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  • 1
    ISSN: 1432-0983
    Keywords: Key wordsSaccharomyces cerevisiae ; Transcription ; Mitochondria ; cis element
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Studies from this laboratory have suggested that mitochondrial (mt) transcription in yeast (Saccharomyces cerevisiae) is governed by changing cellular cAMP levels, and that the mechanism of such transcriptional regulation requires cAMP-dependent protein kinase (PKA) activity; these observations, in turn, suggest a trans-activation process for nucleotide-dependent mt transcriptional control. Here we demonstrate a sequence-specific mtDNA-phosphorylated protein interaction, a requisite part of such a control mechanism, using filter-binding and gel mobility shift assays with mt protein extracts and mtDNA from ρ – strains whose retained mt genes show cAMP-sensitive expression. We demonstrate that the protein-mt DNA interaction depends on PKA activity, that it specifically involves a tripartite GC-rich sequence element on yeast mtDNA, and that it does not involve mt coding or promoter sequences. Sequence analysis indicates that the GC-rich element undergoing protein interaction is present in ten copies on the yeast mt genome, and that each copy is located 5′ to a strong mt promoter; the elements appear in both orientations relative to, and at varying distances upstream from, the putatively associated mt promoter elements. The mt element shows no sequence homology to relevant nuclear cis-elements examined and is unrelated to published vertebrate mt cis-elements. Several lines of evidence and argument strongly suggest that this GC-rich element functions as the cis-regulatory sequence involved in cAMP-mediated transcriptional control in yeast mitochondria.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-1831
    Keywords: Key words Chlamydia ; Alzheimer's disease ; Inflammation ; Dementia ; Infection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We assessed whether the intracellular bacterium Chlamydia pneumoniae was present in post-mortem brain samples from patients with and without late-onset Alzheimer's disease (AD), since some indirect evidence seems to suggest that infection with the organism might be associated with the disease. Nucleic acids prepared from those samples were screened by polymerase chain reaction (PCR) assay for DNA sequences from the bacterium, and such analyses showed that brain areas with typical AD-related neuropathology were positive for the organism in 17/19 AD patients. Similar analyses of identical brain areas of 18/19 control patients were PCR-negative. Electron- and immunoelectron-microscopic studies of tissues from affected AD brain regions identified chlamydial elementary and reticulate bodies, but similar examinations of non-AD brains were negative for the bacterium. Culture studies of a subset of affected AD brain tissues for C. pneumoniae were strongly positive, while identically performed analyses of non-AD brain tissues were negative. Reverse transcription (RT)-PCR assays using RNA from affected areas of AD brains confirmed that transcripts from two important C. pneumoniae genes were present in those samples but not in controls. Immunohistochemical examination of AD brains, but not those of controls, identified C. pneumoniae within pericytes, microglia, and astroglia. Further immunolabelling studies confirmed the organisms' intracellular presence primarily in areas of neuropathology in the AD brain. Thus, C. pneumoniae is present, viable, and transcriptionally active in areas of neuropathology in the AD brain, possibly suggesting that infection with the organism is a risk factor for late-onset AD.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1617-4623
    Keywords: Key wordsChlamydia trachomatis ; Gene expression Ribosomes ; Protein synthesis ; Development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Following binding and internalization into the host cell cytoplasm, elementary bodies (EB) of the obligate intracellular bacterium Chlamydia trachomatis undergo a developmental process resulting in production of reticulate bodies (RB), the vegetative growth form of the organism. EB are metabolically inactive, but EB to RB transformation requires bacterial protein synthesis. Using HeLa cells infected with EB of C. trachomatis serovar C, we examined the time of first appearance of transcripts from several genes whose products are required for assembly and function of the chlamydial protein synthetic system. We monitored appearance of chlamydial RNAs using reverse transcription–polymerase chain reaction assays targeting primary transcripts from the bacterial rRNA operons, and mRNAs encoding the glycyl tRNA synthetase and the ribosomal proteins S5 and L5. Transcripts from the proximal rRNA promoters, and those from the r-protein and tRNA synthetase genes, are detectable as early as 4 h after EB–host binding; transcripts from distal rRNA promoters do not appear until 6 h post-infection. Thus, expression of bacterial genes whose products are required for protein synthesis begins earlier in chlamydial EB to RB development than previously thought.
    Type of Medium: Electronic Resource
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