Biomedical subjects

E Hornes

Publications and source records attributed to E Hornes.

25 records · Page 2Linked to original sources

PubMed1990

A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica.

We compared a synthetically produced 19-mer oligonucleotide probe with a polynucleotide probe consisting of a cloned fragment of the virulence gene yopA for their relative efficiencies in identification and enumeration of virulent Yersinia enterocolitica. The probes were used in DNA-DNA colony hybridization assays to differentiate 70 Yersinia strains with known plasmid profiles. All 19 strains harboring the 40- to 50-megadalton virulence plasmid were positive in the hybridization assay, whereas their isogenic derivatives lacking this plasmid were negative. Both probes correctly identified plasmid-bearing variants of Y. enterocolitica serogroups O:3, O:5,27, O:8, O:9, O:13, and O:21 from three continents. In contrast, none of the probes hybridized with DNA from 32 environmental yersiniae belonging to 26 serogroups not associated with disease. Colony hybridization was used to detect and enumerate virulent Y. enterocolitica in three artificially contaminated food samples. Despite a large background of indigenous bacteria (3 x 10(4) CFU), the efficiency of enumeration ranged from 33 to 82%. The use of nylon filters did not impair the growth of virulent yersiniae. Both probes showed a perfect concordance in their specific differentiation and enumeration of virulent Y. enterocolitica. DNA colony hybridization with these two probes permitted rapid and reliable identification of all common pathogenic serogroups without the need for enrichment or esoteric identification protocols.

Animals↗

PubMed1990

Identification of a double-stranded RNA virus by using polymerase chain reaction and magnetic separation of the synthesized DNA segments.

A double-nested polymerase chain reaction assay (PCR), followed by magnetic separation of the PCR-synthesized DNA segments, was developed to detect a double-stranded RNA virus, infectious pancreatic necrosis virus from salmonid fish. Viral RNA was extracted from cell cultures and used for cDNA synthesis. The cDNA produced was used as a template in a double PCR. The sensitivity of this double PCR was approximately 0.8 pg of template double-stranded RNA. The DNA segment produced from the first PCR was also used as a template in a second PCR with a set of two 5'-labeled primers, one with biotin and the other with 32P. The PCR segment that was then synthesized was separated from the solution by using streptavidin-coated, superparamagnetic beads. The levels of radioactivity measured in the magnetically separated fractions were significantly higher in the positive samples than they were in the negative samples.

Animals↗

PubMed1990

Direct cloning of the human genomic apolipoprotein E gene using magnetic separation of single-stranded DNA.

Solid-phase methods can be used for direct cloning of in vitro-amplified genomic DNA with high efficiency, without the need for restriction enzymes or ligase. Single-stranded DNA fragments are generated by magnetic separation and are simply mixed with single-stranded vector to form gap-duplex molecules that can be readily transformed. This strategy, in combination with direct solid-phase DNA sequencing, was used to analyze individual alleles in the human apolipoprotein E locus.

Amino Acid Sequence↗

PubMed1989

Magnetic DNA affinity purification of yeast transcription factor tau--a new purification principle for the ultrarapid isolation of near homogeneous factor.

We present a new method for rapid purification to near homogeneity of sequence specific DNA binding proteins based on magnetic separation. The method is described for the purification of the yeast transcription factor tau. DNA affinity Dynabeads (monodisperse superparamagnetic particles) specifically bind the protein in the presence of competitor DNA. By magnetic separation, wash and elution, highly enriched transcription factor preparations are obtained within minutes. In less than an hour with three cycles of adsorption, nearly homogeneous factor tau was obtained. The factor preparation contained mainly two polypeptides of 100 and 140 kDa and was fully active in transcription and DNA binding assays. This procedure should work for any high-affinity sequence-specific DNA binding protein with only minor modifications.

Adsorption↗

PubMed1989

Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support.

Approaches to direct solid phase sequencing of genomic and plasmid DNA have been developed using magnetic beads, coated with streptavidin, as solid support. The DNA is immobilized through selective incorporation of biotin into one of the strands. A single stranded template, suitable for sequencing, is obtained through strand-specific elution. Using this concept, in vitro amplified plasmid DNA and chromosomal DNA were sequenced directly from single colonies. The solid phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The system was found to be suitable for sequencing using both isotope- and fluorescent-labelled primers.

Base Sequence↗

PubMed1989

Isolation and in vitro expression of the ricin A-chain gene: effect of deletions on biological activity.

The gene encoding the ricin A-chain was isolated and subcloned into an in vitro expression vector downstream from the SP6 promotor. mRNA encoding the A-chain strongly inhibited the translational activity of reticulocyte lysates. The inhibition correlated with glycosylase activity on rRNA, and could be abolished by addition of antibodies specific for ricin. mRNA generated after linearization of the vector at unique restriction sites within the A-chain coding sequence did not inhibit, except after linearization with ScaI. Also mutants lacking the 28 N-terminal amino acids of native A-chain strongly inhibited the lysates. However, in both cases no glycosylase activity could be observed. We also show that the lack of a stop codon in mRNA does not affect the level of expression as assayed here.

Animals↗

PubMed1988

Assessment of methods for covalent binding of nucleic acids to magnetic beads, Dynabeads, and the characteristics of the bound nucleic acids in hybridization reactions.

Dynabeads are magnetic monosized beads with high stability, high uniformity, unique paramagnetic properties, low particle-particle interaction, and high dispersibility. Different reactive groups; hydroxyl, carboxyl and amino groups can be attached to the surface. Several methods for covalent attachment of DNA or oligonucleotides to the beads were investigated. Best coupling yields were obtained by carbodiimide-mediated end-attachment of 5'-phosphate and 5'-NH2 modified nucleic acids to respectively amino and carboxyl beads. The carboxyl beads showed a low degree of non-specific binding, while a better yield of end-attached nucleic acids was obtained using the amino beads. The DNA-beads worked efficiently in hybridization experiments, and the kinetics of hybridization approach those of solution hybridization.

Chemical Phenomena↗