Effects of some antibiotics on the stringent control of RNA synthesis in Escherichia coli.

Effects of neomycin, spectinomycin, tetracycline and chloramphenicol on the stringent control RNA synthesis and on ppGpp synthesis in the rel+-cells of Escherichia coli having a temperature-sensitive valyl-tRNA synthetase were examined. Without antibiotics, ppGpp began to accumulate and both RNA and protein syntheses were inhibited by transferring the exponentially growing cells from 30 degrees C (permissive temp.) to 40 degrees C (non-permissive temp.). Tetracycline or chloramphenicol, when added after the temperature shift, caused a resumption of RNA synthesis and decay of the accumulated ppGpp, while neomycin or spectinomycin had little effect both on RNA synthesis and the level of ppGpp. When the cells were treated with these antibiotics at permissive temperature, the shift of the temperature to 40 degrees C caused neither inhibition of RNA synthesis nor an accumulation of ppGpp. When neomycin or spectinomycin was added at the beginning of the temperature shift, RNA synthesis continued with an accumulation of ppGpp. Tetracycline or chloramphenicol had no such effect under the same conditions; RNA synthesis continued without an accumulation of ppGpp.     

Specific Sindbis virus-coded function for minus-strand RNA synthesis.

The synthesis of minus-strand RNA was studied in cell cultures infected with the heat-resistant strain of Sindbis virus and with temperature-sensitive (ts) belonging to complementation groups A, B, F, and G, all of which exhibited an RNA-negative (RNA-) phenotype when infection was initiated and maintained at 39 degrees C, the nonpermissive temperature. When infected cultures were shifted from 28 degrees C (the permissive temperature) to 39 degrees C at 3 h postinfection, the synthesis of viral minus-strand RNA ceased in cultures infected with ts mutants of complementation groups B and F, but continued in cultures infected with the parental virus and mutans of complementation groups A and G. In cultures infected with ts11 of complementation group B, the synthesis of viral minus-strand RNA ceased, whereas the synthesis of 42S and 26S plus-strand RNAs continued for at least 5 h after the shift to 39 degrees C. However, when ts11-infected cultures were returned to 28 degrees C 1 h after the shift to 39 degrees C, the synthesis of viral minus-strand RNA resumed, and the rate of viral RNA synthesis increased. The recovery of minus-strand synthesis translation of new proteins. We conclude that at least one viral function is required for alphavirus minus-strand synthesis that is not required for plus-strand synthesis. In cultures infected with ts6 of complementation group F, the syntheses of both viral plus-strand and minus-strand RNAs were drastically reduced after the shift to 39 degrees C. Since ts6 failed to synthesize both plus-strand and minus-strand RNAs after the shift to 39 degrees C, at least one common viral component appears to be required for the synthesis of both minus-strand and plus-strand RNAs.     

Characterization of a temperature-sensitive membrane alteration in chick embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus

The intramembrane particles of freeze-fractured chick embryo fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (TS68) are distributed differently at the permissive and non-permissive temperatures if, and only if, the cells are treated with glycerol before fixation. Few aggregates of intramembrane particles are present in glycerol-treated cells grown at the permissive temperature for transformation (36 degrees C), while numerous large aggregates of particles are present at the non-permissive temperature (41 degrees C). Changes in the distribution of particles after cells are shifted from 36 to 41 degrees C are observed after 20 min, while a temperature shift from 41 to 26 degrees C causes changes in glycerol-induced redistributions after 1 h. The changes observed in temperature shifts from 36 to 41 degrees C and from 41 to 36 degrees D do not require protein synthesis or RNA synthesis.     

Preliminary characterization of coxsackievirus B3 temperature-sensitive mutants.

Prototype temperature-sensitive (ts) mutants of a coxsackievirus B3 parent virus capable of replication to similar levels at 34 or 39.5 degrees C were examined for the nature of the temperature-sensitive event restricting replication in HeLa cells at 39.5 degrees C. The ts mutant prototypes represented three different non-overlapping complementation groups. The ts1 mutant (complementation group III) synthesized less than 1% of the infectious genomic RNA synthesized by the coxsackievirus B3 parent virus at 39.5 degrees C and was designated an RNA- mutant. Agarose gel analysis of glyoxal-treated RNA from cells inoculated with ts1 virus revealed that cell RNA synthesis continued in the presence of synthesis of the small amount of viral RNA. This mutant was comparatively ineffective in inducing cell cytopathology and in directing synthesis of viral polypeptides, likely due to the paucity of nascent genomes for translation. The ts5 mutant (complementation group II) directed synthesis of appreciable quantities of both viral genomes (RNA+) and capsid polypeptides; however, assembly of these products into virions occurred at a low frequency, and virions assembled at 39.5 degrees C were highly unstable at that temperature. Shift-down experiments with ts5-inoculated cells showed that capsid precursor materials synthesized at 39.5 degrees C can, after shift to 34 degrees C, be incorporated into ts5 virions. We suggest that the temperature-sensitive defect in this prototype is in the synthesis of one of the capsid polypeptides that cannot renature into the correct configuration required for stability in the capsid at 39.5 degrees C. The ts11 mutant (complementation group I) also synthesized appreciable amounts of viral genomes (RNA+) and viral polypeptides at 39.5 degrees C. Assembly of ts11 virions at 39.5 degrees C occurred at a low frequency, and the stability of these virions at 39.5 degrees C was similar to that of the parent coxsackievirus B3 virions. The temperature-sensitive defect in the ts11 prototype is apparently in assembly. The differences in biochemical properties of the three prototype ts mutants at temperatures above 34 degrees C may ultimately offer insight into the differences in pathogenicity observed in neonatal mice for the three prototype ts mutants.     

A temperature-sensitive mutant of the mammalian RNA helicase,DEAD-BOX X isoform,DBX, defective in the transition from G1 to S phase

ts ET24 cells are a novel temperature-sensitive (ts) mutant for cell proliferation of hamster BHK21 cells. The human genomic DNA which rescued the temperature-sensitive lethality of ts ET24 cells was isolated and screened for an open reading frame in the deposited human genomic library. X chromosomal DBX gene encoding the RNA helicase, DEAD-BOX X isoform, which is homologous to yeast Ded1p, was found to be defective in this mutant. The single point mutation (P267S) was localized between the Motifs I and Ia of the hamster DBX of ts ET24 cells. At the nonpermissive temperature of 39.5 degrees C, ts ET24 cells were arrested in the G1-phase and survived for more than 3 days. In ts ET24 cells, total protein synthesis was not reduced at 39.5 degrees C for 24 h, while mRNA accumulated in the nucleus after incubation at 39.5 degrees C for 17 h. The amount of cyclin A mRNA decreased in ts ET24 cells within 4 h after the temperature shift to 39.5 degrees C, consistent with the fact that the entry into the S-phase was delayed by the temperature shift.     

Temperature-sensitive Chinese hamster fibroblast mutant with a defect in RNA metabolism

We describe a new temperature-sensitive mutant of Chinese hamster cell fibroblasts. After a shift to the nonpermissive temperature of 40.5 degrees C, the rates of DNA, RNA, and protein synthesis declined rapidly (to < or = 50% within 12 h) and the progression of unsynchronized cells through the cell cycle was affected. We believe that DNA synthesis came to a halt after a short time, because cells no longer entered the S phase. The decrease in protein synthesis at 40.5 degrees C was shown to be a consequence of a decrease in the number of polysomes, whereas free 80S ribosomes accumulated. We concluded that the components of the protein biosynthetic machinery were intact (ribosomes and soluble factors), but synthesis was limited by a shortage of mRNA. The decline in mRNA production had a significant effect on the synthesis of proteins (e.g., heat shock proteins) translated from short-lived messages. We observed that both polyadenylated and nonpolyadenylated RNA syntheses declined at 40.5 degrees C, whereas the synthesis of small RNAs (4 to 5S) was less reduced. The argument is made that the temperature-sensitive phenotype is the result of a defect affecting mRNA synthesis.     

Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses.

Defects in RNA and protein synthesis of seven Sindbis virus and seven Semliki Forest virus RNA-negative, temperature-sensitive mutants were studied after shift to the restrictive temperature (39 degrees C) in the middle of the growth cycle. Only one of the mutants, Ts-6 of Sindbis virus, a representative of complementation group F, was clearly unable to continue RNA synthesis at 39 degrees C, apparently due to temperature-sensitive polymerase. The defect was reversible and affected the synthesis of both 42S and 26S RNA equally, suggesting that the same polymerase component(s) is required for the synthesis of both RNA species. One of the three Sindbis virus mutants of complementation group A, Ts-4, and one RNA +/- mutant of Semliki Forest virus, ts-10, showed a polymerase defect even at the permissive temperature. Seven of the 14 RNA-negative mutants showed a preferential reduction in 26S RNA synthesis. The 26S RNA-defective mutants of Sindbis virus were from two different complementation groups, A and G, indicating that functions of two viral nonstructural proteins ("A" and "G") are required in the regulation of the synthesis of 26S RNA. Since the synthesis of 42S RNA continued, these functions of proteins A and G are not needed for the polymerization of RNA late in infection. The RNA-negative phenotype of 26S RNA-deficient mutants implies that proteins regulating the synthesis of this subgenomic RNA must have another function vital for RNA synthesis early in infection or in the assembly of functional polymerase. Several of the mutants having a specific defect in the synthesis of 26S RNA showed an accumulation of a large nonstructural precursor protein with a molecular weight of about 200,000. One even larger protein was demonstrated in both Semliki Forest virus- and Sindbis virus-infected cells which probably represents the entire nonstructural polyprotein.     

Effect of temperature on RNA synthesis in Escherichia coli CRT 266 (dna Bts) following infection with bacteriophage T3]

Infection of the temperature-sensitive E. coli CRT 266 (dnaBts) with T3-phages at the temperature of 30 degrees C and 35 degrees C, respectively, induced T3-specific RNA synthesis with a maximum rate at 7 min (30 degrees C) and 4.5 min (35 degrees C) after infection. At temperatures above 40 degrees C no T3-induced RNA synthesis could be observed. Infection of E. coli CR 34--45 (dnaB+) with T3 phages at 30 degrees C, 35 degrees C and at temperatures above 40 degrees C, however, produced T3-specific RNA synthesis. The maximum of T3-induced RNA synthesis could be observed between 7 min and 3 min depending on the temperature during infection. The inability to form T3-specific RNA after infection of E. coli CRT 266 at nonpermissive temperatures may be a cause for the absence of the formation of T3 phages and lysis of the host cells.     

Functional complementation between mutations at two distant positions in Escherichia coli RNA polymerase as revealed by second-site suppression

Subunit-subunit interactions are critical for the assembly of the core of Escherichia coli RNA polymerase. The mutant alpha-subunit C131A is unable to complement the temperature-sensitive alpha-R45C mutant strain, which is defective for binding of the beta-subunit. In vitro reconstitution experiments, however, indicate that the alpha-C131A variant is able to form the intermediate alpha2beta, but is defective in contacting the beta'-subunit. We used this alpha-C131A mutant to isolate a suppressor mutation in the beta'-subunit. Genetic and biochemical characterization of the beta' suppressor indicates the allele-specific nature of its effect. Sequence analysis of the suppressor revealed a single substitution of Gly at position 333, an evolutionarily conserved position in the conserved region C of the beta'-subunit, by Asp. However, the crystal structure of the bacterial RNA polymerase indicates that the primary mutation (alpha-C131A) and its suppressor lie far apart. Thus, we propose that long-range interactions, as in this case, may play an important role in the functional assembly of E. coli RNA polymerase.     

Temperature-Sensitive Defect of Mutants Isolated from L Cells Persistently Infected with Newcastle Disease Virus

The temperature-sensitive defects of virus mutants isolated from L cells persistently infected with Newcastle disease virus (NDV) were analyzed. Genetic grouping of the mutants by complementation tests was attempted by using several different methods, including yield analysis, RNA synthesis, and heterozygote formation at 42 to 43 C, the nonpermissive temperature. In each case, specific interference prevented detection of complementation. This interference was shown to occur prior to or at the level of virus RNA synthesis. Temperature-shift experiments with five different NDV(pi) clones showed that virus replication begun at 37 C could not be completed at the nonpermissive temperature. The activity of the NDV-specific RNA-dependent RNA polymerase in the cytoplasm of infected chicken embryo cells was not stable and could not be demonstrated directly. However, indirect measurement of RNA polymerase activity at the nonpermissive temperature was accomplished by studying the kinetics of virus-specific RNA synthesis in infected cells after temperature shift. Two types of response were obtained: with three NDV(pi) clones, virus-specific RNA synthesis ceased immediately upon transfer of infected cells to 42 to 43 C, whereas in cells infected with two other NDV(pi) clones, RNA synthesis continued for several hours at this temperature. These results suggested that there may be two types of ts defects in NDV(pi), both associated with virus-specific RNA polymerase activity.     

Effect of temperature shifts on extracellular proteinase-specific mRNA pools in Pseudomonas fluorescens B52.

The influence of a shift in temperature from 20 to 32 degrees C on extracellular proteinase synthesis by Pseudomonas fluorescens B52 was examined. When cells actively synthesizing proteinase at 20 degrees C were shifted to 32 degrees C, enzyme synthesis ceased immediately. After 30 min at 32 degrees C, cells recovered at 20 degrees C after a lag of 30 min. Rifampin and chloramphenicol prevented recovery of synthesis at 20 degrees C. Rifampin-insensitive proteinase synthesis (an indirect measure of proteinase-specific mRNA pools) decreased after the exposure of cells to 32 degrees C for 30 min but was recovered during incubation at 20 degrees C. Controls not exposed to a temperature shift experienced no loss of rifampin-independent synthesis. Cells experienced a 50% reduction in mRNA pools after 15 min at 32 degrees C. The data support the working hypothesis that the loss of mRNA pools after treatment at 32 degrees C is responsible for the lag before the recovery of extracellular proteinase synthesis.     

Effect of temperature shifts on extracellular proteinase-specific mRNA pools in Pseudomonas fluorescens B52

The influence of a shift in temperature from 20 to 32 degrees C on extracellular proteinase synthesis by Pseudomonas fluorescens B52 was examined. When cells actively synthesizing proteinase at 20 degrees C were shifted to 32 degrees C, enzyme synthesis ceased immediately. After 30 min at 32 degrees C, cells recovered at 20 degrees C after a lag of 30 min. Rifampin and chloramphenicol prevented recovery of synthesis at 20 degrees C. Rifampin-insensitive proteinase synthesis (an indirect measure of proteinase-specific mRNA pools) decreased after the exposure of cells to 32 degrees C for 30 min but was recovered during incubation at 20 degrees C. Controls not exposed to a temperature shift experienced no loss of rifampin-independent synthesis. Cells experienced a 50% reduction in mRNA pools after 15 min at 32 degrees C. The data support the working hypothesis that the loss of mRNA pools after treatment at 32 degrees C is responsible for the lag before the recovery of extracellular proteinase synthesis.     

Further characterization of a murine temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase alpha-activity

tsFT20 cells, which have temperature-sensitive DNA polymerase alpha-activity, were characterized mainly at the cellular level. The cells lost their ability to synthesize DNA immediately after a shift to non-permissive temperature. The extent of decrease in the activity of DNA polymerase alpha in whole-cell extracts was the same as that of the decrease in the DNA replication ability determined by [3H]thymidine incorporation. At 39 degrees C, tsFT20 cells lost most of their colony-forming ability in one doubling time (16 h). The cells could not grow at higher than 38 degrees C, but could grow at 37 degrees C. When tsFT20 cells were synchronized at the G1/S boundary and incubated at 39 degrees C, they could not complete the S phase, ceasing cell cycle progression in mid-S phase. A temperature shift (33 degrees C----39 degrees C) experiment indicated that the whole S phase was temperature-sensitive, whereas the G2 and M phases were not. These results confirmed that DNA polymerase alpha plays a key role in DNA replication in mammalian cells.     

A new gene in E. coli RNA synthesis

A novel spontaneous temperature sensitive mutant of Escherichia coli, which stops synthesizing stable RNA and some proteins immediately upon temperature shift from 30 degrees C to 42 degrees C, is described. Stable RNA species are not preferentially degraded in the mutant at the nonpermissive temperature. The guanine polyphosphate compounds, ppGpp (MS1) and pppGpp (MS2), are not produced at 42 degrees C. The mutant strain does not grow at 42 degrees C in either broth or defined minimal medium supplemented with any of a variety of carbon sources. The temperature sensitive mutation in this strain maps between dap A, E and pts I and defines a new locus affecting RNA synthesis in E. coli.     

Thermosensitive mutations affecting ribonucleic acid polymerases in Saccharomyces cerevisiae.

Among 150 temperature-sensitive Saccharomyces cerevisiae mutants which we have isolated, 15 are specifically affected in ribonucleic acid (RNA) synthesis. Four of these mutants exhibit particularly drastic changes and were chosen for a more detailed study. In these four mutants, RNA synthesis is immediately blocked after a shift at the nonpermissive temperature (37 C), protein synthesis decays at a rate compatible with messenger RNA half-life, and deoxyribonucleic acid synthesis increases by about 40%. All the mutations display a recessive phenotype. The segregation of the four allelic pairs ts-/ts+ in diploids is mendelian, and the four mutants belong to three complementation groups. The elution patterns (diethylaminoethyl-Sephadex) of the three RNA polymerases of the mutants grown at 37 C for 3.5 h show very low residual activities. The in vitro thermodenaturation confirms the in vivo results; the half-lives of the mutant activities at 45 C are 10 times smaller than those of the wild-type enzymes. Polyacrylamide gel electrophoresis shows that the synthesis of all species of RNA is thermosensitive. The existence of three distinct genes, which are each indispensable for the activity of the three RNA polymerases in vivo as well as in vitro, strongly favors the hypothesis of three common subunits in the three RNA polymerases.