PubMed Health⌕ Search

Biomedical subjects

J Höltke

Publications and source records attributed to J Höltke.

3 recordsLinked to original sources
PubMed1990

A highly sensitive, nonradioactive DNA labeling and detection system.

A highly sensitive method for detecting specific nucleotide sequences was recently developed. The method uses digoxigenin-labeled nucleic acid probes for hybridization to immobilized target nucleic acids. Probes can be labeled by the random-primed method, nick translation, oligonucleotide tailing, cDNA synthesis, photodigoxigenin or SP6/T7/T3 polymerase-mediated transcription. Hybrids are detected by an enzyme-linked immunoassay using an anti-digoxigenin antibody conjugate. Visualization of the bound antibody is accomplished by an enzymatic color reaction, enzymatic chemiluminescent reaction or immunofluorescence, depending on the antibody conjugate and enzymatic substrate used. Here we report the successful application of this technology in the detection of specific cloned DNA in colony and plaque hybridizations, specific detection of a single mRNA species in Northern blots and single-copy gene detection in genomic Southern blots.

Bacteriophage lambda↗
PubMed1984

Molecular cloning of the yeast fatty acid synthetase genes, FAS1 and FAS2: illustrating the structure of the FAS1 cluster gene by transcript mapping and transformation studies.

From a Saccharomyces cerevisiae gene bank contained in the novel yeast cosmid shuttle vector pMS201 the fatty acid synthetase (FAS) genes FAS1 and FAS2 were isolated. FAS clones were identified by in situ colony hybridization using two yeast DNA probes apparently capable of producing avian FAS cross-reacting material (J. Carbon, personal communication). Classification as FAS1 or FAS2 clones was achieved by their specific transformation of fas1 and fas2 yeast mutants. By transcription mapping FAS1 was assigned to about 5.3 kb within 14.8 kb of chromosomal DNA covered by two genomically adjacent BamHI fragments. The FAS2 gene was localized on a single BamHI fragment of 25 kb. One of the FAS clones ( FAS2 ) produces immunologically cross-reacting material in Escherichia coli. High frequency transformation of fas1 mutants was only observed with one subclone, pMS3021 , containing the intact FAS1 locus. Other DNA segments cloned in the same self-replicating vector but representing only part of FAS1 exhibited drastically lower transformation rates. As evident from this and from FAS1 /TRP1-cotransformation rates only the intact FAS1 gene in pMS3021 is capable of fas1 -mutant complementation. With partial FAS1 genes, even when coding for an intact equivalent of the mutated domain, their chromosomal integration is necessary for the expression of FAS. In integrative transformants the coexistence of integrated and autonomously replicating plasmid DNA was demonstrated. Both, the extrachromosomal and chromosomally integrated FAS DNA was mitotically unstable. Transformation studies using subcloned FAS1 DNA segments revealed the relative locations of the enoyl reductase and dehydratase domains within this pentafunctional cluster gene.

Cloning, Molecular↗