Potential promoters in the genome of Escherichia coli were searched by pattern recognition software PlatProm and classified on the basis of positions relative to gene borders. Beside the expected promoters located in front of the coding sequences we found a considerable amount of intragenic promoter-like signals with a putative ability to drive either antisense or alternative transcription and revealed unusual genomic regions with extremely high density of predicted transcription start points (promoter ‘islands’), some of which are located in coding sequences. PlatProm scores converted into probability of RNA polymerase binding demonstrated certain correlation with the enzyme retention registered by ChIP-on-chip technique; however, in ‘dense’ regions the value of correlation coefficient is lower than throughout the entire genome. Experimental verification confirmed the ability of RNA polymerase to interact and form multiple open complexes within promoter ‘island’ associated with appY, yet transcription efficiency was lower than might be expected. Analysis of expression data revealed the same tendency for other promoter ‘islands’, thus assuming functional relevance of non-productive RNA polymerase binding. Our data indicate that genomic DNA of E. coli is enriched by numerous unusual promoter-like sites with biological role yet to be understood.
• ~,'ey words." DNA-lipid interaction; Transfection; 1 ipid polymorphism; Membrane ultrastructure Materials and methods Preparation of complexCalf thymus DNA was purchased from Sigma, additionally purified using phenol and chloroform (A26olA2so value being greater than 1.9) and mildly ultrasonicated until the formation of rather homogeneous native fragments with a chain length of about 300-500 bp. The DNA fragments were mixed with a 1 mg/ml suspension of sonicated small unilamellar vesicles of egg PC or DPPC from Avanti in 0.5 mM HEPES solution (pH 7.5) and then a 100 mM stock solution of CaC12 was slowly added with rapid stirring to yield a final Ca 2+ concentration of 20 mM. The mixture was lyophilized and treated with chloroform to disintegrate initially existing membrane bilayer structures and to favour the formation of the inverted phase in organic solvent [19]. After the removal of chloroform under vacuum, the DNA-Ca2+-lipid complex was rehydrated with the initial volume of water. MicrocalorimetryMeasurements were performed using a DASM-4 (Russia) differential scanning microcalorimeter at a heating of rate of 0.25 K/rain. The
Nucleotide sequences of 441 promoters recognized by Escherichia coli RNA polymerase were subjected to a site-specific cluster analysis based on the hierarchical method of classification. Five regions permitting promoter subgrouping were identified. They are located at -54 +/- 4, -44 +/- 3, -35 +/- 3 (-35 element), -29 +/- 2 and -11 +/-4 (-10 element). Promoters were independently subgrouped on the basis of their sequence homology in each of these regions and typical sequence elements were determined. The putative functional significance of the revealed elements is discussed on the basis of available biochemical data. Those promoters that have a high degree of homology with the revealed sequence elements were selected as representatives of corresponding promoter groups and the presence of other sequence motifs in their structure was examined. Both positive and negative correlations in the presence of particular sequence motifs were observed; however, the degree of these interdependencies was not high in all cases, probably indicating that different combinations of the signal elements may create a promoter. The list of promoter sequences with the presence of different sequence elements is available on request by Email: ozoline@venus.iteb. serpukhov.su.
The distribution of deformable base-pair steps in the structure of bacterial promoters is analyzed with respect to their possible structural and functional role. A regular positioning of TA and TG stacks is detected with the best fit period 5.6 bp. This value is interpreted as a half of the sequence period 11.2 bp, somewhat higher than the structural helical repeat of B-DNA (10.55 bp). The difference, +0.65 bp, suggests a sequence-dependent helical writhe of the promoter DNA--a right-handed superhelix. Apparently, to favour rotational setting of DNA on the surface of RNA polymerase the flexible steps deformable largely towards the grooves, follow the half-period spacing. Such rotational setting is consistent with the DNase I footprinting data. Periodical distribution of deformable base-pair stacks shows negative correlation with the presence of -35 canonical hexamer, suggesting the functional significance of this novel element for promoter recognition. The RNA polymerase--DNA recognition is discussed as interaction of distributional type that involves many elements of different nature which are in partially compensatory relations.
Cognitive malfunction, synaptic dysfunction, and disconnections in neural networks are core deficits in Alzheimer's disease (AD). 5xFAD mice, a transgenic model of AD, are characterised by an enhanced level of amyloid-beta and abnormal neurotransmission. The dopaminergic (DA) system has been shown to be involved in amyloid-beta transformations and neuronal plasticity; however, its role in functional network changes in familial AD still remains unclear. In 5xFAD and non-transgenic freely moving mice, electroencephalograms (EEGs) were simultaneously recorded from the secondary motor cortex (MC), superficial layers of the hippocampal CA1 area (HPC), substantia nigra (SN), and ventral tegmental area (VTA). EEGs and their frequency spectra were analysed before and after systemic injection of a DA receptor agonist, apomorphine (APO). In the baseline EEG from MC and HPC of 5xFAD mice, delta and alpha oscillations were enhanced and beta activity was attenuated, compared to control mice. In VTA and SN of 5xFAD mice, delta-theta activity was decreased and beta oscillations dominated. In control mice, APO suppressed delta activity in VTA to a higher extent than in MC, whereas in 5xFAD mice, this difference was eliminated due to attenuation of the delta suppression in VTA. APO increased beta activity in MC of mice from both groups while significant beta suppression was observed in VTA of 5xFAD mice. These mice were characterized by significant decrease of tyrosine hydroxylase immunopositive cells in both VTA and SN and of DA transporter in MC and hippocampal dentate gyrus. We suggest that the EEG modifications observed in 5xFAD mice are associated with alterations in dopaminergic transmission, resulting in adaptive changes in the cerebral networks in the course of familial AD development.
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