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Biomedical subjects

M E Churchill

Publications and source records attributed to M E Churchill.

At least 19 recordsLinked to original sources
PubMed1996

A purified mariner transposase is sufficient to mediate transposition in vitro.

Mariners are a widespread and diverse family of animal transposons. Extremely similar mariners of the irritans subfamily are present in the genomes of three divergent insect host species, which strongly suggests that species-specific host factors are unnecessary for mobility. We tested this hypothesis by examining the activity of a purified transposase from one of these elements (Himar1) present in the horn fly, Haematobia irritans. Himar1 transposase was sufficient to reproduce transposition faithfully in an in vitro inter-plasmid transposition reaction. Further analyses showed that Himar1 transposase binds to the inverted terminal repeat sequences of its cognate transposon and mediates 5' and 3' cleavage of the element termini. Independence of species-specific host factors helps to explain why mariners have such a broad distribution and why they are capable of horizontal transfer between species.

Animals↗
PubMed1995

HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.

The high mobility group (HMG) protein HMG-D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. Finally, we show that HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region.

Animals↗
PubMed1994

Crystal structure of a peptide complex of anti-influenza peptide antibody Fab 26/9. Comparison of two different antibodies bound to the same peptide antigen.

The three-dimensional structure of the complex of a second anti-peptide antibody (Fab 26/9) that recognizes the same six-residue epitope of an immunogenic peptide from influenza virus hemagglutinin (HA1; 75-110) as Fab 17/9 with the peptide has been determined at 2.8 A resolution. The amino acid sequence of the variable region of the 26/9 antibody differs in 24 positions from that of 17/9, the first antibody in this series for which several ligand-bound and free structures have been determined and refined. Comparison of the 26/9-peptide with the 17/9-peptide complex structures shows that the two Fabs are very similar (r.m.s.d. 0.5 to 0.8 A) and that the peptide antigen (101-107) has virtually the same conformation (r.m.s.d. 0.3 to 0.8 A) when bound to both antibodies. A sequence difference in the 26/9 binding pocket (L94; His in 26/9, Asn in 17/9) results in an interaction with a bound water molecule that is not seen in the 17/9 structures. Epitope mapping shows that the relative specificity of 26/9 and 17/9 antibodies for individual positions of the peptide antigen are slightly different. Amino acid substitutions in the peptide, particularly at position SerP107, are tolerated to different extents by 17/9 and 26/9. Structural and sequence analysis suggests that amino acid differences near the peptide-binding site are responsible for altering slightly the specificity of 26/9 for three peptide residues and illustrates how amino acid substitutions can modify antibody-antigen interactions and thereby modulate antibody specificity.

Amino Acid Sequence↗
PubMed1994

The solution structure and dynamics of the DNA-binding domain of HMG-D from Drosophila melanogaster.

BACKGROUND: The HMG-box is a conserved DNA-binding motif that has been identified in many high mobility group (HMG) proteins. HMG-D is a non-histone chromosomal protein from Drosophila melanogaster that is closely related to the mammalian HMG-box proteins HMG-1 and HMG-2. Previous structures determined for an HMG-box domain from rat and hamster exhibit the same global topology, but differ significantly in detail. It has been suggested that these differences may arise from hinge motions which allow the protein to adapt to the shape of its target DNA. RESULTS: We present the solution structure of HMG-D determined by NMR spectroscopy to an overall precision of 0.85 A root mean squared deviation (rmsd) for the backbone atoms. The protein consists of an extended amino-terminal region and three alpha-helices that fold into a characteristic 'L' shape. The central core region of the molecule is highly stable and maintains an angle of approximately 80 degrees between the axes of helices 2 and 3. The backbone dynamics determined from 15N NMR relaxation measurements show a high correlation with the mean residue rmsd determined from the calculated structures. CONCLUSIONS: The structure determined for the HMG-box motif from HMG-D is essentially identical to the structure determined for the B-domain of mammalian HMG-1. Since these proteins have significantly different sequences our results indicate that the global fold and the mode of interaction with DNA are also likely to be conserved in all eukaryotes.

Amino Acid Sequence↗
PubMed1994

Detection of retinoblastoma gene deletions in spontaneous and radiation-induced mouse lung adenocarcinomas by polymerase chain reaction.

A polymerase chain reaction (PCR) technique has been developed to detect deletions in the mouse retinoblastoma gene using histological sections from radiation-induced and spontaneous tumors as the DNA source. Six mouse Rb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. The absence of any of these fragments relative to control PCR products on a Southern blot indicated a deletion of that portion of the mouse Rb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death. Spontaneous tumors as well as those from irradiated mice (5.69 Gy 60Co gamma rays or 0.6 Gy JANUS neutrons, which have been found to have approximately equal radiobiological effectiveness) were analyzed for mouse Rb deletions. Tumors in 6 neutron-irradiated mice had no mouse Rb deletions. However, 1 of 6 tumors from gamma-irradiated mice (17%) and 6 of 18 spontaneous tumors from unirradiated mice (33%) showed a deletion in one or both mouse Rb alleles. All deletions detected were in the 5' region of the mouse Rb gene.

Adenocarcinoma↗
PubMed1993

dHMG-Z, a second HMG-1-related protein in Drosophila melanogaster.

We report the identification of dHMG-Z, a gene related to dHMG-D and encoding a second invertebrate homologue of HMG 1 protein. The encoded proteins is 65% identical to dHMG-D protein, and also contains a single HMG-box as the DNA recognition motif. Analogous to dHMG-D, two transcripts are observed for dHMG-Z which are differentially regulated, and are the product of zygotic transcription unlike the dHMG-D transcripts which arise from both maternal and zygotic transcription. The genes for dHMG-D and dHMG-Z are located on adjacent loci in the genome and each contains two introns. The position of the second intron in the coding region is conserved between the two genes suggesting a common origin via gene duplication.

Animals↗
PubMed1993

A 5-4 pyrimidine-pyrimidone photoproduct produced from mixtures of thymine and 4-thiouridine irradiated with 334 nm light.

The nucleoside 4-thiouridine, present in some bacterial tRNA species, is known to be a chromophore and a target for near-UV light-induced growth delay and also mediates both photoprotection and near-UV cell killing in various bacterial strains. To investigate the photoreaction of 4-thiouridine with DNA or its precursors, we irradiated aqueous mixtures of thymine and 4-thiouridine with 334 nm light and then separated photoproducts using two or more stages of reversed-phase high performance liquid chromatography. The two equally abundant major photoproducts were analyzed by UV absorbance spectrophotometry, fast-atom bombardment and electron-impact mass spectrometry, and 1H- and 13C-NMR spectroscopy, and have been identified as two diastereomers of 6-hydroxy-5-[1-(beta-D-erythro-pentofuranosyl)-4'-pyrimidin-2'- one]dihydrothymine (O6hThy[5-4]Pdo), of molecular weight = 370.32. These two diastereomers, although stable at room temperature or below, are interconvertible by heating (90 degrees C for 5 min) in aqueous solution. The possible biological significance of this photoproduct is discussed, and an application as a crosslinker for oligonucleotides to selectively block replication is suggested.

Magnetic Resonance Spectroscopy↗
PubMed1991

Protein motifs that recognize structural features of DNA.

Many proteins have the ability to bind to a variety of DNA sequences, the recognition of which depends on some general rather than specific feature of the DNA. Some of the protein motifs that are responsible for this type of recognition have been identified in transcription factors and chromosomal proteins. These motifs consist of sequences capable of forming a structural framework in which positively charged residues can be arranged so that binding to a particular DNA structure is preferred.

Amino Acid Sequence↗
PubMed1991

Repair of near-visible- and blue-light-induced DNA single-strand breaks by the CHO cell lines AA8 and EM9.

The induction of single-strand breaks (SSB) and the kinetics of SSB repair were measured in two Chinese hamster ovary cell lines irradiated with monochromatic photons of near-visible radiation (405 nm) and blue light (434 nm). The radiosensitive and UV-A-sensitive mutant line EM9 is known to repair SSB induced by ionizing radiation or 365-nm UV-A more slowly than the parent line AA8. At the 10% survival level, EM9 cells were 1.7- and 1.6-fold more sensitive than AA8 cells to 405 and 434 nm radiation, respectively. This sensitivity was not due to differences in induction of SSB because AA8 and EM9 cells accumulated the same number of initial breaks when irradiated at 0.5 degrees C with either 405 nm (5.9 SSB per MJ/m2) or 434 nm (5.1 SSB per MJ/m2), as measured by alkaline elution. When the cells repaired these SSB at 37 degrees C in full culture medium, biphasic repair kinetics were observed for both cell lines. In both phases of repair, EM9 cells repaired breaks induced by both wavelengths more slowly than did AA8 cells. The t1/2 values for the repair phases for 405-nm-induced SSB were 3.8 and 150 min for EM9, and 1.5 and 52 min for AA8; the corresponding values for repair of 434 nm breaks were 3.7 and 39 min for EM9, and 2.0 and 30 min for AA8. Because of this slower repair, EM9 cells left more SSB unrepaired after 90 min than did AA8 cells for both wavelengths.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Correlation between cell survival and DNA single-strand break repair proficiency in the Chinese hamster ovary cell lines AA8 and EM9 irradiated with 365-nm ultraviolet-A radiation.

Cell survival parameters and the induction and repair of DNA single-strand breaks were measured in two Chinese hamster ovary cell lines after irradiation with monochromatic UVA radiation of wavelength 365 nm. The radiosensitive mutant cell line EM9 is known to repair ionizing-radiation-induced single-strand breaks (SSB) more slowly than the parent line AA8. EM9 was determined to be 1.7-fold more sensitive to killing by 365-nm radiation than AA8 at the 10% survival level, and EM9 had a smaller shoulder region on the survival curve (alpha = 1.76) than AA8 (alpha = 0.62). No significant differences were found between the cell lines in the initial yields of SSB induced either by gamma-radiation (as determined by alkaline sucrose gradient sedimentation) or by 365-nm UVA (as determined by alkaline elution). For measurement of initial SSB, cells were irradiated at 0.5 degrees C to minimize DNA repair processes. Rejoining of 365-nm induced SSB was measured by irradiating cells at 0.5 degrees C, allowing them to repair at 37 degrees C in full culture medium, and then quantitating the remaining SSB by alkaline elution. The repair of these breaks followed biphasic kinetics in both cell lines. EM9 repaired the breaks more slowly (t1/2 values of 1.3 and 61.3 min) than did AA8 (t1/2 values of 0.9 and 53.3 min), and EM9 also left more breaks unrepaired 90 min after irradiation (24% vs 8% for AA8). Thus, the sensitivity of EM9 to 365-nm radiation correlated with its deficiency in repairing DNA lesions revealed as SSB in alkaline elution.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Asymmetric structure of a three-arm DNA junction.

We present here experimental evidence that three-arm branched DNA molecules form an asymmetric structure in the presence of Mg2+. Electrophoretic mobility and chemical and enzymatic footprinting experiments on a three-arm branched DNA molecule formed from three 16-mer strands are described. The electrophoretic mobilities of three species of a three-arm junction in which pairs of arms are extended are found to differ in the presence of Mg2+: one combination of elongated arms migrates significantly faster than the other two. This effect is eliminated in the absence of Mg2+, leading us to suggest that the three-arm DNA junction forms an asymmetric structure due to preferential stacking of two of the arms at the junction in the presence of Mg2+. The pattern of self-protection of each 16-mer strand of the core complex exposed to Fe(II).EDTA and DNase I scission is unique, consistent with formation of an asymmetric structure in the presence of Mg2+. We conclude that three-arm junctions resemble four-arm junctions in showing preferential stacking effects at the branch site. Comparison of the scission patterns of linear duplexes and the branched trimer by the reactive probes methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and Cu(I)-[o-phenanthroline]2 [(OP)2CuI] further indicates that the branch point represents a site of enhanced binding for drugs, as it does in the four-arm case. Reaction with diethyl pyrocarbonate (DEPC), a purine-specific probe sensitive to conformation, is enhanced at the branch site, consistent with loosening of base pairing or unpairing at this point.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

Detection of drug binding to DNA by hydroxyl radical footprinting. Relationship of distamycin binding sites to DNA structure and positioned nucleosomes on 5S RNA genes of Xenopus.

We report the use of hydroxyl radical footprinting to analyze the interaction of distamycin and actinomycin with the 5S ribosomal RNA genes of Xenopus. There is a qualitative difference in the hydroxyl radical footprints of the two drugs. Distamycin gives a conventional (albeit high-resolution) footprint, while actinomycin does not protect DNA from hydroxyl radical attack, but instead induces discrete sites of hyperreactivity. We find concentration-dependent changes in the locations of distamycin binding sites on the somatic 5S gene of Xenopus borealis. A high-affinity site, containing a G.C base pair, is replaced at higher levels of bound drug by a periodic array of different lower affinity sites that coincide with the places where the minor groove of the DNA would face in toward a nucleosome core that is known to bind to the same sequence. These results suggest that distamycin recognizes potential binding sites more by the shape of the DNA than by the specific sequence that is contained in the site and that structures of many sequences are deformable to a shape that allows drug binding. We discuss the utility of hydroxyl radical footprinting of distamycin for investigating the underlying structure of DNA.

Animals↗
PubMed1990

Mode of interaction of the zinc finger protein TFIIIA with a 5S RNA gene of Xenopus.

The zinc finger protein TFIIIA, a positive transcription factor of the 5S RNA gene, binds to an internal control region of 50 nucleotides. Two modes of binding have been considered for the TFIIIA-DNA complex, one of which has been proposed on the basis of nuclease and chemical protection experiments and the other on model building. Since then, evidence has accumulated on the structures of individual components of the complex--for example, zinc finger polypeptides studied by NMR and a segment of the binding site analyzed by x-ray crystallography, but no high-resolution structural data on the TFIIIA-DNA complex itself are available. Probes used previously to study the TFIIIA-DNA complex do not react with every nucleotide of DNA, unlike hydroxyl radical, which cleaves DNA at every backbone position. We describe here the quantitative analysis of high-resolution hydroxyl radical footprints and suggest how the array of zinc fingers might interact with the double helix.

Animals↗
PubMed1990

Photosensitized damage to supercoiled plasmid DNA induced by 334-nm radiation in the presence of 2-thiouracil consists of alkali- and piperidine-labile sites as well as frank strand breaks.

A covalently closed, supercoiled plasmid was irradiated with 334-nm ultraviolet radiation in the presence of the naturally occurring photosensitizer 2-thiouracil (s2Ura). After irradiation, some DNA samples were treated to reveal labile sites. Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed forms, and the DNA bands were quantitated by fluorescence scanning. Irradiation of the plasmid in the absence of s2Ura induced small numbers of frank DNA strand breaks (FSB), alkali-labile sites (ALS), and piperidine-labile sites (PLS). The induction of each of these lesions was enhanced 30 times when s2Ura was present during aerobic irradiation. Anoxia, as well as the hydroxyl radical scavengers acetate and formate, inhibited the formation of all three lesion types. The relative proportions of the three lesion types produced by several DNA damaging treatments were measured. Hydrogen peroxide, gamma-irradiation, and s2Ura photosensitization produced nearly identical damage proportions, with PLS: FSB ratios of 1.25:1, 0.78:1, and 0.84:1, respectively. Treatment with singlet oxygen [data from Blazek et al. (1989) Photochem. Photobiol. 48, 607-613] produced much different proportions, with a PLS:FSB ratio of 4.1:1. These results may indicate a role for hydroxyl radical in s2Ura-photosensitized DNA damage.

DNA Damage↗
PubMed1989

'SPKK' motifs prefer to bind to DNA at A/T-rich sites.

The termini of histone H1 and sea urchin spermatogenous H1 and H2B, which are essential for correct chromatin condensation, often contain repeats of the sequence SPK(R)K(R). A special type of beta-turn structural motif has been proposed for this sequence, and it has been shown that a segment of the sea urchin sperm H1 N terminus, which has six repeats of the motif (S6 peptide), binds to DNA and competes with the DNA binding drug Hoechst 33258. Here, we demonstrate by quantitative analysis of hydroxyl radical footprints that the synthetic oligopeptide, SPRKSPRK (S2), and the S6 peptide prefer to bind to the minor groove of DNA at the same A/T-rich sites. The locations of these binding sites are similar to Hoechst, but the sequence specificity of the oligopeptides is lower than that of Hoechst, and the detailed protection patterns differ slightly. We suggest that these small peptides and Hoechst recognize similar sequence-dependent features of the local architecture of DNA.

Adenine↗
PubMed1988

Construction and analysis of monomobile DNA junctions.

Immobile DNA junctions are complexes of oligomeric DNA strands that interact to yield branched structures in which the branch point cannot migrate. This is achieved by minimizing the sequence symmetry in the flanking arms, so that base pairs lock at the branch site. Here, we report the design, synthesis, and analysis of two semimobile junctions, structures in which a controlled extent of branch point migratory freedom is deliberately introduced. We have constructed two minimally symmetric four-arm semimobile junctions from synthetic deoxy 17-mers. These junctions, termed "monomobile", contain a single pair of base pairs (A-T or C-G) which can migrate at the site of branching, while the rest of the junction is immobile. We have demonstrated by gel electrophoresis techniques that these junctions form and that they have the predicted 1:1:1:1 stoichiometry. We have compared these junctions with the immobile junction on which they are based, by means of hydroxyl radical protection experiments. From these data, both migratory conformers can be seen to coexist in solution. The semimobile junction with the C-G base pair has the same crossover and stacking pattern observed for the immobile junction, while the junction with the A-T base pair has the opposite pattern. We conclude that crossover and stacking patterns are a direct consequence of the base pairs which flank the junction. In addition, the data indicate that the crossover pattern biases for these junctions are much greater than are the migratory biases.

Base Composition↗