Biomedical subjects

S Bergh

Publications and source records attributed to S Bergh.

6 recordsLinked to original sources

PubMed1992

Analysis of a polyketide synthesis-encoding gene cluster of Streptomyces curacoi.

A gene cluster homologous to the beta-ketoacyl synthase-encoding gene, actl, was cloned by Southern blot hybridization from Streptomyces curacoi, and a 5469-bp fragment was sequenced. Analysis of the sequence revealed seven open reading frames (ORFs). A striking similarity to the whiE locus (encoding spore pigment synthesis) of Streptomyces coelicolor was found throughout the whole cluster, with conservation of the size and position of the seven ORFs. The structure of these two gene clusters suggests that they have a common origin, although the lower similarity in the noncoding regions suggests that the regulatory regions have diverged through evolution.

Amino Acid Sequence↗

PubMed1992

Automated magnetic preparation of DNA templates for solid phase sequencing.

An integrated protocol for solid-phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin-biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single-stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)-infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.

Automation↗

PubMed1992

Cloning, analysis, and heterologous expression of a polyketide synthase gene cluster of Streptomyces curacoi.

Streptomyces curacoi produces curamycin, an antibiotic based on a modified orsellinic acid skeleton that is synthesized by the polyketide pathway. We have cloned, characterized, and partly sequenced a polyketide synthase gene cluster of S. curacoi. The sequence data reveal an organization of open reading frames that is similar to those of other polyketide synthetic clusters, although the biosynthetic products differ considerably in size and structure. We propose that one of the predicted open reading frames (curA) encodes polykeptide synthase, on the basis of its homology with other enzymes with similar functions. Expression of the cloned chromosomal fragment in the heterologous host S. lividans leads to the production of a brown pigment in large quantities. The analysis and expression of the cur genes for detailed molecular studies of the mechanism of polyketide biosynthesis is discussed.

Amino Acid Sequence↗

PubMed1991

Colorimetric quantification of in vitro-amplified template DNA to be used for solid phase sequencing.

A protocol for colorimetric determination of DNA amplified by the polymerase chain reaction (PCR) and subsequently immobilized to a solid support is described. The protocol consists of three steps: (i) binding of PCR amplified lac operator-containing DNA to magnetic beads; (ii) binding of a Lac repressor-beta-galactosidase fusion protein to the lac operator and (iii) colorimetric detection of the immobilized beta-galactosidase. In practice, steps (i) and (ii) are performed concurrently. The protocol is well suited both for manual and automated procedures and the immobilized template can, after melting, be used directly for solid phase sequencing. The assay is used to demonstrate that template concentration is important for the quality of sequence data obtained from an automated DNA sequencer.

Base Sequence↗

PubMed1991

Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA.

A solid-phase approach is described for manual and automated sequencing of plasmid DNA obtained directly from bacterial colonies through the polymerase chain reaction. The DNA fragment is selectively immobilized to magnetic beads and after strand-specific elution, the eluted strand, as well as the remaining immobilized strand, is used for bidirectional dideoxy sequencing. The solid-phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The approach is exemplified by fluorescent sequencing of a cloned Streptomyces curacoi gene having a G + C content of more than 70%.

Base Sequence↗

PubMedDate not supplied

Semi-automated solid-phase DNA sequencing.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.

Automation↗