Photofootprinting DNA in vitro.
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Animals↗
Biomedical subjects
M M Becker
Publications and source records attributed to M M Becker.
Origin of ultraviolet damage in DNA.
A novel ultraviolet (u.v.) footprinting technique has been used to analyze the formation of u.v. photoproducts at 250 bases of a 5 S rRNA gene under conditions where the gene is either double or single-stranded. Because many more types of u.v. damage can be detected by the u.v. footprinting technique than has been previously possible, we have been able to examine in detail why certain bases in DNA are damaged by u.v. light while others are not. Our measurements demonstrate that the ability of u.v. light to damage a given base in DNA is determined by two factors, the sequence of the DNA in the immediate vicinity of the photoproduct, and the flexibility of the DNA at the site of the photoproduct. For pyrimidines, the predominant photoreaction in double-stranded DNA involves covalent dimerization between adjacent pyrimidine residues. Dimerization is much easier in melted DNA because the geometrical changes required for adjacent pyrimidine residues to dimerize are easier in single-stranded DNA. The absorption of a u.v. photon cannot simultaneously induce the geometrical changes required for adjacent pyrimidines or other bases to dimerize with one another. Rather, upon the absorption of a u.v. photon, only those thermally excited bases that are in a geometry capable of easily forming a photodimer during excitation, can photoreact. In contrast to adjacent pyrimidines, non-adjacent pyrimidines (pyrimidines flanked on either side by a purine) do not readily form u.v. photoproducts in double-stranded DNA. Because photoreactions at non-adjacent pyrimidine residues are greatly enhanced in single-stranded DNA, their failure to form in double-helical DNA is attributed to torsional constraints imposed by the double helix which make it difficult for non-adjacent pyrimidines to adopt a geometry necessary for photoreaction. Although purines are believed to be resistant to u.v. damage, our measurements demonstrate that at moderate u.v. dosages purines which are flanked on their 5' side by two or more contiguous pyrimidines readily form u.v. photoproducts in double-stranded DNA. Flanking pyrimidines appear to activate purine photoreactions by transferring triplet excitation energy to the purine. Melting of the DNA helix greatly inhibits the ability of flanking pyrimidines to activate purine photoreactions, presumably by disrupting intimate orbital overlap required for triplet transfer.
Base Sequence↗
B----A transitions within a 5 S ribosomal RNA gene are highly sequence-specific.
The UV footprinting technique has been used to detect and map, at single nucleotide resolution, the formation of A conformations within a sea urchin 5S ribosomal RNA gene. Increasing amounts of the dehydrating agent, trifluorethanol, were used to induce the B----A transition. Our measurements argue that the B----A transition is highly sequence-specific. Fourteen different sequences within a fragment of DNA bearing the 5 S gene were found to undergo the B----A transition independently of one another. There is a striking relationship between the midpoint of the B----A transition for each stretch of DNA and its (G+C) content. DNA sequences at the boundary between A and B conformations do not appear to be significantly distorted. A (dAdT)8 tract at the 3' end of the 5 S gene undergoes the B----A transition in two cooperative steps suggesting that for some sequences the B----A transition may actually proceed through the formation of a previously unidentified intermediate. Although the sequence specificity of the B----A transition may be exploited by regulatory proteins when they bind DNA, our measurements argue that binding of the Xenopus laevis transcription factor 111A to 5 S genes does not.
Animals↗
Genomic footprinting in mammalian cells with ultraviolet light.
A simple and accurate genomic primer extension method has been developed to detect ultraviolet footprinting patterns of regulatory protein-DNA interactions in mammalian genomic DNA. The technique can also detect footprinting or sequencing patterns introduced into genomic DNA by other methods. Purified genomic DNA, containing either damaged bases or strand breaks introduced by footprinting or sequencing reactions, is first cut with a convenient restriction enzyme to reduce its molecular weight. A highly radioactive single-stranded DNA primer that is complementary to a region of genomic DNA whose sequence or footprint one wishes to examine is then mixed with 50 micrograms of restriction enzyme-cut genomic DNA. The primer is approximately 100 bases long and contains 85 radioactive phosphates, each of specific activity 3000 Ci/mmol (1 Ci = 37 GBq). A simple and fast method for preparing such primers is described. Following brief heat denaturation at 100 degrees C, the solution of genomic DNA and primer is cooled to 74 degrees C and a second solution containing Taq polymerase (Thermus aquaticus DNA polymerase) and the four deoxynucleotide triphosphates is added to initiate primer extension of genomic DNA. Taq polymerase extends genomic hybridized primer until its polymerization reaction is terminated either by a damaged base or strand break in genomic DNA or by the addition of dideoxynucleotide triphosphates in the polymerization reaction. The concurrent primer hybridization-extension reaction is terminated after 5 hr and unhybridized primer is digested away by mung bean nuclease. Primer-extended genomic DNA is then denatured and electrophoresed on a polyacrylamide sequencing gel, and radioactive primer extension products are revealed by autoradiography. By using this method we demonstrate that it is possible to footprint with ultraviolet light, in intact monkey cells, regulatory protein--DNA interactions along a single copy of a simian virus 40 viral genome integrated into the monkey genome.
Animals↗
[Alkaline calcium hydroxide containing pulp capping material].
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Calcium Hydroxide↗
"Ultraviolet footprinting" accurately maps sequence-specific contacts and DNA kinking in the EcoRI endonuclease-DNA complex.
The "UV footprinting" technique has been used to detect contacts between EcoRI endonuclease and its recognition sequence at single nucleotide resolution. Comparison of the UV-footprinting results to the published crystal structure of the EcoRI endonuclease-DNA complex allows us to determine how UV light detects protein-DNA contacts. We find that kinking of the DNA helix in the complex greatly enhances the UV photoreactivity of DNA at the site of the kink. In contrast to kinking, contacts between the endonuclease and the DNA bases inhibit the UV photoreactivity of DNA. Similar analysis of a proteolytically modified endonuclease that exhibits the same sequence specificity as wild-type enzyme but that does not cleave DNA supports these conclusions. Furthermore, detection of enhanced photoreactivity at the same kink in the modified enzyme-DNA complex allows us to conclude that the loss of cleavage activity by the modified endonuclease is not due to its failure to kink DNA.
Base Sequence↗
Selective visualization of gene structure with ultraviolet light.
The ability of the ultraviolet (UV) "footprinting" technique to detect chromatin has been investigated in vitro. Two basic types of chromatin, a phased nucleosome and a phased nucleosome containing a phased H1 protein, have been reconstituted onto a cloned 5S ribosomal RNA gene from sea urchin. The histone-DNA interactions in each complex have been probed with exonuclease III, DNase I, dimethyl sulfate, and UV light. Whereas DNase I and exonuclease III readily detect interactions between histones and DNA, UV light and dimethyl sulfate do not. In contrast to histone-DNA interactions, we demonstrate that intimate sequence-specific contacts between the same sea urchin 5S DNA and the Xenopus laevis transcription factor IIIA (TFIIIA) are readily detected with UV light. Since the sensitivity of UV light for TFIIIA contacts is similar to its sensitivity for other regulatory protein-DNA contacts, these studies demonstrate the feasibility of using UV light to selectively visualize regulatory protein-DNA interactions in vivo with little or no interference from histone-DNA interactions.
Animals↗
Selenium increases hydrogenase expression in autotrophically cultured Bradyrhizobium japonicum and is a constituent of the purified enzyme.
We have investigated the effect of added selenite on autotrophic growth and the time course of hydrogen oxidation derepression in Bradyrhizobium japonicum 122DES cultured in a medium purified to remove selenium compounds. In addition, hydrogenase was purified to near homogeneity and examined for the specific incorporation of Se into the enzyme. The addition of Se at 0.1 microM significantly increased total cell protein and hydrogenase specific activity of harvested cells. Also, the addition of SeO3(2-) enhanced the time course of hydrogenase derepression by 133%, whereas VO3, AsO2(2-), SO2(2-), and TeO3(2-) failed to substantially affect hydrogenase derepression. During the final chromatographic purification of hydrogenase, a striking coincidence in peaks of protein content, Se radioactivity, and hydrogenase activity of fractions was obtained. The total Se content expressed per milligram of protein increased manyfold during the purification procedure. The mean Se content of the purified hydrogenase was 0.56 +/- 0.13 mol of Se per mol of enzyme. These results indicate that Se is an important element in the H2 metabolism of B. japonicum and that hydrogenase from B. japonicum is a seleno protein.
Culture Media↗
Alterations in the synthesis of proteins in hepatocytes of rainbow trout fed cyclopropenoid fatty acids.
Hepatocytes from rainbow trout reared on a diet containing cyclopropenoid fatty acids were analyzed for alterations in protein composition and synthesis by double label experiments. Both cytosolic and microsomal hepatocyte fractions were investigated. In the cytosolic fraction, the synthesis of proteins in the range of 68,000 to 74,000 daltons were significantly decreased. The identity of these proteins remains uncertain. A pronounced depression in both the mass and apparent synthesis of a 200,000 to 240,000 dalton microsomal protein was also observed. Immunoblotting with antibodies raised against goose acetyl-CoA carboxylase and avidin-peroxidase staining suggest that this protein is acetyl-CoA carboxylase. Moreover, synthesis of this protein as well as mass of the protein in cyclopropenoid fatty acid-fed fish were less than 20% of that found in control fish.
Acetyl-CoA Carboxylase↗