GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. V. THE GROWTH OF COLONIES FROM FREE CELLS ON NUTRIENT AGAR

When angiosperm cells are cultured in a liquid medium they may grow in the form of free, single cells and form small to large groups of cells. This has been shown in earlier papers. This paper deals with the growth of strains of cells (Daucus carota and Haplopappus gracilis cells were used), washed and filtered free from the larger groups, on nutrient agar media in Petri dishes, thus simulating familiar microbiological technique. Each discrete member of a suspension is referred to as a “unit.” On the order of 1–10% of the separate units of a suspension may be induced to grow into viable colonies, depending on the strain and the conditions employed. Whereas at least 30% of the free single carrot cells were shown to be capable of division, only up to about 4% continued their growth to form macroscopically visible colonies when they were widely dispersed. Coconut milk promotes the growth of carrot cells into colonies. Both coconut milk and napthaleneacetic acid, which interact synergistically, arc required for the growth of Haplopappus cells. Various techniques which affect viability (the frequency with which units grow into colonies) were investigated. The viability of carrot units was found (1) to increase with their density on the plates; (2) to decrease upon washing the suspensions prior to plating; (3) to increase with increasing initial size; and (4) to decrease to a vanishingly low value in rigorously filtered suspensions which consist principally of single cells, although the single cells were found to grow with appreciable frequency when the larger units were also present; and (5) to increase dramatically (100-fold) when a rigorously filtered suspension was plated on a medium upon which pieces of growing cultured tissue were placed. Thus, the induction of growth in free cells is enhanced, even in an environment nutritionally optimal for the growth of the larger cell masses, by as yet unknown factors which are contributed by the cells themselves, or by adjacent cells or groups of cells. It is suggested that within a group of cells growing in culture, and perhaps also in the organized growing regions of intact plants, the dividing cells are nourished or stimulated by adjacent but less frequently dividing cells. The implications of these results are discussed.     

AN ANALYSIS OF DIFFERENTIATIVE CAPACITY OF PIGMENTED EPITHELIAL CELLS OF ADULT NEWT IRIS IN CLONAL CELL CULTURE

Singly dissociated cells from dorsal and ventral iris epithelia ( iris iridica ) of adult newts were cultured separately at clonal density to analyse their growth and differentiative capacity. Usually some attached cells began to proliferate on 12th day of culture, and grew with loss of melanosomes to form clonal cell colonies. Up to 30 days after inoculation, most of the clonal colonies formed typical epithelial monolayer sheets which consisted mostly of nonpigmented cells. Then, in some of those colonies, cells piled up together and form typical lens structures containing lens antigens. A month and a half after culturing, 30 to 40% of single iris cells, which had been previously marked, grew to form clonal colonies consisting of more than 100 cells. About 30% of these colonies expressed lens specificity and no significant differences in efficiency of colony formation and differentiation were detected between the dorsal cells and the ventral, suggesting that potent cells capable of transdifferentiating into lens cells are evenly distributed in all parts of the newt iris epithelium.     

PHASE-MICROSCOPIC OBSERVATIONS OF FUSIONS OF NUCLEI IN SOMATIC CELLS OF A HETEROKARYON OF SCHIZOPHYLLUM COMMUNE

Pairs of nuclei in apical cells of a common-B heterokaryon (A41 B41 + A2 B41) of Schizophyllum commune fused prior to the nuclear division. It is assumed that each fusion was followed by a reduction division. Five consecutive fusions and divisions in a single hypha were photographed in situ with a phase microscope. These fusions and reduction divisions are possibly related to the genetically determined phenomenon of somatic recombination as a part of the parasexual cycle in fungi.     

Partial Synchronization of Carrot Cell Culture by Auxin Deprivation

A strain of carrot cells (Daucus carota cv. Kintoki) grew exponentially in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D, 1 mg/1) with a doubling time of about 2 days. When those cells were transferred to a medium lacking 2,4-D, they continued to grow at almost the same rate for about a week. When the cells were again transferred to the auxin-free medium, the rate of cell division gradually decreased. After the cell division had ceased, cells were returned to the ordinary 2,4-D medium. A burst of cell divisions occurred after about 2 days. Timing of DNA synthesis and of mitosis suggested that the cells had been arrested at G₁ phase. In a medium containing indoleacetic acid instead of 2,4-D, the auxin was rapidly degraded and the culture was similarly synchronized as in the auxin-omitted medium.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS IV. THE BEHAVIOR OF THE NUCLEUS

Mitra , J., Marion O. Mapes , and F. C. Steward . (Cornell U., Ithaca, New York.) Growth and organized development of cultured cells. IV. The behavior of the nucleus. Amer. Jour. Bot. 47(5) : 357—368. Illus. 1960.–The nuclei and the chromosomes of carrot cells have been examined at various stages throughout the following sequence: (1) growth of a tissue culture from a preformed explant of secondary phloem from the carrot root; (2) growth and multiplication of carrot cells freely suspended in a liquid medium; (3) growth and re-formation of organs (roots) and whole plants (including flowers) from cells in the freely suspended state. The cells of the carrot are normally diploid (2n = 18), the cells which develop in the explant are also diploid, and the cells of the re-formed organs, and even the flowers developed upon plants grown from cells, are also normal and diploid; normal meioses also occur. Nevertheless, the wide range in growth and form of the freely suspended cells is accompanied by a rich diversity of cytological conditions; these include tetraploid and highly polyploid nuclei which divide, haploidy and such chromosomal aberrations as di- and even tri-centric bridges. Two division figures showing chromosome numbers at different levels of ploidy were seen within the confines of one large cell, and, in another, 2 adjacent division figures were observed with chromosome numbers lower than diploid. Small thick-walled, densely protoplasmic cells divide to form bi- and tetra-nucleate conditions, and in a giant cell a highly multinucleate condition has been seen. Despite this, however, all the regenerated roots and plants yet examined are normally diploid. The implications of these events are discussed.     

Effect of cell cycle stages on the central density of Enterococcus faecium ATCC 9790.

Cultures of Enterococcus faecium growing at various rates were examined for timing of cell division cycle events by using the method of residual divisions and a morphological analysis. Both methods gave essentially the same timing for the onset of D1 (completion of chromosome replication) and of D2 (completion of septation). Frequencies of cells exhibiting a phase-reversed center in bovine serum albumin at various growth rates were determined. The data fit a model in which rapidly growing cells increase in refractive index (which is assumed to represent central density) at completion of the chromosome replication cycle involved in the ongoing division, whereas slowly growing cultures increase in central density at the time of completion of septation. There was no correlation between the timing of increase in central density and the timing of initiation of new sites of surface growth.     

In Vitro Culture of a Root Parasite, Aeginetia indica L. II. The Plane of Cell Division in the Tendril

Factors affecting septation (cell division) of the tendril whichfacilitates the organic connection with the host were studiedin a root parasite Aeginetia indica L. Transverse cell division,which occurs perpendicular to the long axis of the tendril,was promoted by additions of sucrose, glucose and cytokininsto the basal medium. Longitudinal cell division of the tendril,which takes place parallel or obliquely to the long axis, wasstimulated by cytokinins, but not by sucrose. The latter typeof cell division was frequent in basal and sub-basal cells ofthe tendril but was extremely rare in apical cells. The orientationof the planes of these cell divisions was closely related tocell shape. Abnormal growth of the tendril was seen in germinatingseeds grown for six weeks or more in media containing both Miscanthus(a host) root extract and cytokinin. (Received February 23, 1984; Accepted June 12, 1984)     

Cyclic AMP and cell division in Escherichia coli.

We examined several aspects of cell division regulation in Escherichia coli which have been thought to be controlled by cyclic AMP (cAMP) and its receptor protein (CAP). Mutants lacking adenyl cyclase (cya) or CAP (crp) were rod shaped, not spherical, during exponential growth in LB broth or glucose-Casamino Acids medium, and lateral wall elongation was normal; in broth, stationary-phase cells became ovoid. Cell mass was smaller for the mutants than for the wild type, but it remained appropriate for their slower growth rate and thus probably does not reflect early (uncontrolled) septation. The slow growth did not seem to reflect a gross metabolic disorder, since the mutants gave a normal yield on limiting glucose; surprisingly, however, the cya mutant (unlike crp) was unable to grow anaerobically on glucose, suggesting a role for cAMP (but not for CAP) in the expression of some fermentation enzyme. Both cya and crp mutants are known to be resistant to mecillinam, an antibiotic which inhibits penicillin-binding protein 2 (involved in lateral wall elongation) and also affects septation. This resistance does not reflect a lack of PBP2. Furthermore, it was not simply the result of slow growth and small cell mass, since small wild-type cells growing in acetate remained sensitive. The cAMP-CAP complex may regulate the synthesis of some link between PBP2 and the septation apparatus. The ftsZ gene, coding for a cell division protein, was expressed at a higher level in the absence of cAMP, as measured with an ftsZ::lacZ fusion, but the amount of protein per cell, shown by others to be invariable over a 10-fold range of cell mass, was independent of cAMP, suggesting that ftsZ expression is not regulated by the cAMP-CAP complex.     

Effect of Inhibition of Deoxyribonucleic Acid and Protein Synthesis on the Direction of Cell Wall Growth in Streptococcus faecalis

Selective inhibition of protein synthesis in Streptococcus faecalis (ATCC 9790) was accompanied by a rapid and severe inhibition of cell division and a reduction of enlargement of cellular surface area. Continued synthesis of cell wall polymers resulted in rapid thickening of the wall to an extent not seen in exponential-phase populations. Thus, the normal direction of wall growth was changed from a preferential feeding out of new wall surface to that of thickening existing cell surfaces. However, the overall manner in which the wall thickened, from nascent septa toward polar regions, was the same in both exponential-phase and inhibited populations. In contrast, selective inhibition of deoxyribonucleic acid (DNA) synthesis using mitomycin C was accompanied by an increase in cellular surface area and by division of about 80% of the cells in random populations. Little or no wall thickening was observed until the synthesis of macromolecules other than DNA was impaired and further cell division ceased. Concomitant inhibition of both DNA and protein synthesis inhibited cell division but permitted an increase in average cell volume. In such doubly inhibited cells, walls thickened less than in cells inhibited for protein synthesis only. On the basis of the results obtained, a model for cell surface enlargement and cell division is presented. The model proposes that: (i) each wall enlargement site is influenced by an individual chromosome replication cycle; (ii) during chromosome replication peripheral surface enlargement would be favored over thickening (or septation); (iii) a signal associated with chromosome termination would favor thickening (and septation) at the expense of surface enlargement; and (iv) a factor or signal related to protein synthesis would be required for one or more of the near terminal stages of cell division or cell separation, or both.     

异源PDGF-A链表达对CHO细胞生长和转化的作用

用DNA磷酸钙盐沉淀方法把含人PDGF(血小板衍生生长因子)A链cDNA的表达质粒pSV_2neo-A转染CHO细胞(中国仓鼠卵巢细胞),然后经G 418(400-800 μg/ml)筛选分离20个转染细胞株。选出其中At_1和Aot7细胞株所进行的实验结果表明,这些细胞的形态和生长行为均发生明显的变化,PDGF-A链mRNA的表达水平比CHO细胞明显增高,胞质有强阳性的PDGF荧光反应,显示有PDGF样蛋白的合成。这些细胞不但生长速率加快,有高密度持续生长的特性,而且能在软琼脂培基上形成大集落和在裸鼠体内接种形成纤维肉瘤,提示外源PDGF-A链基因的表达有使CHO细胞生长失控和发生细胞恶性转化的作用。     

Unequal cell division, growth regulation and colony size of mammalian cells: a mathematical model and analysis of experimental data.

This work describes mathematically the dynamics of expansion of cell populations from the initial division of single cells to colonies of several hundred cells. This stage of population growth is strongly influenced by stochastic (random) elements including, among others, cell death and quiescence. This results in a wide distribution of colony sizes. Experimental observations of the NIH3T3 cell line as well as for the NIH3T3 cell line transformed with the ras oncogene were obtained for this study. They include the number of cells in 4-day-old colonies initiated from single cells and measurements of sizes of sister cells after division, recorded in the 4-day-old colonies. The sister cell sizes were recorded in a way which enabled investigation of their interdependence. We developed a mathematical model which includes cell growth and unequal cell division, with three possible outcomes of each cell division: continued cell growth and division, quiescence, and cell death. The model is successful in reproducing experimental observations. It provides good fits to colony size distributions for both NIH3T3 mouse fibroblast cells and the same cells transformed with the rasEJ human cancer gene. The difference in colony size distributions could be fitted by assuming similar cell lifetimes (12-13 hr) and similar probabilities of cell death (q = 0.15), but using different probabilities of quiescence, r = 0 for the ras oncogene transformed cells and r = 0.1 for the non-transformed cells. The model also reproduces the evolution of distributions of sizes of cells in colonies, from a single founder cell of any specified size to the stable limit distribution after eight to ten cell divisions. Application of the model explains in what way both random events and deterministic control mechanisms strongly influence cell proliferation at early stages in the expansion of colonies.     

hyp loci control cell pattern formation in the vegetative mycelium of Aspergillus nidulans.

Aspergillus nidulans grows by apical extension of multinucleate cells called hyphae that are subdivided by the insertion of crosswalls called septa. Apical cells vary in length and number of nuclei, whereas subapical cells are typically 40 microm long with three to four nuclei. Apical cells have active mitotic cycles, whereas subapical cells are arrested for growth and mitosis until branch formation reinitiates tip growth and nuclear divisions. This multicellular growth pattern requires coordination between localized growth, nuclear division, and septation. We searched a temperature-sensitive mutant collection for strains with conditional defects in growth patterning and identified six mutants (designated hyp for hypercellular). The identified hyp mutations are nonlethal, recessive defects in five unlinked genes (hypA-hypE). Phenotypic analyses showed that these hyp mutants have aberrant patterns of septation and show defects in polarity establishment and tip growth, but they have normal nuclear division cycles and can complete the asexual growth cycle at restrictive temperature. Temperature shift analysis revealed that hypD and hypE play general roles in hyphal morphogenesis, since inactivation of these genes resulted in a general widening of apical and subapical cells. Interestingly, loss of hypA or hypB function lead to a cessation of apical cell growth but activated isotropic growth and mitosis in subapical cells. The inferred functions of hypA and hypB suggest a mechanism for coordinating apical growth, subapical cell arrest, and mitosis in A. nidulans.     

EVOLUTION OF DEVELOPMENTAL PROGRAMS IN VOLVOX (CHLOROPHYTA)1

The volvocine green algal genus Volvox includes ～20 species with diverse sizes (in terms of both diameter and cell number), morphologies, and developmental programs. Two suites of characters are shared among distantly related lineages within Volvox. The traits characteristic of all species of Volvox—large (>500) numbers of small somatic cells, much smaller numbers of reproductive cells, and oogamy in sexual reproduction—have three or possibly four separate origins. In addition, some species have evolved a suite of developmental characters that differs from the ancestral developmental program. Most multicellular volvocine algae, including some species of Volvox, share an unusual pattern of cell division known as palintomy or multiple fission. Asexual reproductive cells (gonidia) grow up to many times their initial size and then divide several times in rapid succession, with little or no growth between divisions. Three separate Volvox lineages have evolved a reduced form of palintomy in which reproductive cells are small and grow between cell divisions. In each case, these changes are accompanied by a reduction in the rate of cell division and by a requirement of light for cell division to occur. Thus, two suites of characters—those characteristic of all Volvox species and those related to reduced palintomy—have each evolved convergently or in parallel in lineages that diverged at least 175 million years ago (mya).     

Growth and division of carrot cells in suspension culture

In a submerged culture of a strain of carrot cells, cellularmorphology and the mode of cell division were greatly affectedby growth factor(s) added to the medium. In the presence of2,4-D, cells showed two-dimensional growth and often formedtetrad-like structure after a set of two divisions. The sequenceof events was observed microscopically. Orientation of cellgrowth changed after the first division and the second cellplate formed at an oblique angle to the first. When IAA wasadded, instead of 2,4-D, cells showed one-dimensional growthand developed to a filamentous form. (Received June 1, 1970; )     

Change in Molecular Size of Cellulose during Regeneration of Cell Wall on Carrot Protoplasts

Protoplasts isolated from carrot cells were cultured in a chemically defined medium. The majority of them regenerated cell wall and underwent cell division. Cellulose synthesis started without a noticeable lag but the rate of deposition was very low during the initial stage. The degree of polymerization of cellulose was determined by viscosity measurement of the nitrated product. The cellulose formed in the early stage of the wall regeneration consisted mainly of low molecular weight glucan chains. Change in the average molecular weight of cellulose was found during the growth cycle of carrot cells in normal suspension culture.     

Cell kinetics, stomatal differentiation, and diurnal rhythm in Allium cepa

Cell kinetics parameters were obtained for the three mitotic divisions leading to formation of stomata in the epidermis of the cotyledon of Allium cepa seedlings. Analysis of mitotic frequencies throughout the course of development showed that the asymmetrical divisions started at about 50 hr after germination, and the symmetrical divisions were first seen a few hours later. Guard mother cell divisions started around 70 hr after germination. The maximal frequency of both symmetrical and asymmetrical division was found between 3 and 5 days after germination, and the highest frequencies of GMC divisions were observed between 6 and 8.5 days. All divisions ceased after 11 days. The three cell populations analyzed displayed diurnal fluctuations of their mitotic frequencies which were characteristic of the type of cell division measured and seemed independent of the region of the cotyledon in which they took place. The symmetrical divisions displayed two diurnal peaks—one at about 0400 and the other at 1600 hr—and the asymmetrical mitoses showed a single peak at about 2200 hr. Atypical asymmetrical divisions were observed in some guard mother cells, suggesting a different developmental sequence for some of the stomatal complexes.     

Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar

The untransformed mouse fibroblast cells NIH/3T3, C3H/10T1/2, and rat NRK cells do not grow in soft agar in medium supplemented with 10% fetal calf serum. When fetal calf serum in the growth medium was supplemented with less than 1% of sera from mice or other vertebrates, however, these cells responded, forming large colonies. The morphology of soft agar colonies was a function of the treated cell type. In the presence of 10% serum from C57BL/6 mice, NRK cells grew to smooth-surfaced spherical colonies, while NIH/3T3 colonies showed individual round cells on their surface and C3H/10T1/2 cells grew as extended cells forming columns of end to end connected fibroblasts. Mus Musculus Castaneus-Epithelial (MMC- E) cells were not stimulated to grow in soft agar under these conditions. The major fibroblast colony-inducing factor (F-CIF) was partially purified from mouse serum by acid/ethanol-extraction, gel permeation chromatography, and reverse-phase high-pressure liquid chromatography. F-CIF is a polypeptide, which does not compete for binding to epidermal growth factor (EGF) receptors, but stimulates normal fibroblasts to form small colonies in semisolid medium and very large colonies in the presence of added EGF (2 ng/ml). In contrast to unfractionated mouse serum, purified F-CIF did not induce C3H/10T1/2 cells to grow in soft agar, suggesting that serum contains additional cell type-specific agar growth-stimulating activities.     

Utilization of indole analogs by carrot and tobacco cell tryptophan synthase in vivo and in vitro

Twenty-three indole analogs were used to inhibit the growth of carrot and tobacco suspension cultures. The addition of tryptophan or indole partially reversed the inhibition of both cell lines only for 4-fluoroindole, 5-fluoroindole, and 6-fluoroindole. Inhibition of tobacco cell growth by 5-aminoindole, 5-methoxyindole, 6-methoxyindole, or 7-methoxyindole was also partially reversed. Previously selected carrot and tobacco lines, which have high free tryptophan levels, grew in the presence of the analogs for which reversal was noted in all cases except 5-aminoindole and also in some other cases. Growth inhibition caused by all 10 tryptophan analogs studied was partially reversible by tryptophan or indole and the high tryptophan lines were also able to grow in the presence of concentrations inhibitory to the wild type lines.     

A new type of growth exhibited by Trimmatostroma abietis

Trimmatostroma abietis initially grew as hyphae when grown in various media containing yeast extract or bactopeptone. It grew as segmented elements (lumbricoid elements) characterized by bidirectional growth, when grown in Czapek-Dox broth or yeast nitrogen base supplemented with 1% glucose. A lumbricoid element usually was 10–70 m in length, with transverse septation only and contained 3 to 15 cells. Growth and propagation, as revealed by time-lapse photomicrography occurred as follows. Elements usually grew by apical elongation without widening; after simple apical elongation adjacent parts of two central cells eventually started to grow, resulting in the separation of the element into two.Part of this work was presented at the 5th International Mycological Congress, Vancouver, Canada, 1994.     

球形棕囊藻的生长特性及生活史研究

对球形棕囊藻不同的藻株(香港株HK和汕头株ST)在实验室条件下分别进行了形态学,生活史及生长曲线的研究,结果表明:球形棕囊藻具有一个复杂的异型生活史,具有两种不同的生活形态:群体和游离的单细胞,香港株和汕头株二者单细胞呈球形或近球形,直径大多为3-9μm;群体呈中空的球形囊泡,细胞包埋在胶质囊中较均匀分布,但二者群体大小有较大差异。当培养达到一定阶段,群体衰亡破裂释放大量单细胞。批次培养中球形棕囊藻的生长周期约为20-30d,香港株最适生长温度接近25℃,最大比生长率为038;汕头株的最适生长温度接近30℃,最大比生长率为042,这说明温度是重要的生长限制因子之一。