SEARCH · PubMed Health

Results for “duplex sequencing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 667 records · Page 37Linked to original sources

PubMed2005

Amplification of RNAi--targeting HLA mRNAs.

Posttranscriptional suppression of gene expression can be achieved by introduction of sequence-specific small interfering (si) RNA duplexes and by de novo intracellular synthesis of short sequence-specific double-stranded RNAs. However, achieving desired levels of knockdown is a barrier to successful analytic and therapeutic application. We demonstrate that increasing expression of introduced short hairpin RNA (shRNA) can markedly enhance RNA interference (RNAi) and that this approach can be used to achieve maximal target down-regulation, when the choice of optimal siRNA-binding sites is restricted or when multiple genes are simultaneously targeted and the amount of siRNA is limiting. A dose-dependent RNAi effect was accomplished by placing copies of shRNA under control of the Pol III U6 small nuclear RNA promoter in tandem in a DNA vector. Using this system, we achieved simultaneous down-regulation of expression of classical human leukocyte antigen (HLA) class I genes in cultured and primary human T cells, which might be applied to help circumvent T-cell-mediated rejection of immunogenic and/or HLA-disparate allografts.

Base Sequence↗

PubMed1991

Solution structure of an oncogenic DNA duplex containing a G.A mismatch.

The DNA duplex 5'-d(GCCACAAGCTC).d(GAGCTGGTGGC), which contains a central G.A mismatch has been studied by one and two-dimensional NMR techniques. The duplex corresponds to the sequence 29-39 of the K-ras gene. The mismatch position is that of the first base of the Gly12 codon, a hot spot for mutations. The observed NOEs of the nonexchangeable protons show that both of the bases of the mismatched pair are intrahelical over a wide range of pH. However, the structure of the G.A mispair and the conformation of the central part of the duplex change with pH. This structural change shows a pK of 6.0. At low pH, the G.A bases are base paired with hydrogen bonds between the keto group of the G residue and the amino group of the A residue and, secondly, between the N7 of the G and a proton on N1 of A. This causes the G residue to adopt a syn conformation. On raising the pH, the N1-H proton of the protonated A residue is removed, and the base pair rearranges. In the neutral G.A base pair both residues adopt an anti conformation, and the mismatch is stabilized by hydrogen bonds. Our results on the exchangeable and A(H2) protons of the mismatched pair indicate a shift from a classical face-to-face two hydrogen-bonded structure to a slipped structure stabilized by bifurcated hydrogen bonds. This may be a particular characteristics of this oncogenic sequence in which the G.A error is poorly repaired.

Base Composition↗

PubMed1999

Preparation and characterization of DNA containing a site-specific nonadjacent cyclobutane thymine dimer of the type implicated in UV-induced -1 frameshift mutagenesis.

One mechanism for the origin of UV-induced -1 deletion mutations involves the bypass of a nonadjacent cis-syn cyclobutane pyrimidine dimer containing a single intervening nucleotide. To begin to investigate this mechanism, we required a method for obtaining a single, site-specific, nonadjacent dimer. One approach to the preparation of a nonadjacent dimer is to irradiate a DNA duplex containing a centrally located TNT sequence in which the two T's are paired to an AA sequence in an otherwise fully complementary strand. Triplet-sensitized irradiation of the duplex formed between the 13-mer d(GAGTATCTATGAG) and the 12-mer d(CTCATAATACTC) on ice gave a major product that could be reverted to the parent 13-mer by 254 nm irradiation. Proton NMR experiments established the major product to be the nonadjacent cis-syn cyclobutane dimer formed between the two T's of the TCT sequence. Melting temperature studies show that the nonadjacent dimer is more destabilizing to DNA duplex structure than a normal cis-syn dimer and is as stable as the parental bulged DNA duplex. The nonadjacent dimer-containing 13-mer was ligated into a 51-mer and used as a template for primer-extension studies by DNA polymerases. The nonadjacent dimer could not be bypassed by Sequenase Version 2.0 and terminated synthesis primarily prior to and opposite the 3'-T of the dimer. In contrast, approximately 30% of the dimer was bypassed by an exonuclease-deficient (exo-) Klenow fragment, and termination occurred primarily opposite the 3'- and 5'-T's of the dimer. Bypass of the nonadjacent dimer by exo(-) Klenow fragment led primarily to a single-nucleotide deletion mutation as well as small amounts of a full-length product and a four-nucleotide deletion that could be explained by a primer misalignment mechanism.

Acetophenones↗

PubMed2005

Immunomodulatory oligonucleotides containing a cytosine-phosphate-2'-deoxy-7-deazaguanosine motif as potent toll-like receptor 9 agonists.

Bacterial DNA and synthetic oligomers containing CpG dinucleotides activate the immune system through Toll-like receptor (TLR) 9. Here, we compare the immunostimulatory activity of three immunomers with different nucleotide sequences containing a synthetic cytosine-phosphate-2'-deoxy-7-deazaguanosine dinucleotide (CpR), called immunomodulatory oligonucleotides (IMOs), in mouse, human, and monkey systems. IMOs induced IL-12 and IFN-gamma secretion more than a control non-CpG IMO in mice. All three IMOs activated HEK293 cells expressing TLR9 but not TLR3, -7, or -8. IMOs induced human B-cell proliferation and enhanced expression of CD86 and CD69 surface markers on B cells. The three IMOs induced CD86 expression on human plasmacytoid dendritic cells, but only IMOs that contained a 5'-terminal TCR nucleotide sequence induced IFN-alpha secretion. A sequence that forms a duplex structure also was required for IFN-alpha induction in human peripheral blood mononuclear cell cultures. IMOs induced chemokine and cytokine gene expression in human peripheral blood mononuclear cells. In monkeys, all three IMOs induced transient changes in peripheral blood leukocytes and lymphocytes and activated B and T lymphocytes. All three IMOs induced IFN-alpha in vivo in monkeys; the IMO sequence that forms a stable secondary structure induced the highest levels of IFN-alpha. These studies are, to our knowledge, the first comprehensive studies to compare the activity of IMOs containing synthetic stimulatory CpR dinucleotides in mouse, monkey, and human systems. These results suggest that IMOs induce strong and rapid immunostimulation and that the CpR dinucleotide is recognized by TLR9, leading to immune-cell activation and cytokine secretion in vitro and in vivo.

Animals↗

PubMedDate not supplied

Evidence for a DNA triplex in a recombination-like motif: I. Recognition of Watson-Crick base pairs by natural bases in a high-stability triplex.

Data are presented on a triplex type with two parallel homologous strands for which triplex formation is almost as strong as duplex formation at least for some sequences and even at pH 7 and 0.2 M NaCl. The evidence mainly rests upon comparing thermodynamic properties of similar systems. A paperclip oligonucleotide d(A12C4T12C4A12) with two linkers C4 obviously can form a triplex with parallel back-folded adenine strand regions, because the single melting transition of this complex splits in two transitions by introducing mismatches only in the third strand region. Respectively, a hairpin duplex d(A12C4T12) and a single strand d(A12) form a triplex as a 1:1 complex in which the second adenine strand is parallel oriented to the homologous one in the Watson-Crick paired duplex. In this system the melting temperature T(m) of the triplex is practically the same as that of the duplex d(A12)-d(T12), at least within a complex concentration range of 0.2-4.0 microM. The melting behaviour of complexes between triplex stabilizing ligand BePI and the system hairpin duplex plus single strand supports the triplex model. Non-denaturing gel electrophoresis suggests the existence of a triplex for a system in which five of the twelve A-T*A base triads are substituted by C-G*C base triads. The recognition between any substituted Watson-Crick base pair (X-Y) in the hairpin duplex d(A4XA7C4T7YT4) and the correspondingly replaced base (Z) in the third strand d(A4ZA7) is mutually selective. All triplexes with matching base substitutions (Z = X) have nearly the same stability (T(m) values from 29 to 33.5 degrees C), whereas triplexes with non-matching substitutions (Z not equal X) show a clearly reduced stability (T(m) values from 15 to 22 degrees C) at 2microM equimolar oligonucleotide concentration. Most nucleic acid triple helices hitherto known are limited to homopurine-homopyrimidine sequences in the target duplex. A stable triplex formation is demonstrated for inhomogeneous sequences tolerating at least 50% pyrimidine content in the homologous strands. On the basis of the surprisingly similar thermodynamic parameters for duplex and triplex, and of the fact that this triplex type seems to be more stable than many other natural DNA triplexes known, and on the basis of semiempirical and molecule mechanical calculations, we postulate bridging interactions of the third strand with the two other strands in the triplex according to the recombination motif. This triplex, denoted by us 'recombination-like form', tolerates heterogeneous base sequences.

Base Pairing↗

PubMed1987

Differentially expressed bovine cytokeratin genes. Analysis of gene linkage and evolutionary conservation of 5'-upstream sequences.

Cytokeratins are a family of approximately 20 polypeptides which form the intermediate-sized filaments (IFs) characteristic of epithelial cells. They are synthesized co-ordinately as 'pairs' consisting of one representative from each of the two cytokeratin subfamilies, i.e. the acidic (type I) and the more basic (type II) polypeptides, in cell type-specific combinations. We have isolated and characterized the genes coding for four bovine cytokeratins of the basic (type II) subfamily, i.e. cytokeratins Ib, III, IV and 6*, by Southern blot hybridization, hybridization-selection-translation experiments, hetero-duplex mapping, and partial sequencing of the exons coding for the hypervariable carboxy-terminal 'tail' regions of the proteins and the 3'-non-translated ends of the mRNAs which are distinct for the individual cytokeratin polypeptides. Limited 'chromosomal walk' experiments demonstrated that the genes are organized into two tandems, i.e. 6*----Ib and III----IV, in which they are separated by approximately 11 kb. RNA analysis by Northern and dot blots show that both genes of the III----IV tandem are co-expressed in some bovine tissues (muzzle epidermis, hoof pad and tongue mucosa) and cultured cells (BMGE + H) but that in other tissues, cornea for example, only the gene encoding III is expressed. Unexpectedly, the genes linked in the tandem 6*----Ib are not co-expressed in any of the tissues examined. mRNA from gene 6* has been found in tongue mucosa but in none of the other cell lines and tissues examined, whereas mRNA for cytokeratin Ib is expressed in cornea and muzzle epidermis but not in, for example, tongue mucosa and in the epidermis of the heel pad.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1997

Structure of the A-DNA octamer d(GGCATGCC).

The crystal structure of the self-complementary DNA octamer d(GGCATGCC) has been determined to a resolution of 2.38 A and R factor of 17.1%. The structure is an A-DNA octamer duplex with Watson-Crick base pairing along its length. This sequence extends the investigation of octamer duplexes of the type d(GGX(4)CC) which assume P6(1) packing within the crystallographic unit cell. The A-DNA duplex is non-linear with a helix axis curvature of 7 degrees and bending towards the DNA minor groove. In view of the biological importance of protein recognition of some A-DNA sequences the minor-groove geometry and helix-axis curvature for this structure is compared with all others of the type d(GGXATXCC).

Journal Article↗

PubMed1991

P element excision in Drosophila melanogaster and related drosophilids.

The frequency of P element excision and the structure of the resulting excision products were determined in three drosophilid species. Drosophila melanogaster, D. virilis, and Chymomyza procnemis. A transient P element mobility assay was conducted in the cells of developing insect embryos, but unlike previous assays, this mobility assay permitted the recovery of excision products from plasmids regardless of whether the excision event was precise or imprecise. Both quantitative and qualitative differences between the products of excision in the various species studied were observed. The frequency with which P element excision products were recovered from D. melanogaster was 10-fold greater than from D. virilis and C. procnemis; however, the proportion of all excision events resulting in the reversion of a P-induced mutant phenotype was the same. Virtually all excision products recovered, including those resulting in a reversion of the mutant phenotype, did not result in the exact restoration of the original target sequence. Sequence analysis suggested that duplex cleavage at the 3' and 5' termini of the P element, or their subsequent modification, occurred asymmetrically and interdependently. P element-encoded transposase was not absolutely required for P element excision.

Animals↗

PubMed2006

MALDI-TOF mass spectral analysis of siRNA degradation in serum confirms an RNAse A-like activity.

Synthetic siRNA duplexes are used widely as reagents for silencing of mRNA targets in cells and are being developed for in vivo use. Serum stability is a major concern if siRNA is to be used for therapeutic delivery within blood circulation. We have developed the use of MALDI-TOF mass spectrometry as a rapid and convenient analytical tool to identify the most vulnerable sites within siRNA to serum degradation. Using this approach, we found that one siRNA duplex (Dh3) with UpA sequences close to one end was particularly vulnerable to rapid cleavage. This produced a fragment of mass consistent with the presence of a 2',3'-cyclic phosphate that was slowly hydrolysed to a 2'-(3'-)phosphate on extended incubation. Substitution of these sites with 2'-O-methyl U residues prevented cleavage and confirmed that the major pathway for initial degradation is via cleavage by an RNAse A-like activity. Mass spectral analysis was used to follow the serum degradation of siRNA over more prolonged periods to show the accumulation of many fragments, almost all showing cleavage following pyrimidine nucleoside residues. Overall, the MALDI-TOF mass spectral analysis technique should prove useful for preliminary screening of the serum stability of siRNA duplexes and for identification of the most vulnerable cleavage sites.

Base Sequence↗

PubMed1997

Oligonucleotide inhibitors of human thrombin that bind distinct epitopes.

Thrombin, a multifunctional serine protease, recognizes multiple macromolecular substrates and plays a key role in both procoagulant and anticoagulant functions. The substrate specificity of thrombin involves two electropositive surfaces, the fibrinogen-recognition and heparin-binding exosites. The SELEX process is a powerful combinatorial methodology for identifying high-affinity oligonucleotide ligands to any desired target. The SELEX process has been used to isolate single-stranded DNA ligands to human thrombin. Here, a 29-nucleotide single-stranded DNA ligand to human thrombin, designated 60-18[29], with a Kd of approximately 0.5 nM is described. DNA 60-18[29] inhibits thrombin-catalyzed fibrin clot formation in vitro. Previously described DNA ligands bind the fibrinogen-recognition exosite, while competition and photocrosslinking experiments indicate that the DNA ligand 60-18[29] binds the heparin-binding exosite. DNA 60-18[29] is a quadruplex/duplex with a 15-nucleotide "core" sequence that has striking similarity to previously described DNA ligands to thrombin, but binds with 20 to 50-fold higher affinity. The 15-nucleotide core sequence has eight highly conserved guanine residues and forms a G-quadruplex structure. A single nucleotide within the G-quadruplex structure can direct the DNA to a distinct epitope. Additional sequence information in the duplex regions of ligand 60-18[29] contribute to greater stability and affinity of binding to thrombin. A low-resolution model for the interaction of DNA 60-18[29] to human thrombin has been proposed.

Aptamers, Nucleotide↗

PubMed2006

Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins.

The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experiments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be approximately 10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are consistent with the primary function of Gag as a nucleic acid binding and packaging protein and the primary function of the NC proteins as nucleic acid chaperones. Also, our results suggest that NCp7's capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nucleic acid strand annealing by inducing electrostatic attraction between strands, likely optimize the fully processed NC protein to facilitate complex nucleic acid secondary structure rearrangements. In contrast, Gag's stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization.

Capsid Proteins↗

PubMed1985

Nuclear magnetic resonance and circular dichroism studies of a duplex--single-stranded hairpin loop equilibrium for the oligodeoxyribonucleotide sequence d(CGCGATTCGCG).

Nuclear magnetic resonance (NMR) and circular dichroism (CD) studies have been carried out with the oligodeoxyribonucleotide mismatch sequence, d(CGCGATTCGCG), 1. It has been found that 1 exists, in solution, as an equilibrium mixture of slowly interconverting, structured conformational isomers, 1a and 1b. On the basis of the concentration dependence of the 1a-1b equilibrium, the 1H NMR spectrum of the imino protons of the nucleotide bases, and the individual CD spectra of 1a and 1b, it is suggested that the two species correspond to a B-type DNA duplex and a single-stranded, hairpin-loop structure; the portion of the single-stranded species not involved in the loop appears to have a B-type DNA structure (on the basis of the CD measurements). To facilitate 1H NMR resonance assignments, the two possible des-methyl thymidine derivatives of 1 were synthesized; the effect of this substitution on the physical chemical properties of 1 was explored. The 1H NMR spectra of 1, as a function of temperature, showed that, under conditions wherein both species were present to a significant extent, the duplex form melted at a lower temperature than the single-stranded, hairpin loop structure.

Circular Dichroism↗

PubMed2006

Influence of cationic molecules on the hairpin to duplex equilibria of self-complementary DNA and RNA oligonucleotides.

A self-complementary nucleotide sequence can form both a unimolecular hairpin and a bimolecular duplex. In this study, the secondary structures of the self-complementary DNA and RNA oligonucleotides with different sequences and lengths were investigated under various solution conditions by gel electrophoresis, circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy and a ultraviolet (UV) melting analysis. The DNA sequences tended to adopt a hairpin conformation at low cation concentrations, but a bimolecular duplex was preferentially formed at an elevated cationic strength. On the other hand, fully matched RNA sequences adopted a bimolecular duplex regardless of the cation concentration. The thermal melting experiments indicated a greater change in the melting temperature of the bimolecular duplexes (by approximately 20 degrees C) than that of the hairpin (by approximately 10 degrees C) by increasing the NaCl concentration from 10 mM to 1 M. Hairpin formations were also observed for the palindrome DNA sequences derived from Escherichia coli, but association of the complementary palindrome sequences was observed when spermine, one of the major cationic molecules in a cell, existed at the physiological concentration. The results indicate the role of cations for shifting the structural equilibrium toward a nucleotide assembly and implicate nucleotide structures in cells.

Cations↗

PubMed2000

Differential destabilization of the DNA oligonucleotide double helix by a T.G mismatch, 3,N(4)-ethenocytosine, 3,N(4)-ethanocytosine, or an 8-(hydroxymethyl)-3,N(4)-ethenocytosine adduct incorporated into the same sequence contexts.

The T.G mismatch and the exocyclic adduct 3,N(4)-ethenocytosine (epsilonC) are repaired by the same enzyme, the human G/T(U) mismatch-DNA glycosylase (TDG). This enzyme removes the T, U, or epsilonC base from duplex DNA. The rate of cleavage was found to differ with the lesion and was also affected by neighbor sequences [Hang, B., Medina, M., Fraenkel-Conrat, H., and Singer, B. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13561-13566]. Since sequence influences duplex stability, we determined the thermodynamic stability of T.G and epsilonC-containing 15-mer duplexes in which the bases flanking the lesion were systematically varied. The duplexes contained central 5'-TTXTT, 5'-AAXAA, 5'-CCXCC, or 5'-GGXGG sequences, where X is T, epsilonC, or two closely related structural derivatives of epsilonC: 3,N(4)-ethanocytosine (EC) and 8-(hydroxymethyl)-epsilonC (8-HM-epsilonC). Each of the four lesions, incorporated opposite G, decreased both the thermal (T(m)) and thermodynamic stability (DeltaG degrees (37)) of the 15-mer control duplexes. On the basis of the T(m) and DeltaG degrees (37) values, the order of destabilization of the TTXTT sequence in 15-mer duplexes was as follows: 8-HM-epsilonC > EC > epsilonC > T.G. The DeltaT(m) values range from -15.8 to -9.5 degrees C when C(t) = 8 microM. Duplexes with flanking AA or TT neighbors were more destabilized, by an average of 2 degrees C, than those with flanking GG or CC neighbors. The base opposite the modified base also influenced duplex stability. Within the TT context, of the four changed bases opposite the adducts, C had the greatest destabilizing effect, up to -18.4 degrees C. In contrast, a G opposite an adduct was generally the least destabilizing, and the smallest value was -3. 0 degrees C. Destabilizations were enthalpic in origin. Thus, this work shows that independently changing the modified base, the sequence, or the base opposite the lesion each affects the stability of the duplex, to significantly varying extents. The potential contribution of the thermodynamic stability to repair efficiency is discussed.

Base Pair Mismatch↗

PubMedDate not supplied

Sequence-specific targeting of RNA with an oligonucleotide-neomycin conjugate.

The synthesis of neomycin covalently attached at the C5-position of 2'-deoxyuridine is reported. The synthesis outlined allows for incorporation of an aminoglycoside (neomycin) at any given site in an oligonucleotide (ODN) where a thymidine (or uridine) is present. Incorporation of this modified base into an oligonucleotide, which is complementary to a seven-bases-long alpha-sarcin loop RNA sequence, leads to enhanced duplex hybridization. The increase in Tm for this duplex (DeltaTm = 6 degrees C) suggests a favorable interaction of neomycin within the duplex groove. CD spectroscopy shows that the modified duplex adopts an A-type confirmation. ITC measurements indicate the additive effects of ODN and neomycin binding to the RNA target (Ka = 4.5 x 107 M-1). The enhanced stability of the hybrid duplex from this neomycin-ODN conjugate originates primarily from the enthalpic contribution of neomycin {DeltaDeltaHobs = -7.21 kcal/mol (DeltaHneomycin conjugated - DeltaH nonconjugated)} binding to the hybrid duplex. The short linker length allows for selective stabilization of the hybrid duplex over the hybrid triplex. The results described here open up new avenues in the design and synthesis of nucleo-aminoglycoside-conjugates (N-Ag-C) where the inclusion of any number of aminoglycoside (neomycin) molecules per oligonucleotide can be accomplished.

Base Sequence↗

PubMed1997

Effect of base pair A/C and G/T mismatches on the thermal stabilities of DNA oligomers that form B-Z junctions.

The thermal stabilities and structures of B-Z junction forming DNA duplexes possessing A/C or G/T base pair mismatches were compared to those of corresponding duplexes possessing perfect matched base pairs. The upper strands of the duplexes have a generalized sequence 5'-(5meCG)-LMN-GACTG-3', where L stands for A or G while M and N are permutations of pyrimidines. The lower strands were either complementary or were such as to create an A/C or G/T mismatch at the position corresponding to L, M, or N. Optical melting and circular dichroism studies were used to investigate the thermal stabilities and structures of both the mismatched base pair and the perfect matched base pair duplexes. Incorporating mismatched A/C or G/T base pairs did not noticeably affect the conformations of the duplexes in 115 mM Na+ but resulted in perturbed B-Z conformations at 4.5 M Na+. For any mismatched base pair duplex, the B-DNA domain of the hybrid B-Z structure formed at 4.5 M Na+ is significantly perturbed while the Z-DNA domain is less perturbed by the presence of the mismatched base pairs. The presence of a mismatch destabilizes a duplex relative to the perfect matched base pair duplex by 1.7-10.0 kcal/mol depending upon position of the mismatch, type of mismatch base pair involved, and Na+ concentration. The thermodynamic destabilization of a mismatched base pair duplex relative to the perfect matched base pair duplex arises from perturbations in nearest neighbor interactions and hydrogen bonding. In general, we observed that the incorporation of an A/C or G/T base pair mismatch in place of a perfect matched base pair at or near a B-Z junction results in a relatively large change in enthalpy and entropy to produce a significant change in the free energy of the duplex to single strand transition. At 4.5 M Na+, where the duplexes possess perturbed B-Z junctions, the farther away from the junction that the mismatch is, the greater the extent of the destabilization.

Base Composition↗

PubMed1994

Use of oligonucleotides containing ethenoadenine to study the repair of this DNA lesion. Determination of individual and collective repair activities.

Oligonucleotide duplexes of a defined sequence containing one 1,N6-ethenoadenosine (EA) were synthesized and used as substrates to study the repair of this DNA lesion in cell homogenates of peripheral mononuclear blood cells of 39 male and female workers, exposed to vinyl chloride. These data were compared to data from 39 employees of the same company working in other production plants and to data from a control group of 39 persons, living in an area without vinyl chloride production. After incubation of the 5'- and 3'-labeled oligonucleotide duplex with cell homogenate, a specific nicking activity, releasing the deoxyribosyl phosphate originally carrying the EA, was found. This activity was used to determine the individual and collective repair activities for ethenoadenine. The exposed group showed a mean of 158.5 +/- 39.9 (SD) fmol product fragment and did not differ significantly from the mean value of the two control groups with 156.5 +/- 42.9 fmol and 161.2 +/- 53.6 fmol, respectively. Large interindividual variations were found, ranging from 4.9-fold in the exposed to 8.2- and 7.2-fold in the control groups. The development of an assay for ethenoadenine repair is significant for understanding the role of EA repair in eukaryotic cells.

Adenine↗

PubMed2005

AtTBP2 and AtTRP2 in Arabidopsis encode proteins that bind plant telomeric DNA and induce DNA bending in vitro.

Telomeric DNA-binding proteins (TBPs) are crucial components that regulate the structure and function of eukaryotic telomeres and are evolutionarily conserved. We have identified two homologues of AtTBP1 (for Arabidopsis thaliana telomeric DNA binding protein 1), designated as AtTBP2 and AtTRP2, which encode proteins that specifically bind to the telomeric DNA of this plant. These proteins show extensive homology with other known plant TBPs. The isolated C-terminal segments of these proteins were capable of sequence-specific binding to duplex telomeric plant DNA in vitro. DNA bending assays using the Arabidopsis TBPs revealed that AtTBP1 and AtTBP2 have DNA-bending abilities comparable to that of the human homologue hTRF1, and higher than those of AtTRP1 and AtTRP2.

Amino Acid Sequence↗