Biomedical subjects

W Charlton

Publications and source records attributed to W Charlton.

4 recordsLinked to original sources

PubMed2000

Biochemical and molecular approaches to understanding protein import into peroxisomes.

Peroxisomes are eukaryotic organelles that perform diverse and variable functions. Although genetic studies in yeasts and mammals have identified approximately 20 genes (PEX genes) required for the biogenesis of this important organelle, biochemical studies of protein targeting and import have lagged behind and in many cases we have no idea of the function of the PEX gene products (peroxins). Using an import assay in vitro derived from sunflower cotyledon cells and recombinant proteins, we have obtained translocation intermediates on the peroxisome import pathway and are using cross-linking to identify interacting partners. We have also used antibodies raised against human PEX14 to inhibit the import of matrix proteins in this system. To obtain homologous antibodies for inhibition experiments, to immunoprecipitate cross-linked products and to enable us to study the import pathways of peroxins we have cloned and characterized plant orthologues of three PEX genes, PEX6, PEX10 and PEX14.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1993

The Klebsiella pneumoniae nifJ promoter: analysis of promoter elements regulating activation by the NifA promoter.

The nifJ and nifH promoters of Klebsiella pneumoniae are divergently transcribed sigma 54-dependent promoters that are positively activated by the NifA protein. NifA binds to upstream activator sequences (UASs), usually located 60-200 bp upstream of the start of transcription. Bound NifA is presented to the RNA polymerase-sigma 54 complex (E sigma 54) via DNA loop formation, mediated by the binding of integration host factor protein (IHF) between E sigma 54 and NifA. The nifJ promoter sequence contains three potential NifA binding sites (UAS1, 2 and 3) and two potential RNA polymerase-sigma 54-binding sites (downstream promoter elements, DPEs 1 and 2). DPE2 is located 420 bp into the coding region and DPE1 overlaps UAS1 by 5 bp. Mutational and footprinting analyses have shown efficient activation of the nifJ promoter requires that NifA is bound at UAS 2 and 3. Transcription is initiated at DPE1. Only a weak interaction of NifA with the UAS overlapping DPE1 was detected. Footprints demonstrated that E sigma 54 forms a closed complex at DPE1 but not DPE2 and that bound E sigma 54 closely approaches the -15 region of DPE1. Stimulation of nifJ promoter activity by IHF was not as great as that observed for other nif promoters. In the absence of IHF nifH promoter sequences stimulated activation of the nifJ promoter. This appeared to require NifA bound at the nifH UAS. Thus, one additional role of IHF may be to partition NifA between the two promoters by constraining the topology of the DNA.

Amino Acid Sequence↗

PubMed1991

Organization and function of binding sites for the transcriptional activator NifA in the Klebsiella pneumoniae nifE and nifU promoters.

The interaction of the Klebsiella pneumoniae NifA protein, a sigma 54-dependent activator, with the nifE and nifU promoters was analysed. At these promoters NifA established contacts in addition to those predicted by the minimal formulation NifA binding site (5'-TGT-N10-ACA). The positions of the contacts indicate that bound NifA molecules could assemble to form an oligomer. At both promoters contacts with NifA are made predominantly on one face of the DNA helix, and all contacts appear necessary for full activation by NifA. The close contacts made by NifA appear to be made by the DNA-binding domain of NifA. This domain shows specific DNA-binding activity in vitro. The binding of NifA to one site in the nifU promoter depends upon occupancy of additional upstream sequences by NifA. At the nifE promoter NifA binds adjacent to an integration host factor (IHF) binding site, but in contrast to results obtained with the nifU promoter IHF does not diminish nifE promoter occupancy by NifA. The IHF requirement for efficient in vivo activation of the nifU promoter by NifA was greater than that of the nifE promoter. Accordingly, the affinity of IHF for the nifU promoter is higher than for the nifE promoter. Amongst promoters utilizing the sigma 54 holoenzyme, the nifE promoter appears somewhat atypical in having the activator bound at around position -74 rather than the usual 100 base-pairs or more upstream from the transcription start site.

Bacterial Proteins↗

PubMedDate not supplied

Primary angle-closure glaucoma among Alaskan Eskimos.

An epidemiological study of the occurrence of angle-closure glaucoma was made under the Eskimos population of the Norton Sound and Bering Straits region of Alaska. Among 1673 Alaskan Natives examined 14 cases (0.8%) of angle-closure glaucoma were found, in 0.5% of the men and 1.2% of the women; for persons above the age of 40 years these figures were 2.1% and 5.5% respectively. Angle-closure glaucoma was found in 11.8% of women above the age of 60 years. A shallow anterior chamber was seen twice as frequently in women as in men. Above the age of 30 years the chamber angle was considered to be occludable in 2% of men and 7.5% of women on gonioscopic examination. The average intra-ocular pressure of the right eyes of men was 11.7 mm Hg (s.d. 3.3) and of women 12.0 mm Hg (s.d. 3.4). Primary open-angle glaucoma was not seen in the population being studied.

Adolescent↗