Genotype-specific interactions and the trade-off between host and parasite fitness

Background  

Evolution of parasite traits is inextricably linked to their hosts. For instance one common definition of parasite virulence is the reduction in host fitness due to infection. Thus, traits of infection must be viewed in both protagonists and may be under shared genetic and physiological control. We investigated these questions on the oomycete Hyaloperonospora arabidopsis (= parasitica), a natural pathogen of the Brassicaceae Arabidopsis thaliana.     

Putative candidate genes for blood pressure control in the SHR.BN-RNO8 congenic substrains

The SHR-Lx congenic strain carrying a differential segment of chromosome 8 of BN and PD origin was recently shown to exhibit a significant decrease in blood pressure as compared to the SHR strain. There were two positional candidate genes for blood pressure control mapped to the differential segment: the rat kidney epithelial potassium channel gene (Kcnj1) and brain dopamine receptor 2 gene (Drd2). Bot these genes were separated into SHR.BN-RNO8 congenic substrains. In this communication, we are presenting the assignment of two further putative candidate genes, which might be involved in blood pressure control to the BN/PD differential segment of the SHR-Lx congenic strain. These are: the gene coding for smooth muscle cell specific protein 22 (Sm22) defined by the D8Mcw1 marker and neuronal nicotinic acetylcholine receptor gene cluster, defined by the D8Bord1 marker. Moreover, the glutamate receptor gene Grik4 which also maps to the differential segment of the SHR-Lx should be taken into account. The genetic separation of all these putative candidate genes of blood pressure control is being performed by recombinations and subsequent selection using (SHR×SHR-Lx) intercross population.     

Active site mutations in DNA topoisomerase I distinguish the cytotoxic activities of camptothecin and the indolocarbazole, rebeccamycin.

DNA topoisomerase I (Top1p) catalyzes topological changes in DNA and is the cellular target of the antitumor agent camptothecin (CPT). Non-CPT drugs that target Top1p, such as indolocarbazoles, are under clinical development. However, whether the cytotoxicity of indolocarbazoles derives from Top1p poisoning remains unclear. To further investigate indolocarbazole mechanism, rebeccamycin R-3 activity was examined in vitro and in yeast. Using a series of Top1p mutants, where substitution of residues around the active site tyrosine has well-defined effects on enzyme catalysis, we show that catalytically active, CPT-resistant enzymes remain sensitive to R-3. This indolocarbazole did not inhibit yeast Top1p activity, yet was effective in stabilizing Top1p-DNA complexes. Similar results were obtained with human Top1p, when Ser or His were substituted for Asn-722. The mutations altered enzyme function and sensitivity to CPT, yet R-3 poisoning of Top1p was unaffected. Moreover, top1delta, rad52delta yeast cells expressing human Top1p, but not catalytically inactive Top1Y723Fp, were sensitive to R-3. These data support hTop1p as the cellular target of R-3 and indicate that distinct drug-enzyme interactions at the active site are required for efficient poisoning by R-3 or CPT. Furthermore, resistance to one poison may potentiate cell sensitivity to structurally distinct compounds that also target Top1p.     

The cortical actomyosin system of cytochalasin D-treated lymphoblasts

Global cytoskeleton dynamics is likely to exist in animal cells and some experimental evidence for this has recently been obtained in cells from the human lymphoblastic cell line KE37. We have further investigated the dramatic and reversible microtubule-dependent cell elongation which occurs upon treatment of KE37 cells with cytochalasin D. This phenomenon results in a non-locomotory cell with definite polarity. It involves a sustained equatorial myosin II-dependent contraction of cortical, most of the myosin II being accumulated on segments of the main cellular extension. We report here that such a cell lengthening is energy-dependent and can be inhibited, or suppressed, by surface ligands such as wheat germ agglutinin but not by concanavalin A. Suppression of the cytochalasin D effect by wheat germ agglutinin is rapid and appears to be collapse of the cell extension and relocalization of the contracted actomyosin as a whole. It suggests that the binding of the wheat germ agglutinin to the cell surface results in the transient disassembly of microtubules, a possibility also raised by the potent antagonist effect of taxol on wheat germ agglutinin action. Taken together, the data are consistent with a specific role of microtubules in the control of the activity of the cortical actomyosin system.     

Complexation of copper(II) by a peptide hybrid of bleomycin and amsacrine. Circular dichroism and electron spin resonance studies

A model incorporating the metal chelating moiety of bleomycin and an anilinoacridine ring able to intercalate in DNA has been synthesized. The copper(II) complex of that molecule has been studied using circular dichroism and electron spin resonance by comparison with bleomycin. The introduction of the anilinoacridine ring involves a modification in the geometry of the complex. A distortion of the square-pyramidal form (type II complex) gives rise to a type I complex in which the metallic atom is drawn out of the plane of the four square-planar ligands and displaced slightly towards the fifth ligand.     

Gene expression of D-beta-hydroxybutyrate dehydrogenase. II. Incorporation of pre-BDH into mitochondria

beta-hydroxybutyrate dehydrogenase (BDH), a major protein located in the inner mitochondrial membrane is encoded, as most of mitochondrial proteins, in the nuclear genome. It is synthetized on the free polysomes and post-translationally imported into the mitochondria. The neosynthesized protein is a higher molecular weight precursor. The presequence is cleaved by the matrix protease to give the mature protein. The translocation across the mitochondrial membranes needs energy. The results also indicate that cytosolic factors with low molecular weight are essential in the recognition of precursor by mitochondria and to sort out newly synthetized nuclear encoded mitochondrial proteins from others nuclear encoded proteins.     

Gene expression of D-beta-hydroxybutyrate dehydrogenase. III. Molecular cloning of a DNAc

In order to continue the molecular studies of D-beta-hydroxybutyrate dehydrogenase (BDH) undertaken in our laboratory for several years, we have initiated a genetic approach which consists in the BDH cDNA cloning from a rat liver cDNA library. The immunoscreening method allowed to isolate a clone which exhibits a DNA insert shorter than the expected full length BDH cDNA.     

Mechanism of DNA strand nicking at apurinic/apyrimidinic sites by Escherichia coli [formamidopyrimidine]DNA glycosylase.

Escherichia coli [formamidopyrimidine]DNA glycosylase catalyses the nicking of both the phosphodiester bonds 3' and 5' of apurinic or apyrimidinic sites in DNA so that the base-free deoxyribose is replaced by a gap limited by 3'-phosphate and 5'-phosphate ends. The two nickings are not the results of hydrolytic processes; the [formamidopyrimidine]DNA glycosylase rather catalyses a beta-elimination reaction that is immediately followed by a delta-elimination. The enzyme is without action on a 3'-terminal base-free deoxyribose or on a 3'-terminal base-free unsaturated sugar produced by a beta-elimination reaction nicking the DNA strand 3' to an apurinic or apyrimidinic site.     

Molecular recognition between oligopeptides and nucleic acids. DNA sequence specificity and binding properties of an acridine-linked netropsin hybrid ligand

The binding to DNA of a mixed function ligand (NETGA) is described, in which a potential intercalating group, an acridine moiety, is incorporated at the carboxyl terminus of the minor groove binding oligopeptide netropsin skeleton. Scatchard analysis of absorption data provided evidence of two modes of binding to DNA with K1 = 9.1 x 10(5) M-1 at low r values (0.003-0.1), and a binding site size n = 10, indicative of binding of both moeities. At high binding ratios (greater than 0.1), K2 = 0.9 x 10(5) M-1 and n = 5 corresponding to external binding. Complementary strand MPE footprinting on a pBR322 restriction fragment showed NETGA binds to 5'-AAAT like netropsin. It causes enhanced cleavage by MPE, particularly at G-C rich sequences and remote from the preferred binding sites. Viscometry measurements provided evidence for biphasic modes of the two binding portions of NETGA. Fluorescence polarization and linear dichroism measurements were in accord with distinct modes of interaction of the acridine (intercalation) and oligopeptide (minor groove binding) portions of NETGA. LD measurements on NETGA indicate that the oligopeptide moiety (netropsin-like) has an orientation typical of minor groove binders, whereas the degree of intercalation of the acridine group is decreased by association of the oligopeptide moiety.