Regulation of Ribonucleic Acid Synthesis in Escherichia coli During Diauxie Lag: Accumulation of Heterogeneous Ribonucleic Acid

The synthesis of ribonucleic acid (RNA) and of protein in Escherichia coli during glucose-lactose diauxie lag have been examined. The rate of RNA synthesis is about 7%, of the corresponding rate during exponential growth and the rate of protein synthesis 10 to 15%. Inhibition of RNA synthesis occurs to the same extent in both rel and rel(+) strains. The RNA which accumulates during 20 min in diauxie lag is composed of about 50% ribosomal and transfer RNA species and about 50% of a fraction which resembles messenger RNA (mRNA) in its heterogeneous sedimentation properties. Decay of the heterogeneous fraction occurs in the presence of glucose and actinomycin D with a half-life of 3 min, the same as that of pulse-labeled mRNA; however, during the diauxie lag, the half-life of this RNA is about 25 min. Accumulation of the heterogeneous RNA is further increased when protein synthesis is blocked by chloramphenicol. The data suggest that the disproportionate accumulation of mRNA during diauxie lag and energy source shift-down may be attributed at least in part to increased stability of mRNA, but do not rule out a preferential synthesis of mRNA.     

Developmental expression of crystallin genes: in situ hybridization reveals a differential localization of specific mRNAs

The time and place of the accumulation of alpha A-, beta B1- and gamma-crystallin RNA in the developing rat lens have been studied by in situ hybridization. alpha A- and gamma-crystallin RNA were first detected in the lens vesicle, while beta B1-crystallin RNA could be seen only after elongation of the primary fiber cells. Both beta B1- and gamma-crystallin RNA were confined to the fiber cells of fetal lenses, while alpha A-crystallin mRNA could also be detected in the epithelial cells. A quantification of the hybridization pattern obtained in the differentiation zone of the newborn rat lens showed that alpha A-crystallin RNA is concentrated in the cortical zone. alpha B-crystallin mRNA has the same distribution pattern. beta B1-crystallin RNA was relatively poorly detectable by in situ hybridization in both fetal and newborn rat lenses. The grain densities obtained with this probe increased from the periphery of the lens toward the interior, indicating that beta B1-crystallin RNA accumulated during differentiation of the secondary fiber cells. A similar accumulation pattern was obtained for gamma-crystallin mRNA, but, unexpectedly, this RNA could also be detected in the elongating epithelial cells. Our results show that gamma-crystallin RNA starts to accumulate as soon as visible elongation of epithelial cells occurs, during differentiation of the primary as well as the secondary fiber cells.     

Topography of the insertion of LamB protein into the outer membrane of Escherichia coli wild-type and lac-lamB cells

The appearance of newly induced LamB protein at the cell surface of Escherichia coli was followed topographically by immuno-electron microscopy. LamB protein was induced in E. coli wild-type or lac-lamB cells for a short period of time (4 to 6 min), such that the overall level of LamB protein in induced cells was at least twofold higher than that in uninduced cells. Antibodies bound to LamB protein exposed at the cell surface were labeled with a protein A-gold probe, and the probe distribution in briefly induced cells was compared to that in uninduced cells. Analysis of large numbers of cells showed that newly inserted LamB protein appeared homogeneously over the entire cell surface, both in wild-type cells and in lac-lamB cells. A peak of insertion which was observed at the division site of the cell was also observed in the absence of induction and in control experiments in which a nonspecific probe was used. It is concluded therefore that insertion of LamB protein into the cell envelope of E. coli occurs at multiple sites over the entire cell surface. The average amount of LamB protein which appeared at the cell surface after induction was determined for various cell size classes. It was found that cells of various size classes all synthesized LamB protein after induction, indicating that synthesis of the protein was not restricted to cells in a particular stage of the cell cycle. However, the rate of LamB synthesis was found to vary during the cell cycle: this rate was constant regardless of cell size in nondividing cells, whereas it increased in dividing cells. It is concluded that the accumulation of newly induced LamB protein follows a linear pattern.     

Tobacco embryogenesis: Storage-protein-accumulating cells of embryo, suspensor, and endosperm are able to undergo cytokinesis

Summary It is widely accepted that seed storage proteins accumulate only in cells which have entered the cell expansion phase and do not continue to divide. Here we present data indicating that the accumulation of storage globulins in tobacco begins already during early embryogenesis in a period of sustained mitotic activity. Western blot analysis revealed that polypeptides of the legumin-like 12S globulins (M_r 60000, 40000, 20000) appear at mid/late globular stage, whereas the vicilin-like 7S globulin (M_r 50000) follows during the transition from heart to torpedo stage. The accumulation of legumin-like polypeptides begins first in the endosperm during the mid globular stage followed in the embryo-suspensor complex during the heart-shaped stage. The vicilin-related fraction appears first in the endosperm and three days later in the embryo. Examination of individual cells from squash preparations revealed that protein bodies are not confined to intermitotic cells, but are also present in cells undergoing mitosis. Protein bodies of dividing cells situated outside the mitotic apparatus are not metabolized during cytokinesis. The only cell type which loses its protein bodies completely prior to the first mitotic division is the primary hypophysis cell. Our finding that storage proteins can occur in dividing cells independent of their origin and developmental capacity indicates that the cell expansion hypothesis of storage protein accumulation has to be revised.     

Transformation of Lipid Bodies Related to Hydrocarbon Accumulation in a Green Alga,Botryococcus braunii (Race B)

The colonial microalga Botryococcus braunii accumulates large quantities of hydrocarbons mainly in the extracellular space; most other oleaginous microalgae store lipids in the cytoplasm. Botryococcus braunii is classified into three principal races (A, B, and L) based on the types of hydrocarbons. Race B has attracted the most attention as an alternative to petroleum by its higher hydrocarbon contents than the other races and its hydrocarbon components, botryococcenes and methylsqualenes, both can be readily converted into biofuels. We studied race B using fluorescence and electron microscopy, and clarify the stage when extracellular hydrocarbon accumulation occurs during the cell cycle, in a correlation with the behavior and structural changes of the lipid bodies and discussed development of the algal colony. New accumulation of lipids on the cell surface occurred after cell division in the basolateral region of daughter cells. While lipid bodies were observed throughout the cell cycle, their size and inclusions were dynamically changing. When cells began dividing, the lipid bodies increased in size and inclusions until the extracellular accumulation of lipids started. Most of the lipids disappeared from the cytoplasm concomitant with the extracellular accumulation, and then reformed. We therefore hypothesize that lipid bodies produced during the growth of B. braunii are related to lipid secretion. New lipids secreted at the cell surface formed layers of oil droplets, to a maximum depth of six layers, and fused to form flattened, continuous sheets. The sheets that combined a pair of daughter cells remained during successive cellular divisions and the colony increased in size with increasing number of cells.     

Seasonal variations in the pattern of RNA metabolism of tuber tissue in response to excision and culture

Summary Between December 1975 and June 1976 expiants excised from Jerusalem artichoke tubers were cultured both in the presence and in the absence of 2,4-D, the cells in the tissue dividing only in the presence of 2,4-D, in which the length of the first cell cycle increased nonlinearly from 18 hours to 40 hours as the tubers aged in storage at 4 °C. Simultaneously the amount of RNA in the tissue declined linearly from 8 to 5 g RNA per explant. Detailed examination of the RNA metabolism in dividing and in non-dividing cells during February and June 1976 revealed superimposed but independent responses to wounding during excision and to stimulation into growth by 2,4-D. The responses to wounding involved only a very low level of metabolic activity, were complete within a few hours of excision and changed very little with the storage of the tubers. Tissue treated with 2,4-D showed a much higher level of metabolic activity including the periodic accumulation of RNA coupled to its discontinuous synthesis. The features of these growth-related responses changed considerably during the investigation.     

Morphological changes in staphylococcal cytoplasmic membrane due to action of non-ionic detergent Triton X-100

Ribosomal RNA, labelled with uridine and methionine, from dividing and post-mitotic mouse prenatal muscle cell cultures, has been characterised on polyacrylamide gels. Progress of differentiation in cultures was monitored by recording changing proportions of nuclei in myotubes, and increases in creatine kinase activity. Ribosomal RNA synthesis in myotubes is reduced relative to that in dividing cell cultures and considerable wastage of processed ribosomal RNA occurs. This changed pattern of ribosomal RNA production appears to be established in the post-mitotic myoblasts prior to fusion.     

Studies of oogenesis in Urechis caupo. II. Accumulation during oogenesis of carbohydrate, RNA, microtubule protein, and soluble, mitochondrial, and lysosomal enzymes

In the echiuroid worm Urechis caupo, the oocytes develop independently as single cells in the coelomic fluid. The accumulation of oocyte constituents during oogenesis was determined in different size classes of coelomic oocytes, separated by fractionation on a Ficoll density gradient. Each size class was assayed for protein, carbohydrate, RNA, microtubule protein, glucose-6-phosphate dehydrogenase, acid phosphatase, and cytochrome c oxidase. All the constituents investigated accumulated continuously during oogenesis, and the rates of accumulation paralleled the volume increase of the oocytes. The electrophoretic pattern of soluble egg proteins supported this finding. These data suggest that the genes involved in the synthesis of all the oocyte constituents studied are activated early in oogenesis.     

Distributionof actin filaments in differentiating cells of Equisetum hyemale root tips

The distribution of F-actin was studied in various cell types in root tips of Equisetum hyemale using rhodamine labelled phalloidin as a probe. In dividing cells no cables could be observed, although a strong diffuse staining, which has the same distribution as the microtubules, occurs in the phragmoplast. During cell development the cables become longer and form meshworks but no relationship with cell expansion and microtubule patterns was observed. The distribution apparently follows the pattern of plasma streaming. In expanded cells in particular the nucleus is surrounded by a web of filaments connected with the larger cables in the cell.     

Accumulation of potassium by human red cells

1. A method is described for measuring the accumulation of K at 37°C. by washed human red cells in glucose-containing systems in which the pH is kept constant, the K content of the cells being compared with that of the cells of systems which contain no added glucose but which are otherwise treated similarly. 2. In systems containing added glucose, the accumulation of K begins shortly after the cells have been warmed to 37°C., proceeds to a maximum which is reached after about 10 hours, and then falls exponentially. The maximum rate of accumulation is found during the first 3 hours. In systems which contain no added glucose, the K content of the cells appears to decrease exponentially with time for about 18 to 24 hours; thereafter the K content of the cells may decrease rapidly and the systems may show considerable hemolysis. Sometimes a small accumulation effect is observed during the first 2 to 3 hours; this may be the result of the washed cells not having been completely freed of glucose. 3. The accumulation process proceeds at its maximum rate at pH 7.4 to 7.6, which is also the pH at which the K loss from the red cells is at a minimum in systems containing no added glucose. 4. When red cells are stored at 4°C. for increasing lengths of time, the storage is accompanied by increasing K loss and the maximum rate of accumulation observed when the cells are warmed to 37°C. at first becomes greater. If the storage at 4°C. is continued for more than 3 to 4 days, the rate of the accumulation which occurs at 37°C decreases again, the accumulation mechanism showing progressive deterioration with time even at low temperatures. This deterioration has a counterpart in the progressive deterioration (deduced from the analysis of the curves relating K content and time) of the accumulation mechanism with time at 37°C. 5. The accumulation of K occurs at a maximum rate when the concentration of glucose in the system is between 50 and 200 mg./100 ml. Its temperature coefficient over the range 27–37°C. is 2.4. In the presence of glucose and at pH 7.6, accumulation of K takes place from isotonic mixtures of KCl and LiCl or of KCl and CsCl only a little less actively than from mixtures of KCl and NaCl; i.e., the accumulation of K under optimum conditions seems to be an active process which is at least partly independent of the excretion of Na.     

Accumulation of 70S Monoribosomes in Escherichia coli After Energy Source Shift-Down

When Escherichia coli is shifted from glucose-minimal to succinate-minimal medium, a transient inhibition of protein synthesis and a time-dependent redistribution of ribosomes from polysomes to 70S monosomes occurs. These processes are reversed by a shift-up with glucose. In a lysate made from a mixture of log-phase and down-shifted cells, the 70S monosomes are derived solely from the down-shifted cells and are therefore not produced by polysome breakage during preparation. This conclusion is supported by the absence of nascent proteins from the 70S peak. The monosomes are not dissociated by NaCl or by a crude ribosome dissociation factor, so they behave as "complexed" rather than "free" particles. When down-shifted cells are incubated with rifampin to block ribonucleic acid (RNA) synthesis, the 70S monosomes disappear with a half-life of 15 min. When glucose is also added this half-life decreases to 3 min. The 70S particles are stable in the presence of rifampin when chloramphenicol is added to block protein synthesis. We interpret these data to mean that the existence of the 70S monosomes depends on the continued synthesis of messenger RNA and their conversion to free ribosomes (which dissociate under our conditions) is a result of their participation in protein synthesis. Finally, a significant fraction of the RNA labeled during a brief pulse of (3)H-uracil is found associated with the 70S peak. These results are consistent with the hypothesis that the 70S monosomes are initiation complexes of single ribosomes and messenger RNA, which do not initiate polypeptide synthesis during a shift-down.     

Accumulation of activated lymphocytes in liver of endotoxin treated rabbits.

Intravenous administration of endotoxin in doses, 1.0 mg/kg, 0.1 mg/kg or 0.01 mg/kg caused activation (in the sense of nucleolar RNA synthesis) of the lymphocytes in peripheral blood, kidney and liver in rabbits. The evaluation of the ratio of lymphocyte count to the number of organ tissue cells leads to the conclusion that the accumulation of endotoxin activated lymphocytes occurs in liver.     

Isolation and characterization of a ColE1 plasmid containing the entire bio gene cluster of Escherichia coli K12.

Summary Temperature-sensitive mutants of Tetrahymena pyriformis which had previously been selected for their inability to grow at 38°C but which grew normally (or near normally) at 30°C were characterized with respect to their patterns of RNA and protein accumulation at both the permissive and nonpermissive temperatures. Out of 116 such mutants, the majority (72) acted like wild type for these accumulations during a 3 h labelling period although some of them stopped dividing during this time. The remainder exhibited a variety of altered phenotypes for the rate, extent, and timing of RNA and/or protein accumulation. Those mutants which exhibited selective inhibition of RNA accumulation, and were thus potential ribosomal RNA (rRNA) mutants, were further characterized by examining patterns of protein and RNA synthesis in cells starved at the permissive temperature, but re-fed at the permissive and non-permissive temperatures. At least five different types of mutants as defined by patterns of protein and RNA synthesis in refed cells were identified. Direct analysis of the RNA synthesized in cells from 2 of these types of mutants showed that in 5 out of 6 cases rRNA synthesis and/or processing was inhibited within 30 min after shifting to the non-permissive temperature. The other mutant examined was found to show a delayed inhibition of rRNA synthesis.     

THE SYNTHESIS OF DNA, RNA, AND NUCLEAR PROTEIN IN NORMAL AND TUMOR STRAIN CELLS : I. Fresh Embryo Human Cells

Interferometric and photometric measurements have been made on replicating embryo human cell cultures. From a study of the relations between successive physical measurements on individual cells, it was found that the net syntheses of DNA, nuclear RNA, nuclear protein, and cytoplasmic RNA are closely associated during interphase. Additional measurements of DNA and cytoplasmic RNA on freshly prepared replicating monkey kidney cells gave similar results. In auxiliary experiments with embryo human cells, an inhibition of the onset of DNA synthesis (produced by a dose of X-rays) was found to block the majority of the accumulation of nuclear protein and RNA and about half the accumulation of cytoplasmic RNA. These results are consistent with others previously reported in dividing cell cultures freshly prepared from normal tissues.     

Accumulation and localisation of maternal RNA in oocytes of Japanese quail

Accumulation of total RNA and poly(A+)RNA was determined in the oocytes of Japanese quail (Coturnix coturnix japonica) during oogenesis, by a standard spectrophotometric method, after RNA extraction. Intensive RNA accumulation was observed in the oocytes 0.25-2.0 mm in diameter (correlated with the presence of lampbrush chromosomes), followed by a plateau in 2.0-3.0 mm oocytes (correlated with the condensation of lampbrush chromosomes). Resumption of the RNA accumulation in the 3.0-5.0 mm oocytes is interpreted as a result of external RNA delivery by the granulosa cells. Most of the RNA (approximately 3/4, 3-4 micrograms) contained in the ovulated oocyte was found under the vitelline membrane surrounding the yolk. This RNA has been designated as 'extraembryonic RNA', as it is located outside the germinal disc region where the embryo is formed. The extraembryonic RNA is rapidly degraded within 24 h, from ovulation until oviposition, during egg passage through the oviduct, while the RNA present in a germinal disc (approximately 1.1 micrograms) is stable. In bird oocytes the presence of two, territorially separated pools of RNA has been postulated: one contained in a germinal disc and needed for early embryo development, and the second present in the cytoplasmic layer around the yolk supporting oocyte growth and development during oogenesis.     

A general method for modeling cell populations undergoing G1----G0 transitions during development.

The transition from the dividing state to a non-dividing, terminally differentiated state is common to the history of most populations of cells during development. Quantifying such transitions and events related to them is often difficult, even in those cases for which there is a good tissue culture model, because the process is asynchronous and occurs against a background of continued extensive growth. A general model for analyzing these complex population changes is presented here. In the absence of definitive data, the model provides projections of the possible range, under a given set of boundary values, for the rate of terminal differentiation, the overall growth rate, and the degree of cell death. On the other hand, given data on the rate of DNA accumulation, dividing cell fraction, and generation time, the model provides the effective partitioning coefficient between the dividing and non-dividing states averaged over the population, at a given time. These data also allow for an assessment of the degree of actual cell death against a background in which significant numbers of cells are withdrawing from the cell cycle. The types of data required with respect to the model's ability to resolve the nature of a G0 transition "window" within the cell cycle are also discussed.     

Patterns of wall synthesis inSaccharomyces cerevisiae

Wall formation inSaccharomyces cerevisiae seems to be the result of two main patterns of wall material deposition: (i) around the whole periphery of the cell in nonbudding ones, and (ii) mainly at the tip of the daughter cell or at the cross wall that separates dividing cells. This interpretation has been obtained following experiments in which RNA or protein synthesis has been inhibited. Under these conditions, glucan formation takes place, and wall thickening is probably due to the accumulation of this polysaccharide. Furthermore, once a pattern of wall deposition has been established, it is not modified by inhibition of RNA or protein synthesis.