Macromolecular composition of a Cellulomonas sp. cultivated in continuous culture under glucose and zinc limitation.

The mutant strain of Cellulomonas sp. (ATCC 21399) was cultivated under glucose and zinc limitation at a variety of growth rates in continuous culture. The growth characteristics and macromolecular composition of the population varied with the limitation imposed and the growth rate. Glucose- and zinc-limited cultures maintained a constant relative protein content. The relative ribonucleic acid content increased, whereas the carbohydrate and deoxyribonucleic acid contents decreased with an increase in the population growth rate in glucose-limited cultures. Free unbound lipid remained constant. The maximum population growth rate in zinc-limited cultures was directly proportional to the zinc concentration and demonstrated a traditional steady-state function. The nucleic acid content increased with increased growth rate; however, the relative nucleic acid content was significantly depressed when compared to glucose limited cells. This manner of cultivation may prove to be a useful tool for the production of single cell protein with lowered nucleic acid content and the elucidation of micronutrient involvement in growth-related processes.     

Macromolecular composition of a Cellulomonas sp. cultivated in continuous culture under glucose and zinc limitation.

The mutant strain of Cellulomonas sp. (ATCC 21399) was cultivated under glucose and zinc limitation at a variety of growth rates in continuous culture. The growth characteristics and macromolecular composition of the population varied with the limitation imposed and the growth rate. Glucose- and zinc-limited cultures maintained a constant relative protein content. The relative ribonucleic acid content increased, whereas the carbohydrate and deoxyribonucleic acid contents decreased with an increase in the population growth rate in glucose-limited cultures. Free unbound lipid remained constant. The maximum population growth rate in zinc-limited cultures was directly proportional to the zinc concentration and demonstrated a traditional steady-state function. The nucleic acid content increased with increased growth rate; however, the relative nucleic acid content was significantly depressed when compared to glucose limited cells. This manner of cultivation may prove to be a useful tool for the production of single cell protein with lowered nucleic acid content and the elucidation of micronutrient involvement in growth-related processes.     

Stalk Formation and Its Inhibition in Caulobacter crescentus

Estimates of average rates of stalk formation over several generations of growth in Caulobacter crescentus showed that long-stalked Sk1 mutant and phosphate-starved wild-type cultures produce stalk material at about twice the rate of wild-type C. crescentus grown with adequate nutrients. Thus, the long stalks of Sk1 or phosphate-starved caulobacters are not merely a function of their longer doubling times. Inhibition of cell division of Sk1 418 with mitomycin C (MC) caused production of cellular filaments and resulted in inhibition of stalk formation. There was no appreciable decrease in total cell mass or in rates of ribonucleic acid and protein synthesis in the MC-treated cultures as compared with controls, but stalk formation, which is normally dependent on these processes, was severely retarded. Average stalk lengths in MC-treated Sk1 cultures were 30% of those found in control cultures. MC-produced cellular filaments were also subjected to deoxyribonucleic acid analysis and ultrastructural examination. The deoxyribonucleic acid content of MC-treated bacteria was about 50 to 60% that of untreated bacteria. Hydroxyurea also was found to produce some cellular filaments and shorter stalks, but with accompanying decreases in growth rate and yield.     

Inhibition of Nucleic Acid and Protein Synthesis in Mouse Spleen Cells In Vitro by Azathioprine

The effect of azathioprine on macromolecular biosynthesis was studied in mouse spleen cells cultured in vitro. The rate of incorporation of (3)H-thymidine, (3)H-uridine, and (14)C-leucine into acid-insoluble material was used to measure deoxyribonucleic acid, ribonucleic acid, and protein synthesis. Results indicate that azathioprine inhibited nucleic acid and protein synthesis at levels which did not decrease cell viability.     

The Isolation and Characterization of Adenosine Monophosphate-rich Polynucleotides Synthesized by Soybean Hypocotyl Cells: Their Relation to Messenger Ribonucleic Acid

Plant ribonucleic acids which have high adenosine monophosphate concentrations were studied. Purified deoxyribonucleic acid-like ribonucleic acid and tenaciously bound ribonucleic acid fractions both contained poly-adenosine monophosphate sequences (those from the latter being longer than those from the former); without these poly-adenosine monophosphate sequences their base compositions were the same. The average poly-adenosine monophosphate sequence from purified tenaciously bound ribonucleic acid was 160 residues long, as measured by gel electrophoresis. However, base hydrolysis and chromatography indicated one 3′-nucleoside (adenosine) per 71 nucleotides, giving a chain length of 72 residues. The dominant species in the cytoplasm, as measured by radioactive precursor incorporation, was tenaciously bound ribonucleic acid, whereas deoxyribonucleic acid-like ribonucleic acid was present in greater amounts in the nucleus. This work provides evidence that deoxyribonucleic acid-like ribonucleic acid and tenaciously bound ribonucleic acid represent forms of messenger ribonucleic acid in soybean, with deoxyribonucleic acid-like ribonucleic acid residing in the nucleus, perhaps as the messenger ribonucleic acid precursor, and tenaciously bound ribonucleic acid residing, as the active messenger ribonucleic acid, in the cytoplasm.     

Absence of Adenine-Rich Ribonucleic Acid from Purified Infectious Reovirus 3

The basic properties of released and cell-associated reovirus are the same. Both contained as their total nucleic acid complement only double-stranded ribonucleic acid (RNA) with an adenine content of 28%. Preparations of purified cell-associated virus, but not released virus, contained adenine-rich RNA which could be separated from the virus with little or no loss of infectivity. These adenine-rich ribonucleic acids were present in the virus preparations either as free RNA or associated with some structures of molecular weight less than 25 x 10(6) daltons. In contrast to our previous report, double-stranded reovirus RNA possessed little or no template activity for the Escherichia coli deoxyribonucleic acid and RNA polymerases.     

Quantitation of lymphocyte response to antigen by flow cytofluorometry.

The response of human peripheral blood lymphocytes to antigenic stimulation has been studied in vitro using flow cytofluorometry and an acridine orange (AO) staining technique for cellular deoxyribonucleic acid and ribonucleic acid (RNA). Antigen-stimulated "pyroninophillic" immunoblasts, identified by an increase in cellular content of RNA (red fluorescence with AO), were quantitated in triplicate cultures incubated up to 7 days with and without bacterial antigen. These results were similar to 14C-thymidine incorporation into identical cultures incubated in parallel. Cytofluorometric analysis showed a peak in percentage of immunoblasts after 6 days in culture, while maximum thymidine incorporation was seen on day 7. Cells from patients with depressed immune response secondary to cancer showed lower than normal antigen response by cytofluorometry. Kinetic studies revealed both a lower percentage of immunoblasts when compared to normal and a lower average per cell RNA content of the stimulated cells. AO cytofluorometry is suggested as a convenient method of simultaneously assessing lymphocyte proliferative and nonproliferative response to antigen.     

Changes in RNA and protein metabolism preceding onset of hemoglobin synthesis in cultured Friend leukemia cells.

We have studied the hematopoietic process which is induced by dimethyl sulfoxide (Me₂SO) in the Ostertag FSD-1 line of Friend erythroleukemia (FL) cells and have observed several changes that precede the onset of hemoglobin synthesis at 48 hr. Although cellular viability, mitotic rate, and deoxyribonucleic acid content are unaffected by our induction procedure, the induced cells become progressively smaller, and by 96 hr contain only 55% as much ribonucleic acid and 60–70% as much protein as control cells. The decline in ribonucleic acid content is significant by 24 hr and affects 4S and ribosomal ribonucleic acids in a noncoordinated manner throughout the hematopoietic process. Furthermore, incorporation of radioactive uridine into the 45S precursor of ribosomal ribonucleic acid is specifically inhibited by 1–2 hr after first treating FL cultures with 1% Me₂SO. This earliest known effect of Me₂SO on FL cells is followed by a decline in synthesis of protein. The basic sequence of macromolecular and cell size changes are similar to those that occur during normal erythropoiesis.     

Rapid multiparameter analysis of cell stimulation in mixed lymphocyte culture reactions.

A flow-cytofluorometric method, based on the differential stability of deoxyribonucleic acid versus ribonucleic acid with the metachromatic dye, acridine orange, simultaneously measures the following parameters of stimulation in mixed lymphocyte cultures: (a) number of nonstimulated cells; (b) total number of stimulated lymphocytes; (c) number of stimulated lymphocytes in G1, S and G2 + M phases of the cell cycle; (d) number of macrophages; (e) number of dead cells. The progress of lymphocyte stimulation may also be measured by a parameter representing ribonucleic acid accumulation per cell. The method is rapid, avoids cell rinsing, fixation and centrifugation and is applicable to microcultures. Multiparameter analysis of cell stimulation which provides simultaneous measurements of lymphocyte proliferation and accumulation of ribonucleic acid per cell may prove to be a more sensitive assay of histocompatibility than tests based only on cell proliferation (tritiated thymidine incorporation).     

Method for reproducible large-volume production and purification of Rauscher murine leukemia virus.

Rauscher murine leukemia virus was produced in roller-bottle cultures of chronically infected JLS-V9 cells. Virus from this culture fluid was concentrated and purified by two semi-isopycnic bandings in sucrose gradients. Virus material obtained from young, nonconfluent cultures (early-harvest virus) yielded products characteristically containing endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with high specific activity (400 to 1,000 pmol of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour). Fluids obtained from older confluent cultures (late-harvest virus) yielded products with endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with little or no specific activity (200 pmol or less of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour), but with higher virus particle counts and greater amounts of protein and gs antigen than the early-harvest products.     

Method for reproducible large-volume production and purification of Rauscher murine leukemia virus.

Rauscher murine leukemia virus was produced in roller-bottle cultures of chronically infected JLS-V9 cells. Virus from this culture fluid was concentrated and purified by two semi-isopycnic bandings in sucrose gradients. Virus material obtained from young, nonconfluent cultures (early-harvest virus) yielded products characteristically containing endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with high specific activity (400 to 1,000 pmol of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour). Fluids obtained from older confluent cultures (late-harvest virus) yielded products with endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with little or no specific activity (200 pmol or less of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour), but with higher virus particle counts and greater amounts of protein and gs antigen than the early-harvest products.     

Properties and Mode of Action of a Bactericidal Compound (= Methylglyoxal) Produced by a Mutant of Escherichia coli

A lethal product (BPG) produced by a glycerol kinase mutant of Escherichia coli was purified, and its mode of action on E. coli was studied. At concentrations where BPG strongly inhibits in vivo deoxyribonucleic acid, ribonucleic acid, and protein synthesis, it produces small effects on other functions: slight inhibition of respiration and small changes in intracellular pools of substrates, nucleic acids degradation, and adenosine triphosphate levels. BPG also inhibits in vitro protein synthesis and produces inactivation of bacteriophage T4. The bactericidal product has been identified in another laboratory as methylglyoxal (MG). By comparing BPG and MG, we confirmed this observation and concluded that the activity found in our BPG preparation is due to its MG content. We also observed that MG is able to react with guanosine triphosphate. According to these results, it is interpreted that MG could act directly on macromolecular synthesis by reacting with the guanine residues of nucleic acids and its precursors.     

In vitro production of Rauscher murine leukemia virus: influence of culture age on biological properties.

Large-scale production and concentration procedures have been standardized to study the biological properties of Rauscher leukemia virus produced from the high-passaged JLS-V9-H mouse bone marrow cell line. Virus produced early (days 4 to 6) in the harvest and refeed cycle contained higher levels of ribonucleic acid-directed deoxyribonucleic acid polymerase activity and was more infectious than Rauscher leukemia virus produced later (days 7 to 10) in the growth period. The peak of virus production as detected by physical assays (virus particle count, protein, and p30 antigen) was highest at day 6, whereas the optimum biological and ribonucleic acid-directed deoxyribonucleic acid polymerase activity occurred 24 h earlier. When product characterization values of each concentrate were adjusted to a specific activity (i.e., per milligram of protein) basis, virus particle counts averaged 4 x 10(11) through days 5 to 9, and the peak infectivity occurred at day 4, whereas ribonucleic acid-directed deoxyribonucleic acid polymerase activity was highest at day 4 (endogenous) and 5 (exogenous). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed only slight differences in the polypeptide pattern of Rauscher leukemia virus harvested from cultures of varying age, although Rauscher leukemia virus produced between days 3 and 5 contained more glycoprotein than either earlier or later harvests.     

In vitro production of Rauscher murine leukemia virus: influence of culture age on biological properties.

Large-scale production and concentration procedures have been standardized to study the biological properties of Rauscher leukemia virus produced from the high-passaged JLS-V9-H mouse bone marrow cell line. Virus produced early (days 4 to 6) in the harvest and refeed cycle contained higher levels of ribonucleic acid-directed deoxyribonucleic acid polymerase activity and was more infectious than Rauscher leukemia virus produced later (days 7 to 10) in the growth period. The peak of virus production as detected by physical assays (virus particle count, protein, and p30 antigen) was highest at day 6, whereas the optimum biological and ribonucleic acid-directed deoxyribonucleic acid polymerase activity occurred 24 h earlier. When product characterization values of each concentrate were adjusted to a specific activity (i.e., per milligram of protein) basis, virus particle counts averaged 4 x 10(11) through days 5 to 9, and the peak infectivity occurred at day 4, whereas ribonucleic acid-directed deoxyribonucleic acid polymerase activity was highest at day 4 (endogenous) and 5 (exogenous). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed only slight differences in the polypeptide pattern of Rauscher leukemia virus harvested from cultures of varying age, although Rauscher leukemia virus produced between days 3 and 5 contained more glycoprotein than either earlier or later harvests.     

Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli

Luzzati, Denise (Institut de Biologie Physico-Chimique, Paris, France). Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 92:1435-1446. 1966.-During the course of thymine starvation, the rate of synthesis of messenger ribonucleic acid (mRNA, the rapidly labeled fraction of the RNA which decays in the presence of dinitrophenol or which hybridizes with deoxyribonucleic acid) decreases exponentially, in parallel with the viability of the thymine-starved bacteria. The ability of cell-free extracts of starved bacteria to incorporate ribonucleoside triphosphates into RNA was determined; it was found to be inferior to that of extracts from control cells. The analysis of the properties of cell-free extracts of starved cells shows that their decreased RNA polymerase activity is the consequence of a modification of their deoxyribonucleic acid, the ability of which to serve as a template for RNA polymerase decreases during starvation.     

Regulation of tropomyosin gene expression during myogenesis.

In skeletal muscle, tropomyosin has a critical role in transduction of calcium-induced contraction. Presently, little is known about the regulation of tropomyosin gene expression during myogenesis. In the present study, qualitative and quantitative changes in the nucleic acid populations of differentiating chicken embryo muscle cells in culture have been examined. Total nucleic acid content per nucleus increased about fivefold in fully developed myotubes as compared to mononucleated myoblasts. The contribution of deoxyribonucleic acid to the total nucleic acid population decreased from 24% in myoblasts to 5% of total nucleic acid in myotubes. Concomitant with the decrement in deoxyribonucleic acid contribution to total nucleic acid was an increase in polyadenylated ribonucleic acid (RNA) content per cell which reached levels in myotubes that were 17-fold higher than those of myoblasts. Specific changes in the RNA population during myogenesis were further investigated by quantitation of the synthetic capacity (messenger RNA levels) per cell for alpha- and beta-tropomyosin. Cell-free translation and immunoprecipitation demonstrated an approximately 40-fold increase in messenger RNA levels per nucleus for alpha- and beta-tropomyosin after fusion in the terminally differentiated myotubes. Indirect immunofluorescence with affinity-purified tropomyosin antibodies demonstrated the presence of tropomyosin-containing filaments in cells throughout myogenesis. Thus, the tropomyosin genes are constitutively expressed during muscle differentiation through the production of tropomyosin messenger RNA and translation into tropomyosin protein.     

Specific Neisseria gonorrhoeae DNA-probes derived from ribosomal RNA

Eighteen sequences complementary to less-conserved regions within the 16S and 23S ribosomal ribonucleic acid (rRNA) of Neisseria gonorrhoeae were subcloned or chemically synthesized and used as probes in a dot-spot deoxyribonucleic acid (DNA): DNA hybridization format. Some of these probes exclusively detected Neisseria gonorrhoeae nucleic acid, whereas others also showed hybridization signals with nucleic acid from other bacterial species. Our results indicate that rRNA-derived DNA-probes can be used to differentiate between very closely related taxa without the use of Southern-blot analysis.     

Effect of Putative Deoxyribonucleic Acid Inhibitors on Macromolecular Synthesis in Saccharomyces cerevisiae

The effects of inhibitors of bacterial deoxyribonucleic acid (DNA) synthesis upon logarithmically growing cultures of Saccharomyces cerevisiae were investigated. Cell division, ribonucleic acid (RNA) synthesis, and DNA synthesis were measured after addition of nalidixic acid, fluorodeoxyuridine, or phenethyl alcohol to cultures of yeast growing in defined and complex media. Both nalidixic acid and fluorodeoxyuridine had only temporary effects on nucleic acid synthesis in cultures growing in defined medium, and little or no observable effect on cultures growing in complex medium. Neither compound inhibited colony formation on complex solid medium, although growth was slow on defined solid medium. Phenethyl alcohol caused complete inhibition of DNA synthesis, RNA synthesis, and cell division in cultures growing in defined medium. In cultures growing in complex medium, RNA synthesis and cell division were inhibited to a lesser extent. A slight increase in DNA was observed in the presence of the inhibitor.     

Deoxyribonucleic Acid, Ribonucleic Acid, Protein and Uncombined Amino Acid Content of Legume Seeds During Embryogeny

The nucleic acid, protein and uncombined amino acid contentof seeds of soya-bean (Glycine max L. Merr.), garden pea (Pisumsativum L.), kidney bean (Phaseolus vulgaris L.) and peanut(Arachis hypogaea L.) were measured at various times duringseed formation in an effort to understand why the soya-beanhas nearly twice as much protein as the other legume seeds.In all these species the concentration of deoxyribonucleic acid,ribonucleic acid and uncombined amino acids decreased duringseed formation. The protein level of kidney bean was relativelyconstant during development whereas the protein levels of pea,peanut and soya-bean increased during development. The proteincontent of the soya-bean increased throughout development whereasthe protein increase in peanut took place early and that inpea took place later in development. The ratio of protein toribonucleic acid was highest in peanut, less in soya-bean, andlowest in pea and kidney bean. Similarly, the ratio of proteinto deoxyribonucleic acid was higher in kidney bean than in soya-bean.Soya-beans had a lower amino acid content than any of the otherseeds at all stages of development. These results indicate thatneither total deoxyribonucleic acid, ribonucleic acid nor uncombinedamino acid content is responsible for the higher protein contentof soya-beans.     

Serological Analysis of the Deoxyribonucleic Acid Polymerase of Avian Oncornaviruses I. Preparation and Characterization of Monospecific Antiserum with Purified Deoxyribonucleic Acid Polymerase

Monospecific antiserum was prepared against purified deoxyribonucleic acid (DNA) polymerase from avian myeloblastosis virus (AMV). Immunodiffusion assay with purified DNA polymerase revealed that the anti-DNA polymerase serum formed one precipitation band, whereas no reaction with any of the seven major structural proteins of AMV was observed. The antiserum also demonstrated enzyme-neutralizing antibody activity that was associated with the immunoglobulin G fraction. There was no difference in the neutralization of DNA polymerase activity directed by ribonucleic acid (RNA), DNA, or RNA-DNA hybrid templates.