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M J Fournier

Publications and source records attributed to M J Fournier.

At least 55 records · Page 3Linked to original sources
PubMed1990

Loss of 4.5S RNA induces the heat shock response and lambda prophage in Escherichia coli.

During depletion of 4.5S RNA, cells of Escherichia coli displayed a heat shock response that was simultaneous with the first detectable effect on ribosome function and before major effects on cell growth. Either 4.5S RNA is involved directly in regulating the heat shock response, or this particular impairment of protein synthesis uniquely induces the heat shock response. Several hours later, lambda prophage was induced and the cells lysed.

Bacteriophage lambda↗
PubMed1990

Escherichia coli 4.5S RNA gene function can be complemented by heterologous bacterial RNA genes.

The essential 4.5S RNA gene of Escherichia coli can be complemented by 4.5S RNA-like genes from three other eubacteria, including both gram-positive and gram-negative organisms. Two of the genes encode RNAs similar in size to the E. coli species; the third, from Bacillus subtilis, specifies an RNA more than twice as large. The heterologous genes are expressed efficiently in E. coli, and the product RNAs resemble those produced by cognate cells. We conclude that the heterologous RNAs can replace E. coli 4.5S RNA and that the essential function of 4.5S RNA is evolutionarily conserved. A consensus structure is presented for the functionally related 4.5S RNA homologs.

Base Sequence↗
PubMed1990

Depletion of U14 small nuclear RNA (snR128) disrupts production of 18S rRNA in Saccharomyces cerevisiae.

Repression of an essential nucleolar small nuclear RNA (snRNA) gene of Saccharomyces cerevisiae was shown to result in impaired production of 18S rRNA. The effect, observed for an snRNA species of 128 nucleotides (snR128), was evident within one generation after the onset of SNR128 gene repression and correlated well with depletion of the snRNA. The steady-state mass ratio of 18S RNA to 25S RNA decreased eightfold over the course of the analysis. Results from pulse-chase assays revealed the basis of the imbalance to be underaccumulation of 18S RNA and its 20S precursor. This effect appears to result from impairment of processing of the 35S rRNA transcript at sites that define the 20S species coupled with rapid turnover of unstable intermediates. Possible bases for the effects observed are discussed. A common U14 designation is proposed for the structurally related yeast snRNA and 4.5S hybRNAs from amphibians and mammals.

Base Sequence↗
PubMed1989

Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoprotein.

Expression of antigenic fragments of the Japanese encephalitis virus envelope protein (E) in Escherichia coli has been used to define the boundaries of an antigenic domain that contains the binding sites for 10 anti-E monoclonal antibodies (MAbs). All of these antibodies neutralized the virus in vitro and some of them passively protected mice from a fatal virus challenge. We have shown previously that nine of these antibodies react with the antigenic determinants encoded by a 405 bp fragment of viral cDNA. To determine the amino acid sequences of specific determinants, truncated polypeptides were expressed as fusion proteins in E. coli following progressive Bal 31 exonuclease digestion of the 5' and 3' ends of the cDNA fragment. Examination of the immunoreactivity of these polypeptides revealed that the region from methionine 303 to tryptophan 396 was the shortest sequence capable of reacting with any of the 10 MAbs or with a polyclonal, antiviral hyperimmune mouse ascitic fluid. Biochemical tests showed that an intramolecular disulphide cross-linkage between cysteine 304 and cysteine 335 of the E protein sequence was required for presentation of the binding site(s) for these MAbs. Although this 95 amino acid antigenic domain appeared to be capable of forming several conformational neutralizing epitopes, it was not an effective immunogen for inducing neutralizing or protective antibodies in mice.

Amino Acid Sequence↗
PubMed1988

Sequence and genetic analysis of a dispensible 189 nucleotide snRNA from Saccharomyces cerevisiae.

The structure of a Saccharomyces cerevisiae gene that encodes a small nuclear RNA (snRNA) of 189 nucleotides is described. This gene, designated SNR189, is located 400 base pairs upstream of the CRY1 gene on yeast chromosome III. Gene replacement analysis revealed the SNR189 gene to be dispensable for growth under a variety of culture conditions. The snR189 sequence lacks homology with other sequenced yeast or metazoan snRNAs.

Base Sequence↗
PubMed1988

Characterization of an SNR gene locus in Saccharomyces cerevisiae that specifies both dispensible and essential small nuclear RNAs.

A genetic locus is described that specifies two Saccharomyces cerevisiae small nuclear RNAs (snRNAs). The genes specifying the two snRNAs are separated by only 67 base pairs and are transcribed in the same direction. The product RNAs contain 128 and 190 nucleotides and are designated snR128 and snR190, respectively. These RNAs resemble snRNAs of other eucaryotes in nuclear localization and possession of a 5' trimethylguanosine cap. Neither snRNA is related in sequence to previously described vertebrate or yeast snRNAs. Both RNAs exhibit properties consistent with nucleolar organization and hydrogen bonding to pre-rRNA species, suggesting possible roles in ribosome biogenesis. The snR128 species cosediments with deproteinized 27S pre-rRNA, whereas snR190 is associated with a 20S intermediate. Gene disruption in vitro followed by replacement of the chromosomal alleles reveals that SNR128 is essential, whereas SNR190 is not.

Base Sequence↗
PubMed1987

Sequence of the dengue-1 virus genome in the region encoding the three structural proteins and the major nonstructural protein NS1.

Sequence results are presented for a 3745-nucleotide region at the 5' end of the dengue type-1 virus (DEN-1) genome. The strain characterized is a Western Pacific isolate from Nauru Island. The sequenced region contains the beginning of a continuous open reading frame which specifies the capsid (C), membrane (M), and envelope (E) structural proteins and the nonstructural protein NS1. The sequences are compared with corresponding segments for seven other flaviviruses, including two of the three remaining dengue serotypes, DEN-2 and DEN-4. The results show the DEN-1 genome size and organization to be similar to those of other characterized flaviviruses and that major features of the individual proteins are conserved. It is of special interest that comparisons of the E glycoprotein sequences between the dengue serotypes (DEN-1, -2, -4) reveal only moderately greater sequence relatedness (63-68%) than occurs in comparisons of DEN-1 with five other flaviviruses (46-54%). For the other structural proteins, C and M, the relatedness values are 59-74% for comparisons between DEN-1 and the other dengue serotypes and 31-45% for comparisons between DEN-1 and the five other flaviviruses.

Base Sequence↗
PubMed1987

Partial nucleotide sequence of the Japanese encephalitis virus genome.

Approximately 10 kb of the estimated 10.9-kb genome of the Japanese encephalitis virus (JE; Nakayama strain) has been cloned as cDNA; the uncloned portion includes 430 bases at the 5'-terminus and 450 bases at the 3'-end. A map of the genome has been developed through nucleotide sequencing and in vivo expression with the Escherichia coli expression vector lambda gt11 and immunological identification. Sequence results for 4320 nucleotides suggest the JE genome organization is very similar to those of three other flaviviruses for which sequence information is available. Like the other flaviviruses, the JE proteins are encoded by a single open reading frame that continues uninterrupted throughout the region sequenced. Considerable homology exists between the JE RNA and protein sequences and those of the other characterized flaviviruses. Comparative nucleotide and (amino acid) homology values for the M-E-NS1-ns2 segment of JE are approximately MVE, 70% (80%), WN, 68% (76%), and YF, 50% (45%). Even greater homology is suggested when the protein hydrophobicity profiles are compared. The molecular relationships are consistent with the established serological relationships among JE, MVE, and WN viruses and argue that these flaviviruses may have been derived from a common evolutionary ancestor.

Amino Acid Sequence↗
PubMed1987

Expression of Japanese encephalitis virus antigens in Escherichia coli.

The expression of Japanese encephalitis virus (JE) cDNA in Escherichia coli has been used to study the functional organization of the viral genome. JE protein coding sequences were expressed in E. coli by subcloning random fragments of cloned cDNA (P.C. McAda, P.W. Mason, C.S. Schmaljohn, J.M. Dalrymple, T.L. Mason, and M.J. Fournier, 1987, Virology 158, 348-360) into the bacteriophage lambda gt11 expression vector. Over 120 lambda gt11 recombinants expressing viral protein sequences as beta-galactosidase fusion proteins were identified immunologically with monoclonal antibodies (MAbs) and polyclonal hyperimmune mouse ascites fluid (HMAF). This expression and immunological detection strategy has been used to (1) map viral protein coding sequences to the JE genome; (2) demonstrate that contiguous viral protein coding regions can be expressed as single polypeptides in E. coli, providing functional confirmation for a long viral open reading frame; (3) localize important antigenic domains within the envelope protein E; and (4) identify in JE-infected cells a form of the glycosylated nonstructural protein NS1 that contains a hydrophobic C-terminal extension encoded by portions of the "ns2a" region of the JE genome.

Antigens, Viral↗
PubMed1986

Escherichia coli B/r leuK mutant lacking pseudouridine synthase I activity.

Escherichia coli B/r strain EB146 containing mutation leuK16 has elevated levels of enzymes involved in the synthesis of leucine, valine, isoleucine, histidine, and tryptophan (Brown et al., J. Bacteriol. 135:542-550, 1978). We show here that strain EB146 (leuK16) has properties that are similar to those of E. coli and Salmonella typhimurium hisT strains. In tRNA1Leu from both hisT and leuK strains, positions 39 and 41 are uridine residues rather than pseudouridine residues. Furthermore, in tRNA3Leu and tRNA4Leu from a leuK strain, uridine residues at positions 39 and 40, respectively, are unmodified. Pseudouridine synthase I activity is missing in extracts of strain EB146 (leuK16), and extracts of strain EB146 (leuK16) and of a hisT strain do not complement one another in vitro. Four phenotypes of strain EB146 (leuK16), leucine excretion, wrinkled colony morphology, and elevated levels of leu and his enzymes, are complemented by a plasmid having a 1.65-kilobase DNA fragment containing the E. coli K-12 hisT locus. These results indicate that either leuK codes for pseudouridine synthase I (and is thus a hisT locus in reality) or, less likely, it codes for a product that affects the synthesis or activity of pseudouridine synthase I.

Amino Acids↗
PubMed1985

Escherichia coli 6S RNA is not essential for growth or protein secretion.

The function of the stable 6S RNA of Escherichia coli is not known. Recently, it was proposed that the 6S RNA is a component of a bacterial signal recognition particle required for protein secretion. To test this proposal, we isolated a mutant that lacks the 6S RNA. Studies of the mutant show that the 6S RNA is not essential for growth or for protein secretion. The gene for the 6S RNA (ssr) maps near serA at 63 min on the E. coli genetic map.

Bacterial Proteins↗
PubMed1985

Escherichia coli 6S RNA gene is part of a dual-function transcription unit.

The gene coding for the metabolically stable 6S RNA of Escherichia coli has been cloned, sequenced, and partially characterized in expression analyses. The DNA sequence results confirm the accuracy of the previously established RNA sequence and, with genomic hybridization data, reveal that there is only one copy of the 6S DNA in the chromosome. Consistent with its relaxed mode of expression, the promoter region of the 6S RNA gene was found to lack the hypothetical GC-rich discriminator domain common to other stable RNA genes under stringent control. The sequence results also revealed the occurrence of a 540-base-pair open reading frame immediately downstream from the 6S RNA coding region. Results from the expression analyses show that the protein and RNA coding regions are cotranscribed in vitro and that the open reading frame is translated in vivo.

Base Sequence↗
PubMed1984

Cloning and sequence analysis of the Escherichia coli 4.5 S RNA gene.

The structure of the Escherichia coli gene coding for the metabolically stable 4.5 S RNA has been determined by cloning and DNA sequence analysis. Results from Southern hybridization assays carried out prior to cloning show the 4.5 S DNA to be limited to a single locus in the E. coli K12 genome. A 5.4 X 10(3) base DNA fragment containing the 4.5 S DNA was cloned into plasmid pBR322 for restriction, hybridization and sequence analyses. Cells harboring the cloned gene overproduce the 4.5 S RNA by 15-fold under normal culturing conditions; however, no effect on growth rate is observed. DNA sequencing revealed only one copy of the 4.5 S RNA gene, with a deduced RNA sequence both longer at 114 bases and slightly different from the RNA sequence reported earlier. A promoter structure immediately preceding the structural gene shows good agreement with the prokaryotic consensus sequence at both the -35 and -10 regions. In addition, a G + C-rich sequence between the Pribnow box and the start of transcription agrees well with an apparent consensus sequence found for other stable RNA genes also under stringent control. No clearly recognizable termination signal was found immediately downstream from the 3' terminus of the 4.5 S DNA, although structural elements with that potential appear to occur. A potential coding sequence for a protein occurs about 100 bases downstream from the 4.5 S DNA, suggesting the possibility of a dual function 4.5 S RNA-mRNA transcript.

Base Sequence↗
PubMed1984

The 4.5 S RNA gene of Escherichia coli is essential for cell growth.

The Escherichia coli gene coding for the metabolically stable 4.5 S RNA (ffs) has been shown to be required for cell viability. Essentiality was demonstrated by examining the recombination behavior of substitution mutations of ffs generated in vitro. Substitution mutants of ffs are able to replace the chromosomal allele only in the presence of a second, intact copy of ffs. Independent evidence of essentiality and the finding that 4.5 S RNA is important for protein synthetic activity came from characterization of cells dependent on the lac operon inducer isopropyl-beta-D-thiogalactoside for ffs gene expression. Here, a strain dependent on isopropyl-beta-D-thiogalactoside for 4.5 S RNA synthesis was developed by inactivation of the chromosomal ffs allele and lysogenization by a lambda phage containing 4.5 S DNA fused to a hybrid trp-lac promoter. Withdrawal of the thiogalactoside leads to a deficiency in 4.5 S RNA, a dramatic loss in protein synthesis activity, and eventual cell death. Tagging of the chromosomal ffs region with a kanamycin-resistance gene allowed mapping of the 4.5 S RNA gene. Results from this analysis place ffs near lon at approximately ten minutes on the E. coli linkage map.

Bacterial Proteins↗
PubMed1984

Physical properties of the E. coli 4.5S RNA: first results suggest a hairpin helix of unusual thermal stability.

Hyperchromicity measurements and quasi-elastic laser light scattering (QELS) have been used to assess the solution structure of the metabolically stable E. coli 4.5S RNA. Results from thermal denaturation measurements revealed the 4.5S species to be markedly more stable than most other RNAs characterized thus far. Optical Tm's range from 79 degrees to 88 degrees with transitions approximately 25 degrees C wide. The Tm values show little dependence on ionic strength, but stability is enhanced considerably by Mg+2. In the QELS experiments the diffusion coefficient does not decrease until T greater than 70 degrees C. Neither the diffusive melting nor the diffusion coefficient at infinite dilution (D0(20,w)) show dependencies on ionic strength but both are influenced by Mg+2. The diffusion behavior is in agreement with that predicted for a rigid cylindrical molecule 125 to 160 A long and 37 to 26 A in diameter. Taken together these results are consistent with the more stable hypothetical secondary structures that can be formed, in which 70-75% of the 114 bases are paired to form a single extended hairpin helix.

Base Sequence↗
PubMed1984

Structure of an Escherichia coli tRNA operon containing linked genes for arginine, histidine, leucine, and proline tRNAs.

A plasmid containing a gene for the most abundant Escherichia coli leucine isoacceptor tRNA, tRNALeu1 (anticodon CAG) was isolated from the Clarke-Carbon bank of cloned E. coli DNA. The clone contains a 12.3-kilobase DNA insert which was mapped by F' DNA hybridization analysis to the region 82 to 89 min on the chromosome. The cloned tDNALeu corresponds to the minor of two chromosomal regions containing different amounts of DNA complementary to tRNALeuCAG . Sequencing of the tDNA region revealed it to contain a multimeric transcription unit consisting of four different tRNA genes. The genes are in the arrangement 5'-leader- tRNAArgCCG -57 base pairs- tRNAHisGUG -20 base pairs- tRNALeuCAG -42 base pairs- tRNAProUGG -3'. Coordinate expression of the component tRNAs in vivo and the absence of intercistronic promoters indicated that all four tDNAs reside in the same operon. The tDNA sequence is bounded by a promoter element showing good agreement with the procaryotic consensus sequence and a GC-rich stem-loop element that corresponds to a rho-independent terminator. The promoter region contains a GC-rich sequence that agrees with a suggested consensus stringency control element and two domains possessing dyad symmetry which flank the Pribnow box and include the putative stringency control region.

Base Sequence↗