Selective inhibition of DNA gyrase in vitro by a GC specific eight-ring hairpin polyamide at nanomolar concentration

The influence of an eight-ring hairpin DNA minor groove binder on the gyrase mediated DNA supercoiling and cleavage reaction step of the enzyme was investigated. The results demonstrate that supercoiling is affected by the hairpin polyamide in the millimolar concentration range while the enzyme catalyzed cleavage of a 162 bp fragment of pBR322 containing a single strong gyrase site is effectively inhibited at nanomolar concentration. As demonstrated by footprint analysis the latter effect is caused by a specific binding of the hairpin forming polyamide to the enzyme recognition site (GGCC), which indicates that the gyrase activity to produce a double strand break is blocked at this site. The pyrrole-imidazole hairpin polyamide is the most potent inhibitor of the gyrase mediated cleavage reaction compared to other known anti-gyrase active DNA binding agents.     

Cell-mediated DNA transport between distant inflammatory sites following intradermal DNA immunization in the presence of adjuvant

After intradermal genetic immunization, naked DNA is transported from the site of injection to regional lymph nodes. Little is known on how inflammation influences this process and whether DNA is transported beyond local lymph nodes. In the experiments herein reported, we injected naked DNA in the presence of adjuvant to address questions related to 1) the fate of naked DNA in the presence of inflammation; 2) the generation of immune responses to the encoded protein during inflammation; and, more in general, 3) the fate of ingested molecules beyond regional lymph nodes during inflammation. Two sites of inflammation were induced in vivo in mice. Naked DNA was injected in the nape together with adjuvant, and adjuvant only was injected at a distant peritoneal site. Injected DNA, uptaken at the primary dermal site of inflammation, was transported beyond regional lymph nodes to distant organs such as the spleen and to the distant peritoneal site of inflammation. This transport, mediated by CD11b+ cells, was cumulative during chronic inflammation. These results indicate a novel route of transport of DNA beyond regional lymph nodes and may have specific implications for DNA-based immune modulation.     

Reproductive isolation in Caenorhabditis: terminal phenotypes of hybrid embryos

SUMMARY Several interspecific combinations of the "elegans" group of Caenorhabditis species are cross-fertile. Most F1 hybrids from these crosses arrest during embryogenesis. Developmental defects observed in hybrid embryos include defects in gastrulation initiation, defects in embryonic compaction, and defects in embryonic elongation. These reproductive barriers have arisen multiple times in the evolution of Caenorhabditis.     

Steady-state and rapid kinetic analysis of topoisomerase II trapped as the closed-clamp intermediate by ICRF-193

DNA topoisomerase II uses a complex, sequential mechanism of ATP hydrolysis to catalyze the transport of one DNA duplex through a transient break in another. ICRF-193 is a catalytic inhibitor of topoisomerase II that is known to trap a closed-clamp intermediate form of the enzyme. Using steady-state and rapid kinetic ATPase and DNA transport assays, we have analyzed how trapping this intermediate by the drug perturbs the topoisomerase II mechanism. The drug has no effect on the rate of the first turnover of decatenation but potently inhibits subsequent turnovers with an IC(50) of 6.5 +/- 1 microM for the Saccharomyces cerevisiae enzyme. This drug inhibits the ATPase activity of topoisomerase II by an unusual, mixed-type mechanism; the drug is not a competitive inhibitor of ATP, and even at saturating concentrations of drug, the enzyme continues to hydrolyze ATP, albeit at a reduced rate. Topoisomerase II that was specifically isolated in the drug-bound, closed-clamp form continues to hydrolyze ATP, indicating that the enzyme clamp does not need to re-open to bind and hydrolyze ATP. When rapid-quench ATPase assays were initiated by the addition of ATP, the drug had no effect on the sequential hydrolysis of either the first or second ATP. By contrast, when the drug was prebound, the enzyme hydrolyzed one labeled ATP at the uninhibited rate but did not hydrolyze a second ATP. These results are interpreted in terms of the catalytic mechanism for topoisomerase II and suggest that ICRF-193 interacts with the enzyme bound to one ADP.     

Mutant RBL mast cells defective in Fc epsilon RI signaling and lipid raft biosynthesis are reconstituted by activated Rho-family GTPases

Characterization of defects in a variant subline of RBL mast cells has revealed a biochemical event proximal to IgE receptor (Fc epsilon RI)-stimulated tyrosine phosphorylation that is required for multiple functional responses. This cell line, designated B6A4C1, is deficient in both Fc epsilon RI-mediated degranulation and biosynthesis of several lipid raft components. Agents that bypass receptor-mediated Ca(2+) influx stimulate strong degranulation responses in these variant cells. Cross-linking of IgE-Fc epsilon RI on these cells stimulates robust tyrosine phosphorylation but fails to mobilize a sustained Ca(2+) response. Fc epsilon RI-mediated inositol phosphate production is not detectable in these cells, and failure of adenosine receptors to mobilize Ca(2+) suggests a general deficiency in stimulated phospholipase C activity. Antigen stimulation of phospholipases A(2) and D is also defective. Infection of B6A4C1 cells with vaccinia virus constructs expressing constitutively active Rho family members Cdc42 and Rac restores antigen-stimulated degranulation, and active Cdc42 (but not active Rac) restores ganglioside and GPI expression. The results support the hypothesis that activation of Cdc42 and/or Rac is critical for Fc epsilon RI-mediated signaling that leads to Ca(2+) mobilization and degranulation. Furthermore, they suggest that Cdc42 plays an important role in the biosynthesis and expression of certain components of lipid rafts.     

The rug3 locus of pea encodes plastidial phosphoglucomutase

Two cDNA clones were isolated from pea (Pisum sativum L.) and their deduced amino acid sequences shown to have significant homology to phosphoglucomutases from eukaryotic and prokaryotic sources. The longer cDNA contained a putative transit-peptide-encoding sequence, supporting the hypothesis that the isolated clones represent the cytosolic and plastidial isoforms of phosphoglucomutase in pea. Plastid protein import assays confirmed that the putative plastidial isoform was targeted to the plastid stroma where it was proteolytically processed. Expression, co-segregation, linkage, and molecular analyses have confirmed that the rug3 locus of pea encodes plastidial phosphoglucomutase. Mutations at this locus result in a near-starchless phenotype of the plant.     

The evolution of ADHD: a disorder of communication?

Attention deficit hyperactivity disorder (ADHD) is the most commonly diagnosed psychiatric condition. Many believe that the central disability is impaired inhibition, which leads to reduced abilities in social skills, self-control, organization and time management. The behaviors identified by clinicians as problematic--inattention, hyperactivity and impulsivity--have been incorporated into several evolutionary models as selectively adaptive cognitive skills for surviving the challenges of a variable Pleistocene environment. We propose that the "disabilities" exhibited by individuals with ADHD are maladaptive, and we concur with Barkley that there is a central impairment in the behavioral inhibition system. The underlying neural anatomy and physiology support the possibility that neurotransmitter pathology may have an impact on other interlinked systems (including language), and may also account for the frequent comorbidity of aggression, anxiety, depression, and learning disabilities (many of which are language-related). Language skills compete with other cognitive activities for the attentional system, and thus the evolution of language could not in fact be independent of the evolution of attention. If language represents the ultimate expression of the attentional system, and some individuals with ADHD are seriously impaired in the coordination of interlinked neural systems (including language), then ADHD fits Jerome Wakefield's definition of "harmful dysfunction," and communication impairments should be investigated more thoroughly by clinicians.     

High levels of sequence polymorphism and linkage disequilibrium at the telomere of 12q: implications for telomere biology and human evolution

The human Xp/Yp telomere-junction region exhibits high levels of sequence polymorphism and linkage disequilibrium. To determine whether this is a general feature of human telomeres, we have undertaken sequence analysis at the 12q telomere and have extended the analysis at Xp/Yp. A total of 22 single-nucleotide polymorphisms (SNPs) and one 30-bp duplication were detected in the 1,870 bp adjacent to the 12q telomere. Twenty polymorphic positions were in almost complete linkage disequilibrium, creating three common diverged haplotypes accounting for 80% of 12q telomeres in the white population. A further 6% of 12q telomeres contained a 1,439-bp deletion in the DNA flanking the telomere. The remaining 13% of 12q telomeres did not amplify with the primers used (nulls). The distribution of telomere (TTAGGG) and variant repeats within 12q telomeres was hypervariable, but alleles with similar distribution patterns were associated with the same haplotype in the telomere-adjacent DNA. These data suggest that 12q telomeres, like Xp/Yp telomeres, exhibit low levels of homologous recombination and evolve along haploid lineages. In contrast, high levels of homologous recombination occur in the adjacent proterminal regions of human chromosomes. This suggests that there is a localized telomere-mediated suppression of recombination. In addition, the genetic characteristics of these regions may provide a source of deep lineages for the study of early human evolution, unaffected by both natural selection and recombination. To explain the presence of a few diverged haplotypes adjacent to the Xp/Yp and 12q telomeres, we propose a model that involves the hybridization of two archaic hominoid lineages ultimately giving rise to modern Homo sapiens.     

Structural effects of DNA sequence on T.A recognition by hydroxypyrrole/pyrrole pairs in the minor groove

Synthetic polyamides composed of three types of aromatic amino acids, N-methylimidazole (Im), N-methylpyrrole (Py) and N-methyl-3-hydroxypyrrole (Hp) bind specific DNA sequences as antiparallel dimers in the minor groove. The side-by-side pairings of aromatic rings in the dimer afford a general recognition code that allows all four base-pairs to be distinguished. To examine the structural consequences of changing the DNA sequence context on T.A recognition by Hp/Py pairs in the minor groove, crystal structures of polyamide dimers (ImPyHpPy)(2) and the pyrrole counterpart (ImPyPyPy)(2) bound to the six base-pair target site 5'-AGATCT-3' in a ten base-pair oligonucleotide have been determined to a resolution of 2.27 and 2.15 A, respectively. The structures demonstrate that the principles of Hp/Py recognition of T.A are consistent between different sequence contexts. However, a general structural explanation for the non-additive reduction in binding affinity due to introduction of the hydroxyl group is less clear. Comparison with other polyamide-DNA cocrystal structures reveals structural themes and differences that may relate to sequence preference.     

Hybridization,introgression, and linkage evolution

Genetic mapping methods provide a unique opportunity to study the interactions of differentiated genes and genomes in a hybrid genetic background. After a brief discussion of theoretical and analytical concerns, we review the application of these methods to a wide range of evolutionary issues. Map-based studies of experimental hybrids indicate that most postzygotic reproductive barriers in plants are polygenic and that the expression of extreme or novel traits in segregating hybrids (transgressive segregation) results from the complementary action of divergent parental alleles. However, genetic studies of hybrid vigor do not concur in their interpretations of the relative roles of dominance, overdominance, and epistasis. Map-based studies of natural hybrids are much rarer, but the few existing studies confirm the polygenic basis of postzygotic barriers and demonstrate the utility of genetic linkage for detecting cryptic introgression. In addition, studies of experimental and natural hybrid lineages provide compelling evidence that homoploid hybrid speciation has occurred in nature, and that it represents a rapid and repeatable mode of speciation. Data further indicate that this mode is facilitated by strong fertility selection and high chromosomal mutation rates. We recommend that future studies of hybrid genomes focus on natural hybrids, not only because of the paucity of data in this area, but also because of the availability of highly recombinant hybrid genotypes in hybrid zones. Of particular value will be studies of long-lived or difficult-to-propagate organisms, which previously have not been amenable to genetic study.