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Biomedical subjects

R Brousseau

Publications and source records attributed to R Brousseau.

At least 73 records · Page 4Linked to original sources
PubMed1989

Human metallothionein-II is synthesized as a stable membrane-localized fusion protein in Escherichia coli.

A synthetic gene encoding human metallothionein-II (HMT) was cloned into the specially constructed high-copy-number expression vector, pUA7, and expressed in Escherichia coli. The plasmid construct includes the promoter/operator and regulatory sequences of the Salmonella typhimurium ara operon and part of the 5'-coding and all of the 3'-noncoding regions of the E. coli lpp. Upon induction with arabinose, the resulting Lpp::HMT fusion protein was produced 75,000-fold over uninduced cells, with a relatively stable mRNA (T1/2 of 8.3 min) and a completely stable protein. In addition, over 95% of the final fusion protein was localized in the outer membrane and was capable of binding heavy metals (especially cadmium) in vitro. Cells producing Lpp::HMT bioaccumulated heavy metals (e.g., cadmium) 66-fold over nonproducing cells.

Arabinose↗
PubMed1989

Transformation of Bacillus thuringiensis vegetative cells by electroporation.

A highly efficient procedure for the transformation of Bacillus thuringiensis and Bacillus subtilis using covalently closed circular plasmid DNA was developed by using the small Staphylococcus aureus plasmid pC194 and electroporation. We have achieved transformation efficiencies in B. thuringiensis subsp. kurstaki (HD-73) greater than 5 x 10(6) transformants/micrograms plasmid DNA. The electro-transformation (or electroporation) procedure also worked with B. subtilis 168 although at a 200-fold less level of efficiency. The results indicated that the plasmid exists in double and single-stranded forms both in B. subtilis and B. thuringiensis. A second single-stranded species was also observed in both species. This technique may prove to be applicable to other members of the genus Bacillus.

Bacillus thuringiensis↗
PubMed1989

Retrovirus-induced murine motor neuron disease: mapping the determinant of spongiform degeneration within the envelope gene.

The Cas-Br-E murine leukemia virus (MuLV) induces a degenerative myeloencephalopathy leading to hind-limb paralysis when inoculated into newborn mice. To map the viral DNA sequences encoding the determinant of neurological degeneration, we constructed chimeric viruses in vitro with parental genomes from Cas-Br-E MuLV and from nonparalytogenic MuLVs. We found that a 1.5-kilobase-pair env Cas-Br-E fragment was sufficient to confer the full paralysis-inducing potential to chimeric viruses. This region encodes the 19 carboxyl-terminal residues of the leader sequence, all of gp70, and the 45 amino-terminal residues of the transmembrane protein (p15E). Within this env region, we identified a 372-base-pair fragment which was necessary for the full paralysis-inducing potential of the virus and which influenced the development of the disease in a strain-dependent manner. This domain encodes the 19 carboxyl-terminal residues of the leader peptide and the first 67 amino-terminal residues of gp70. We propose that Cas-Br-E MuLV induces spongiform degeneration through binding of its gp70 to a specific cellular receptor.

Animals↗
PubMed1988

Scrambling of bands in gel electrophoresis of DNA.

Under certain conditions of agarose gel electrophoresis, larger DNA molecules migrate faster than smaller ones. This anomalous mobility of DNA, which can lead to serious errors in the measurement of DNA fragment lengths, is related to near-zero velocity conformations which can trap DNA chains during electrophoresis. Intermittent electric fields can be used to alter the chain conformations so as to restore the monotonic mobility-size relationship which is necessary for a correct interpretation of the gel. These data are in agreement with the results of a computer simulation based on a theoretical model of electrophoresis.

Bacteriophage lambda↗
PubMed1988

Bacillus thuringiensis insecticidal crystal toxins: gene structure and mode of action.

Thanks to the techniques of recombinant DNA, there is now abundant sequence information on several endotoxin genes of Bacillus thuringiensis. The task of correlating this sequence information with the economically important aspects of the toxins such as insect specificity, LD(50) and speed of kill is now under worldwide investigation. Progress has also been made on understanding the mechanism of action of the toxins and on identifying the parts of the protoxin which are important in toxicity. Taken together, the mechanistic data and the sequence information allow the first attempts at rational design of mutant endotoxin genes and greatly facilitate the transfer of those genes to other organisms such as plants. More information is still needed, however, as to the nature of the binding site of the toxin and on the three-dimensional structure of the activated toxins.

Journal Article↗
PubMed1987

Use of oligonucleotide probes to study the relatedness of delta-endotoxin genes among Bacillus thuringiensis subspecies and strains.

Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.

Bacillus thuringiensis↗
PubMed1986

Ligation of single-stranded oligodeoxyribonucleotides by T4 RNA ligase.

Despite its unique ability to ligate single-stranded DNA molecules, T4 RNA ligase has so far seen little use in molecular biology due to long reaction times, modest yields, and apparent inability to promote ligation of long oligodeoxyribonucleotides. We describe here a set of reaction conditions which dramatically shorten the reaction time and give reproducible 40 to 60% ligation of DNA fragments of up to 40 bases in length. These improvements open promising new fields of application to T4 RNA ligase.

Adenosine Triphosphate↗
PubMed1984

Construction of a family of universal expression plasmid vectors.

A family of universal expression vectors based on the pUC8 and pUC9 plasmids has been constructed. These vectors cover all three possible reading frames in both directions, allowing any synthetic DNA, genomic DNA or cDNA to be expressed under control of the lac promoter. The four new vectors retain the useful features of the pUC plasmids, including the blue to white color change on X-gal plates indicating the presence of an insert. This family of expression vectors is expected to be quite useful in allowing direct immunological screening of cDNA or genomic DNA banks.

Cloning, Molecular↗
PubMed1984

Synthesis of a human insulin gene. VII. Synthesis of preproinsulin-like human DNA, its cloning and expression in M13 bacteriophage.

A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M 13mp8 vector. A clone pNB82 -121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25-bp affinity leader sequence coding for (glu)7 was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of beta-galactosidase. Preproinsulin was expressed under lac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide.

Base Sequence↗
PubMed1984

The human preproinsulin gene: synthesis, cloning, gene modification, and expression studies.

A 355 base pair (bp) DNA sequence coding for human preproinsulin has been assembled by joining a synthetic DNA leader sequence coding for 24 preregion amino acids to the previously synthesized DNA duplex of 277 bp constituting the sequence of BCA chain. It was next cloned in M13 mp8 single-stranded bacteriophage and subjected to site-specific mutagenesis and phase shifting to allow its inducible expression under lac operator control. An affinity leader sequence of 25 bp has been added in an attempt to facilitate purification of the preproinsulin.

Bacteriophages↗
PubMed1983

Synthesis of a human insulin gene. VI. Expression of the synthetic proinsulin gene in yeast.

The construction of plasmid vectors for the controlled expression of a synthetic human proinsulin gene in the yeast Saccharomyces cerevisiae is described. Attempts to express the proinsulin gene using the yeast ADH1 promoter alone did not yield detectable levels of proinsulin. Successful expression was achieved when the proinsulin gene was fused with the promoter and protein leader sequence of the GAL1 gene (coding for yeast galactokinase) in the yeast-Escherichia coli plasmid vector pYT7810. Two different-length leader sequences were employed; the longer leader (about 280 amino acids, fusion plasmid pPS13) gave about five times greater expression than the shorter leader fusion (30 amino acids, plasmid pPS5). Both fusions gave soluble protein products, and the proinsulin could be cleaved by cyanogen bromide treatment from the leader polypeptide. Proinsulin was detected by radioimmunoassay for human C-peptide only in cells induced with galactose, and was not detected in the gene fusions that were out of phase with the GAL1 leader sequence. Methods of improving the level of expression of the proinsulin gene in yeast using this system are discussed.

Amino Acid Sequence↗
PubMed1982

Synthesis of a human insulin gene. V. Enzymatic assembly, cloning and characterization of the human proinsulin DNA.

To form a 258-bp sequence coding for human proinsulin, 41 synthetic deoxyribo-oligonucleotide fragments of 11 to 15 nucleotides in length were assembled by enzymatic methods. The coding sequence is preceded by ATG and following by TGA for translation start and stop signals, and terminated in an EcoRI and a BamHI recognition sequence. The complete synthetic sequence was ligated to a plasmid and cloned in Escherichia coli. The cloned DNA was shown to have the correct human proinsulin coding sequence.

Base Sequence↗
PubMed1980

Synthesis of the human insulin gene. Part III. Chemical synthesis of 5'-phosphomonoester group containing deoxyribooligonucleotides by the modified phosphotriester method. Its application in the synthesis of seventeen fragments constituting human insulin C-chain DNA.

A method for phosphorylating a protected deoxyribooligonucleotide containing phosphotriester linkages is described. The modified phosphotriester method of chemical synthesis is further refined in terms of (i) better final deblocking conditions and (ii) new chromatography solvent systems containing acetone-water-ethyl acetate to yield pure oligomers. The effectiveness of these improvements has been demonstrated in the rapid and efficient synthesis of seventeen fragments constituting the sequence of human insulin C-chain DNA.

Base Sequence↗
PubMed1980

Sequence determination of protected oligodeoxyribonucleotides containing phosphotriester linkages by californium-252 plasma desorption mass spectrometry.

A mass spectrometric method for determining the sequence and molecular weight of protected oligodeoxyribonucleotides is described. By using the method of 252Cf plasma desorption mass spectrometry [Macfarlane, R. D. & Torgerson, D. F. (1976) Science 191, 920--925], positive-ion mass spectra were obtained for a series of protected oligonucleotides extending to a decanucleotide; the spectra were dominated by the presence of the oligonucleotide molecular ion. The negative-ion mass spectra were characterized by a nested set of fragment ions extending from the 3'- or 5'-terminal nucleotide to the opposite terminal nucleotide, thereby identifying the sequence. The utility of this method has been demonstrated by the sequence determination of protected tetra-, penta-, and hexanucleotides synthesized by the improved phosphotriester method.

Base Sequence↗
PubMed1980

Synthesis of the human insulin gene. Part IV. New synthetic deoxyribooligonucleotide adaptors and primer for DNA cloning and sequence analysis.

The chemical synthesis of four new deoxyribooligonucleotides to be used as adaptors in molecular cloning of DNA for expression studies is described. These are (i) start-adaptor, (ii) stop-adaptor, (iii) conversion adaptor to insert ribosomal binding site, and (iv) retrieving adaptor. We have also synthesized a 19-bases-long primer, 5'-TTGTAAAACGACGGCCAGT-3' to increase the speed of DNA sequence analysis and also demonstrated its application using single-stranded M13 DNa vector.

Base Composition↗