Biomedical subjects

J D Hoheisel

Publications and source records attributed to J D Hoheisel.

57 records · Page 4Linked to original sources

PubMed1989

Control of partial digestion combining the enzymes dam methylase and MboI.

A method is described which allows the preparation of reproducible partial digests without previous establishment of the incubation conditions. It is based on a combined application of dam methylase and the restriction endonuclease MboI, both recognizing the sequence 5'-GATC-3' but MboI unable to cut the methylated site. Due to their competition for the same substrate the DNA is partially digested, with the size of the resulting fragments strongly dependent on the ratio of enzymes. The Km of the dam methylase was determined to be 115 ng DNA/microliters indicating a variance in fragment sizes generated at low DNA-concentrations. This effect is minimized above 150 ng/microliters. Any influence of digestion time is avoided, because the reaction runs until complete modification of all sites. The dependence on enzyme concentration and presence of agarose was checked. Knowledge of these parameters allows an accurate prediction of fragment sizes generated at different conditions. The technique was successfully used to construct libraries from different sources, in particular chromosome-specific libraries from small amounts of flow-sorted material.

DNA↗

PubMed1989

A cassette with seven unique restriction sites, including octanucleotide sequences: extension of multiple-cloning-site plasmids.

A 27-bp synthetic DNA cassette was constructed which contains the restriction sites of the two rare-cutter enzymes NotI and SfiI and, in an overlapping arrangement, those of five enzymes with 6-bp recognition sequences: ApaI, BalI, NdeI, SacII, XmaIII. The protruding termini of the fragment allow its insertion into any EcoRI-cut DNA creating a new EcoRI site at one side of the cassette only. This fragment was integrated into the pUC18-like multiple-cloning-site (MCS) plasmids pTZ18R and pTZ19R, producing a set of vectors which carry seven additional unique restriction sites (giving a total of 17) within their MCS. They still provide the capabilities of simple recombinant selection by blue/white coloured colonies, creation of single-stranded DNA in the presence of a helper phage, and in vitro transcription of cloned DNA using T7 RNA-polymerase. Plasmids with two copies of the DNA cassette inserted into their MCS were also constructed. Beside the advantages they provide in some cloning procedures, these latter plasmids, which carry a tandem repeat, are valuable sources of related 27-bp fragments, with features similar to the original but with different cloning termini.

Base Sequence↗

PubMed1987

Searching for potential Z-DNA in genomic Escherichia coli DNA.

The Clarke-Carbon library with Escherichia coli DNA cloned into plasmid ColE1 was partially screened for Z-DNA with the monoclonal antibody Z-D11 using the retardation of the covalently closed circular DNA-protein complex by nitrocellulose filters. About 85% of the plasmids tested at "natural" supercoil density bound to the filter. Together with binding studies of the iodinated antibody, one Z-DNA segment per about 18,000 base-pairs of E. coli DNA is observed. One clone containing the region around the lactose operon, pLC20-30, was studied in detail. Subcloning a partial Sau3A digest and selection with antibodies gave three different Z-forming sites. They were mapped to within about +/- 20 base-pairs by preparing unidirectional deletion clones, selection of protein binding plasmids on nitrocellulose filters and subsequent sizing on agarose gels. The size of the Z-DNA-forming segments was estimated from two-dimensional gels of topoisomer mixtures. Together with results from sequencing of the plasmid DNA using exonuclease III to create single-stranded templates, stretches of alternating purine-pyrimidine tracts of 12 to 15 base-pairs were found to be responsible for Z-DNA formation. One of the sites was found in the middle of the lacZ gene, where it might be an obstacle for RNA polymerase. The methods used here should also be helpful for studying other DNA-protein sites, especially if they exist only in supercoiled DNA.

Antibodies, Monoclonal↗