Nucleotide sequence and stability of the RNA component of RNase P from a temperature-sensitive mutant of E. coli.

The gene coding for the RNA component of RNase P was cloned from a temperature-sensitive mutant of Escherichia coli defective in RNase P activity (ts709) and its parental wild-type strain (4273), and the complete nucleotide sequences of the gene and its flanking regions were determined. The 5'- and 3'-terminal sequences of the RNA component were determined and mapped on the DNA sequence. The mutant gene has GC-to-AT substitutions at positions corresponding to 89 and 365 nucleotides downstream from the 5' terminus of the RNA sequence. Comparing to the wild-type RNA, the mutant RNA is less stable and rapidly degraded in vivo and in vitro.     

Mutations affecting two distinct functions of the RNA component of RNase P.

The effect of structural changes on the functions of the RNA component (M1 RNA) of ribonuclease P (RNase P) of Escherichia coli has been studied using the thermosensitive mutants of the rnpB gene. One of the mutants, ts709, has two G--A substitutions at positions 89 and 365 from the 5' end of M1 RNA. Of these substitutions, the one at position 89 from the 5' end is responsible for the phenotype of this mutant. Although the RNase P activity of ts709 is thermosensitive, the mutant M1 RNA has the same catalytic activity as the wild-type RNA. M1 RNA of another mutant, ts2418, has a G--A substitution at position 329. This mutant RNA has extremely low catalytic activity. The upstream mutational site of ts709 appears to play a role in the association with the protein subunit, whereas the mutational site of ts2418 is related to the catalytic function of M1 RNA.     

The transfer RNA processing defect in Escherichia coli strains BN and CAN is not due to a mutation in RNAase D or RNAase II

Escherichia coli strains BN and CAN are unable to support the growth of bacteriophage T4 psu₁⁺-amber double mutants. For strain BN, this phenotype has been attributed to a defect in 3′ processing of the precursor to psu₁⁺ tRNA^Ser. Since RNAase D and RNAase II are the only well-characterized 3′ exoribonucleases to be implicated in tRNA processing, the status of these activities and their genes in the mutant strains was investigated. Although extracts of strains BN and CAN were defective for hydrolysis of the artificial tRNA precursor, tRNA-C-U, these strains contained normal levels of RNAase D and RNAase II, and purified RNAase D or RNAase II could only partially complement the mutant extracts. Introduction of the wild-type RNAase D gene into strains BN and CAN did not correct the mutant phenotype. Likewise, strains defective in RNAase D and/or RNAase II plated T4psu₁⁺-amber phage normally. These results indicate that the tRNA processing defect in strains BN and CAN is not due to a mutation in either RNAase U or RNAase II. The possibility that the mutation in these strains affects another exoribonuclease or a factor influencing the activity and specificity of RNAase D or RNAase II is discussed.     

Phenotypic characterization of temperature-sensitive mutants of vaccinia virus with mutations in a 135,000-Mr subunit of the virion-associated DNA-dependent RNA polymerase

The phenotypic defects of three temperature-sensitive (ts) mutants of vaccinia virus, the ts mutations of which were mapped to the gene for one of the high-molecular-weight subunits of the virion-associated DNA-dependent RNA polymerase, were characterized. Because the virion RNA polymerase is required for the initiation of the viral replication cycle, it has been predicted that this type of mutant is defective in viral DNA replication and the synthesis of early viral proteins at the nonpermissive temperature. However, all three mutants synthesized both DNA and early proteins, and two of the three synthesized late proteins as well. RNA synthesis in vitro by permeabilized mutant virions was not more ts than that by the wild type. Furthermore, only one of three RNA polymerase activities that was partially purified from virions assembled at the permissive temperature displayed altered biochemical properties in vitro that could be correlated with its ts mutation: the ts13 activity had reduced specific activity, increased temperature sensitivity, and increased thermolability under a variety of preincubation conditions. Although the partially purified polymerase activity of a second mutant, ts72, was also more thermolabile than the wild-type activity, the thermolability was shown to be the result of a second mutation within the RNA polymerase gene. These results suggest that the defects in these mutants affect the assembly of newly synthesized polymerase subunits into active enzyme or the incorporation of RNA polymerase into maturing virions; once synthesized at the permissive temperature, the mutant polymerases are able to function in the initiation of subsequent rounds of infection at the nonpermissive temperature.     

Characterization in vitro of the defect in a temperature-sensitive mutant of the protein subunit of RNase P from Escherichia coli.

We have studied the assembly of Escherichia coli RNase P from its catalytic RNA subunit (M1 RNA) and its protein subunit (C5 protein). A mutant form of the protein subunit, C5A49, has been purified to apparent homogeneity from a strain of E. coli carrying a thermosensitive mutation in the rnpA gene. The heat inactivation kinetics of both wild-type and mutant holoenzymes are similar, an indication of equivalent thermal stability. However, when the catalytic efficiencies of the holoenzymes were compared, we found that the holoenzyme containing the mutant protein had a lower efficiency of cleavage than the wild-type holoenzyme at 33, 37, and 44 degrees C. We then explored the interaction of M1 RNA and C5 protein during the assembly of the holoenzyme. The yield of active holoenzyme obtained by reconstitution with wild-type M1 RNA and C5A49 protein in vitro can be considerably enhanced by the addition of excess M1 RNA, just as it can be in vivo. We concluded that the Arg-46----His-46 mutation in the C5A49 protein affects the ability of the protein to participate with M1 RNA in the normal assembly process of RNase P.     

Taurocyamine-utilizing Mutant of Pseudomonas aeruginosa with Altered Induction of 3-Guanidinopropionate Amidinohydrolase

A mutant, strain M-6, capable of utilizing taurocyamine (2-guanidinoethanesulfonate) as a nitrogen source was isolated from the parent strain, Pseudomonas aeruginosa GB-4, a derivative of wild-type P. aeruginosa PAO1 lacking the ability to produce guanidinobutyrase (EC 3.5.3.7). 3-Guanidinopropionate amidinohydrolase (EC class 3.5.3), which acts slowly on taurocyamine, was induced effectively by only 3-guanidinopropionate in the parent strain, while the enzyme of strain M-6 was induced by taurocyanime, guanidinoacetate, 3-guanidinopropionate, 4-guanidinobutyrate, and guanidinosuccinate. Strain M-6 synthesized a slight amount of the enzyme constitutively. The enzyme partially purified from strain M-6 exhibited substrate specificity similar to that of the wild-type strain. The mutant could grow also on 4-guanidinobutyrate, unlike the parent strain. These results indicate that strain M-6 acquired the ability to grow on taurocyamine by virtue of a mutation at the regulatory gene for 3-guanidinopropionate amidinohydrolase, which led to alteration of the specificity of the regulatory protein.     

Detection of long eukaryote-specific pyrimidine runs in repetitive DNA sequences and their relation to single-stranded regions in DNA isolated from sea urchin embryos.

Precursor molecules for Escherichia coli tRNAs that accumulated in a temperature-sensitive mutant defective in tRNA synthesis (TS709) were investigated. More than 20 precursors were purified by two-dimensional polyacrylamide gel electrophoresis. The purified molecules were analyzed by RNA fingerprint analysis and/or in vitro processing after treatment with E. coli cell-free extracts. The molecular sizes of most of the precursors identified were in the range of 4 to 5 S RNAs, although several larger ones were also detected. Fingerprint analysis revealed that the precursors generally differ from the corresponding mature tRNAs in the 5′ termini, having extra nucleotides. Thus, the genetic block in TS709 was shown to affect the trimming of the 5′ side of tRNA by impairing the function of RNAase P. Although this mutant had been isolated as a conditional mutant defective in the synthesis of su⁺ 3 tRNA₁^Tyr, the synthesis of many tRNA species was affected at high temperature. On the basis of their mode of maturation in vivo, the precursor molecules were discussed as intermediates in tRNA biosynthesis in E. coli. Accumulation of these intermediates was accounted for as a common feature of E. coli mutants defective in RNAase P function.     

Genetic detoxification of an aroA Salmonella enterica serovar Typhimurium vaccine strain does not compromise protection against virulent Salmonella and enhances the immune responses towards a protective malarial antigen

Live Salmonella vaccines are limited in use by the inherent toxicity of the lipopolysaccharide. The waaN gene encodes a myristyl transferase required for the secondary acylation of lipid A in lipopolysaccharide. A waaN mutant exhibits reduced induction of the inflammatory cytokines associated with lipopolysaccharide toxicity. Here the characteristics of a Salmonella enterica serovar Typhimurium aroA waaN mutant (SK100) in vitro and in vivo compared with its parent aroA strain (SL3261) were described. Phenotypic analysis of purified lipopolysaccharide obtained from SK100 confirmed that the physical and biological activities of the lipopolysaccharide had been altered. Nevertheless both strains had similar patterns of colonization and persistence in mice and significantly the aroA waaN mutant was equally as effective as the parent at protecting against challenge with wild-type S. Typhimurium. Furthermore, a SK100 strain was constructed expressing both tetanus toxin fragment C and the circumsporozoite protein of a malaria parasite. In marked contrast to its isogenic parent, the new attenuated strain induces significantly enhanced immune responses against the circumsporozoite protein. The waaN mutation enhances the ability of this strain to elicit immune responses towards guest antigens. This study provides important insights into the development of safe and effective multivalent Salmonella vaccines.     

Cloning and nucleotide sequence of the firA gene and the firA200(Ts) allele from Escherichia coli.

The Escherichia coli gene firA, previously reported to code for a small, histonelike DNA-binding protein, has been cloned and found to reside immediately downstream from skp, a gene previously identified as the firA locus. firA encodes a 36-kDa protein. The mutant firA200(Ts) allele was also cloned and shown to contain three mutations, each mutation giving rise to a single amino acid change. Partially purified wild-type FirA (from a firA+ strain) and mutant FirA [from a firA200(Ts) strain] proteins have amino-terminal sequences predicted from their common DNA sequences. Both proteins lack an N-terminal methionine. Modest overexpression of wild-type or mutant FirA restored wild-type growth to firA200(Ts) strains at 43 degrees C, whereas high-level expression of wild-type FirA was required for more complete suppression of the rifampin sensitivity of firA200(Ts) rpoB double mutants. High-level expression of mutant FirA did not suppress this rifampin sensitivity.     

Thermosensitive mutants of Escherichia coli K-12 altered in the catalytic Subunit and in a Regulatory factor of the glutamy-transfer ribonucleic acid synthetase.

The glutamyl-transfer ribonucleic acid synthetase (GluRS) of a partial revertants (ts plus or minus) of the thermosensitive (ts) mutant strain JP1449 (LOcus gltx) and of a ts mutant strain EM111-ts1 with a lesion in or near the locus gltx have been studied to find the relation between these two genetic loci known to influence the GluRS activity in vitro and the presence of a catalytic subunit and of a regulatory subunit in the GluRS purified from Escherichia coli K-12. The ts character of strain JP1449-18ts plus or minus is co-transduced with the marker dsdA at the same frequency as is the ts character of strain JP1449. Its purified GluRS is very thermolabile and its Km for glutamate is higher than that of a wild-type GluRS. These results indicate that the locus gltX is in the structural gene for the catalytic subunit of this enzyme. The location of the mutation causing the partial ts reversion in strain JP1449-18ts plus or minus is discussed. The GluRS purified from the ts mutant strain EM111-ts1 has the same stability as the wild-type enzyme, but its Km forglutamate increases with the temperature, suggesting that the locus gltE codes for a regulatory factor, possibly for the polypeptide chain that is co-purified with the catalytic subunit.     

Characterization of RNA synthesis in an Escherichia coli mutant with a temperature-sensitive lesion in stable RNA synthesis

Previous experiments with Escherichia coli strain 2S142 have shown that the synthesis of stable RNA is preferentially blocked at the restrictive temperature. In this paper, we have examined the capacity of this mutant strain to synthesize RNA in vitro. Growth of the strain for as short a period as 10 min at 42 degrees C resulted in a 40 to 60% loss of RNA synthetic capacity and a fourfold decrease in percent rRNA synthesized in toluenized cell preparations. The time course for the loss and recovery of this RNA synthetic capacity correlated very well with the changes in RNA synthesis observed in vivo. We found no difference in temperature sensitivity of the purified RNA polymerase from the mutant and the parental strains. Moreover, there was no detectable alteration in the amount of enzyme, specific activity of the enzyme, or electrophoretic mobility of the subunits when the mutant strain was grown at 42 degrees C. The capacity for rRNA synthesis was also measured with the Zubay in vitro system (Reiness et al., Proc. Natl. Acad. Sci. 72:2881-2885, 1975). Supernatant fractions (S-30) prepared from cells grown at 30 degrees C were capable of up to 31.2% rRNA synthesis, using phi 80d3 DNA as template. S-30 fractions from cells grown at 42 degrees C synthesized 8.6% rRNA. The bottom one-third of the S-100 fraction and the ribosomal salt wash from 30 degrees C cells contained one or more factors which partially restored preferential rRNA synthesis in S-30 fractions from cells grown at 42 degrees C. Preliminary evidence suggests that the factor(s) is protein in nature.     

Reconstitution of RNAase P activity using inactive subunits from E. coli and HeLa cells

HeLa cell RNAase P activity found in the flow-through of anti-Sm affinity columns can be separated into inactive RNA and protein components. These components can be used to reconstitute active hybrid enzyme complexes with purified subunits from E. coli RNAase P. The RNA in the HeLa cell fractions employed is enriched for species between 85 and 115 nucleotides long. This reconstitution assay is a convenient means of purifying the functional RNA and protein of HeLa cell RNAase P. Probes derived from the genes for the subunits of E. coli RNAase P hybridize to genomic DNA of gram-negative prokaryotic organisms, but no positive signals are seen with genomic DNA from a variety of eukaryotic organisms.     

Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: biochemical and genetic characterization.

Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.     

Genetic and molecular analysis of the temperature-sensitive mutant un-17 carrying a mutation in the gene encoding poly(A)-polymerase in Neurospora crassa

The un-17 mutant was originally isolated as an irreparable temperature-sensitive (ts) mutant in Neurospora crassa. Early experiments showed that cells of this mutant immediately stopped growing and died when the temperature of the culture was shifted from a permissive temperature (25 degrees C) to non-permissive temperature (35 degrees C). This ts phenotype is suppressed by addition of cycloheximide or in some conditions of growth repression. Even at the permissive temperature, it shows a female sterile phenotype and is deficient in production of exocellular superoxide dismutase SOD4 (EC 1.15.1.1). By searching for a DNA fragment that complements the ts phenotype of the un-17 mutant from a N. crassa genome library, we found the un-17 gene. The cloned un-17 gene encodes a homolog of the Saccharomyces cerevisiae poly(A) polymerase (PAP). The un-17 mutant had a one-base substitution mutation in the gene. The cloned un-17 genes from the wild-type strain and the un-17 mutant were introduced into both the un-17 mutant and wild-type strain. The un-17 mutant introduced by un-17 DNA from the wild-type strain showed recovery of both the ts and female sterile phenotypes. Moreover, the purified product derived from the wild-type strain showed PAP activity in vitro. These findings indicate that the un-17 mutant carries a ts mutation in the gene encoding PAP.