Poly(L-lysine)-graft-dextran copolymer is a novel stabilizer of triplex DNA (I): stabilization of poly(dA).2poly(dT) triplex

Comb-type polylysine copolymer having grafted hydrophilic side chains was newly designed as a novel stabilizer of triplex DNAs. The comb-type copolymer elevated melting temperature of poly(dA).2poly(dT) triplex by 50 degrees C without affecting reversibility, melting and reassociation, of the triplex in buffer with physiological salt concentrations. The stabilizing effect of the copolymer was greater than spermine. Our results indicate that the molecular designing of polycation with comb-type structure is a successful strategy for creating an effective triplex stabilizer.     

Polymorphism and disorder of poly(dA).poly(dT) in fibers

OBJECTIVE: The purpose of this report is to describe the clinical history, treatment, pathology, and imaging in two cases of rare CNS infection caused by free-living amebas. The Naegleria fowleri and Acanthamoeba species cause primary amebic meningoencephalitis and granulomatous amebic encephalitis, respectively. We describe the neuroimaging findings of a case involving a nonspecific cerebral edema pattern in primary amebic meningoencephalitis and a case involving focal enhancing lesions in granulomatous amebic encephalitis. CONCLUSION: Primary amebic meningoencephalitis and granulomatous amebic encephalitis have a grave prognosis and, although rare, should be considered in the differential diagnosis for patients who present with appropriate histories and imaging findings, including nonspecific brain edema on CT in primary amebic meningoencephalitis and focal punctate enhancing lesions in the posterior cranial fossa on T1-weighted MR imaging in granulomatous amebic encephalitis.     

Temperature effects on the structure of poly(dA).poly(dT): an X-ray fiber diffraction study

X-ray fiber diffraction of poly(dA).poly(dT) subjected to variation in the relative humidity, has allowed us to demonstrate the effects of temperature on the conformation of the polynucleotide. When the temperature of the poly(dA).poly(dT) is greater than 30 degrees C and the relative humidity near 80%, a new diffraction pattern is obtained. We observe a transition between the classical alpha B' form of poly(dA).poly(dT) and a double helical structure, B*, which remains stable at a temperature up to 70 degrees C. This new conformation of poly(dA).poly(dT) is a right-handed double helix with 11.4 nucleotide pairs per turn and a pitch of 36.7 A.     

Specific excision of the inserted DNA segment from hybrid plasmids constructed by the poly(dA). poly (dT) method

The hybrid plasmid PBETAG, consisting of plasmid PMB9 DNA with an insert of rabbit globin DNA (about 600 base pairs) flanked by poly(dA) poly(dT) regions (Maniatis, T., Kee, S.G., Efstratiadis, A. and Kafatos, F.C. (1976) Cell 8, 163-182), was cleaved into two fragments by endonuclease S1 under conditions of partial denaturation. Only the smaller fragment (575 base pairs) contained globin-specific sequences, showing that excision had occurred in the A-T-rich regions. This method of cleavage provides a useful procedure for assessing the length of inserts in hybrid plasmids prepared by the poly(dA)-POLY(DT) tail method, and allows the preparative recovery of the insert.     

Synergistic effects in the melting of DNA hydration shell: melting of the minor groove hydration spine in poly(dA).poly(dT) and its effect on base pair stability

We propose that water of hydration in contact with the double helix can exist in several states. One state, found in the narrow groove of poly(dA).poly(dT), should be considered as frozen to the helix, i.e., an integral part of the double helix. We find that this enhanced helix greatly effects the stability of that helix against base separation melting. Most water surrounding the helix is, however, melted or disassociated with respect to being an integral part of helix and plays a much less significant role in stabilizing the helix dynamically, although these water molecules play an important role in stabilizing the helix conformation statically. We study the temperature dependence of the melting of the hydration spine and find that narrow groove nonbonded interactions are necessary to stabilize the spine above room temperature and to show the broad transition observed experimentally. This calculation requires that synergistic effects of nonbonded interactions between DNA and its hydration shell affect the state of water-base atom hydrogen bonds. The attraction of waters into narrow groove tends to retain waters in the groove and compress or strain these hydrogen bonds.     

Termination within oligo(dT) tracts in template DNA by DNA polymerase gamma occurs with formation of a DNA triplex structure and is relieved by mitochondrial single-stranded DNA-binding protein

Xenopus laevis DNA polymerase gamma (pol gamma) exhibits low activity on a poly(dT)-oligo(dA) primer-template. We prepared a single-stranded phagemid template containing a dT41 sequence to test the ability of pol gamma to extend a primer through a defined oligo(dT) tract. pol gamma terminates in the center of this dT41 sequence. This replication arrest is abrogated by addition of single-stranded DNA-binding protein or by substitution of 7-deaza-dATP for dATP. These features are consistent with the formation of a T.A*T DNA triplex involving the primer stem. Replication arrest occurs under conditions that permit highly processive DNA synthesis by pol gamma. A similar replication arrest occurs for T7 DNA polymerase, which is also a highly processive DNA polymerase. These results suggest the possibility that DNA triplex formation can occur prior to dissociation of DNA polymerase. Primers with 3'-oligo(dA) termini annealed to a template with a longer oligo(dT) tract are not efficiently extended by pol gamma unless single-stranded DNA-binding protein is added. Thus, one of the functions of single-stranded DNA-binding protein in mtDNA maintenance may be to enable pol gamma to successfully replicate through dT-rich sequences.     

Gene stability in transgenic aspen (Populus). I. Flanking DNA sequences and T-DNA structure

The stability of transgenes in the genome of transformed plants depends strongly on their correct physical integration into the host genome as well as on flanking target DNA sequences. For long-lived species like trees, however, no information is available so far concerning inactivation or loss of transgenes due to gene silencing or somatic genome rearrangement events. In this study, four independently transformed 35S-rolC transgenic hybrid aspen plants (Populus tremula L. x tremuloides Michx.), each harbouring one copy of the transgene, were investigated during continuous growth in the greenhouse. In one of these transgenic lines (Esch5:35S-rolC-#1) individuals frequently show phenotypic reversions, while in the remaining three lines (Esch5:35S-rolC-#3, -#5, -#16) the gene was essentially stable. Molecular analysis including PCR, Southern and Northern assays clearly showed that the transgene had been lost in the revertant tissue of the unstable line. Sequencing of T-DNA right and left borders, and flanking DNA regions, in all four transgenic aspen lines revealed no differences either in the type of flanking DNA (G-C to A-T ratio) or with respect to the presence of enhancers or MAR (matrix associated repeats)-like structures. Primers located within the left and right flanking regions in the three stable lines could be used to recover the target sites from the untransformed plants. This was not possible, however, with the unstable line, indicating that at least one flanking sequence does not derive from the plant target DNA but is of unknown origin. PCR using other primer pairs, and inverse PCR analysis, revealed an additional truncated T-DNA copy of 1050 nucleotides adjacent to the left border of the complete copy in this line. Sequencing of this truncated T-DNA revealed that it represented an inverted copy of part of the right half of the original construct. This special feature would allow the inverted repeat to pair with right border sequences of the complete copy. This would explain the frequently observed reversion resulting in transgene loss as due to intrachromosomal base-pairing leading to double-stranded loops of single-stranded DNA during mitotic cell divisions.     

Solution structure of an intramolecular DNA triplex containing 5-(1-propynyl)-2'-deoxyuridine residues in the third strand

Incorporation of the modified base 5-(1-propynl)-2'-deoxyuridine (propynylU) in the third strand of a triplex leads to enhanced triplex stabilization. To investigate effects of the propyne nucleotide on triplex structure and the factors underlying the increased stability, we have determined the solution structure of the intramolecular DNA pyrmidine-purine-pyrimdine d(AGAGAGAA-(EG)6-TTCTCTCT-(EG)6-PCPCPCPP) (PDD-EG), which contains 5-(1-propynl)-2'-deoxyuridine (P) in the third strand and hexakis(ethylene glycol) linkers [(EG)6]. The structure was calculated using X-PLOR with distance and dihedral angle restraints obtained from two-dimensional NMR experiments and refined with the direct relaxation matrix method. The structures show that the extended aromatic electron cloud of the propynylU nucleotide stacks well over the 5'-neighboring nucleotides, resulting in increased stabilization. The propynylU nucleotides also affect the overall structure of the triple helix. A comparison of the structure to that of the nonmodified intramolecular DNA triplex of the same sequence, d(AGAGAGAA-(EG)6-TTCTCTCT-(EG)6-TCTCTCTT) (DDD-EG), shows that PDD-EG has a more A-DNA like X displacement and inclination than DDD-EG yet still maintains predominantly S-type sugar puckers as found in DDD-EG and other DNA triplexes.     

The crystal structure of the RNA/DNA hybrid r(GAAGAGAAGC). d(GCTTCTCTTC) shows significant differences to that found in solution

The crystal structure of the RNA/DNA hybrid r(GAAGAGAAGC). d(GCTTCTCTTC) has been solved and refined at 2.5 A resolution. The refinement procedure converged at R = 0.181 for all reflections in the range 20.0-2.5 A. In the crystal, the RNA/DNA hybrid duplex has an A' conformation with all but one of the nucleotide sugar moieties adopting a C3'- endo (N) conformation. Both strands in the double helix adopt a global conformation close to the A-form and the width of the minor groove is typical of that found in the crystal structures of other A-form duplexes. However, differences are observed between the RNA and DNA strands that make up the hybrid at the local level. In the central portion of the duplex, the RNA strand has backbone alpha, beta and gamma torsion angles that alternate between the normal gauche -/ trans / gauche + conformation and an unusual trans / trans / trans conformation. Coupled with this so-called 'alpha/gamma flipping' of the backbone torsion angles, the distance between adjacent phosphorous atoms on the RNA strand systematically varies. Neither of these phenomena are observed on the DNA strand. The structure of the RNA/DNA hybrid presented here differs significantly from that found in solution for this and other sequences. Possible reasons for these differences and their implications for the current model of RNase H activity are discussed.     

Evidence that the extracytoplasmic function sigma factor sigmaE is required for normal cell wall structure in Streptomyces coelicolor A3(2)

The sigE gene of Streptomyces coelicolor A3(2) encodes an RNA polymerase sigma factor belonging to the extracytoplasmic function (ECF) subfamily. Constructed sigE deletion and disruption mutants were more sensitive than the parent to muramidases such as hen egg white lysozyme and to the CwlA amidase from Bacillus subtilis. This correlated with an altered muropeptide profile, as determined by reverse-phase high-performance liquid chromatography analysis of lytic digests of purified peptidoglycan. The sigE mutants required high levels of magnesium for normal growth and sporulation, overproducing the antibiotic actinorhodin and forming crenellated colonies in its absence. Together, these data suggest that sigE is required for normal cell wall structure. The role of sigmaE was further investigated by analyzing the expression of hrdD, which is partially sigE dependent. The hrdD gene, which encodes the sigmaHrdD subunit of RNA polymerase, is transcribed from two promoters, hrdDp1 and hrdDp2, both similar to promoters recognized by other ECF sigma factors. The activities of hrdDp1 and hrdDp2 were reduced 20- and 3-fold, respectively, in sigE mutants, although only hrdDp1 was recognized by EsigmaE in vitro. Growth on media deficient in magnesium caused the induction of both hrdD promoters in a sigE-dependent manner.     

Hemoglobin Sydney--alpha beta 2 67 (E11) Val-Ala and hemoglobin Olomouc alpha 2 beta 2 86 (F 2) Ala-Asp in Czech families. DNA sequence analysis for a more accurate diagnosis of hemoglobinopathies

The paper demonstrates the importance of sequence analysis of DNA for the identification of Hb-Sydney [alpha2-beta2 67 (E11) Val-Ala]. The latter was considered erroneously, based on results of biochemical analyses to be Hb-M-Milwaukee [alpha2 beta2 67 (E 11) Val-Glu]. With the unstable Hb-Sydney correspond also phenotypical manifestations of disease (haemolytic anaemia with Heinz bodies in red blood cells). Sequence analysis of DNA of patients with Hb-Olomouc [alpha 2 beta 2 (F 2) Ala-Asp] revealed that mutation of Ala-Asp in position 86 (F 2) of the beta globin chain is coded by mutation C-->A (GCC-GAC).     

A correlation between stability of compounds and structure of hydrogen-induced amorphous alloys in GdM2 (M = Fe,Co, Ni)

Enthalpy changes ΔH for hydrogen absorption and desorption, hydrogen-induced amorphization (HIA) in C15 Laves phases c-GdM₂ (M = Fe, Co, Ni) and crystallization of the resultant amorphous alloys a-GdM₂H_x have been measured by differential scanning calorimetry (DSC). Furthermore, structures of a-GdM₂H_x have been investigated by X-ray diffractometry (XRD) to correlate their structures with the stabilities of the original compounds. Both a-GdFe₂H_3.0 and a-GdCo₂H_3.4 prepared from the less stable compounds show the strong tendency for the same kinds of metal atoms to cluster and crystallize to GdH₂ and Fe(Co). Correspondingly, the sum of ΔH for all reactions become equal to that for the formation of GdH₂. On the contrary, a-GdNi₂H_3.5 prepared from the stable compound shows a rather homogeneous structure and crystallizes to GdH₂ and GdH₂ and GdNi₅. As a result, the sum of ΔH shows a large negative value in comparison with that for the formation of GdH₂.     

The Saccharomyces cerevisiae protein YJR043C (Pol32) interacts with the catalytic subunit of DNA polymerase alpha and is required for cell cycle progression in G2/M

We have analysed the YJR043c gene of Saccharomyces cerevisiae, previously identified by systematic sequencing. The deletion mutant (yjr043cdelta) shows slow growth at low temperature (15 degrees C), while at 30 degrees C and 37 degrees C the growth rate of mutant cells is only moderately affected. At permissive and nonpermissive temperatures, mutant cells were larger and showed a high proportion of large-budded cells with a single duplicated nucleus at or beyond the bud neck and a short spindle. This phenotype was even more striking at low temperature, the mutant cells becoming dumbbell shaped. All these phenotypes suggest a role for YJR043C in cell cycle progression in G2/M phase. In two-hybrid assays, the YJR043c gene product specifically interacted with Pol1, the catalytic subunit of DNA polymerase alpha. The pol1-1 /yjr043cdelta double mutant showed a more severe growth defect than the pol1-1 single mutant at permissive temperature. Centromeric plasmid loss rate elevated in yjr043cdelta. Analysis of the sequence upstream of the YJR043c ORF revealed the presence of an MluI motif (ACGCGT), a sequence associated with many genes involved in DNA replication in budding yeast. The cell cycle phenotype of the yjr043cdelta mutant, the evidence for genetic interaction with Pol1, the presence of an MluI motif upstream and the elevated rate of CEN plasmid loss in mutants all support a function for YJR043C in DNA replication.     

Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27Kip1 are limited to cell cycle control at the G1/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G1/S transition, contributes to regulation of the G2/M transition. Both G1- and G2-arrested cells were observed in all cell types, with different preponderances. Preponderant G1 arrest in response to p21 expression correlated with the presence of functional pRb. G2 arrest was more prominent in pRb-negative cells. The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest distributions [corrected], DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G2-arrested cells. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G1 arrest as well as to blockade of DNA replication from either G1 or G2 phase is the pRb regulatory system. Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway.