Search PubMed⌕ Search

Biomedical subjects

A Rich

Publications and source records attributed to A Rich.

At least 127 records · Page 7Linked to original sources
PubMed1993

Conformational influence of the ribose 2'-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes.

We have crystallized three double-helical DNA-RNA chimeric duplexes and determined their structures by X-ray crystallography at resolutions between 2 and 2.25 A. The two self-complementary duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC).r(GCG)d(TATACCC), were found to adopt A-type conformations. The crystal structures are non-isomorphous, and the crystallographic environments for the three chimeras are different. A number of intramolecular interactions of the ribose 2'-hydroxyl groups contribute to the stabilization of the A-conformation. Hydrogen bonds between 2'-hydroxyls and 5'-oxygens or phosphate oxygens, in addition to the previously observed hydrogen bonds to 1'-oxygens of adjacent riboses and deoxyriboses, are observed in the DNA-RNA chimeric duplexes. The crystalline chimeric duplexes do not show a transition between the DNA A- and B-conformations. CD spectra suggest that the Okazaki fragment assumes an A-conformation in solution as well. In this molecule the three RNA residues may therefore lock the complete decamer in the A-conformation. Crystals of an all-DNA strand with the same sequence as the self-complementary chimeras show a morphology which is different from those of the chimera crystals. Moreover, the oligonucleotide does not match any of the sequence characteristics of DNAs usually adopting the A-conformation in the crystalline state (e.g., octamers with short alternating stretches of purines and pyrimidines). In DNA-RNA chimeric duplexes, it is therefore possible that a single RNA residue can drive the conformational equilibrium toward the A-conformation.

Base Sequence↗
PubMed1993

B-DNA to Z-DNA structural transitions in the SV40 enhancer: stabilization of Z-DNA in negatively supercoiled DNA minicircles.

During replication and transcription, the SV40 control region is subjected to significant levels of DNA unwinding. There are three, alternating purine-pyrimidine tracts within this region that can adopt the Z-DNA conformation in response to negative superhelix density: a single copy of ACACACAT and two copies of ATGCATGC. Since the control region is essential for both efficient transcription and replication, B-DNA to Z-DNA transitions in these vital sequence tracts may have significant biological consequences. We have synthesized DNA minicircles to detect B-DNA to Z-DNA transitions in the SV40 enhancer, and to determine the negative superhelix density required to stabilize the Z-DNA. A variety of DNA sequences, including the entire SV40 enhancer and the two segments of the enhancer with alternating purine-pyrimidine tracts, were incorporated into topologically relaxed minicircles. Negative supercoils were generated, and the resulting topoisomers were resolved by electrophoresis. Using an anti-Z-DNA Fab and an electrophoretic mobility shift assay, Z-DNA was detected in the enhancer-containing minicircles at a superhelix density of -0.05. Fab saturation binding experiments demonstrated that three, independent Z-DNA tracts were stabilized in the supercoiled minicircles. Two other minicircles, each with one of the two alternating purine-pyrimidine tracts, also contained single Z-DNA sites. These results confirm the identities of the Z-DNA-forming sequences within the control region. Moreover, the B-DNA to Z-DNA transitions were detected at superhelix densities observed during normal replication and transcription processes in the SV40 life cycle.

DNA, Superhelical↗
PubMed1993

Polynucleotide binding to macrophage scavenger receptors depends on the formation of base-quartet-stabilized four-stranded helices.

Macrophage scavenger receptors exhibit unusually broad, but circumscribed, polyanionic ligand-binding specificity. For example, the polyribonucleotides poly(I) and poly(G) are ligands but poly(A) and poly(C) are not. To further investigate the molecular basis of this polynucleotide-binding specificity, we tested the capacity of various oligodeoxyribonucleic acids to inhibit the scavenger receptor-mediated degradation of 125I-labeled acetylated low density lipoprotein by Chinese hamster ovary cells expressing the type I bovine scavenger receptor. A series of short oligodeoxyriboguanines (dGn, where 5 < or = n < or = 37) were effective inhibitors. The dG6, dG12, and dA5G37 members of this series were shown by circular dichroism and UV spectroscopy to be assembled into four-stranded helices stabilized by G-quartets. [32P]dA5G37 bound directly to scavenger receptors. Partial or complete denaturation of the quadruplex structures of these oligonucleotides by boiling destroyed their inhibitory activity. Receptor activity was also inhibited by d(T4G4)4, a telomere-like oligonucleotide which forms an intramolecular quadruplex. In addition, conversion of the four-stranded potassium salt of poly(I) to the single-stranded lithium salt dramatically reduced its inhibitory activity. Addition of KCl to the Li+ salt resulted in the reformation of poly(I)'s quadruplex structure and restoration of its inhibitory activity. A variety of single-stranded and double-stranded oligo- and polydeoxyribonucleotides (e.g. dA37, HaeIII restriction fragments of phi X174) exhibited very little or no inhibitory activity. Thus, a base-quartet-stabilized four-stranded helix appears to be a necessary structural determinant for polynucleotide binding to and inhibition of scavenger receptors. This conformational requirement accounts for the previously unexplained polyribonucleotide-binding specificity of scavenger receptors. The spatial distribution of the negatively charged phosphates in polynucleotide quadruplexes may form a charged surface which is complementary to the positively charged surface of the collagenous ligand-binding domain of the scavenger receptor.

Animals↗
PubMed1993

Dihydropyridine-sensitive calcium channels expressed in canine colonic smooth muscle cells.

Experiments were performed to identify and characterize the types of calcium channels that regulate inward calcium current in canine colonic smooth muscle. Freshly dispersed smooth muscle cells from the circular layer of the canine proximal colon were used. Single-channel currents were measured with 80 mM Ba2+ as the charge carrier. Small-conductance (10 +/- 2 pS, EBa = 46 +/- 11 mV, n = 9) and large-conductance (21 +/- 1 pS, EBa = 52 +/- 3 mV, n = 19) single-channel currents were observed during depolarizing voltage steps positive to -30 mV. Both types of single-channel currents were inhibited by the addition of 10(-6) M nifedipine to the bath solution. The smaller current was infrequently observed and therefore was not further characterized. Open probability (P(o)) of the larger current amplitude was strongly dependent on voltage. Activation curves were well described by a Boltzmann function with half activation occurring at 4 mV, and a 5-mV increase in membrane potential resulted in an e-fold increase in P(o). BAY K 8644 (1 microM) shifted the activation curve to the left while nifedipine (1 microM) resulted in a right shift. Molecular analysis showed that only the C class of Ca2+ channel alpha 1-subunit is expressed in this tissue. Furthermore, only a single splice variant (rbc-II) was observed. The results suggest that a single class of dihydropyridine-sensitive calcium channels regulates inward calcium current in canine colonic smooth muscle cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Enhanced proliferation of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes in a plasma-derived cell culture fluid.

The proliferation and activation of murine and human T-lymphocytes in a high-protein lymphocyte culture fluid (LCF) is compared to that of cells cultured in 10% fetal bovine serum (FBS). Tumor-infiltrating lymphocytes (TIL) proliferate exponentially in the LCF for up to 46 days and generate cell numbers that are nearly 100,000-fold greater than cells cultured in FBS. This rapid growth of T cells in LCF could have an important impact in adoptive immunotherapy and gene therapy since cell growth is a limiting factor in these technologies.

Animals↗
PubMed1992

Large scale chemical synthesis, purification and crystallization of RNA-DNA chimeras.

RNA-DNA chimeras, in which both DNA and RNA monomers are site-specifically substituted in the same strand, may be prepared only by chemical synthesis. Biochemical studies have revealed a number of surprising and subtle effects resulting from the insertion of either a ribonucleotide into a DNA strand or a deoxyribonucleotide into an RNA strand. The availability of large quantities of these chimeras allows for their crystallization and subsequent x-ray structure determination. We describe a flexible and efficient method for the large-scale preparation of these compounds, their purification, and their crystallization. The methodology is based on a combination of existing DNA phosphoramidite synthons and those recently introduced for the preparation of biochemically active RNA1. We demonstrate that these two different synthons are compatible, produce large quantities of nucleic acid needed for physical studies, and that high resolution diffraction quality crystals may be grown from these chimeras. Of the duplex chimeras synthesized and crystallized, [r(G)d(CGTATACGC)]2, [d(GCGT)r(A)d(TACGC)]2 and [r(GCG)d(TATACCC) + d(GGGTATACGC)] form A-helices and d(CG)r(CG)d(CG)]2 forms a left-handed Z-helix.

Base Sequence↗
PubMed1992

Crystal structure of four-stranded Oxytricha telomeric DNA.

The sequence d(GGGGTTTTGGGG) from the 3' overhang of the Oxytricha telomere has been crystallized and its three-dimensional structure solved to 2.5 A resolution. The oligonucleotide forms hairpins, two of which join to make a four-stranded helical structure with the loops containing four thymine residues at either end. The guanine residues are held together by cyclic hydrogen bonding and an ion is located in the centre. The four guanine residues in each segment have a glycosyl conformation that alternates between anti and syn. There are two four-stranded molecules in the asymmetric unit showing that the structure has some intrinsic flexibility.

Animals↗
PubMed1992

Crystal structure of an Okazaki fragment at 2-A resolution.

In DNA replication, Okazaki fragments are formed as double-stranded intermediates during synthesis of the lagging strand. They are composed of the growing DNA strand primed by RNA and the template strand. The DNA oligonucleotide d(GGGTATACGC) and the chimeric RNA-DNA oligonucleotide r(GCG)d(TATACCC) were combined to form a synthetic Okazaki fragment and its three-dimensional structure was determined by x-ray crystallography. The fragment adopts an overall A-type conformation with 11 residues per turn. Although the base-pair geometry, particularly in the central TATA part, is distorted, there is no evidence for a transition from the A- to the B-type conformation at the junction between RNA.DNA hybrid and DNA duplex. The RNA trimer may, therefore, lock the complete fragment in an A-type conformation.

Base Sequence↗
PubMed1992

Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae.

A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.

Amino Acid Sequence↗
PubMed1992

Transcription of human c-myc in permeabilized nuclei is associated with formation of Z-DNA in three discrete regions of the gene.

When human U937 cells are placed in agarose microbeads and treated with a detergent, the cytoplasmic membrane is lysed and the nuclear membrane is permeabilized. However, the nuclei remain intact and maintain both replication and transcription. Biotin labeled monoclonal antibodies against Z-DNA have been diffused into this system and used to measure the amount of Z-DNA present in the nuclei. It has previously been shown that the amount of Z-DNA present decreases due to relaxation by topoisomerase I and increases as the level of transcription increases. Here we measure the formation of Z-DNA in the c-myc gene by crosslinking the antibodies to DNA using laser radiation at 266 nm for 10 ns. The crosslinked DNA is isolated by restriction digestion, separation of antibody labeled fractions through the biotin residue, and subsequent proteolysis to remove the crosslinked antibody. Three AluI restriction fragments of the c-myc gene are shown to form Z-DNA when the cell is transcribing c-myc. The Z-DNA forming segments are near the promoter regions of the gene. However, when U937 cells start to differentiate and transcription of the c-myc gene is down-regulated, the Z-DNA content goes to undetectable levels within 30-60 min.

Base Sequence↗
PubMed1992

Oral ulceration.

Oral ulceration is a common problem. The presentation is often very similar, irrespective of the underlying cause. This paper illustrates some of the more commonly encountered forms of oral ulceration.

Diagnosis, Differential↗
PubMed1991

Structure of the pure-spermine form of Z-DNA (magnesium free) at 1-A resolution.

We describe the three-dimensional X-ray structure of a complex of spermine bound to a Z-DNA duplex, [d(CGCGCG)]2, in the absence of any inorganic polyvalent cations. We have crystallized the DNA hexamer d(CGCGCG) in the exclusion of magnesium and other polyvalent ions and solved its structure at 1.0-A resolution. In the crystal of this pure-spermine form of Z-DNA, the relative orientation, position, and interactions of the DNA differ from the arrangement uniformly observed in over a dozen previously reported Z-DNA hexamers. Moreover, the conformation of the Z-DNA hexamer in this structure varies somewhat from those found in earlier structures. The DNA is compressed along the helical axis, the base pairs are shifted into the major groove, and the minor groove is more narrow. The packing of spermine-DNA complexes in crystals suggests that the molecular basis for the tendency of spermine to stabilize compact DNA structures derives from the capacity of spermine to interact simultaneously with several duplexes. This capacity is maximized by both the polymorphic nature and the length of the spermine cation. The length and flexibility of spermine and the dispersion of charge-charge, hydrogen-bonding, and hydrophobic bonding potential throughout the molecule maximize the ability of spermine to interact simultaneously with different DNA molecules.

Base Sequence↗
PubMed1991

Cloning and characterization of Rrp1, the gene encoding Drosophila strand transferase: carboxy-terminal homology to DNA repair endo/exonucleases.

We previously reported the purification of a protein from Drosophila embryo extracts that carries out the strand transfer step in homologous recombination (Lowenhaupt, K., Sander, M., Hauser, C. and A. Rich, 1989, J. Biol. Chem. 264, 20568). We report here the isolation of the gene encoding this protein. Partial amino acid sequence from a tryptic digest of gel purified strand transfer protein was used to design a pair of degenerate oligonucleotide primers which amplified a 635 bp region of Drosophila genomic DNA. Recombinant bacteriophage were isolated from genomic and embryo cDNA libraries by screening with the amplified DNA fragment. These bacteriophage clones identify a single copy gene that expresses a single mRNA transcript in early embryos and in embryo-derived tissue culture cells. The cDNA nucleotide sequence contains an open reading frame of 679 amino acids within which are found 5 tryptic peptides from the strand transfer protein. Expression of this cDNA in E. coli produces a polypeptide with the same electrophoretic mobility as the purified protein. The deduced protein sequence has two distinct regions. The first 427 residues are basic, rich in glutamic acid and lysine residues and unrelated to known proteins. The carboxy-terminal 252 residues are average in amino acid composition and are homologous to the DNA repair proteins, Escherichia coli exonuclease III and Streptococcus pneumoniae exonuclease A. This protein, which we name Rrp1 (Recombination Repair Protein 1), may facilitate recombinational repair of DNA damage.

Amino Acid Sequence↗
PubMed1991

Synthesis of RNA containing inosine: analysis of the sequence requirements for the 5' splice site of the Tetrahymena group I intron.

Two protected derivatives of the ribonucleoside inosine have been prepared to serve as building blocks for phosphoramidite-based synthesis of RNA. Two different synthetic routes address the unusual solubility characteristics of inosine and its derivatives. The final products of the different synthetic pathways, 5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsiyl) inosine 3'-O-(beta-cyanoethyldiisopropylamino) phosphoramidite 5a, and O6-p-nitrophenylethyl-5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsilyl) inosine 3'-O-(methyldiisopropylamino) phosphoramidite 5b, were chemically incorporated into short oligoribonucleotides which also contained the four standard ribonucleoside bases. The oligomers were chosen to study base-specific interactions between an RNA substrate and an RNA enzyme derived from the Group I Tetrahymena self-splicing intron. The oligomers were shown to be biochemically competent using a trans cleavage assay with the modified Tetrahymena intron. The results confirm the dependence of the catalytic activity on a wobble base pair, rather than a Watson-Crick base pair, in the helix at the 5'-splice site. Furthermore, comparison of guanosine and inosine in a wobble base pair allows one to assess the importance of the guanine 2-amino group for biological activity. The preparation of the inosine phosphoramidites adds to the repertoire of base analogues available for the study of RNA catalysis and RNA-protein interactions.

Animals↗
PubMed1991

Drosophila Rrp1 protein: an apurinic endonuclease with homologous recombination activities.

A protein previously purified from Drosophila embryo extracts by a DNA strand transfer assay, Rrp1 (recombination repair protein 1), has an N-terminal 427-amino acid region unrelated to known proteins, and a 252-amino acid C-terminal region with sequence homology to two DNA repair nucleases, Escherichia coli exonuclease III and Streptococcus pneumoniae exonuclease A, which are known to be active as apurinic endonucleases and as double-stranded DNA 3' exonucleases. We demonstrate here that purified Rrp1 has apurinic endonuclease and double-stranded DNA 3' exonuclease, activities and carries out single-stranded DNA renaturation in a Mg(2+)-dependent manner. Strand transfer, 3' exonuclease, and single-stranded DNA renaturation activities comigrate during column chromatography. The properties of Rrp1 suggest that it could promote homologous recombination at sites of DNA damage.

Amino Acid Sequence↗
PubMed1991

Triplex RNA.

Explore the source record for details and available documents.

Molecular Structure↗
PubMed1991

Detection of an unusual distortion in A-tract DNA using KMnO4: effect of temperature and distamycin on the altered conformation.

The chemical probes potassium permanganate (KMnO4) and diethylpyrocarbonate (DEPC) can be used to study the conformational flexibility of short tracts of adenine (A-tracts) present in DNA. With these probes, we demonstrate that a novel distortion is induced in a 5 base pair A-tract at low temperature. Formation of this distorted A-tract structure, which occurs in a DNA fragment from the promoter region of the plasmid pBR322, is distinguished by a dramatic increase in the KMnO4 reactivity of the central thymines in this tract at 12 degrees C. This alteration occurs in the absence of any detectable rearrangement in the conformation of the adenines in the complementary strand. Induction of this low temperature A-tract structure is blocked by the minor groove binding drug distamycin. Hydroxyl radical footprinting of distamycin binding to the fragment containing the d(A)5 tract at 12 degrees C suggests that this drug has two different modes of binding to DNA in agreement with recent NMR data. These experiments show that short A-tracts are capable of forming more than one structural variant of B DNA in solution. The possible relationship between the intrinsic bending of DNA containing short phased A-tracts and the low temperature A-tract conformation is discussed.

Adenine↗