Population structure and its effect on haplotype diversity and linkage disequilibrium surrounding the xa5 locus of rice (Oryza sativa L.)

To assess the usefulness of linkage disequilibrium mapping in an autogamous, domesticated species, we have characterized linkage disequilibrium in the candidate region for xa5, a recessive gene conferring race-specific resistance to bacterial blight in rice. This trait and locus have good mapping information, a tractable phenotype, and available sequence data, but no cloned gene. We sampled 13 short segments from the 70-kb candidate region in 114 accessions of Oryza sativa. Five additional segments were sequenced from the adjacent 45-kb region in resistant accessions to estimate the distance at which linkage disequilibrium decays. The data show significant linkage disequilibrium between sites 100 kb apart. The presence of the xa5 resistant reaction in two ecotypes and in accessions with different haplotypes in the candidate region may indicate multiple origins or genetic heterogeneity for resistance. In addition, genetic differentiation between ecotypes emphasizes the need for controlling for population structure in the design of linkage disequilibrium studies in rice.     

Genome-wide analysis of mariner-like transposable elements in rice reveals complex relationships with stowaway miniature inverted repeat transposable elements (MITEs)

Stowaway is a superfamily of miniature inverted repeat transposable elements (MITEs) that is widespread and abundant in plant genomes. Like other MITEs, however, its origin and mode of amplification are poorly understood. Several lines of evidence point to plant mariner-like elements (MLEs) as the autonomous partners of the nonautonomous Stowaway MITEs. To better understand this relationship, we have taken advantage of the nearly complete genome sequences of two rice subspecies to generate the first inventory of virtually all MLEs and Stowaway families coexisting in a single plant species. Thirty-four different MLEs were found to group into three major clades and 25 families. More than 22,000 Stowaway MITEs were identified and classified into 36 families. On the basis of detailed sequence comparisons, MLEs were confirmed to be the best candidate autonomous elements for Stowaway MITEs. Surprisingly, however, sequence similarity between MLE and Stowaway families was restricted to the terminal inverted repeats (TIRs) and, in a few cases, to adjacent subterminal sequences. These data suggest a model whereby most of the Stowaway MITEs in rice were cross-mobilized by MLE transposases encoded by distantly related elements.     

The protozoan parasite, Theileria annulata, induces a distinct acute phase protein response in cattle that is associated with pathology

Acute phase proteins (APP) are synthesised in the liver in response to the systemic presence of high levels of pro-inflammatory cytokines. Bacteria are considered to be strong inducers of APP whereas viruses are weak or non-inducers of APP. Very few reports have been published on APP induction by parasites. Here, we report that the tick-borne protozoan parasite of cattle, Theileria annulata, induced an atypical acute phase response in cattle. Following experimental infection, serum amyloid A (SAA) appeared first, followed by a rise in alpha(1) acid glycoprotein (alpha(1)AGP) in all animals, whereas haptoglobin, which is a major APP in cattle, only appeared in some of the animals, and generally at a low level. All three APP only became elevated around or after the appearance of schizonts in draining lymph nodes and after the first observed temperature rise. Increased alpha(1)AGP levels coincided with the appearance of piroplasms. The production of SAA and alpha(1)AGP correlated strongly with each other, and also with some clinical measures of disease severity including the time to fever, development of leucopaenia, parasitaemia and mortality. These results are consistent with the hypothesis that T. annulata causes severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines.     

Identification of the F1-binding surface on the delta-subunit of ATP synthase

The stator function in ATP synthase was studied by a combined mutagenesis and fluorescence approach. Specifically, binding of delta-subunit to delta-depleted F(1) was studied. A plausible binding surface on delta-subunit was identified from conservation of amino acid sequence and the high resolution NMR structure. Specific mutations aimed at modulating binding were introduced onto this surface. Affinity of binding of wild-type and mutant delta-subunits to delta-depleted F(1) was determined quantitatively using the fluorescence signals of natural delta-Trp-28, inserted delta-Trp-11, or inserted delta-Trp-79. The results demonstrate that helices 1 and 5 in the N-terminal domain of the delta-subunit provide the F(1)-binding surface of delta. Unexpectedly, mutations that impaired binding between F(1) and delta were found to not necessarily impair ATP synthase activity. Further investigation revealed that inclusion of the soluble cytoplasmic domain of the b subunit substantially enhanced affinity of binding of delta-subunit to F(1). The new data show that the stator is "overengineered" to resist rotor torque during catalysis.     

Structure of an integrin-ligand complex deduced from solution x-ray scattering and site-directed mutagenesis

The structural basis of the interaction of integrin heterodimers with their physiological ligands is poorly understood. We have used solution x-ray scattering to visualize the head region of integrin alpha 5 beta 1 in an inactive (Ca2+-occupied) state, and in complex with a fragment of fibronectin containing the RGD and synergy recognition sequences. Shape reconstructions of the data have been interpreted in terms of appropriate molecular models. The scattering data suggest that the head region undergoes no gross conformational changes upon ligand binding but do lend support to a proposed outward movement of the hybrid domain in the beta subunit. Fibronectin is observed to bind across the top of the head region, which contains an alpha subunit beta-propeller and a beta subunit vWF type A domain. The model of the complex indicates that the synergy region binds on the side of the beta-propeller domain. In support of this suggestion, mutagenesis of a prominent loop region on the side of the propeller identifies two residues (Tyr208 and Ile210) involved in recognition of the synergy region. Our data provide the first view of a complex between an integrin and a macromolecular ligand in solution, at a nominal resolution of approximately 10 A.     

Phosphatidylinositol 4-OH kinase is a downstream target of neuronal calcium sensor-1 in enhancing exocytosis in neuroendocrine cells

Neuronal calcium sensor-1 (NCS-1), the mammalian orthologue of frequenin, belongs to a family of EF-hand-containing Ca(2+) sensors. NCS-1/frequenin has been shown to enhance synaptic transmission in PC12 cells and Drosophila and Xenopus, respectively. However, the precise molecular mechanism for the enhancement of exocytosis is largely unknown. In PC12 cells, NCS-1 potentiated exocytosis evoked by ATP, an agonist to phospholipase C-linked receptors, but had no effect on depolarization-evoked release. NCS-1 also enhanced exocytosis triggered by ionomycin, a Ca(2+) ionophore that bypasses K(+) and Ca(2+) channels. Overexpression of NCS-1 caused a shift in the dose-response curve of inhibition of ATP-evoked secretion using phenylarsine oxide, an inhibitor of phosphatidylinositol 4-OH kinase (PI4K). Plasma membrane phosphatidylinositol 4,5-bisphosphate pools were increased upon NCS-1 transfection as visualized using a phospholipase C-delta pleckstrin homology domain-green fluorescent protein construct. NCS-1-transfected cell extracts displayed increased phosphatidylinositol-4-phosphate biosynthesis, indicating an increase in PI4K activity. Mutations in NCS-1 equivalent to those that abolish the interaction of recoverin, another EF-hand-containing Ca(2+) sensor, with its downstream target rhodopsin kinase, lost their ability to enhance exocytosis. Taken together, the present data indicate that NCS-1 modulates the activity of PI4K, leading to increased levels of phosphoinositides and concomitant enhancement of exocytosis.     

Molecular and functional characterization of genes encoding horse MHC class I antigens

Sequence and functional analyses were undertaken on two cDNAs and a genomic clone encoding horse major histocompatibility complex (MHC) class I molecules. All of the clones were isolated from a single horse that is homozygous for all known horse MHC class I and class II antigens. The two cDNAs (clones 8-9 and 1-29) were isolated from a lymphocyte library and encode polymorphic MHC antigens from two loci. The genomic cosmid clone, isolated from a sperm library, contains the 8-9 gene. All three genes were expressed in mouse L-cells and were recognized by alloantisera and, for the cDNAs, by alloreactive cytotoxic T lymphocytes. A total of 3815 bp of the genomic clone were sequenced, extending from 429 bp upstream (5') of the leader peptide through the 3' untranslated region. Promoter region motifs and an intron-exon structure characteristic of MHC class I genes of other species were found. A subclone containing 407 bp of the promoter region was inserted into a chloramphenicol acetyl transferase reporter plasmid, tested in transient transfection assays, and found to have promoter activity in heterologous cells. This genomic clone will enable detailed studies of MHC class I gene regulation in horse trophoblasts, and in horse retroviral infections.     

Heart rate-arterial blood pressure relationship in conscious rat before vs. after spinal cord transection

This experiment quantified the initial disruption and subsequent adaptation of the blood pressure (BP)-heart rate (HR) relationship after spinal cord transection (SCT). BP and HR were recorded for 4 h via an implanted catheter in neurally intact, unanesthetized rats. The animals were then anesthetized, and their spinal cords were severed at T(1)-T(2) (n = 5) or T(4)-T(5) (n = 6) or sham lesioned (n = 4). BP was recorded for 4 h daily over the ensuing 6 days. The neurally intact rat showed a positive cross correlation, with HR leading BP at the peak by 1.8 +/- 0.8 (SD) s. The cross correlation in unanesthetized rats (n = 2) under neuromuscular blockade was also positive, with HR leading. After SCT at T(1)-T(2), the cross correlation became negative, with BP leading HR, and did not change during the next 6 days. The cross correlation also became negative 1-3 days after SCT at T(4)-T(5), but in four rats by day 6 and thereafter the cross correlation progressively reverted to a positive value. We propose that the positive cross correlation with HR leading BP in the intact rat results from an open-loop control that depends on intact supraspinal input to sympathetic preganglionic neurons in the spinal cord. After descending sympathetic pathways were severed at T(1)-T(2), the intact vagal pathway to the sinoatrial node dominated BP regulation via the baroreflex. We suggest that reestablishment of the positive correlation after SCT at T(4)-T(5) was attributable to the surviving sympathetic outflow to the heart and upper vasculature reasserting some effective function, perhaps in association with decreased spinal sympathetic hyperreflexia. The HR-BP cross correlation may index progression of sympathetic dysfunction in pathological processes.     

Molecular differentiation of the heterocystous cyanobacteria,Nostoc and Anabaena,based on complete NifD sequences

The segregation of Nostoc and Anabaena into separate genera has been debated for some time. The nitrogen fixation gene nifD was completely sequenced from representatives of these genera and analyzed phylogenetically, by using the representatives of other genera of the heterocystous cyanobacteria as outgroups. We were clearly able to differentiate between Nostoc and Anabaena in all analyses used. Our data suggest that Nostoc and Anabaena should remain as separate genera. Received: 16 November 2001 / Accepted: 14 December 2001