THE SYNTHESIS OF 5S RIBOSOMAL RNA DURING POLLEN DEVELOPMENT

Tradescantia paludosa 5S ribosomal RNA (rRNA) has been characterized with respect to its base composition and relative electrophoretic mobility in comparison with that of E. coli . The period of 5S rRNA synthesis during pollen grain development was determined by pulse labeling the RNA synthesized during a 24 hr period of development with ³²P and then chasing in cold medium until pollen maturity. The period of highest 5S rRNA synthesis was found to occur prior to microspore mitosis. During and following mitosis over a period of 4 days there was a sharp decrease in the amount of 5S RNA synthesized and during the last 48 hr of pollen maturation, no 5S rRNA was synthesized.     

BIOCHEMICAL AND CYTOLOGICAL EXAMINATION ON THE INITIATION OF RIBOSOMAL RNA SYNTHESIS DURING GASTRULATION OF XENOPUS LAEVIS

Embryos of Xenopus laevis at stage 6 were labeled with ¹⁴CO₂ for 4 hr and then allowed to develop under nonradioactive conditions until they reached stage 9, 10, 11 or 12. RNA was extracted and electrophoresed on a polyacrylamide-agarose gel. From the pattern of newly synthesized RNAs, the incorporation into 18S and 28S ribosomal RNAs was measured. At the same time, the specific radioactivity of nucleoside triphosphates in the acid-soluble fraction was determined. On the basis of the results obtained, the absolute amounts of the RNAs synthesized were calculated. The results show that the synthesis of the ribosomal RNAs begins, or is at least markedly activated, around stage 10. Moreover, cytological examination has shown that cells with nucleolated nuclei appeared between stages 9 and 10 and increased thereafter.
Thus, from the results of these studies along two different lines, it can safely be concluded that the initiation of 18S and 28S RNA synthesis takes place around stage 10.     

RNA SYNTHESIS IN CULTURED CHICK EMBRYO CELLS IN GROWING AND CONFLUENT PHASES

RNA synthesis at the growing phase in monolayer cultures of chick embryo fibroblasts was compared with that at confluent phases by zonal sedimentation, base composition and hybridization experiments. The nuclei were isolated by treatment with Nonidet p-40. The ratio of RNA/DNA in isolated nuclei was higher at the growing phase than that of confluent. The rate of RNA synthesis was reduced in the cells at confluent phase to 15.1% of that at the growing phase. The sucrose density gradient sedimentation pattern of nuclear RNA was on the whole the same in both phases. According to the distribution of ¹⁴C-uridine incorporated into nuclear RNA, 45S ribosomal precursor RNA was more distinct for the growing cell, while the radioactivities were found to be polydispersed, including the RNA which sedimented faster than 28S RNA in the cells at confluent phase. The base compositions and hybridization analyses indicated that ribosomal RNA was synthesized more actively in the growing cells. About 50% of newly synthesized RNA was ribosomal in the growing cells but 35% in the confluent.
It was found that newly synthesized 18S and 28S ribosomal RNAs appeared in cytoplasm after 21 and 33 min lag periods respectively. These times were exactly same in both growing and confluent phases.     

Polyamine stimulation of ribosomal synthesis and activity in a polyamine-dependent mutant of Escherichia coli

A polyamine-dependent mutant of Escherichia coli KK101 was isolated by treatment of E. coli MA261 with N-methyl-N'-nitro-N-nitrosoguanidine. In the absence of putrescine, doubling time of the mutant was 496 min. The mutation was accompanied by a change in the nature of the 30 S ribosomal subunits. Addition of putrescine to the mutant stimulated the synthesis of proteins and subsequently, this led to stimulation of RNA and DNA synthesis. Under these conditions, we determined which proteins were preferentially synthesized. Putrescine stimulated the synthesis of ribosomal protein S1 markedly, but stimulated ribosomal proteins S4, L20, and X1, and RNA polymerase slightly. The amounts of initiation factors 2 and 3 synthesized were not influenced significantly by putrescine. The preferential stimulation of the synthesis of ribosomal protein S1 occurred as early as 20 min after the addition of putrescine, while stimulation of the synthesis of the other ribosomal proteins and RNA polymerase appeared at 40 min. The stimulation of the synthesis of ribosomal RNA also occurred at 40 min after addition of putrescine. Our results indicate that putrescine can stimulate both the synthesis and the activity of ribosomes. The increase in the activity of ribosomes was achieved by the association of S1 protein to S1-depleted ribosomes. The early stimulation of ribosomal protein S1 synthesis after addition of putrescine may be important for stimulation of cell growth by polyamines.     

用双参数流式细胞光度术(FCM)研究阿克拉霉素对L_(1210)白血病细胞周期的影响

本文用双参数FCM技术,对同一个细胞的DNA和RNA含量进行相关测量,比较了ACM B对小鼠L_(1210)白血病细胞周期和RNA含量的影响.结果发现在一次给药后8小时可导致早、中期S的积累,并抑制S期细胞的DNA合成;到24小时DNA合成恢复正常,并进入G_2期,但由于G_2期细胞进入M期受阻,造成G_2期细胞的积累,这时被阻断在G_2期的细胞RNA含量显著增加,形成正不平衡生长,而给药剂量较大的实验组(1/1.5LD_(50))S期细胞的RNA含量不随着DNA含量的增加而增加,形成负不平衡生长,ACM A和ACM B对体内Li_(210)细胞周期作用相同.     

Precursor Ribosomal Ribonucleic Acid and Ribosome Accumulation In Vivo During the Recovery of Salmonella typhimurium from Thermal Injury

When cells of S. typhimurium were heated at 48 C for 30 min in phosphate buffer (pH 6.0), they became sensitive to Levine Eosin Methylene Blue Agar containing 2% NaCl (EMB-NaCl). The inoculation of injured cells into fresh growth medium supported the return of their normal tolerance to EMB-NaCl within 6 hr. The fractionation of ribosomal ribonucleic acid (rRNA) from unheated and heat-injured cells by polyacrylamide gel electrophoresis demonstrated that after injury the 16S RNA species was totally degraded and the 23S RNA was partially degraded. Sucrose gradient analysis demonstrated that after injury the 30S ribosomal subunit was totally destroyed and the sedimentation coefficient of the 50S particle was decreased to 47S. During the recovery of cells from thermal injury, four species of rRNA accumulated which were demonstrated to have the following sedimentation coefficients: 16, 17, 23, and 24S. Under identical recovery conditions, 22, 26, and 28S precursors of the 30S ribosomal subunit and 31 and 48S precursors of the 50S ribosomal subunit accumulated along with both the 30 and 50S mature particles. The addition of chloramphenicol to the recovery medium inhibited both the maturation of 17S RNA and the production of mature 30S ribosomal subunits, but permitted the accumulation of a single 22S precursor particle. Chloramphenicol did not affect either the maturation of 24S RNA or the mechanism of formation of 50S ribosomal subunits during recovery. Very little old ribosomal protein was associated with the new rRNA synthesized during recovery. New ribosomal proteins were synthesized during recovery and they were found associated with the new rRNA in ribosomal particles. The rate-limiting step in the recovery of S. typhimurium from thermal injury was in the maturation of the newly synthesized rRNA.     

Nature of ribonucleic acid stimulated by streptomycin in the absence of protein synthesis

Freda, Celia E. (University of Pennsylvania School of Medicine, Philadelphia), and Seymour S. Cohen. Nature of ribonucleic acid stimulated by streptomycin in the absence of protein synthesis. J. Bacteriol. 92:1680-1688. 1966.-The ribonucleic acid (RNA) synthesized in a thymineless, arginineless, uracil-less Escherichia coli strain 15 in the absence of arginine was characterized by sucrose density gradient centrifugation. About 60% of this RNA had sedimentation rates in the range between 4S and 16S, and the remainder was comprised of the 23S and 16S ribosomal components. On addition of streptomycin for 1 hr in the absence of the amino acid, there was an inhibition of synthesis of material of 4S to 16S, whereas 16S RNA was slightly stimulated. Between 1 and 3 hr after addition of the antibiotic, during the precipitous killing of the bacteria in the arginine-deficient culture, the synthesis of 16S ribosomal RNA was specifically and sharply stimulated.     

Synthesis of reovirus ribonucleic acid in L cells

Kudo, Hajime (The Wistar Institute of Anatomy and Biology, Philadelphia, Pa.), and A. F. Graham. Synthesis of reovirus ribonucleic acid in L cells. J. Bacteriol. 90:936-945. 1965.-There is no inhibition of protein or deoxyribonucleic acid (DNA) synthesis in L cells infected with reovirus until the time that new virus starts to form about 8 hr after infection. At this time, both protein synthesis and DNA synthesis commence to be inhibited. Neither the synthesis of ribosomal ribonucleic acid (RNA) nor that of the rapidly labeled RNA of the cell nucleus is inhibited before 10 hr after infection. Actinomycin at a concentration of 0.5 mug/ml does not inhibit the formation of reovirus, although higher concentrations of the antibiotic do so. Pulse-labeling experiments with uridine-C(14) carried out in the presence of 0.5 mug/ml of actinomycin show that, at 6 to 8 hr after infection, two species of virus-specific RNA begin to form and increase in quantity as time goes on. One species is sensitive to ribonuclease action and the other is very resistant. The latter RNA is probably double-stranded viral progeny RNA, and it constitutes approximately 40% of the RNA formed up to 16 hr after infection. The function of the ribonuclease-sensitive RNA is not yet known. Synthesis of both species of RNA is inhibited by 5 mug/ml of actinomycin added at early times after infection. Added 6 to 8 hr after infection, when virus-specific RNA has already commenced to form, 5 mug/ml of actinomycin no longer inhibit the formation of either species of RNA.     

Control of Protein Synthesis in Acanthamoeba castellanii

During development of Acanthamoeba castellanii in a non-nutrient medium, the pattern of synthesis of proteins changes. Comparison of in vivo and in vitro patterns of protein synthesis reveals concomitant relative increases of five proteins, indicating a control of synthesis of these proteins at the level of the RNA content. The decrease in the overall rate of protein synthesis and relative decreases in the synthesis of actin and ribosomal proteins during development, not accompanied by equivalent changes in the content of mRNA, suggest control mechanisms also at the level of translation. Patterns of ribosomal proteins do not change qualitatively during encystation, indicating that the inhibition in the overall rate of protein synthesis and the formation of inactive monosomes is not controlled by this mechanism; however, phosphorylation of one ribosomal protein, S 3, is observed occasionally during encystation. Phosphorylation of S 3 is also detected after transfer of stationary phase cells into fresh nutrient medium. It was found that only such cells having RNA of aberrant properties are able to phosphorylate S 3 after transfer into fresh nutrient medium. Since these changes in the property of RNA are never observed in cysts, in which phosphorylation of S 3 sometimes occurs, it is concluded that either other alterations in the properties of RNA than those detected or other parameters are responsible for changes in phosphorylation of S 3.     

Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus: I. Patterns of Ribonucleic Acid Synthesis in Productively Infected Cells

HEp-2 cells were pulse-labeled at different times after infection with herpes simplex virus, and nuclear ribonucleic acid (RNA) and cytoplasmic RNA were examined. The data showed the following: (i) Analysis by acrylamide gel electrophoresis of cytoplasmic RNA of cells infected at high multiplicities [80 to 200 plaque-forming units (PFU)/cell] revealed that ribosomal RNA (rRNA) synthesis falls to less than 10% of control (uninfected cell) values by 5 hr after infection. The synthesis of 4S RNA also declined but not as rapidly, and at its lowest level it was still 20% of control values. At lower multiplicities (20 PFU), the rate of inhibition was slower than at high multiplicities. However, at all multiplicities the rates of inhibition of 18S and 28S rRNA remained identical and higher than that of 4S RNA. (ii) Analysis of nuclear RNA of cells infected at high multiplicities by sucrose density gradient centrifugation showed that the synthesis and methylation of 45S rRNA precursor continued at a reduced but significant rate (ca. 30% of control values) at times after infection when no radioactive uridine was incorporated or could be chased into 28S and 18S rRNA. This indicates that the inhibition of rRNA synthesis after herpesvirus infection is a result of two processes: a decrease in the rate of synthesis of 45S RNA and a decrease in the rate of processing of that 45S RNA that is synthesized. (iii) Hybridization of nuclear and cytoplasmic RNA of infected cells with herpesvirus DNA revealed that a significant proportion of the total viral RNA in the nucleus has a sedimentation coefficient of 50S or greater. The sedimentation coefficient of virus-specific RNA associated with cytoplasmic polyribosomes is smaller with a maximum at 16S to 20S, but there is some rapidly sedimenting RNA (> 28S) here too. (iv) Finally, there was leakage of low-molecular weight (4S) RNA from infected cells, the leakage being approximately three-fold that of uninfected cells by approximately 5 hr after infection.     

CYTOKINETICS OF RAT TRACHEAL EPITHELIUM STIMULATED BY MECHANICAL TRAUMA

Mild abrasion of rat tracheal epithelium results in irreversible damage to the superficial cells and stimulates the viable basal cells to participate in a nearly synchronous wave of DNA synthesis and mitosis. For the growth population as a whole, DNA synthesis started at 14 hr after injury and persisted for 16 hr. The duration of S in individual cells was determined autoradiographically by identifying the time at which a second pulse of DNA precursor (¹⁴C-TdR) was no longer incorporated by cells labelled with ³H-TdR at the onset of S. S was found to be 8–9 hr long. It was also determined that cells entering S at later times synthesized DNA for the same 8–9 hr period. T_G2 was calculated to be 21/2–31/2 hr by subtraction of T_s and 1/2T_M from the period from onset of DNA synthesis to metaphase. By making a second denuding lesion adjacent to the first injury, the cells were stimulated through at least another period of S. At the peak of the second wave of DNA synthesis (50 hr after injury) ¹⁴C-TdR was present in the same cells which had incorporated ³H-TdR administered at the mid-point of the preceding synthetic phase. The 28-hr interval between these two peaks of synthesis is the measure of cell cycle duration for these regenerating tracheal epithelial cells.     

Regulation of RNA synthesis in plant cell culture: delayed synthesis of ribosomal RNA during transition from the stationary phase to active growth

Ribosomal RNA synthesis was studied during the early phases of growth activation in a cell suspension culture derived from peanut (Arachis hypogaea, L.) cotyledon. Upon dilution from stationary phase, these cells show a characteristic lag of 3 days before the commencement of cell division. An analysis of the nature of RNA synthesized during this early period of growth showed that the cells obtained immediately upon dilution from stationary phase synthesize primarily messenger RNA and essentially no ribosomal RNA. The synthesis of ribosomal RNA is delayed for about 24 hr after which it rises sharply resulting in a 2- to 3-fold accumulation of ribosomal RNA per cell during the subsequent 24-hr period. Both the messenger RNA and the ribosomal RNA were characterized by their cellular localization; by sucrose and CsCl gradient analyses, and by the determination of their base ratios.It would appear that a major facet of the lag phase in the cell growth is the diversion of a significant part of the RNA biosynthetic apparatus from the synthesis of messenger RNA to that of ribosomal RNA.     

THE REGULATION OF RNA SYNTHESIS AND PROCESSING IN THE NUCLEOLUS DURING INHIBITION OF PROTEIN SYNTHESIS

The effect of protein synthesis inhibition by cycloheximide on nucleolar RNA synthesis and processing has been studied in HeLa cells. Synthesis of 45S RNA precursor falls rapidly after administration of the drug. However, the nucleolar content of 45S RNA remains relatively constant for at least 1 hr because the time required for cleavage of the precursor molecule into its products is lengthened after treatment with cycloheximide. The efficiency of transformation of 45S RNA to 32S RNA remains constant with approximately one molecule of the 32S RNA produced for each cleavage of a molecule of 45S RNA. However, shortly after the cessation of protein synthesis the formation of 18S RNA becomes abortive. The amount of 32S RNA present in the nucleolus remains relatively constant. After long periods of protein synthesis inhibition the 28S RNA continues to be synthesized and exported to the cytoplasm but at a greatly reduced rate. When the protein synthesis inhibitor is removed, a prompt, although partial, recovery in the synthesis rate of 45S RNA occurs. The various aspects of RNA synthesis regulation and processing are discussed.     

Effect of Chloramphenicol on the Synthesis and Stability of Ribonucleic Acid in Bacillus subtilis

The effect of chloramphenicol on the synthesis and accumulation of ribonucleic acid (RNA) in Bacillus subtilis was studied. In the presence of chloramphenicol, transfer RNA and ribosomal RNA were synthesized as rapidly 2 to 3 hr after challenge as they were just prior to the addition of the antibiotic. However, under the same conditions, net RNA accumulation ceased after only 30 to 45 min. The failure to accumulate RNA after this time resulted from a rapid degradation of ribosomal RNA synthesized in the presence of chloramphenicol and a slow degradation of mature ribosomes. Since transfer RNA was not appreciably degraded, the ratio of transfer RNA to total RNA increased during the challenge.     

Biochemical studies of mammalian oogenesis: synthesis and stability of various classes of RNA during growth of the mouse oocyte in vitro

The synthesis of various classes of RNA in mouse oocytes at different stages of growth has been examined after incubating follicles in medium containing radiolabeled uridine. After fractionation on poly(U)-Sepharose of radiolabeled oocyte RNA, of which about 83% is associated with the nucleus after a 5-hr labeling period, revealed that about 40–50% of the radiolabeled RNA behaved as poly(A)-containing RNA. This value remained fairly constant during the period of oocyte growth in which oocyte diameter increased from about 35 to about 55 μm. After a 5-hr labeling, the percentage of radiolabeled poly(A)-containing RNA in either the fully grown dictyate oocyte, metaphase II oocyte, or one-cell embryo was about 20%. After a 5-hr labeling, agarose gel electrophoretic analysis of the radiolabeled species of oocyte RNA obtained after fractionation on poly(U)-Sepharose revealed the presence of a putative ribosomal RNA precursor, ribosomal (28 and 18 S) RNA, transfer plus 5 S RNA and heterodisperse poly(A)-containing RNA. A significant fraction of the radiolabeled RNA species was quite large (>40 S). The ratios of the relative proportions of the radiolabeled ribosomal RNAs and transfer plus 5 S RNA remained essentially constant during oocyte growth. The stability of various classes of RNA was examined by incubating follicles with radiolabeled uridine, washing the follicles free of radioactivity and culturing the follicles under conditions which support oocyte growth in vitro (Eppig, 1977). Under these conditions, total oocyte radiolabeled RNA was quite stable as determined by retention of acid-insoluble radioactive material ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 28}\text{days}$). However, under conditions in which oocytes are viable but do not grow, the half-life of total RNA was about 4.5 days. Poly(A)-containing RNA was also very stable; after 8 days in culture, about 50% of the radiolabeled poly(A)-containing RNA present after 5 hr of labeling was still present. Agarose gel electrophoretic analysis of radiolabeled RNA in oocytes after 4 days of culture and after fractionation on poly(U)-Sepharose revealed the presence of ribosomal (28 and 18 S) RNA, transfer plus 5 S RNA, and heterodisperse poly(A)-containing RNA. At this time, these RNAs are located in the oocyte cytoplasm. In addition, the molecular weight distribution of poly(A)-containing RNA was significantly lower than that after 5 hr of labeling. The ratios of the relative proportions of radiolabeled ribosomal RNAs and transfer plus 5 S RNA were quite similar to those after 5 hr of labeling.     

Arrest of cell cycle progression of HeLa cells in the early G1 phase in K+-depleted conditions and its recovery upon addition of insulin and LDL

Cell cycle progression of synchronized HeLa cells was studied by measuring labeling of the nuclei with [³H]thymidine. The progression was arrested in a chemically defined medium in which K⁺ was replaced by Rb⁺ (Rb-CDM) but was restored upon addition of insulin and/or low density lipoprotein (LDL). Cells started DNA synthesis 12 hr after addition of insulin and/or LDL, regardless of the time of arrest, suggesting their arrest early in the G1 phase. After incubation of cells in Rb-CDM containing insulin or LDL singly for 3, 6, or 9 hr, replacement of the medium by that without an addition resulted in marked delay in entry of cells into the S phase, but in its replacement by medium containing both agents, the delay was insignificant. Synthesis of bulk protein, estimated as increase in the cell volume, was not strongly inhibited. From these results we conclude that cell cycle progression of HeLa cells in K^?-depleted CDM is arrested early in the G1 phase and that the arrest is due to lack of some protein(s) required for entry into the S phase that is synthesized in the early G1 phase.