Synthesis and repair of RNA-containing supercoiled plasmid ColE1 DNA in Escherichia coli

Supercoiled plasmid molecules sensitive to nicking by RNase or alkali have been shown to accumulate during replication of colicinogenic factor E1 (ColE1) in Escherichia coli in the presence of chloramphenicol. The possibility that this sensitivity is due to the covalent integration of RNA molecules during the synthesis of plasmid DNA is supported by the demonstration that (a) strands of supercoiled ColE1 newly replicated in the presence of chloramphenicol exhibit sensitivity to RNase and alkali treatment, while (b) RNase- and alkali-resistant circular strands of plasmid DNA synthesized either before or after the addition of chloramphenicol remain resistant during subsequent replication of the plasmid in the presence of chloramphenicol. Furthermore, newly made plasmid DNA strands cannot act as templates for further rounds of replication if they possess an RNA segment. The existence of a repair mechanism for the removal of the RNA segment from supercoiled ColE1 DNA molecules was demonstrated by pulse-chase experiments. It was observed that the proportion of RNase-sensitive molecules is considerably higher in pulse-labeled as compared to continuously labeled ColE1 DNA synthesized in the presence of chloramphenicol, and the proportion of pulse-labeled ColE1 DNA that is RNase sensitive is greatly reduced during a chase period. Removal of the RNA segment is also carried out effectively at the restrictive temperature in temperature-sensitive DNA polymerase I mutants. In a survey of other bacterial mutants defective in the repair of damaged DNA, a substantial increase in the rate of accumulation of RNase-and alkali-sensitive supercoiled ColE1 DNA in the presence of chloramphenicol was observed in recBC and uvrA mutants in comparison with the wild-type strains.     

Isolation and properties of a plasmid which expresses the E. coli Su+7 amber suppressor tRNA gene.

Summary The gene of the amber suppressor tRNA derived from tRNA^Try, Su⁺7, has been inserted into a col E₁-derived vehicle by selecting for its expression. Despite selection for a suppressor phenotype, and the plasmid's stable presence at ca. 180 copies/cell during balanced growth, the level of mature tRNA maintained by the gene is less than that of the normal haploid tRNA^Try locus in the bacterial chromosome. Transfer RNA genes, both the plasmid Su⁺7 gene and chromosomal tRNA's are expressed during inhibition of protein synthesis. During, e.g. chloramphenicol inhibition, Su^-7 and Su⁺7 tRNA can be elevated similarly in the plasmid-containing cell; Su⁺7 reaches levels of molecules/cell which ordinarily characterize a major tRNA.The recombinant plasmid, but not the cloning vehicle alone, has a more general effect on tRNA levels; accumulation of tRNA from three chromosomal tRNA loci including tRNA^Try, continues during extensive isoleucine limitation. The plasmid therefore contains a locus which probably alters the relaxedstringent circuit, whose effects is disseminated to at least 3 widely separated loci.     

THE BIOGENESIS OF MITOCHONDRIA IN SACCHAROMYCES CEREVISIAE : A Comparison between Cytoplasmic Respiratory-Deficient Mutant Yeast and Chloramphenicol-Inhibited Wild Type Cells

The effects of chloramphenicol on S. cerevisiae and on a cytoplasmic respiratory-deficient mutant derived from the same strain are compared. In the normal yeast, high concentrations of chloramphenicol in the growth medium completely inhibit the formation of cytochromes a, a₃, b, and c₁ and partially inhibit succinate dehydrogenase formation, whereas they do not affect cytochrome c synthesis. This has been correlated with the marked reduction of mitochondrial cristae formation in the presence of the drug. In glucose-repressed normal yeast, chloramphenicol has little effect on the formation of outer mitochondrial membrane, or on the synthesis of malate dehydrogenase and fumarase. However, both these enzymes, as well as the number of mitochondrial profiles, are markedly decreased when glucose de-repressed yeast is grown in the presence of chloramphenicol. The antibiotic did not appear to affect the cytoplasmic respiratory-deficient mutant. The results have been interpreted to indicate that chloramphenicol inhibits the protein-synthesizing system characteristic of the mitochondria. Since the drug does not prevent the formation of cytochrome c, of several readily solubilized mitochondrial enzymes, or of outer mitochondrial membrane, it is suggested that these are synthesized by nonmitochondrial systems.     

Transfer RNA genes in the mitochondrial DNA of cytoplasmic petite mutants of Saccharomyces cerevisiae

Hybridization saturation analyses of mitochondrial DNA from 11 petite clones genetically characterized with respect to chloramphenicol and erythromycin resistance markers, have been carried out with 11 individual mitochrondrial transfer RNAs. Mitochondrial tRNA cistrons were lost, retained, or amplified in different petite strains. In some cases hybridization levels corrected for kinetic complexity of the mtDNA³ were two- to threefold greater than that for grande mtDNA indicating selective amplification, or increased number of copies, of the segment of mtDNA containing that tRNA cistron. Hybridization levels corrected for reduced kinetic complexity of petite mtDNAs in many cases were only 1 to 10% of that for grande mtDNA suggesting a low level of intracellular molecular heterogeneity of mtDNA with respect to tRNA cistrons. Some petite clones that retained tRNA genes continued to transcribe mitochondrial tRNAs, since tRNA isolated from these strains could be aminoacylated with Escherichia, coli synthetases and hybridized with mtDNA. Hybridization data allow us to order several of the tRNA cistrons on the mitochondrial genome with respect to the chloramphenicol and erythromycin antibiotic resistance markers.     

Changes in rate of RNA synthesis and ribosomal gene number during oogenesis of Drosophila melanogaster.

A new method for separating Drosophila egg chambers into different developmental classes (Jacobs-Lorena and Crippa, 1977) made it possible to study changes in the rate of ribosomal RNA (rRNA), 5S RNA, and tRNA synthesis and the changes in ribosomal gene number during oogenesis. Synthesis of RNA was measured by [³H]uridine incorporation in vivo and subsequent analysis on sucrose gradients or gel electrophoresis. Specific radioactivity of nucleotide pools has also been determined. Ribosomal gene number has been measured by hybridization of egg chamber DNA to rRNA of high specific radioactivity. Our findings led us to conclude that in Drosophila melanogaster: (i) rRNA, 5S RNA, and tRNA are synthesized in all stages of oogenesis. (ii) In every stage, rRNA is the main RNA species synthesized. (iii) The rate of rRNA, 5S RNA, and tRNA synthesis increases greatly during oogenesis and is paralleled by a similar increase in ribosomal gene number resulting from the polyploidization of the nurse cell nuclei.     

Formation of the yeast mitochondrial membrane. 3. Accumulation of mitochondrial proteins synthesized in both the cytoplasm and mitochondria in yeast undergoing glucose depression

The inhibitors of protein synthesis, chloramphenicol and cycloheximide, were added to cultures of yeast undergoing glucose derepression at different times during the growth cycle. Both inhibitors blocked the increase in activity of coenzyme QH₂-cytochrome c reductase, suggesting that the formation of complex III of the respiratory chain requires products of both mitochondrial and cytoplasmic protein synthesis.The possibility that precursor proteins synthesized by either cytoplasmic or mitochondrial ribosomes may accumulate was investigated by the sequential addition of cycloheximide and chloramphenicol (or the reverse order) to cultures of yeast undergoing glucose derepression. When yeast cells were grown for 3 hr in medium containing cycloheximide and then transferred to medium containing chloramphenicol, the activity of cytochrome oxidase increased at the same rate as the control during the first hour in chloramphenicol. These results suggest that some accumulation of precursor proteins synthesized in the mitochondria had occurred when cytoplasmic protein synthesis was blocked during the growth phase in cycloheximide. In contrast, essentially no products of mitochondrial protein synthesis accumulated as precursors for either oligomycin-sensitive ATPase or complex III of the respiratory chain during growth of the cells in cycloheximide.When yeast were grown for 3 hr in medium containing chloramphenicol followed by 1 hr in cycloheximide, the activities of cytochrome oxidase and succinate-cytochrome c reductase increased at the same rate as the control, while the activities of oligomycin-sensitive ATPase and NADH or coenzyme QH₂-cytochrome c reductase were nearly double that of the control. These data suggest that a significant accumulation of mitochondrial proteins synthesized in the cytoplasm had occurred when the yeast cells were grown in medium containing sufficient chloramphenicol to block mitochondrial protein synthesis. The possibility that proteins synthesized in the cytoplasm may act to control the synthesis of mitochondrial proteins for both oligomycin-sensitive ATPase and complex III of the respiratory chain is discussed.     

The methylation of transfer ribonucleic acid during regeneration of the liver

Transfer ribonucleic acid¹ is methylated after the molecule is synthesized; at least eight enzymes are involved in the transfer of methyl groups (derived from methionine). The time courses of methylation and synthesis of tRNA during rat liver regeneration have been compared in an in vivo radioisotopic study, using 6-orotic acid-¹⁴C and ³H-methyl-L-methionine as precursors in double label pulses. Liver regeneration is a synchronized system in which biochemical events of the cell cycle are separable. Transfer RNA methylation increase precedes by several hours tRNA synthesis during regeneration, although the curves overlap. A ratio of the relative rate of methylation to the relative rate of synthesis has been made; that curve positively correlates with the rise and fall of protein synthesis during regeneration. It is clear that methylation and synthesis of tRNA are only weakly coupled; changing methyl content of the tRNA "pool" resulting from differential tRNA methylase and polymerase activities may regulate the rate of protein synthesis in the cell cycle at the translational level. The "pool sizes" of uridine monophosphate (UMP) and S-adenosylmethionine (SAM) were measured indirectly; UMP and SAM were isolated from perchloric acid supernatants and their specific activities were computed. Differential changes in radioactivity available to tRNA methylases and polymerases are not a source of artifact. That is, the control of both the synthesis and methylation of tRNA is at the enzyme level in vivo, rather than at some enzymatic step prior to those enzymatic reactions.     

Permeability of Xenopus laevis embryos: Specific incorporation of precursors into eukaryotic proteins and nucleic acids

All amino acids and several nucleic acid precursors are taken up by Xenopus laevis embryos. The embryos are completely intact and not modified in any way. These precursors are directly incorporated into the macromolecules of Xenopus embryos and not prokaryotic contaminants as has been previously claimed. Radioactive leucine is incorporated into Xenopus laevis ribosomal proteins as characterized by sucrose gradient centrifugation. The uptake of the amino acids is cycloheximide sensitive and unaffected by chloramphenicol. Radioactive adenosine and orotic acid are taken up and incorporated into tRNA and rRNA at high levels as characterized by sucrose gradients and electrophoresis. These characterizations of labeled macromolecules unequivocally show that normal Xenopus laevis embryos will take up and incorporate labeled precursors to levels which are sufficient to study cellular biochemical events at such early stages of development.     

Sequential folding of transfer RNA: A nuclear magnetic resonance study of successively longer tRNA fragments with a common 5′ end

Most folding studies on proteins and nucleic acids have been addressed to the transition between the folded and unfolded states of an intact molecule, where an entire residue sequence is present during the folding event. However, since these polymers are synthesized sequentially from one terminus to the other in vivo, their folding pathways may be influenced greatly by the sequential appearance of the residues as a function of time.The three-dimensional structure of yeast tRNA^Phe in the crystalline state is correlated with 360 MHz proton nuclear magnetic resonances from three fragments plus an intact molecule of the tRNA that share a common 5′ end and are in a solution condition similar to that of the crystal structure. This has allowed identification of folded structures present in the fragments and presumably present in the growing tRNA molecule as it is being synthesized from the 5′ end. The experiments show that only the correct stems are formed in the fragments; no additional or competing helical region is produced. This suggests that in the biosynthesis of this tRNA, correct folding of helical stems occurs before the entire molecule is formed. Further, some of the tertiary interactions (hydrogen bonds) found in the crystal structure are also probably present before the synthesis is completed. These findings are generalized to consider the precursor of the tRNA as well as other tRNAs.     

Differential rates of synthesis of glycerokinase and glycerophosphate dehydrogenase in Neurospora crassa during induction.

The synthesis of the enzymes of the glycerophosphate pathway in Neurospora has been examined during exponential growth of cells on acetate as the sole carbon source. After the addition of glycerol to the media, increases in the levels of both glycerokinase and a mitochondrial glycerol-3-phosphate dehydrogenase are observed within 1 h and fully induced levels are reached within one and a half mass doublings for glycerokinase and two and a half mass doublings for glycerol-3-phosphate dehydrogenase. The increase in glycerokinase activity represents de novo synthesis of enzyme as evidenced by the absence of immunologically related protein in uninduced cell extracts. The synthesis of both glycerokinase and glycerol-3-phosphate dehydrogenase can be totally inhibited by treatment of cells with 20 μg/ml cycloheximide. During incubation with 4 mg/ml chloramphenicol, there is normal synthesis of glycerokinase but a 30–50% inhibition of mitochondrial glycerol-3-phosphate dehydrogenase synthesis. However, under these conditions, in the cytosol fraction there is a significant increase in glycerol-3-phosphate dehydrogenase specific activity, suggesting that precursors are synthesized and accumulated in the cytosol prior to incorporation into mitochondria. Upon removal of chloramphenicol, the rate of appearance of glycerol-3-phosphate dehydrogenase into the mitochondria is up to four times greater than observed in untreated controls. It is concluded that both glycerokinase and glycerol-3-phosphate dehydrogenase are synthesized on cytoplasmic ribosomes, but that final assembly of glycerol-3-phosphate dehydrogenase into mitochondria is dependent on concomitant synthesis of mitochondrial inner membrane.     

Tissue inhibitor of metalloproteinases. Identification of precursor forms synthetized by human fibroblasts in culture

Precursor forms of the glycoprotein tissue inhibitor of metalloproteinases (TIMP) synthesized by human fibroblasts in culture have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of specific immunoprecipitates. Translation of mRNA extracted from fibroblasts in the cell-free rabbit reticulocyte lysate system yielded a single immunoprecipitable precursor of tissue inhibitor of metalloproteinases, M_r 22 000. Intact fibroblasts cultured in the presence of tunicamycin synthesized an M_r 20 000 form of tissue inhibitor of metalloproteinases, detectable intracellularly and extracellularly. This is in contrast to the predominantly intracellular M_r 24 000.form synthetized during monensin treatment of cells and the normal secreted form of tissue inhibitor of metalloproteinases, M_r 29 000. Isoelectric focusing of the various immunoprecipitable precursor forms showed a progressive increase in positive charge and microheterogeneity of the protein during cellular processing. The data suggest that the inhibitor protein core, of basic pI, is glycosylated initially by the addition of mostly neutral sugars and subsequently by acidic sugars, prior to secretion.     

Activation of several tRNAs of Escherichia coli by the phenoxyacetyl derivative of N-hydroxysuccinimide

Escherichia coli 15T^? treated with chloramphenicol produces tRNA^phe which is deficient in minor nucleosides. Undermodified tRNA^phe chromatographs as two new peaks from a benzoylated diethylaminoethyl-cellulose column. Chloramphenicol tRNA^phe was purified by phenoxyacetylation of phenylalanyl-tRNA and subsequent chromatography on benzoylated diethylaminoethyl-cellulose. Purified tRNA^phe had an altered Chromatographie profile as a result of the purification procedure. Phenoxyacetylation of an unpurified tRNA preparation, which was either charged with phenylalanine or kept discharged, resulted in a permanent alteration of tRNA^phe which was similar to the alteration of the purified tRNA^phe. The altered tRNAs eluted with higher salt or ethanol concentrations from benzoylated diethylaminoethyl-cellulose. The alteration was also shown for tRNA^phe of phenoxyacetylated tRNA from late log phase E. coli 15T?. tRNA^glu and tRNA^Leu were not changed, but both tRNA^Arg and tRNA^Ile were altered. tRNA₂^Val and tRNA^Met shifted in the elution profile; tRNA₁^Val and tRNA_f^Met were not affected.Comparison of the primary structures of the alterable and nonalterable tRNA's revealed that all alterable tRNA's have the undefined nucleoside X in the extra loop. Phenoxyacetylation of nucleoside X probably was the cause of the altered profiles.tRNA^phe from E. coli 15T^? treated with chloramphenicol was less reactive towards phenoxyacetylation than normal tRNA, possibly because of a different conformation of the modification-deficient molecule relative to the normal tRNA^phe. tRNA^phe from E. coli 15T^?, starved for cysteine and methionine and treated with chloram-phenicol, is more deficient in minor nucleosides and showed even less reactivity.Acceptor capacities of the altered tRNA species were not changed significantly; only the acceptor capacity for tRNA^Ile decreased approximately 25%. The recognition site for the aminoacyl-tRNA synthetases probably is not affected.     

New archaeal methyltransferases forming 1-methyladenosine or 1-methyladenosine and 1-methylguanosine at position 9 of tRNA

Two archaeal tRNA methyltransferases belonging to the SPOUT superfamily and displaying unexpected activities are identified. These enzymes are orthologous to the yeast Trm10p methyltransferase, which catalyses the formation of 1-methylguanosine at position 9 of tRNA. In contrast, the Trm10p orthologue from the crenarchaeon Sulfolobus acidocaldarius forms 1-methyladenosine at the same position. Even more surprisingly, the Trm10p orthologue from the euryarchaeon Thermococcus kodakaraensis methylates the N¹-atom of either adenosine or guanosine at position 9 in different tRNAs. This is to our knowledge the first example of a tRNA methyltransferase with a broadened nucleoside recognition capability. The evolution of tRNA methyltransferases methylating the N¹ atom of a purine residue is discussed.     

Similarities in properties and a functional difference in purified leucyl-tRNA synthetase isolated from two developmental stages of Tenebrio molitor

In Tenebrio molitor, as well as in other biological systems, there are indications that differences in leucyl-tRNA synthetase activity may play a role in translational control. However, it has not been clear whether the difference in activity is due to the appearance of a multiplicity of enzymes during development or to the alteration of a single enzyme.The purification of leucyl-tRNA synthetase from day 1 and day 7 after the larval pupal molt of Tenebrio molitor is described. The enzyme from both developmental stages was purified over a 1000-fold. The two enzyme preparations are identical in molecular weight (99,000). They show the same characteristics after aging. The pH optimum, heat inactivation behavior, and dependency on divalent cations are the same for both enzymes. They also show identical kinetics with similar values of Km for leucine, ATP, Mg²⁺, and tRNA day 1. However, leucyl-tRNA synthetase purified from day 7 exhibits an additional function in recognizing a new species of isoaccepting tRNA in day 7 tRNA. We have tentatively concluded that the two enzymes are probably different forms of the same enzyme and the additional activity is due to alteration of the enzyme at the macromolecular level during development.     

A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture.

A method is presented for determining the extent of methylation of tRNAs synthesized in mammalian and bacterial cell systems and is based upon determining the distribution of radioactivity associated with the guanine constituents of total cellular tRNA preparations previously labeled with [2-¹⁴C]guanosine and with [methyl]-³H or -¹⁴C]methionine. Whereas labeling with guanosine provides a means of assessing the extent of methylation of the [2-¹⁴C]guanine residues incorporated into tRNA, methionine labeling provides a measure of the percentage of [methyl-³H or -¹⁴C]methylated constituents that are methylated guanines. Analyses such as the above reveal that the tRNA of KB cells acquires approximately three times as many methyl groups as that of E. coli B tRNA. Coupled with the knowledge that both mammalian and bacterial tRNA preparations contain an average of 24 guanine residues per molecule, the above analyses further reveal that 7.2 and 2.4 methyl groups are incorporated into each tRNA molecule synthesized in exponentially growing KB- and E. coli B-cells, respectively. Additional information regarding the extent of formation of individual methylated constituents per tRNA molecule synthesized is presented.     

Drosophila RNase Z processes mitochondrial and nuclear pre-tRNA 3' ends in vivo

Although correct tRNA 3′ ends are crucial for protein biosynthesis, generation of mature tRNA 3′ ends in eukaryotes is poorly understood and has so far only been investigated in vitro. We report here for the first time that eukaryotic tRNA 3′ end maturation is catalysed by the endonuclease RNase Z in vivo. Silencing of the JhI-1 gene (RNase Z homolog) in vivo with RNAi in Drosophila S2 cultured cells causes accumulation of nuclear and mitochondrial pre-tRNAs, suggesting that JhI-1 encodes both forms of the tRNA 3′ endonuclease RNase Z, and establishing its biological role in endonucleolytic tRNA 3′ end processing. In addition our data show that in vivo 5′ processing of nuclear and mitochondrial pre-tRNAs occurs before 3′ processing.