Interactions between lac repressor protein and site-specific bromodeoxyuridine-substituted operator DNA. Ultraviolet footprinting and protein-DNA cross-link formation.

K L Wick; K S Matthews

Home/Archive/PubMed

PubMed · 2007568

Interactions between lac repressor protein and site-specific bromodeoxyuridine-substituted operator DNA. Ultraviolet footprinting and protein-DNA cross-link formation.

K L Wick·K S Matthews

View original source ↗Export metadata ↓

Abstract

Specific contacts between the lac repressor and operator have been explored using 5-bromodeoxyuridine-substituted DNA. Substitution of BrdU for single thymidine positions in a synthetic 40-base pair operator provides substrate for ultraviolet irradiation; upon irradiation, strand scission occurs at the BrdU residues. When bound, lac repressor protein provides protection against UV-induced breakage depending on the nature of the sites and type of interaction. We have confirmed 13 unique sites of inducer-sensitive protection along the operator sequence using this method compared to complete substitution with BrdU; differences were observed at two positions for singly substituted versus completely substituted DNAs (Ogata, R., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4973-4976). The ability of these photosensitive DNAs to form short range cross-links to bound protein has been used to determine the efficiency with which cross-linked protein-DNA complexes are generated at each individual site of BrdU substitution. Five sites of high efficiency cross-linking to the repressor protein have been identified. At one site, cross-linking without protection from strand scission was observed; this result suggests an unusual mechanism of strand scission and/or cross-linking at this site. Comparison of the UV protection results and the cross-linking data show that these processes provide complementary tools for identifying and analyzing individual protein-DNA contacts.

Explore related subjects

Autoradiography Base Sequence Bromodeoxyuridine Cross-Linking Reagents DNA Fingerprinting DNA, Bacterial DNA-Binding Proteins Electrophoresis, Polyacrylamide Gel Escherichia coli Molecular Sequence Data Operator Regions, Genetic Repressor Proteins Ultraviolet Rays Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

K L Wick, K S Matthews. 1991-04-05. Interactions between lac repressor protein and site-specific bromodeoxyuridine-substituted operator DNA. Ultraviolet footprinting and protein-DNA cross-link formation.. https://pubmed.ncbi.nlm.nih.gov/2007568/

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

Analysis of neurotransmitter receptor distribution patterns in the cerebral cortex.

The concentration of transmitter receptors varies between different locations in the human cerebral cortex, but also between the different cortical layers within the same area. Analyzing the regional differences in the laminar distribution patterns of various neurotransmitter receptor binding sites by means of quantitative receptor autoradiography may thus provide a functionally relevant insight into the organization of the cortex. Here we introduce an approach to the analysis of in vitro receptor autoradiographic data, providing a framework for the assessment of differences in both mean concentration and laminar distribution patterns across multiple subjects. Initially, laminar receptor distribution patterns for cortical areas are quantified by sampling density profiles in a series of regions of interest (ROIs) from digitalized autoradiographs and computing a mean profile per ROI. These ROI mean profiles are then corrected for distortions in the laminar pattern introduced by cortical folding and averaged to yield a mean profile per area, receptor and hemisphere. Differences in mean binding site concentration between areas are quantified by the asymmetry coefficient which is the difference of the mean concentrations divided by their sum. To quantify differences in laminar receptor distribution patterns between areas, the effects of absolute binding site concentration are first removed by dividing each profile by its mean value. Differences in the laminar pattern were then quantified by calculating the Euclidean distance between these mean corrected profiles. For single subject analysis, we propose a permutation test, comparing the differences between the mean profiles for two areas to differences between groups of profiles randomly assembled from all ROIs sampled in either area. Group inference can then be based on a between-subject conjunction analysis after correcting p-values to control for multiple comparisons. The feasibility of the presented approach is demonstrated by an exemplary analysis of the neurochemical differences between the ventral parts of the second and the third visual area.

Autoradiography↗

PubMed2006

Fate of cadmium in Elodea canadensis.

Elodea canadensis is a submersed macrophytes, widely distributed in stormwater treatment ponds and able to remove heavy metals from water. This study examines the Cd uptake, translocation, and efflux patterns in Elodea. Several experiments were set up in a climate chamber. To study the root and shoot Cd uptake, living and dead roots and shoots were treated with (109)Cd in one- and two-compartment systems. Furthermore, to examine Cd translocation and distribution, either roots or shoots were treated with (109)Cd. Finally, the efflux of Cd from roots and shoots, respectively, to the external solution was studied after loading whole plants with (109)Cd. Results from the two compartment studies show that Cd is accumulated via direct uptake by both roots and shoots of Elodea. The Cd accumulation proved not to be metabolically dependent in Elodea, and the apoplastic uptake in particular was decreased by Cd pretreatment. In one week, up to 23% of the root uptake was translocated to the shoots, while about 2% of the Cd accumulated by shoots was translocated to the roots. Thus, slight dispersion of Cd is possible, while metal immobilization will not be directly mediated via the Elodea plant. The efflux experiment proved that both shoots of dead plants and roots of living plants had a faster efflux than did shoots of living plants. This information is relevant for an understanding of the fate of Cd in stormwater treatment ponds with Elodea.

Autoradiography↗

PubMed2006

Microautoradiographic localisation of [3H]sucrose and [3H]mannitol in Robinia pseudoacacia pulvinar tissues during phytochrome-mediated nyctinastic closure.

We have analysed the incorporation of [(3)H]sucrose and [(3)H]mannitol in pulvinar motor cells of Robinia pseudoacacia L. during phytochrome-mediated nyctinastic closure. Pairs of leaflets, excised 2 h after the beginning of the photoperiod, were fed with 50 mM [(3)H]sucrose or [(3)H]mannitol, irradiated with red (15 min) or far-red (5 min) light and placed in the dark for 2-3 h. Label uptake was measured in whole pulvini by liquid scintillation counting. The distribution of labelling in pulvinar sections was assessed by both light and electron microautoradiography. [(3)H]Sucrose uptake was twice that of [(3)H]mannitol incorporation in both red- and far-red-irradiated pulvini. In the autoradiographs, [(3)H]sucrose and [(3)H]mannitol labelling was localised in the area from the vascular bundle to the epidermis, mainly in vacuoles, cytoplasm, and cell walls. Extensor and flexor protoplasts displayed a different distribution of [(3)H]sucrose after red and far-red irradiation. Far-red light drastically reduced the [(3)H]sucrose incorporation in extensor protoplasts and caused a slight increase in internal flexor protoplasts. After red light treatment, no differences in [(3)H]sucrose labelling were found between extensor and flexor protoplasts. Our results indicate a phytochrome control of sucrose distribution in cortical motor cells and seem to rule out the possibility of sucrose acting as an osmoticum.

Autoradiography↗