Studies on ribosomal protein biosynthesis in an RNA polymerase temperature sensitive E. coli mutant.

Summary In E. coli strain XH56 the synthesis of all RNA species is blocked upon shifting the culture to the non-permissive temperature. The decay of specific messenger RNA species coding for individual ribosomal (r) proteins was followed by measuring the rate of r-protein synthesis by pulse labelling at various times after the shift. The half-lives of the average 30S r-protein and 50S r-protein mRNA species are identical (1.75 min) and shorter than those of the average messenger coding for total cell proteins (2.75 min). Most individual r-protein messengers have a half-life in the same range (1.50–2.00). Only a few r-protein messengers have significantly longer half-lives: S1 (2.80 min), S17 (3.29 min), L29 (2.30 min), L31 (2.30 min), L32 (2.33 min) and L16 (2.60 min). The results indicate that the degradation of most individual r-protein mRNA species is not specifically controlled.After a few min at the non-permissive temperature, all protein synthesis is blocked. The restart of r-protein synthesis was followed after shifting the culture back to the permissive temperature. The recovery of cell growth is very slow. During this period preferential r-protein synthesis was observed. Moreover differential rates of biosynthesis of r-proteins was obtained, it may be indicative of specific regulatory process(es).     

Escherichia coli mutant temperature sensitive for group I RNA bacteriophages.

The temperature-sensitive conjugational transfer-deficient mutant Escherichia coli JCFL39, carrying a traD(Ts) mutation, is herein described as also being temperature sensitive for group I RNA phages (MS2, f2, and R17) but not for Q beta. Temperature shift experiments showed that the growth of group I phage MS2 in the mutant could be inhibited by a post-penetration event at high temperature. A possible role for the traD cistron of sex factor F in the intracellular development of MS2 is suggested.     

Purineless death: ribosomal RNA turnover in a purine-starved ade- mutant of Chinese hamster cells.

When V79 pur 1, a purine-requiring auxotroph of a Chinese hamster cell line, is deprived of adenine, nucleic acid and protein synthesis decline rapidly. However, on continuous starvation RNA and DNA synthesis recommences to reach approximately 30% of the normal level between 12 to 24 h starvation. This is accompanied by a rise in the intracellular nucleotide pool. Utilizing mengovirus, which gives a productive infection in V79 pur 1 cells even under conditions of starvation, we can show that rRNA is preferentially degraded and provides the nucleotides for RNA synthesis. Thus "purineless" death in mammalian cells is accompanied by turnover of stable RNA.     

Temperature sensitive phosphoproteins in rat cells transformed by a temperature sensitive mutant of rous sarcoma virus

The localization of the src-encoded protein kinase was examined by fractionating cellular extracts from rat cells transformed by a wild type and a temperature-sensitive mutant of Rous sarcoma virus (SR-A 3Y1 and ts68 3Y1 cells). It was found to be specifically localized in the post-microsomal supernatant (PMS) fraction. Furthermore, it was noticed that a protein with a molecular weight of 16,000 (16K-protein) in the PMS fraction was phosphorylated in vitro when the PMS fraction from ts68 3Y1 cells was preincubated at 33 degrees C, but not at 42 degrees C. This protein was phosphorylated when the fraction from SR-A 3Y1 cells was preincubated at 33 degrees C and at 42 degrees C. Similar temperature-sensitive phosphorylation of 16K-protein was also observed in the PMS fraction from ts68 3Y1 cells labeled in vivo with [32P]orthophosphate at 33 degrees C. These results suggest that this 16K-protein might be a candidate for the endogenous acceptor for the src-encoded protein kinase.     

A temperature sensitive mutant of Escherichia coli which does not allow replication of RNA phage at a high temperature.

A conditional mutant, referred to as RepR43, was isolated from Escherichia coli W2252 by N-methyl-N'-nitro-N-nitroso-guanidine mutagenesis. Although RepR43 does not permit growth of RNA phage beta at the restrictive temperature, 43 degrees C, cell growth and synthesis of macromolecules such as RNA and protein continue at a somewhat reduced rate. Several lines of evidence indicate that a RepR43 function is indispensable for normal phage RNA replication. In addition, this function appears to be involved in the maintenance of the perpetuated phage genome. The addition of 10% sucrose to the medium at the restrictive temperature resulted in the production of the phage, suggesting that the mutant cell might have an altered membrane organization which interferes with normal viral replication.     

Characterization of the genomic RNA from a Rous sarcoma virus mutant temperature sensitive for cell transformation.

We have found that the LA23 t/s mutant of Rous sarcoma virus (phenotype Prague B), even when passaged repeatedly at high multiplicity of infection, does not give rise to transformation defective deletion mutants comparable to those derived from RSV. In view of this fact and of the high rate of production of this mutant at 41 degrees C, we have undertaken a detailed analysis of the genome of this virus by ordering all large T1 oligonucleotides and by determining their nucleotide sequences. The results indicate a high degree of mutation in the onc gene as compared to that of Pr-A or Pr-B.     

Escherichia coli traD(Ts) mutant temperature sensitive for assembly of RNA bacteriophage MS2.

We report here a study on the temperature-sensitive conjugational transfer-deficient mutant Escherichia coli JCFL39, carrying a traD(Ts) mutation, which is also temperature sensitive for group I RNA phages (MS2, f2, and R17). It is shown that, when the mutant was infected with MS2 at 42 degrees C, phage RNA replicated; a 27S MS2 RNA and phage proteins were synthesized. However, neither PFU nor physical MS2 particles were formed, showing that phage assembly was inhibited. In addition, the high temperature affected the membranes of the host mutant: the mutant was hypersensitive to chemicals, and the electrophoretic pattern of the membranal proteins was modified. We suggest that the pleiotropic effects of the traD mutation on MS2 assembly and DNA transfer during conjugation were a result of the changes in the membrane of the mutant.     

Secondary structure maps of ribosomal RNA. II. Processing of mouse L-cell ribosomal RNA and variations in the processing pathway

Secondary structure mapping in the electron microscope was applied to ribosomal RNA and precusor ribosomal RNA molecules isolated from nucleoli and the cytoplasm of mouse L-cells. Highly reproducible loop patterns were observed in these molecules. The polarity of L-cell rRNA was determined by partial digestion with 3′-exonuclease. The 28 S region is located at the 5′-end of the 45 S rRNA precursor. Together with earlier experiments on labeling kinetics, these observations established a processing pathway for L-cell rRNA. The 45 S rRNA precursor is cleaved at the 3′-end of the 18 S RNA sequence to produce a 41 S molecule and a spacer-containing fragment (24 S RNA). The 41 S rRNA is cleaved forming mature 18 S rRNA and a 36 S molecule. The 36 S molecule is processed through a 32 S intermediate to the mature 28 S rRNA. This pathway is similar to that found in HeLa cells, except that in L-cells a 36 S molecule occurs in the major pathway and no 20 S precusor to 18 S RNA is found. The processing pathway and its intermediates in L-cells are analogous to those in Xenopus laevis, except for a considerable size difference in all rRNAs except 18 S rRNA.The arrangement of gene and transcribed spacer regions and of secondary structure loops, as well as the shape of the major loops were compared in L-cells, HeLa cell and Xenopus rRNA. The over-all arrangement of regions and loop patterns is very similar in the RNA from these three organisms. The shapes of loops in mature 28 S RNA are also highly conserved in evolution, but the shapes of loops in the transcribed spacer regions vary greatly. These observations suggest that the sequence complementarity that gives rise to this highly conserved secondary structure pattern may have some functional importance.     

The temperature sensitive mutant 72c

Summary The spontaneous temperature sensitive mutant 72c is shown to be more tolerant to fusidic acid, but less tolerant to trimethoprim on plates at permissive temperature, than is the parental strain. The poor growth of the mutant on amino acids supplemented plates, as well as its inability to grow on broth plates at 40°, can be compensated by sublethal amounts of chloroamphenicol. Also some mutations to Rif-R or Str-R improve growth of the mutant under certain conditions.Reversion and other genetic analysis strongly suggest, that the pleiotropic behaviour of the mutant is due to a single mutation in a gene, which is designated fusB and is closely cotransducible with lip at min 14 of the E. coli chromosome. The gene order is lip-fusB-supE.