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Introduction: Evidence indicates children who suffer from ill-health are less likely to attend or complete schooling. Malaria is an important cause of morbidity and mortality in school-age children. However, they are less likely to receive malaria treatment at health facilities and evidence for how to improve schoolchildren's access to care is limited. This study aimed to evaluate the impact of a programme of school-based malaria case management on schoolchildren's attendance, health and education., Methods: A cluster randomised controlled trial was conducted in 58 primary schools in Zomba District, Malawi, 2011-2015. The intervention, implemented in 29 randomly selected schools, provided malaria rapid diagnostic tests and artemisinin-based combination therapy to diagnose and treat uncomplicated malaria as part of basic first aid kits known as 'Learner Treatment Kits' (LTK). The primary outcome was school attendance, assessed through teacher-recorded daily attendance registers and independent periodic attendance spot checks. Secondary outcomes included prevalence of Plasmodium spp infection, anaemia, educational performance, self-reported child well-being and health-seeking behaviour. A total of 9571 children from standards 1-7 were randomly selected for assessment of school attendance, with subsamples assessed for the secondary outcomes., Results: Between November 2013 and March 2015, 97 trained teachers in 29 schools provided 32 685 unique consultations. Female schoolchildren were significantly more likely than male to seek a consultation (unadjusted OR=1.78 (95% CI 1.58 to 2.00). No significant intervention effect was observed on the proportion of child-days recorded as absent in teacher registers (n=9017 OR=0.90 (95% CI 0.77 to 1.05), p=0.173) or of children absent during random school visits-spot checks (n=5791 OR=1.09 (95% CI 0.87 to 1.36), p=0.474). There was no significant impact on child-reported well-being, prevalence of Plasmodium spp, anaemia or education scores., Conclusion: Despite high community demand, the LTK programme did not reduce schoolchildren's absenteeism or improve health or education outcomes in this study setting., Trial Registration Number: ClinicalTrials.gov NCT02213211., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Background: The World Health Organization recommends that persons of all ages suspected of malaria should receive a parasitological confirmation of malaria by use of malaria rapid diagnostic test (RDT) at community level, and that rectal artesunate should be used as a pre-referral treatment for severe malaria to rapidly reduce parasitaemia. This paper reports on findings from a pilot study that assessed the feasibility, acceptability and effects of integrating RDTs and pre-referral rectal artesunate into the integrated Community Case Management programme in Malawi., Methods: This study used mixed methods to collect information for this survey. Pre- and post-intervention, cross-sectional, household surveys were carried out. A review of integrated community case management reports, including supervision checklists was conducted. Quantitative data were collected in tablets running on open data kit software, and then data were transferred to STATA version 12 for analysis. For key indicators, proportions were calculated at 95% confidence intervals. Qualitative data were recorded onto digital recorders, translated into English and transcribed for analysis., Results: Out of 86 observed RDT performances, a total of 83 (97%) were performed correctly with a proper disposal of sharps and biohazard wastes. Only two (2%) febrile children who had an RDT negative result were treated with artemether-lumefantrine, contrary to malaria treatment guidelines. Utilization of community health workers (CHWs) as a first source of care increased from (33.9%) (95% CI; 25.5-42.3) at baseline to (89.7%) (95% CI; 83.5-95.5) at end line in the intervention villages. There was a corresponding decrease in the proportion of caregivers that first sought care from informal sources from 12.9% (95% CI; 6.9-18.9) to 1.9% (95% CI; 0.9-4.4) in the intervention villages. Acceptability of the use of RDTs and pre-referral rectal artesunate at the community level was relatively high., Conclusion: Integration of RDTs and pre-referral rectal at artesunate community level is both feasible and acceptable. The strategy has the potential to increase and improve utilization of child health services at community level. However, this depends on the CHWs' skills and their availability in remote areas.

Objective: Malaria is a major cause of morbidity and mortality in tropical and subtropical regions, affecting mostly the impoverished sections of the population. Pregnant women living in malaria-endemic areas are at higher risk of malaria infection with higher density of parasitaemia than non-pregnant women. The aim of this study was to assess factors affecting the uptake of IPT among women attending antenatal clinics at Bondo District Hospital, Western Kenya., Methods: This study was a hospital-based cross-sectional survey among pregnant women attending clinics. Malaria is endemic in Bondo district. Both women from Bondo town (urban) and greater Bondo District (rural) who had been pregnant for at least 35 weeks or had delivered not more than 6 weeks prior to the survey), and had ANC cards were included in the study. The main outcomes were ANC attendance, IPT doses received and client and provider factors., Results: Results showed that women's knowledge on ANC and IPT was high. The uptake of IPT was low among pregnant women with those from urban areas more likely to make more ANC visits and to get more IPT doses than women from the rural areas. ANC attendance was hampered by the fear of being tested for HIV at the clinic. Perceived side effects associated with IPT-SP hindered IPT uptake and were linked to HIV-related symptoms. Negative attitude among health workers towards pregnant women also adversely impacted IPT uptake. Women suggested that IPT drugs be distributed through community health workers instead of the health facility for improved uptake., Conclusions: Retraining of health workers on the administration of IPT, harmonization of health messages, and assessment of alternative community-based IPT distribution channels ought to be urgently considered. More evidence on the influence of HIV pandemic on perceptions and attitudes toward and uptake of other health interventions is urgently needed.

The vibrational circular dichroism (VCD) and infrared absorption (IR) spectra of the mismatched octamer oligonucleotides d(CGTGCGCG)(2) (CGT) and d(CGCGTGCG)(2) (CGC) and their complexes with the antitumor drug daunomycin were measured in D(2)O, interpreted, and compared to the octamer d(CGCGCGCG)(2) (CG). The IR spectra of the mismatched octamers in the carbonyl-stretching region are similar to those of the parent CG, whereas the VCD spectra differ in several respects between each other. The main VCD feature due to carbonyl stretching is informative for the mismatches and CG. Vibrational modes in the sugar-phosphate region remain essentially unchanged especially for PO(2) (-) symmetric stretching. Differences between the free and complexed mismatch octamers occurred mainly in the carbonyl-stretching region (1,700-1,600 cm(-1)). The absorption intensity of the C==O peak of G is more prominent for CGC than CGT and resembles CG in this respect. The detailed composition of this doublet is clearly visible, indicating the geometric rearrangement of the base pairs in the presence of the mismatch and upon forming the daunomycin complex.

The four-stranded intercalated DNA structure exemplified by the oligonucleotide 5'-d(CCCCCCCCCCCC) (d(C)12) was studied at acidic pH by infrared absorption (IR) and vibrational circular dichroism (VCD) spectroscopy and compared with spectra of the same oligonucleotide at neutral pH to establish distinct VCD markers for the intercalation motif. The most striking feature is a new absorption at 1694 cm(-1) and its corresponding VCD couplet with reversed sign. These are unique for the intercalated structure and have not been observed for other parallel stranded duplexes. Significant characteristic features resulting from the spatial arrangement of the sugar-phosphate backbone are also clearly present for d(C)12 at acidic pH. An extensive network of CH...O bonds twists the backbone such that multiple through-space vibrational coupling occurs among neighbouring sugar-phosphate residues resulting in unusual VCD signals.

The vibrational circular dichroism (VCD) and IR absorption spectra of a dodecamer d(CCTCTGGTCTCC).d(GGAGACCAGAGG) coordinated with cisplatin are distinct compared to those of the dodecamer without cisplatin. Although the intensity of PO(2)/deoxyribose absorptions (1150-850 cm(-1)) increases noticeably relative to those of the carbonyl and ring deformations of the bases (1750-1500 cm(-1)), the VCD spectra differ to a much greater extent. Overlapping positive and negative bands can be assigned relatively easily to individual vibrational modes. The effect of platination on the dodecamer duplex is expressed most prominently in VCD arising solely from the vibrations of the guanines bound to the platinum atom. The effect on the VCD features of other bases leads to minute wavenumber shifts at most. These observations are in agreement with previous NMR and X-ray experiments on the same oligonucleotide. The assignment of the absorption and VCD bands strongly resembles those of the octamer duplex d(CCTGGTCC).d(GGACCAGG) when coordinated with platinum. The spectra of the dodecamer did not indicate any isomerization of the complex with time, as is clearly the case for the octamer., (Copyright 2003 Wiley Periodicals, Inc.)

Vibrational circular dichroism (VCD) spectroscopy and simultaneous IR absorption measurements are applied to study the interaction of natural calf thymus DNA with Cu2+ ions at room temperature in a Cu2+ concentration range of 0-0.4M (a Cu2+/phosphate molar ratio [Cu]/[P] of 0-0.7). In some important instances, VCD provides more detailed insights than previous IR investigations whereas in several others it leads to the same interpretations. The Cu2+ ions bind to phosphate groups at a low metal concentration. Upon increasing the ion concentration, chelates are formed in which Cu2+ binds to the N7 of guanine (G) and a phosphate group. Detectable only by VCD, significant distortion of most guanine-cytosine (GC) base pairs occurs at a [Cu]/[P] ratio of 0.5 with only a minor affect on adenine-thymine (AT) base pairs, which favors a "sandwich" complex in which a Cu2+ ion is inserted between two adjacent guanines in a GpG sequence. The AT base pairs become significantly distorted when the metal concentration is increased to 0.7 [Cu]/[P]. A number of GC base pairs, which are possibly involved in sandwich complexes, remain stacked and paired even at 0.7 [Cu]/[P], preventing complete strand separation. The DNA secondary structure changes considerably from the standard B-form geometry at a [Cu]/[P] ratio of 0.4 and higher. A further transition to some intermediate conformation that is inconsistent with either the A- or Z-form or a completely denatured state is suggested in agreement with other works. In general, VCD proves to be a reliable indicator of the 3-dimensional structure of the DNA-metal ion complexes, which reveals structural details that cannot be deduced from the IR absorption spectra alone., (Copyright 2003 Wiley Periodicals, Inc.)

Phase transitions were studied of the sodium salt of poly(rA).poly(rU) induced by elevated temperature without Ni(2+) and with Ni(2+) in 0.07 M concentration in D(2)O (approximately 0.4 [Ni]/[P]). The temperature was varied from 20 degrees C to 90 degrees C. The double-stranded conformation of poly(rA).poly(rU) was observed at room temperature (20 degrees C-23 degrees C) with and without Ni(2+) ions. In the absence of Ni(2+) ions, partial double- to triple-strand transition of poly(rA).poly(rU) occurred at 58 degrees C, whereas only single- stranded molecules existed at 70 degrees C. While poly(rU) did not display significant helical structure, poly(rA) still maintained some helicity at this temperature. Ni(2+) ions significantly stabilized the triple-helical structure. The temperature range of the stable triple-helix was between 45 degrees C and 70 degrees C with maximum stability around 53 degrees C. Triple- to single-stranded transition of poly(rA).poly(rU) occurred around 72 degrees C with loss of base stacking in single-stranded molecules. Stacked or aggregated structures of poly(rA) formed around 86 degrees C. Hysteresis took place in the presence of Ni(2+) during the reverse transition from the triple-stranded to the double-stranded form upon cooling. Reverse Hoogsteen type of hydrogen-bonding of the third strand in the triplex was suggested to be the most probable model for the triple-helical structure. VCD spectroscopy demonstrated significant advantages over infrared absorption or the related electronic CD spectroscopy.

We employ NMR structure determination, thermodynamics, and enzymatics to uncover the structural, thermodynamic and enzymatic properties of alpha/beta-ODNs containing 3'-3' and 5'-5' linkages. RNase H studies show that alpha/beta-gapmers that are designed to target erbB-2 efficiently elicit RNase H activity. NMR structures of DNA.DNA and DNA.RNA duplexes reveal that single alpha-anomeric residues fit well into either duplex, but alter the dynamic properties of the backbone and deoxyriboses as well as the topology of the minor groove in the DNA.RNA hybrid.

The B-Z transition of the synthetic oligonucleotide, (dG-dC)20, induced by Mn2+ ions at room temperature, was investigated by absorption and Vibrational Circular Dichroism (VCD) spectroscopy in the range of 1800-800 cm(-1). Metal ion concentration was varied from 0 to 0.73 M Mn2+ (0 to 8.5 moles of Mn2+ per mole of oligonucleotide phosphate, [Mn]/[P]). While both types of spectra showed considerable changes as the Mn2+ concentrations were raised, differences between the two were often complementary in their expression and extent, those displayed by VCD being more clearly evident due to the inversion of the opposite helical sense from the right-handed to the left-handed conformation. The main phase of the transition occurred in the metal ion concentration between 0.8-1.1 [Mn]/[P]. Gradual changes that took place in the spectra were interpreted in terms of simultaneous processes that depended on metal ion concentration, namely B-Z transformation, binding of Mn2+ to phosphates and to nitrogen bases, and partial denaturation. Below approximately 0.6 [Mn]/[P], only a small portion of the oligonucleotide adopted the Z conformation within a 3 hour period, whereas conversion was completed in the same time interval for concentrations between 0.9-1.2 [Mn]/[P]. At [Mn]/[P] >1.7, complete transition to the Z-form took place immediately on adding Mn2+. Applying VCD spectroscopy in combination with conventional infrared absorption proved most useful for corroborating changes in the absorption spectra, and for detecting in an unique manner, not attainable by absorption methods, conformational changes that lead to the inversion of the helical sense of the oligonucleotide.

Alternating polypurine sequences exhibit remarkable polymorphism. In this study, we report that dGA.dGT sequences form parallel stranded duplex DNA at neutral pH. Using two model hairpins, 3'-d(GT)3-5'5'-T4(AG)3-3' (I) and 3'-d(GT)4-5'5'-T4(AG)4-3' (II), containing 5'5' linkages which direct parallel strand formation, we systematically explored the spectroscopic and thermodynamic properties of parallel stranded d(GA)n.d(GT)n. The parallel stranded hairpins are remarkably stable structures with TM's of 41.5 and 47.5 degreesC (in 0.4 M NaCl) for the shorter and longer hairpins, respectively. The van't Hoff enthalpies of 80.7 and 114 kJ mol-1 are relatively low but are comparable to a parallel stranded d(GA)n duplex. On the basis of the spectroscopic and electrophoretic characteristics, we conclude that parallel strand formation is not restricted to hairpin systems, but also readily occurs in unconstrained dimeric duplexes with the appropriate sequence homologies. Both melting curves and electrophoretic analyses of parallel stranded heteroduplexes in which the sequence enforces specific base pairing demonstrate that G-G and A-T base pairs are formed in d(GA)n.d(GT)n segments.

Alternating polypurine d(GA)n, sequences exhibit a considerable polymorphism. Here we report that alpha d(GA) x d(GA) sequences form an antiparallel stranded duplex DNA at neutral pH. The spectroscopic, electrophoretic and thermodynamic properties of the alpha/beta chimeric oligodeoxynucleotide, 5'-d(GA)4(T)4 alpha d(AG)4T-3', support the formation of a hairpin structure with antiparallel strands in the stem. The optical properties of this novel antiparallel structure are different from the parallel stranded homoduplex formed by d(GA)G7. This alpha/beta hairpin has a remarkably high Tm of 44.5 degrees C in 0.4 M NaCl with a van't Hoff enthalpy comparable to that of a parallel d(GA)n duplex. Base pairing was confirmed by T4 polynucleotide ligase catalyzed joining of the alpha/beta hairpin to an antiparallel bimolecular duplex and by non-denaturing gel electrophoresis using duplexes containing sequence constraints. Both support the presence of alphaG-G and alphaA-A base pairing in the antiparallel 5'-d(GA)4(T)4 alpha d(AG)4T-3' intramolecular duplex. This study adds to the polymorphic nature of alternating d(GA)n sequences as well as providing novel homopurine base pairing approaches for probing polypurine polypyrimidine sequences.

Human papillomavirus (HPV) type 16 E6 and E7 inactivate the tumor suppressors p53 and pRB, respectively. Both viral oncoproteins play important roles in maintaining the transformed phenotype of cells. In this study, we examine the effects of antisense oligodeoxynucleotides with polarity and anomeric center reversal (alpha/beta-ODNs). ODNs of the general structure 5'alphaN3'3'NNN5'5'alphaN3'3'NNNN5'5'alphaN3+ ++'3'N5' were synthesized using phosphoramidite DNA chemistry. These alpha/beta-ODNs were complementary in sequence to regions flanking the start codons of HPV type 16 E6 and E7 genes. The anti-HPV type 16 alpha/beta-ODNs were able to form stable duplexes with their complementary RNA, which then serve as substrates for RNase H hydrolysis. Anti-HPV type 16 alpha/beta-ODNs also specifically inhibited the growth of two cervical carcinoma cell lines, CaSki and SiHa, both of which harbor HPV type 16 DNA. A decrease in E7 protein expression was also observed. Injection of nude mice with SiHa cells induces tumors. Treatment of these tumor-bearing mice with anti-HPV type 16 alpha/beta-ODNs led to substantially smaller tumors. These results show that alpha/beta-ODNs can exert antisense activities both in vitro and in vivo on the E6 and E7 genes of HPV type 16.

We have correlated the structural perturbations caused by DNA mismatches with the enzymatic data of the interaction of the restriction endonuclease EcoRI with DNA. Oligonucleotides d(CGAGAATTCTCA5GAXAATTCT) (X = G, A, T) and d(CGCGAATTYGCGT4CGCXAATTCGCG) (Y = C, X = G, T and Y = A, X = T) containing single mismatches within the EcoRI recognition site were characterized by NMR spectroscopy and by their EcoRI substrate properties. UV melting and gel electrophoresis studies confirm that the oligonucleotides form hairpin structures. The presence of either a CT or a CA mismatch results in markedly lower Tm and van't Hoff enthalpies compared with the fully base paired control. NMR imino proton spectra of these hairpins demonstrate that the perturbation caused by the two mispairs or a noncanonical AT pair is localized and limited to one or two base pairs on either side of the perturbation. The DNA hairpin structures containing single mismatches, and to a lesser extent also sequences with a single noncanonical base pair, are substrates for the restriction endonuclease. In addition to the strand scission at the nonperturbed GpA phosphodiester bond some cleavage is observed at the mismatched position. The interactions of the CA and CT mismatched hairpin with the enzyme are characterized by binding constants that are only 33 and 57 times lower, respectively, than that for the canonical sequence, corresponding to 8-10 kJ x mol(-1) less favorable free binding energy. This, taken together with the NMR data, indicates that the CA and CT mismatches have only small effects on the EcoRI recognition of the DNA substrate. We conclude that two out of the three hydrogen bonds that characterize the interaction of EcoRI with the CG base pair in the canonical sequence can still be formed for either the CT or CA mismatched recognition site.

Uracil-DNA glycosylase (UDG) is the enzyme responsible for the first step in the base-excision repair pathway that specifically removes uracil from DNA. Here we report the isolation of the cDNA and genomic clones for the mouse uracil-DNA glycosylase gene (ung) homologous to the major placental uracil-DNA glycosylase gene (UNG) of humans. The complete characterization of the genomic organization of the mouse uracil-DNA glycosylase gene shows that the entire mRNA coding region for the 1.83-kb cDNA of the mouse ung gene is contained in an 8.2-kb SstI genomic fragment which includes six exons and five introns. The cDNA encodes a predicted uracil-DNA glycosylase (UDG) protein of 295 amino acids (33 kDa) that is highly similar to a group of UDGs that have been isolated from a wide variety of organisms. The mouse ung gene has been mapped to mouse chromosome 5 using fluorescence in situ hybridization (FISH).

We report a comparative spectroscopic study of a novel self-complementary duplex decamer, d(GCGAAT-3'-3'-(alpha T)-5'-5'-CGC)2, in which an alpha-anomeric nucleotide has been inserted into the sequence in a parallel orientation via 3'-3' and 5'-5' phosphodiester bonds, and its unmodified B-DNA analog, d(GCGAATTCGC)2. Plots of the hyperchromicity and circular dichroism of these oligonucleotides are virtually identical, indicating that the overall base stacking and handedness are preserved in the alpha duplex. Thermodynamic parameters extracted from UV melting experiments show that the alpha duplex is only slightly less stable than the control. A near complete set of 1H and 31P nuclear magnetic resonance (NMR) assignments were obtained for both duplexes using classical one- and two-dimensional approaches. Several lines of evidence, in particular, imino 1H, 31P, nuclear Overhauser enhancement, and deoxyribose ring proton spin-spin coupling data, convincingly demonstrate that the overall structural integrity of the alpha and control duplexes are quite comparable, with any perturbations in the former localized to the regions of the construct encompassing the alpha-nucleotide and the unique backbone linkages. Specifically, the alpha duplex exhibits normal Watson-Crick type base pairing, it remains antiparallel except at the inverted nucleotide, all bases are in the anti orientation, and the sugar ring puckering is predominantly "S"-type. However, the J-coupling information for the alpha-nucleotide and the neighboring (3') cytidine are notably different, and reflect a decrease in the amplitude of the sugar pucker in alpha T7, and a significant shift in the conformational equilibrium of the furanose ring in C8 toward the "N"-type pucker. The feasibility of synthesizing oligodeoxynucleotides containing a combination of alpha sugars and short parallel stranded segments, their propensity for forming stable duplexes, and the structural insights into such complexes reported here are of potential importance in the area of antisense therapy.

The DNA repair enzyme Uracil-DNA Glycosylase (UDG) can be used to investigate three different features of protein-DNA interactions. Complexes can be probed by simple protection experiments ('footprinting') or by two kinds of interference assays: a missing thymine site (MT-site) experiment and a missing thymine methyl site (MTM-site) experiment. The three probing methods are assessed using the well-characterized in vitro systems of lambda repressor and lac repressor binding to their respective operator sites. The results obtained with UDG probing agree well with previous probing experiments on the same systems and, in certain cases, extend previous interpretations: for example, comparison of the results obtained with the two interference assays shows that formation of the lac repressor-operator complex requires interactions with the methyl group of one particular thymine residue (T-13) in the operator but also requires interactions with other parts of the thymine base at operator positions 7, 8, 9, 21, 23 and 24. Overall, the properties of UDG recommend it as a versatile and convenient method to investigate DNA-protein interactions both in vitro and possibly in vivo.

Systemic lupus erythematosus (SLE) sera, two classes of serum lipoproteins, and IgG antibodies from SLE and normal sera were tested for their reactivity with a Z-DNA polymer, Br-poly (dG-dC). In all cases preferential binding to Z-DNA over B-DNA was observed. This interaction, for the most part, could be inhibited by the negatively charged phospholipid, cardiolipin, which suggests that most of the anti-Z-DNA activity associated with sera arises from relatively non-specific ionic interactions between proteins and polyanionic molecules. An assay has been described that can eliminate proteins cross-reactive with negatively charged phospholipids.

The structure of the oligodeoxynucleotide (3')T8(5')-(5')C4A8(3') hairpin in aqueous solution was studied by two-dimensional (2D) proton and phosphorus nuclear magnetic resonance (NMR) spectroscopy. At 2.5 mM and 10 degrees C, the molecule exists predominantly as a monomolecular hairpin with a C4 loop. At higher concentrations and lower temperatures, NMR signals from multimers are obvious. They account for approximately 25% of the total population at 4 mM and 10 degrees C. Nearly all of the proton NMR signals for the hairpin could be assigned using 2D COSY, HOHAHA, and NOESY experiments. 2D 1H-31P correlation experiments were used to assign all the phosphorus resonances and to provide an additional check for the sequential assignments. A parallel-stranded T8.A8 stem can be formed in the hairpin due to the presence of the unusual 5'-5' linkage in the loop. 2D NOESY experiments indicate that the A H2 and its 5'-end neighbor base pair T methyl protons are within 5 A of each other. This is in accord with reverse Watson-Crick base pairing between T and A, which locates the A H2 and the T methyl protons in the same groove of the duplex. The chemical shifts of A H1', H2', and H2" sugar and the H2 base protons are quite different compared to normal B-DNA. Analysis of the 2D COSY and NOESY cross peak patterns indicates that the deoxyribose rings are mainly in the C2'-endo conformation and that the stem forms a right-handed helix, with the two strands held together by eight reverse Watson-Crick A.T base pairs to form a parallel-stranded duplex. The backbone torsion angles, as determined from the 31P chemical shifts, are slightly different for the A and the T residues. A molecular model was constructed, using a total of 336 proton NOE cross peak intensities as proton-proton distance constraints. In the refined structure, the conformations of the sugar-phosphate linkage, the deoxyribose rings, and the glycosyl bonds for the two parallel strands of the hairpin are close to a regular B-DNA structure. The base-stacking and the hydrogen-bonding interactions are well optimized; however, the two grooves are of approximately equal width. Thus, compared to B-DNA, the parallel-stranded duplex has a very different surface shape, and because of the reverse Watson-Crick base pairing, it has different groups exposed in each groove.

The ability of recA protein to interact with a Z-DNA polymer, Br-poly(dG-dC), or M13 bacteriophage single-stranded DNA was investigated. RecA protein binds more avidly to Z-DNA than to single-stranded DNA in the absence of a nucleotide cofactor. This binding pattern changes in the presence of adenosine 5'-(gamma-thio)triphosphate (ATP[S]), however, such that the binding to Z-DNA decreases while binding to single-stranded DNA increases roughly 2-fold. When present together, the two forms of DNA compete with each other in the presence of ATP[S]. Experiments involving recA protein binding to recombinant plasmids showed neither a preferential binding of recA protein to the plasmid containing Z-DNA nor a similar effect of ATP[S] to that observed with the Z-DNA polymer. In contrast, maximal binding was obtained with a plasmid (linear or supercoiled) containing a polypurine.polypyrimidine insert, thus suggesting that recA protein displays sequence preferences in its interaction with DNA. The results of the present study provide no evidence that recA protein specifically interacts with or stabilizes the Z-DNA insert of a recombinant plasmid in the left-handed conformation.

Uracil DNA glycosylase excises uracil residues from DNA that can arise as a result of deamination of cytosine or incorporation of dUMP residues by DNA polymerase. We have carried out a detailed study to define the specificities and the kinetic parameters for its substrates by using a number of synthetic oligodeoxyribonucleotides of varying lengths and containing uracil residue(s) in various locations. The results show that the Escherichia coli enzyme can remove a 5'-terminal U from an oligomer only if the 5'-end is phosphorylated. The enzyme does not remove U residues from a 3'-terminal position, but U residues can be excised from oligonucleotides with either pd(UN)p or pd(UNN) 3'-termini. The oligomer d(UUUUT) can have the second or third U residues from the 5'-end excised even when the neighboring site is an abasic site (3' or 5', respectively). On the basis of these findings, pd(UN)p was anticipated to be the smallest size substrate. Results show detectable amounts of U release from the substrate pd(UT)p; however, significantly higher amounts of U release were observed from pd(UT-sugar) or pd(UTT). Determinations of the Km and Vmax values show that the different rates of U excision from oligomers of different sizes (trimeric to pentameric) but containing U in the same position are largely due to the differences in the Km values, whereas the different rates of U excision from the substrates of the same size but containing U in different positions are largely due to different Vmax values.

A group of single-stranded d(TC)n specific binding proteins has been detected in the nuclear extracts of several mammalian species that included mouse, human, African green monkey, chimpanzee, and Chinese muntjac. Southwestern analysis of 500 mM KCI nuclear extracts has shown that these proteins cluster in a similar size range, 55.5 to 57 kD. An additional 54 kD band was present for the three primate species examined. The single-stranded d(TC)n binding activity was confirmed with bandshift assay. Specific double-stranded binding activity for duplex d(TC)n.d(GA)n or single-stranded d(GA)n was not detected. The conservation of size distribution and d(TC)n-binding activity across the species examined indicates that this class of single-stranded binding proteins may have an important biological function in vivo.

The RecA protein of Escherichia coli binds specifically to acidic phospholipids such as cardiolipin and phosphatidylglycerol. This binding appears to be affected by the presence of divalent cations such as Ca2+ and Mg2+. The interaction leads to the inhibition of RecA binding to at least two different conformations of DNA, single-stranded DNA and left-handed Z-DNA, thus suggesting that the phospholipids interact at the DNA-binding site of the RecA protein. Inclusion of a nucleotide cofactor [adenosine 5'-O-(gamma-thiotriphosphate)] in the reactions did not prevent the inhibition of DNA-binding activities of RecA protein by the phospholipids. The interaction of RecA protein with cardiolipin and phosphatidylglycerol, which represent two of the three major phospholipids of the E. coli membrane, may be physiologically important, as it provides a possible mechanism for the RecA-membrane association during the SOS response. These observations raise the possibility that the Z-DNA-binding activity of RecA protein is merely a manifestation of its phospholipid-binding property.

Parallel-stranded DNA can be formed from alternating AT segments and is not restricted exclusively to homooligomeric AT sequences. DNA oligonucleotides 3'-d(AT)nxC4(AT)n-3' (where x indicates the location of the 5'-5' phosphodiester linkage) form parallel-stranded hairpin structures at micromolar strand concentration for n = 4 or 5 but not for n = 6, 7. The spectral properties of the parallel-stranded structures are similar to those of the hairpin structures containing homooligomeric AT stems. However, parallel-stranded structures formed in alternating AT segments are significantly less stable than either their corresponding antiparallel control or the homooligomeric parallel AT hairpins as evidenced by their lower helix-coil transition enthalpy, melting temperature, and stability constant. This results in a remarkable polymorphism which is most pronounced for 3'-d(AT)5xC4(AT)5-3'. This oligonucleotide can exist as a parallel-stranded hairpin, coil, or concatameric antiparallel structure(s), depending on temperature and strand concentration. These results suggest simple guidelines for the design of parallel-stranded DNA. In addition, we present a model for the assessment of the stability of parallel-stranded duplex structures formed from AT base pairs based on their sequence.

The solution structures of two alternating purine-pyrimidine octamers, [d(G-T-A-C-G-T-A-C)]2 and the reverse sequence [d(C-A-T-G-C-A-T-G)]2, are investigated by using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Chemical shift assignments are obtained for non-exchangeable protons by a combination of two-dimensional correlation and nuclear Overhauser enhancement (NOE) spectroscopy experiments. Distances between protons are estimated by extrapolating distances derived from time-dependent NOE measurements to zero mixing time. Approximate dihedral angles are determined within the deoxyribose ring from coupling constants observed in one and two-dimensional spectra. Sets of distance and dihedral determinations for each of the duplexes form the bases for structure determination. Molecular dynamics is then used to generate structures that satisfy the experimental restraints incorporated as effective potentials into the total energy. Separate runs start from classical A and B-form DNA and converge to essentially identical structures. To circumvent the problems of spin diffusion and differential motion associated with distance measurements within molecules, models are improved by NOE-based refinement in which observed NOE intensities are compared to those calculated using a full matrix analysis procedure. The refined structures generally have the global features of B-type DNA. Some, but not all, variations in dihedral angles and in the spatial relationships of adjacent base-pairs are observed to be in synchrony with the alternating purine-pyrimidine sequence.

The distribution of d(CT)-rich pyrimidine tracts in the karyotypes of a variety of vertebrates was studied by in situ hybridization. The probe for these studies was a 56bp homopyrimidine/homopurine sequence obtained from a mouse genomic library constructed with DNA prepared from a restriction enzyme digestion of metaphase chromosomes. Single-stranded DNA nuclease digestions and two-dimensional gel analysis of topoisomers of this sequence indicated that it is capable of adopting a triplex conformation in vitro. In situ hybridization with this probe to the karyotypes of ten different vertebrate species revealed a highly conserved chromosomal distribution of d(CT)-rich tracts. These tracts are found throughout the chromosomal arms and in some karyotypes they are clustered, producing a banding pattern. However, at the resolution of the light microscope these tracts appeared to be absent from the centromeric regions of all chromosomes examined except those of chicken. The non-random distribution of these tracts to the chromosomal arm regions implies an organizational or functional role for this repeat class. It is unlikely that the 56 bp sequence type contributed to the formation of the triplex DNA structure previously detected in centromeric domains of mouse.

We have investigated loop-induced structural perturbation of the stem structure in hairpins d(GAATTCXnGAATTC) (X = A, T and n = 3, 4, 5 and 6) that contain an EcoRI restriction site in close proximity to the hairpin loop. Oligonucleotides containing either a T3 or a A3 loop were not hydrolyzed by the restriction enzyme and also showed only weak binding to EcoRI in the absence of the cofactor Mg2+. In contrast, hairpins with larger loops are hydrolyzed by the enzyme at the scission site next to the loop although the substrate with a A4 loop is significantly more resistant than the oligonucleotide containing a T4 loop. The hairpin structures with 3 loop residues were found to be thermally most stable while larger hairpin loops resulted in structures with lower melting temperatures. The T-loop hairpins are thermally more stable than the hairpins containing the same number of A residues in the loop. As judged from proton NMR spectroscopy and the thermodynamic data, the base pair closest to the hairpin loop did form in all cases studied. The hairpin loops did, however, affect the conformation of the stem structure of the hairpins. From 31P and 1H NMR spectroscopy we conclude that the perturbation of the stem structure is stronger for smaller hairpin loops and that the extent of the perturbation is limited to 2-3 base pairs for hairpins with T3 or A4 loops. Our results demonstrate that hairpin loops modulate the conformation of the stem residues close to the loop and that this in turn reduces the substrate activity for DNA sequence specific proteins.

We used a Z-DNA affinity column to isolate a collection of Z-DNA binding proteins from a high salt extract of Escherichia coli. We identified one of the major Z-DNA binding proteins of this fraction, not as a protein involved in gene regulation or genetic recombination, but rather as an outer membrane porin protein. We then showed that several other known phospholipid-binding proteins (bovine lung annexins and human serum lipoproteins) also bind much more tightly to Z-DNA than to B-DNA. In all cases, this Z-DNA binding was strongly blocked by competition with acidic phospholipids, such as cardiolipin. Our results raise the question whether many of the Z-DNA binding proteins previously isolated are actually phospholipid-binding proteins.

The partial self-complementary 24-mer oligodeoxynucleotide d(C-G)5T4(C-G)5 forms a hairpin which can be enzymatically dimerized to a dumbbell structure. The blunt-ended nature of the hairpin is indicated by its ability to inhibit the T4 DNA ligase catalyzed joining of phi X174 HaeIII fragments. The hairpin monomer and dimer (dumbbell) undergo a reversible B to Z transition as shown by ultraviolet, circular dichroism, and 31P NMR spectroscopy. The Z form of the hairpin monomer and dimer is supported by monovalent ions (Na+), divalent ions (Mg2+ but not Mn2+), and dehydrating (ethanol) conditions. The conformational transition of d(C-G)5T4(C-G)5 monomer requires higher ionic or dehydrating conditions than those necessary for the corresponding linear oligomer d(C-G)5. The contribution of the loop (-T4-) of the hairpin to the apparent free energy change for the B to Z conformational transition at the midpoint was calculated to be 3.8 kJ mol-1.

The ability of right- vs. left-handed alternating purine/pyrimidine copolymers to support the formation of nucleosomes has been examined by using a trout testis assembly factor. The protein, which is thermostable, has a molecular weight of 29000 and will assemble nucleosomes onto both SV40 and calf thymus DNA. This assembly factor has been used to assemble nucleosomes onto the B and Z conformations of poly[d(Gm5C)] and the B conformation of poly[d(GC)]. The isolated B-form particles, which sediment at approximately 11 S in a sucrose density gradient, contain DNA of 140-200 bases in length and the four core histones. The isolated Z-form particles, which also sediment at approximately 11 S, contain the four core histones and DNA of 170-250 bases in length. Physical analysis of the particles by absorbance and circular dichroic spectroscopy indicates that the DNA remains in the original conformation throughout the isolation procedure. Further, the particles reconstituted onto left-handed DNA compete effectively for an anti-Z DNA antibody, while the corresponding right-handed particles do not. Analytical sedimentation velocity determinations indicate that the B-form poly[d(Gm5C)] and poly[d(GC)] particles sediment at 11.2 and 11.1 S, respectively. In contrast, the poly[d(Gm5C)] Z-form particles have an S20,w of 10.6 S. The differences in the sedimentation velocity and the density of the cores, and in the lengths of DNA associated with the particles, suggest that the conformation of the DNA affects the manner in which it associates with the histone octamer.

The phenanthridine dye ethidium bromide (EB) intercalates with double-stranded DNA (dsDNA) resulting in an enhancement of fluorescence. Single-stranded DNA (ssDNA) does not show this fluorescent enhancement. Purified IgG from patients with Systemic Lupus Erythematosus (SLE) containing anti-dsDNA antibodies competes with EB for binding to DNA resulting in a decrease in fluorescence. This study has shown that antibodies which bind ssDNA in the Millipore filter radioimmunoassay displace EB from dsDNA showing that antigenic determinants are available for binding in the double-stranded molecule. This study introduces the EB assay and presents a comparison with the Millipore filter assay.

The complete nucleotide sequence of the Escherichia coli ung gene is described. Transcription initiation and termination sites were determined by S1 nuclease and RNase mapping. The common prokaryotic -35, -10, and the ribosome binding site sequences are represented by TGTTCTGTA, TAAGCTA, and AGGAGAG at their respective locations. A putative hairpin transcription terminator structure is present at the major transcription terminator sites. The open reading frame of the ung gene codes for a protein of 229 amino acids (25,664 daltons). The molecular weight, amino acid composition, and the N-terminal amino acid sequence of the uracil DNA glycosylase purified from E. coli cells match with the open reading frame of the ung gene. The protein sequence analysis shows that the N-terminal methionine is cleaved off in the mature protein. The in vitro transcription coupled translation of the ung gene directs the synthesis of a protein which comigrates with uracil DNA glycosylase. Also, the CNBr cleavage of the protein synthesized in vitro confirms the positions of the methionines deduced from the DNA sequence. The levels of ung gene expression remain constant up to the early stationary phase, but decline in the late stationary phase of the E. coli culture. The E. coli gene showed a strong sequence homology to Shigella, a weak sequence homology to Salmonella and Citrobacter, and a very weak sequence homology to Proteus genes. No sequence homologies were seen for Pseudomonas, Clostridium, Micrococcus, and several eukaryotic genomes.

The poly(dA) dependent T4 polynucleotide ligase catalyzed polymerization of oligodeoxythymidylates is dependent upon duplex stability. The antibiotics ethidium bromide, netropsin and Hoechst 33258 stabilize the duplex poly(dA) . P(dT)n (n = 6-10) to thermal denaturation. Ethidium bromide to DNA ratio of 1.25 and netropsin or Hoechst 33258 to DNA ratio of 0.1 the Tm of d(pT) 10 . poly (dA) was increased by 10 degrees and 25 degrees C respectively. The T4 polynucleotide ligase activity was not inhibited under these conditions and temperature optimum of joining of d(pT) 10 . poly(dA) was increased 5 degrees to 10 degrees by the binding of the antibiotics. Duplexes containing shorter oligodeoxythymidylates required lower concentrations of the antibiotics netropsin or Hoechst 33258 to show no inhibition of T4 polynucleotide ligase. The temperature optima of joining the duplexes d(pT)6 . POLY(DA) and d(pT) 8 . poly(dA) were increased by 5 degrees C upon binding of the antibiotics. Polyacrylamide gel analysis of the T4 polynucleotide ligase catalyzed joining of the oligodeoxythymidylates showed that the presence of antibiotics affected the product distribution of the polymerized oligomers.

Yolk proteins purified from the nematode Caenorhabditis elegans, from the frog Xenopus laevis, and from chicken eggs all have the unexpected property of binding strongly and preferentially to a left-handed Z-DNA probe, brominated poly(dG-dC). We estimate that the nematode proteins bind to Z-DNA with an association constant of at least 10(4) (M-1) and that this association constant is at least 40-50-fold higher than the association constant to B-DNA. Thus, yolk proteins have a higher Z-DNA specificity than most of the Z-DNA binding proteins previously isolated from other sources. Although yolk protein binding to Z-DNA is poorly competed by a wide variety of nucleic acids, the interaction is strongly competed by the phospholipids cardiolipin and phosphatidic acid (500-1000-fold better than by the same mass of B-DNA). We suggest that Z-DNA interacts with the yolk protein phospholipid binding site. In general, our results emphasize the danger of using physical properties to infer biological function. In particular, our results should raise serious questions about the biological relevance of previously isolated Z-DNA binding proteins.

Several antibiotics, netropsin, distamycin A, actinomycin D, Hoechst 33258 and olivomycin, which demonstrate base specificity in their DNA binding properties have been found to alter the electrophoretic mobility of DNA restriction fragments in native polyacrylamide gels. The antibiotics mostly reduced the migration of larger DNA fragments, but netropsin and Hoechst 33258 were observed to increase the migration rate of several DNA fragments of intermediate size. DNA fragments of similar molecular weight which comigrate as a single gel band can at times be separated as the result of differential mobility shifts promoted by antibiotic DNA complex formations.

The effects of the first-row transition metal ions on the right(B)- to left(Z)-handed helical transition of poly[d(G-C)] have been determined. The Z conformation is induced by MnCl2 at submillimolar concentrations. The forward reaction has a very large activation energy (440 kJ/mol) so that a facile conversion occurs only at temperatures above 45 degrees C. However, the left-handed form remains stable upon cooling. The addition of ethanol (20% v/v) eliminates the requirement for elevated temperature. The transition is highly co-operative and is accompanied by spectral changes (absorption, circular dichroism) characteristic for the B----Z conformational transition. NiCl2 and CoCl2 also induce the B----Z transition in poly[d(G-C)] but the activation energies and thus the temperature requirements for the forward reaction are lower than those observed with MnCl2. The left-handed DNA formed in the presence of Mn2+ is similar to 'Z DNA' previously described in Mg2+-EtOH (van de Sande and Jovin , 1982): (a) it readily sediments out of solution at low speed as a consequence of intermolecular association which, however, is not accompanied by turbidity; and (b) it supports the binding of ethidium bromide although this drug interacts preferentially with the B form of DNA. With Ni2+, the B----Z isomerization step can be separated from the subsequent specific Z----Z* association. Mn2+, Ni2+, and Co2+ also promote the B----Z transition of poly[d(G-m5C)] at substoichiometric concentrations with respect to DNA nucleotide.

Nucleosomal core particles containing the right- and left-handed conformations of DNA were examined for their ability to support the B----Z or Z----B transition. Nucleosomes were assembled onto the B-and Z-conformations of poly[d(Gm5C)] and the B-conformation of poly[d(GC)] as previously described. Absorbance and circular dichroic spectroscopy indicated that the DNA on all three core particle populations could undergo the conformational B----Z transition. Further, the right- to left-handed transition for both poly[d(Gm5C)] and poly[d(GC)] appeared to be facilitated by the DNAs association with the histone octamer. The DNA remained associated with the protein core subsequent to the transition, and electron microscopy and sedimentation velocity analysis indicated that there were no gross changes in nucleosomal structure. However, a change in the sedimentation value of the poly[d(Gm5C)] core particles was detected when the conformation of the DNA was altered from B to Z, resulting in a lower S20,w value for the Z-form particles than for the corresponding B-form particles.