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M J Fournier

Publications and source records attributed to M J Fournier.

At least 37 records · Page 2Linked to original sources
PubMed1996

Expression and purification of a spider silk protein: a new strategy for producing repetitive proteins.

Synthetic genes were constructed based on the known sequence of the spider dragline silk protein MaSp 2. The genes had 8, 16, or 32 contiguous units of the consensus repeat sequence of the protein. These artificial genes were constructed using a strategy involving compatible but nonregenerable restriction sites, which allowed construction of very large inserts in a precisely controlled manner. This strategy should have general utility in the controlled construction of repetitive proteins composed of identical or different repeat units. The protein from the 16-unit repeat was produced in Escherichia coli at levels up to 10 mg/g wet wt of cells although yields of 1-2 mg/g were more typical. The protein was easily purified with high recovery using an affinity column. The purified protein had the predicted amino acid composition and N-terminal sequence after cleavage of a leader sequence. The methodology described will allow production of sufficient quantities of protein for basic structure/function studies including production of synthetic fibers.

Amino Acid Sequence↗
PubMed1996

Differential clearance of glycoforms of IgG in normal and autoimmune-prone mice.

In order to understand better the origins of the elevated levels of the glycoform of IgG that lacks galactose on both arms of the oligosaccharide chain (G0%) located in the Fc, which occurs in man and mouse with age, and in particular in autoimmune disease, we investigated the clearance of two glycosylated forms of IgG2a and IgG1 in normal (BALB/c) and autoimmune-prone (MRL/1pr, MRL/+, and non-obese diabetic (NOD)) mice. To investigate the possibility of different rates of catabolism, enzymatically generated glycoforms of monomeric IgG1 and IgG2a (fully glycosylated or G0%), were iodinated and injected into the tail vein of the mice. We found that the G0% IgG2a remained in circulation significantly longer than the fully glycosylated variants, in all of the mouse strains tested. In contrast, the two forms of IgG1 had similar kinetics in all the autoimmune-prone mice, whereas in BALB/c, there was a longer half-life (t1/2) for G0% IgG1. These data suggest that there may be differences in the ability of the IgG glycoforms to bind to the Fc gamma receptors, in particular Fc gamma RI. The clearance rates were found to vary among the strains studied, with MRL/1pr having the fastest catabolic rates for all glycoforms and IgG subclasses tested. This appeared to be due to the presence of circulating IgG and IgM rheumatoid factors (RF). There were significantly increased frequencies and titres for both IgM and IgG RF in MRL/1pr mice compared with the other strains. In contrast, interferon-gamma, known to induce the Fc gamma RI, was found to be similar in the sera, in all of the strains of mice examined. These results suggest that RF probably play an important biological function in the MRL/1pr mice and aid in the clearance of circulating IgG. Our study shows that the state of glycosylation of IgG affects the t1/2 in vivo, and that by removing the terminal sugars (sialic acid and galactose), the antibody (IgG2a) will remain in circulation significantly longer. These observations may thus provide a partial explanation for the increase in relative percentage of this glycoform that occurs with age.

Animals↗
PubMed1995

U14 base-pairs with 18S rRNA: a novel snoRNA interaction required for rRNA processing.

U14 is a conserved small nucleolar RNA (snoRNA) required for processing of yeast 18S rRNA. The presence of two long sequences (13 and 14 nucleotides) with strong complementarity to 18S rRNA suggests that U14 base-pairs with pre-rRNA. Evidence of direct binding was developed by showing that mutations in these U14 elements mimic U14 depletion and that function can be rescued by a compensatory sequence change in 18S RNA. The U14 elements are functionally interdependent, indicating that both participate in binding. Folding models predict that binding might occur through both rRNA elements simultaneously. Potential roles of U14 in rRNA folding, maturation, and ribosome assembly are discussed. U14 is one of several snoRNAs with long complementarities to rRNA and the first snoRNA in this class shown to interact directly with rRNA.

Anti-Bacterial Agents↗
PubMed1995

Characterization of three new snRNAs from Saccharomyces cerevisiae: snR34, snR35 and snR36.

Genes for three novel snRNAs of Saccharomyces cerevisiae have been isolated, sequenced and tested for essentiality. The RNAs encoded by these genes are designated snR34, snR35 and snR36 respectively and contain 203, 204 and 182 nucleotides. Each RNA is derived from a single copy gene and all three RNAs are believed to be nucleolar, i.e. snoRNAs, based on extraction properties and association with fibrillarin. SnR34 and snR35 contain a trimethylguanosine cap, but this feature is absent from snR36. The novel RNAs lack elements conserved among several other snoRNAs, including box C, box D and long sequence complementarities with rRNA. Genetic disruption analyses showed each of the RNAs to be dispensable and a haploid strain lacking all three RNAs and a previously characterized fourth snoRNA (snR33) is also viable. No differences in the levels of precursors or mature rRNAs were apparent in the four gene knock-out strain. Possible roles for the new RNAs in ribosome biogenesis are discussed.

Alleles↗
PubMed1995

Rheumatoid factor avidity in patients with rheumatoid arthritis: identification of pathogenic RFs which correlate with disease parameters and with the gal(0) glycoform of IgG.

The standard ELISA for measuring rheumatoid factor (RF) binding was modified by treatment after the RF-Fc interaction with 2 M guanidine, which allowed a measurement of the avidity of the interaction. Incubation with 4 M guanidine eliminated RF binding. There was a direct correlation (r = 0.99) between the avidity as measured by the modified guanidine ELISA, and the dissociation constant for monoclonal RFs, as measured by competitive ELISA. Of the seropositive rheumatoid arthritis (RA) patients tested, 47% had high-avidity RFs (> or = 8% RF binding remaining after guanidine treatment). Tender joint count scores were significantly higher in the high avidity group (p = 0.05), whereas there was no significant difference in the ages, disease duration, sedimentation rate, RF titer or serum Ig levels compared to those with low-avidity RFs. Additionally 58% of those with high-avidity RFs had subcutaneous nodules, compared to 40% of the low-avidity group. A significantly higher number of nodules was present in the high-avidity RF group compared to those with low-avidity RFs (p = 0.03). Interestingly, the RF avidity was significantly higher in isolated immune complexes (IC), compared to that in circulating IgM RFs (p = 0.01). The RF avidity correlated with the presence of the glycoform of IgG lacking galactose in both circulating and IC-derived IgG (p = 0.003 and 0.009 respectively). Information about the strength of binding to Fc identifies a subgroup of IgM RFs that are likely pathological in patients with RA, as well as a specific glycoform of the target antigen.

Antibodies↗
PubMed1995

Antisense snoRNAs: a family of nucleolar RNAs with long complementarities to rRNA.

A growing subset of small nucleolar RNAs (snoRNAs) contains long stretches of sequence complementarity to conserved sequences in mature ribosomal RNA (rRNA). This article reviews current knowledge about these complementarities and proposes that these antisense snoRNAs might function in pre-rRNA folding, base modification and ribosomal ribonucleoprotein assembly, in some cases acting as RNA chaperones.

Animals↗
PubMed1995

The small nucleolar RNAs.

The present review summarizes key progress made in characterizing the small nucleolar RNAs (snoRNAs) of eukaryotic cells. Recent studies have shown snoRNA populations to be substantially more complex than anticipated initially. Many newly discovered snoRNAs are synthesized by an intron-processing pathway, which provides a potential mechanism for coordinating nuclear RNA synthesis. Several snoRNAs and snoRNP proteins are known to be needed for processing of ribosomal RNA, but precise functions remain to be defined. In principle, snoRNAs could have several roles in ribosome synthesis including: folding of pre-rRNA, formation of rRNP substrates, catalyzing RNA cleavages, base modification, assembly of pre-ribosomal subunits, and export of product rRNP particles.

Animals↗
PubMed1994

Chemical sequence control of beta-sheet assembly in macromolecular crystals of periodic polypeptides.

A family of uniform periodic polypeptides has been prepared by bacterial expression of the corresponding artificial genes, with the objective of exploring the potential for control of supramolecular organization in genetically engineered protein-based polymeric materials. The repeating units of the polypeptides consist of oligomeric alanyl-glycine sequences interspersed with glutamic acid residues inserted at intervals of 8 to 14 amino acids. Crystallization of such materials from formic acid produces beta-sheet structures in the solid state, as shown by vibrational spectroscopy, nuclear magnetic resonance spectroscopy, and wide-angle x-ray diffraction. The diffraction results, together with observations from electron microscopy, are consistent with the formation of needle-shaped lamellar crystals whose thickness is controlled by the periodicity of the primary sequence. These results can be used to control solid-state structure in macromolecular materials.

Amino Acid Sequence↗
PubMed1994

Saccharomyces cerevisiae U14 small nuclear RNA has little secondary structure and appears to be produced by post-transcriptional processing.

Yeast U14 small nuclear (sn) RNA is required for normal processing of rRNA. The sequence and folding properties of U14 were analyzed in the present study, with the aim of defining the structures of natural U14 subspecies and characterizing the folding properties of free U14 RNA. Natural U14 was determined to consist of four subspecies of 125-128 nucleotides, none containing a 5'-cap structure. Length heterogeneity occurs at both ends and is presumed to reflect post-transcriptional processing of U14 precursors. Results from nuclease and chemical probing revealed that U14 has surprisingly little secondary structure overall. Three essential sequence elements conserved among all U14 RNAs occur in regions that are largely single-stranded, i.e. box C, box D, and a 13-nucleotide segment complementary to 18 S rRNA; a non-essential 14-nucleotide sequence complementary to 18 S rRNA is also unpaired. Two non-conserved segments required for activity are part of a stably folded 32-base domain that is unique to yeast U14. Finally, a 5'-, 3'-stem shown earlier to be required for U14 accumulation appears to exist only in precursors to U14 and not in protein-free mature RNA. The implications of these results are discussed in terms of U14 synthesis and function.

Base Sequence↗
PubMed1993

SnR31, snR32, and snR33: three novel, non-essential snRNAs from Saccharomyces cerevisiae.

Genes for three novel yeast snRNAs have been identified and tested for essentiality. Partial sequence information was developed for RNA extracted from isolated nuclei and the respective gene sequences were discovered by screening a DNA sequence database. The three RNAs contain 222, 188 and 183 nucleotides and are designated snR31, snR32 and snR33, respectively. Each RNA is derived from a single copy gene. The SNR31 gene is adjacent to a gene for an unnamed protein associated with the cap-binding protein eIF-4E. The SNR32 gene is next to a gene for ribosomal protein L41 and the gene for SNR33 is on chromosome III, between two open reading frames with no known function. Genetic disruption analyses showed that none of the three snRNAs is required for growth. The new RNAs bring the number of non-spliceosomal snRNAs characterized thus far in S. cerevisiae to 14, of which only three are essential.

Base Sequence↗
PubMed1993

The nucleolar snRNAs: catching up with the spliceosomal snRNAs.

Despite their early discovery, research into the small RNAs associated with the eukaryotic nucleolus (snoRNAs) has lagged behind that of their cousins, the small nuclear RNAs which are known to function in mRNA splicing (spliceosomal snRNAs). Recent progress has now shown that the snoRNAs also occupy a vital niche in the RNA world, participating in the processing of ribosomal RNA. Like the spliceosomal snRNAs, the snoRNAs exist as ribonucleoprotein (RNP) particles which appear to assemble into a large multi-RNA RNP complex for pre-rRNA maturation.

Animals↗
PubMed1992

U14 function in Saccharomyces cerevisiae can be provided by large deletion variants of yeast U14 and hybrid mouse-yeast U14 RNAs.

The functional equivalency of yeast and mouse U14 RNAs was examined in Saccharomyces cerevisiae. The test RNAs included mouse U14 and several yeast-mouse bi- and tri-partite hybrid RNAs, all transcribed from yeast U14 gene signals. The ability of the heterologous RNAs to provide essential U14 function was assessed in a test strain containing a single glucose-repressible wild-type U14 gene. Mouse U14 was not functional in yeast. However, wild-type growth was supported by hybrid RNAs that included universal sequence elements from either source, two yeast-specific segments and a 5',3' terminal stem domain. The universal sequences include box C, box D and a sequence complementary to 18S rRNA, all shown previously to be required for function of yeast U14. Deletion and substitution mapping defined the yeast-specific elements and showed that a major portion of neighboring non-conserved RNA is dispensible. The results are discussed with a view to defining a minimal consensus U14 molecule.

Animals↗
PubMed1992

Accumulation of U14 small nuclear RNA in Saccharomyces cerevisiae requires box C, box D, and a 5', 3' terminal stem.

U14 is one of several nucleolar small nuclear RNAs required for normal processing of rRNA. Functional mapping of U14 from Saccharomyces cerevisiae has yielded a number of mutants defective in U14 accumulation or function. In this study, we have further defined three structural elements required for U14 accumulation. The essential elements include the U14-conserved box C and box D sequences and a 5', 3' terminal stem. The box elements are coconserved among several nucleolar small nuclear RNAs and have been implicated in binding of the protein fibrillarin. New mutational results show that the first GA bases of the box C sequence UGAUGA are essential, and two vital bases in box D have also been identified. An intragenic suppressor of a lethal box C mutant has been isolated and shown to contain a new box C-like PyGAUG sequence two bases upstream of normal box C. The importance of the terminal stem was confirmed from new compensatory base changes and the finding that accumulation defects in the box elements can be complemented by extending the terminal stem. The results suggest that the observed defects in accumulation reflect U14 instability and that protein binding to one or more of these elements is required for metabolic stability.

Base Composition↗
PubMed1991

Japanese encephalitis virus-vaccinia recombinants produce particulate forms of the structural membrane proteins and induce high levels of protection against lethal JEV infection.

Four recombinant vaccinia viruses were engineered for expression of different portions of the Japanese encephalitis virus (JEV) open reading frame. All four recombinant vaccinias contained the NS1 and NS2A genes, and each of these viruses specified the synthesis, glycosylation, and secretion of the nonstructural glycoprotein (NS1). All four recombinants also contained the E gene, and each virus correctly directed the synthesis and glycosylation of the envelope glycoprotein (E). Interestingly, two of these viruses (vP555 and vP650), which expressed the prM gene in addition to E and NS1, produced an extracellular hemagglutinin containing M and E that migrated in sucrose gradients similarly to the slowly-sedimenting hemagglutinin found in the culture fluid of JEV-infected cells. Immunization of 3-week-old mice with the recombinant viruses vP555 and vP658 resulted in immune responses to NS1, whereas only the virus that directed the synthesis of extracellular forms of E (vP555) induced an immune response to E. Both viruses provided protection against lethal challenge with JEV. Animals given two inoculations with vP555 were fully protected from greater than 10,000 LD50 of JEV. This high level of protection was correlated with the production of high titers of neutralizing and hemagglutination-inhibiting antibodies.

Animals↗
PubMed1990

Identification of essential elements in U14 RNA of Saccharomyces cerevisiae.

The U14 RNA of Saccharomyces cerevisiae is a small nucleolar RNA (snoRNA) required for normal production of 18S rRNA. Depletion of U14 results in impaired processing of pre-rRNA, deficiency in 18S-containing intermediates and marked under-accumulation of mature 18S RNA. The present report describes results of functional mapping of U14, by a variety of mutagenic approaches. Special attention was directed at assessing the importance of sequence elements conserved between yeast and mouse U14 as well as other snoRNA species. Functionality was assessed in a test strain containing a galactose dependent U14 gene. The results show portions of three U14 conserved regions to be required for U14 accumulation or function. These regions include bases in: (i) the 5'-proximal box C region, (ii) the 3'-distal box D region, and (iii) a 13 base domain complementary to 18S rRNA. Point and multi-base substitution mutations in the snoRNA conserved box C and box D regions prevent U14 accumulation. Mutations in the essential 18S related domain do not effect U14 levels, but do disrupt synthesis of 18S RNA, indicating that this region is required for function. Taken together, the results suggest that the box C and box D regions influence U14 expression or stability and that U14 function might involve direct interaction with 18S RNA.

Animals↗
PubMed1990

The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coli.

The antigenic structures of the envelope protein, E, and the non-structural protein, NS1, of dengue type 1 virus (DEN1) have been studied in the form of recombinant fusion proteins expressed in Escherichia coli. Deletion analysis was used to identify two distinct antigenic domains in E that reacted with subsets of antiviral monoclonal antibodies (MAbs). Domain I of E extends from amino acid residues (aa) 76 to 93 of E; domain II extends from aa 293 to 402 and contains an essential disulphide bridge. MAbs also reacted with several determinants clustered near the N terminus of the NS1 protein (aa 57 to 126). Recombinant fusion proteins containing E. coli trpE sequences and most of the sequences for either E or NS1 were immunogenic in mice. The antibodies elicited by the E fusion protein reacted with a portion of the protein containing domain II, whereas antibodies elicited by the NS1 fusion protein did not react with the antigenic determinants defined by our MAbs.

Animals↗