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Biomedical subjects

D B Danner

Publications and source records attributed to D B Danner.

26 records · Page 2Linked to original sources
PubMed1982

Plasmid cloning vectors resistant to ampicillin and tetracycline which can replicate in both E. coli and Haemophilus cells.

We have constructed two plasmid vectors, pHCV5 and pHVTl, which will replicate both in Haemophilus and in Escherichia coli. Both contain the ampicillin-resistance gene and the replication origin from a Haemophilus plasmid, pRSF0885. Both also contain the pBR322 origin and therefore can be amplified in E. coli by chloramphenicol treatment. The plasmid pHCV5 contains the tetracycline-resistance gene of pBR322, and pHVT1 contains the analogous region from the transposon Tn10.

Cloning, Molecular↗
PubMed1982

Construction of DNA recognition sites active in Haemophilus transformation.

Competent Haemophilus cells recognize and preferentially take up Haemophilus DNA during genetic transformation. This preferential uptake is correlated with the presence on incoming DNA of an 11-base-pair (bp) sequence, 5'-A-A-G-T-G-C-G-G-T-C-A-3'. To prove that this sequence is the recognition site that identifies Haemophilus DNA to the competent cell, we have now constructed a series of plasmids, each of which contains the 11-bp sequence. Using two different assay systems we have tested the ability of fragments from these plasmids to compete with cloned Haemophilus DNA fragments that naturally contain the 11-bp sequence. We find that the addition of the 11-bp sequence to a DNA fragment is necessary and sufficient for preferential uptake of that fragment. However, plasmid DNAs containing this sequence may vary as much as 48-fold in uptake activity, and this variation correlates with the A+T-richness of the DNA flanking the 11-mer.

Base Sequence↗
PubMed1980

An eleven-base-pair sequence determines the specificity of DNA uptake in Haemophilus transformation.

Only certain DNA fragments are taken up efficiently by component Haemophilus cells; this implies that efficient uptake requires the presence of a specific nucleotide sequence on the incoming DNA (Sisco and Smith, 1979). To determine the structure of this "uptake site", we have isolated and sequenced four small fragments of cloned H. parainfluenzae DNA which retain the ability to be taken up by cells. These fragments have a sequence of eleven base pairs in common, 5'-AAGTGCGGTCA-3' and ethylation of certain phosphoryl groups in this sequence causes significant decreases in fragment uptake. We conclude that this is the sequence of the uptake site.

Base Sequence↗
PubMedDate not supplied

DNA damage and repair with age in individual human lymphocytes.

Previous biochemical studies on DNA repair competence and aging have been limited to techniques, such as alkaline elution or nucleoid sedimentation, involving mass cell populations. These techniques provide no information about the distribution of DNA damage and repair among individual cells and are unlikely to detect age-dependent changes affecting a minor fraction of the cell population. We have recently described a microgel electrophoretic assay (Singh et al., 1988) that measures, at the level of the individual cell, single-strand DNA breaks and alkali-sensitive sites. Here, we employ this method to analyze DNA damage and repair in lymphocytes isolated from the peripheral blood of 31 subjects (23 males and 8 females aged 25-91 years) and exposed in vitro to 200 rads of X-irradiation. While basal (pre-irradiation) levels of damage were independent of the age of the donor, an age-dependent increase in DNA damage was observed immediately following irradiation. For all subjects, the mean level of DNA damage was restored to pre-irradiation control levels within 2 h of incubation at 37 degrees C. However, a distribution analysis of DNA damage among cells within each sample indicated the presence of a few highly damaged cells (4-16%) in the 2-h sample, the occurrence of which was significantly more common among aged individuals. These data indicate an age-related decline in DNA repair competence among a small subpopulation of lymphocytes.

Adult↗