aging studies, because the number of bud scars (stage) proportionally correlates with age. Its maximum stages are known to reach at 20-30 stages on an isolated agar medium. However, their stage dynamics in a liquid culture is virtually unknown. We investigate the population dynamics by counting scars in each cell. Here one cell division produces one new cell and one bud scar. This simple rule leads to a conservation law: "The total number of bud scars is equal to the total number of cells." We find a large discrepancy: extremely fewer cells with over 5 scars than expected. Almost all cells with 6 or more scars disappear within a short period of time in the late log phase (corresponds to the inflection point). This discrepancy is confirmed directly by the microscopic observations of broken cells. This finding implies apoptosis in older cells (6 scars or more).     

NUCLEAR SYNCHRONY IN VALONIA MACROPHYSA(CHLOROPHYTA): LIGHT MICROSCOPY AND FLOW CYTOMETRY1

The coenocytic alga Valonia macrophysa Kützing was selected for an investigation of nuclear synchrony in the order Siphonocladales. Light microscopy reveals that nuclear synchrony is evident as patches of nuclei dividing simultaneously. Flow cytometry was utilized for the first time with a macroalga for cell-cycle analysis. Results indicate that nuclei in the entire cell exhibit a high degree of synchrony throughout the cell cycle. Also it appears that cells within a clonal culture are synchronous with each other, in their progression through the cell cycle. The advantages of using flow cytometry for cell-cycle analysis of coenocytic algae include the rapid collection of quantitative data on relative DNA content for a large number of nuclei.     

Timing of swarmer cell cycle morphogenesis and macromolecular synthesis by Hyphomicrobium neptunium in synchronous culture.

The swarmer cycle of Hyphomicrobium neptunium consists of a temporal sequence of discrete developmental events. To time morphogenesis and to investigate modulations in macromolecular synthesis, we attempted methods for synchronous culture. During synchrony, swarmer maturation occurred over 32%, hyphal growth occurred over 36%, and bud maturation occurred over 32% of the time required to complete the swarmer cycle. Daughter cells were released after 265 min. Deoxyribonucleic acid replication was discontinuous, having a G1 period of approximately 180 min. In addition, ribonucleic acid and protein syntheses were depressed during the earlier phases of development.     

Studies on the behavior of meiotic protoplasts II. Induction of a high fusion frequency in protoplasts from liliaceous plants

A method for the induction of high fusion frequencies in meioticprotoplasts of a lily and Trillium is described. Fusion wasinduced during the rapid isolation of protoplasts by quicklybringing about physical contact between protoplasts. Fused protoplastswere obtained in 50 to 90% yields from prophase cells and in30 to 60% yields from cells in later phases. Large protoplastswith more than 10 nuclei were frequently produced. During culture,meiotic development was not synchronous in these protoplasts;each multinudeate maintaining a high degree of synchrony. (Received February 28, 1973; )     

A study of the budding ofSaccharomyces uvarum Beijerinck with the scanning electron microscope

Vegetative cells ofSaccharomyces uvarum Beijerinck in the exponential growth phase were examined with the scanning electron microscope. The existence of two types of scars — birth scars and bud scars — was confirmed. Birth scars had larger diameters than bud scars; both remained visible on old cells. The distribution of the buds on the mother cell did not appear to be a random one: there seemed to be a more or less emphasized cell polarity. The author wishes to thank Mr. Bert for technical assistance in the use of the scanning electron microscope.     

The chitin-glucan complex inSaccharomyces cerevisiae

The content of glucosamine in the walls of daughter (without bud scars) and mother (multiscar) cells ofSaccharomyces cerevisiae was examined in a control and after treatment with dilute alkali, acid and buffer. The occurrence of chitin in the bud and birth scars is discussed. The results of IR and X-ray analysis of cell-wall fractions indicate the presence of α-chitin which is a part of the chitin-glucan complex. The size of the crystallite of α-chitin in this complex is about 60 Å.     

Synchrony induced by double phosphate starvation in a suspension culture of Catharanthus roseus

A synchronous cell division system was established using the double phosphate starvation method, based on the observation that one of the limiting factors in the growth of a suspension culture of Catharanthus roseus (L.) G. Don cells in the medium of Murashige and Skoog was phosphate. In the system, an increase in cell number took place in a short period of only 4 h, while the cell number remained almost constant during other periods of the cell cycle. The synchrony of the culture was confirmed by changes in mitotic index, which increased sharply prior to the increase in cell number. The S phase was determined by measuring incorporation of [³H]-thymidine into the DNA fraction during the cell cycle and synchrony of DNA synthesis was verified likewise. Synchronization by phosphate starvation is discussed in relation to the function of phosphate as a nutrient. The synchronous system thus established will be useful in biochemical studies of the cell cycle in higher plants.     

Mass isolation and culture of sea urchin micromeres

Summary A procedure is described for large-scale isolation of micromeres from 16-cell stage sea urchin embryos. One to two grams of >99% pure, viable micromeres (2.3 to 4.6 × 10⁸ cells) are routinely isolated in a single preparation. In culture, these cells uniformly proceed through their normal development, in synchrony with micromeres in whole embryos, ultimately differentiating typical larval skeletal structures. The attributes of this procedure are: (a) the very early time of isolation of the cells, directly after the division that establishes the cell line; (b) the large yield of cells; (c) the purity of the preparation of cell; and (d) their synchronous development in culture through skeletogenesis. The procedure greatly aids in making sea urchin micromeres a favorable material for molecular analysis of development. This work was supported in part by the following grants from the National Institutes of Health: Grant HL-10312 to A.H.W., Grant GM-20784 to Helen R. Whiteley, Grant ES-02190 to N. Karle Mottet, M.D., and Training Grants ES-07032 and HD-00266.     

Drifts in DNA level in the cyanobacterium Anacystis nidulans during synchrony induction and in synchronous culture

Cultures of the cyanobacterium Anacystis nidulans were synchronized by using alternating light-dark cycles. The DNA level in the cells was determined, at intervals, during pre-synchrony treatment and subsequent synchronous growth. The DNA content/cell gradually increased during synchrony induction and reached a maximum value after about 9–10 dark-light cycles, coinciding with the minimum length of pre-synchrony treatment necessary for obtaining good synchrony of cell division in our system. DNA synthesis was found to be discontinuous in the synchronous cultures. The results suggest two gaps in DNA synthesis, one occurring before and one after cell division. The results are compared with the relevant data published on the life cycle of other prokaryotic microorganisms.     

Effects of enzyme isolation on the basal lamina of developing avian limb bud epithelia

Summary The developing avian limb bud is a classic example of an epithelial-mesenchymal interaction. Numerous attempts at maintenance of the epithelia in culture have been predominantly unsuccessful. The fate of the isolated epithelial sheet of the limb bud [including the apical ectodermal ridge (AER)] in culture may depend at least in part on the integrity of its basal lamina following isolation. In this study the distal epithelium of the stage 23 limb bud was isolated utilizing trypsin and Dispase II in a variety of procedures. The integrity of the basal lamina of limb epithelium immediately upon isolation and after 2 h in culture was determined by immunofluorescent staining for laminin, and electron microscopy. In epithelial sheets isolated with Dispase II a direct relationship was observed between maintenance of the extracellular matrix at isolation and the preservation of the tissue structure and cytoarchitecture following 2 h in culture. In contrast, there was an accelerated deterioration during incubation of the tissue isolated with trypsin, independent of isolation conditions and integrity of basal lamina after isolation. Short-term maintenance of limb bud epithelial structure and cytoarchitecture after enzymatic isolation seems correlative to the maintenance of extracellular matrix at the epithelial basal surface.     

Age-related distribution of cells in cell cycle phases in dependence on specific rate of growth in chemostat cultures ofCandida utilis

Individual cells in chemostat cultures ofCandida utilis (D=0.05, 0.1, 0.2, 0.25, 0.3 and 35/h) were analyzed according to the number of bud scars and according to their position in the cell cycle. The distribution in the cell cycle phases depends both on the genealogical age and the specific rate of growth.     

SPHEROPLAST FORMATION AND ASSOCIATED ULTRASTRUCTURAL CHANGES IN A SYNCHRONOUS CULTURE OF ANACYSTIS NIDULANS TREATED WITH LYSOZYME1,2

Cells of Anacystis nidnlans were grown in synchronous culture using a light-dark alternation to obtain synchronization. Two synchronous cycles were obtained with, decay of synchrony beginning with the third cycle. Cells of various ages in the growth cycle were treated with lysozyme to form spheroplasts. The percentage of spheroplast formation varied with age of the cells. After extended periods of lysozyme treatment, up to 90% of the cells of all ages showed spheroplast formation. Some cells were resistant to the action of lysozyme regardless of age or length of treatment. An ultrastructure study of the spheroplast was made. The electron-dense inner layer of the cell wall was removed by the action of lysozyme on the glucosamine residues of the cell wall, indicating true spheroplast formation. The photosynthetic apparatus became more pronounced with extended treatment with lysozyme.     

Synchrony of butterfly populations across species' geographic ranges

Understanding the mechanisms by which global climate change and habitat loss impact upon biodiversity is essential in order to mitigate any negative impacts. One such impact may be changes to population synchrony (defined as correlated fluctuations in the density of separate populations). It is well established that synchrony depends on both dispersal ability and correlated environmental conditions, for example shared climate. However, what is not clear is whether differences in habitat or position within a species' range also mediate synchrony. Since synchronous metapopulations are thought to be more extinction‐prone, establishing the drivers of synchrony has clear conservation implications. Using three butterfly species (Maniola jurtina, Pyronia tithonus and Aphantopus hyperantus) we investigated the effects of habitat similarity and range position on population synchrony, after accounting for the effects of distance and climate. Range position was present in all minimum adequate models, though non‐significant using Mantel randomization tests in one case. We show that M. jurtina and P. tithonus synchrony is not consistent across species' ranges, with marginal populations showing more synchronous dynamics. Increased climatic constraints on marginal populations, leading to a narrower range of suitable microhabitats may be responsible for this, which is supported by the result that habitat similarity between sites was also positively correlated with population synchrony. As the landscape becomes increasingly homogeneous, overall population synchrony may be expected to rise. We conclude that habitat modification and climate change have the capacity to drive changes in population synchrony that could make species more vulnerable to extinction.     

Life history strategies affect climate based spatial synchrony in population dynamics of West African freshwater fishes

Spatial synchrony in species abundance is a general phenomenon that has been found in populations representing virtually all major taxa. Dispersal among populations and synchronous stochastic effects (the so called "Moran effect") are the mechanisms most likely to explain such synchrony patterns. Very few studies have related the degree of spatial synchrony to the biological characteristics of species. Here we present a case where specific predictions can be made to relate river fish species characteristics and synchrony determined exclusively by a Moran effect through the expected sensitivity of species to the regional component of environmental stochasticity. By analyzing 23-year time series of abundance estimates in two isolated localities we show that species associated with synchronized reproduction during the wet season, high fecundity, small egg size and high gonado-somatic index (the so called "periodic" strategy) have a higher degree of spatial synchrony in population dynamics than species associated with the opposite traits (the so called "equilibrium" strategy). This is supported by significant relationships (P values <0.01) between species traits and the levels of synchrony after removing taxonomical relatedness. Spatial synchrony computed from summed annual total catches by groups of species, separated into strategy types also showed a significantly higher degree of synchrony for the periodic (r=0.83) than the equilibrium (r=0.46) group. Regional hydrological variability is likely to be partly responsible for the observed synchrony pattern and a regional discharge index showed better relationships with the periodic group, supporting the expected differential effect of regional environmental correlation on population dynamics.     

Induction and stabilization of synchrony in the cell division of Escherichia coli

Escherichia coli strains B5 and B/r/1 were grown under conditions of periodic glucose starvation in a minimal medium. Such conditions of growth give rise to two synchronous populations that are out of phase regarding their time of division, one dividing shortly after a new supply of fresh medium and the other at a later stage of the feeding cycle. Preferential selection of one of the two populations using heat treatment resulted in a homogeneous synchronized culture that exhibited in a non-limiting medium a high degree of synchrony that was long lasting. Synchrony and its persistence could survive preservation of such a synchronized culture by freeze drying. An explanation of the synchrony persistence was put forward and the practical implications of these findings were discussed.     

Synchrony,leadership, and association in male Indo-pacific bottlenose dolphins (Tursiops aduncus)

Male Indo-pacific bottlenose dolphins in Shark Bay, Western Australia, have converged with humans in the formation of nested male alliances and the use of synchrony in alliance behavior. Further, the strength of association among allied male dolphins varies and the stability of alliances correlates with the rate that males consort with estrus females (and is thus a possible indicator of dominance). To examine the possibility that synchrony reflects alliance association strength and dominance relationships, we analyzed videotapes from focal follows of two groups of males that reflect the range of alliance size and the strength of association between individuals in the population. We examined two variables: leadership during synchronous behaviors, based on which animal in a synchronously surfacing pair surfaced first, and the degree of synchrony, based on temporal differences in synchronous surfacing. We predicted that closer associates would exhibit a greater degree of synchrony and that one dolphin in a dyad would consistently lead. Contrary to our predictions, the degree of synchrony was inversely related to strength of association within alliances. This surprising result suggests that individuals with less secure bonds may strive more to achieve synchrony. We found no evidence of leadership during synchronous surfacing or between synchrony and other behavioral variables. Proximate mechanisms for synchronous behavior, such as entrainment and mutual motor imitation (“the mirror game” paradigm), may inhibit leadership in this context. Our results show that synchrony during surfacing is not a useful behavior to examine for dominance relationships in wild dolphins but it may be a useful tool to examine variation in alliance relationships.     

Aggression,nesting synchrony,and reproductive fitness in ring-billed gulls

The relationship between synchronous nesting and aggressive behaviour between adults was studied in a colony of ring-billed gulls (Larus delawarensis) from 1976 to 1978. By exchanging eggs and creating more or less synchronous hatching in two of three study plots, I tested the hypothesis that synchronous hatching among neighbouring nests reduces aggression between neighbours. The third plot served as a control. Aggression was lowest in the synchronous plot and highest in the asynchronous plot. More synchronous hatching also reduced aggression when each pair's average synchrony with its neighbours and each neighbour-neighbour interaction were considered. The relationships between neighbour-neighbour hatching synchrony, aggression, and fledging success suggested that gulls that engaged in more aggression had lower reproductive fitness. I discuss the importance of this phenomenon in colonial birds.