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Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.

Huntington's disease (HD) is caused by an expansion of a poly glutamine (polyQ) stretch in the huntingtin protein (HTT) that is necessary to cause pathology and formation of HTT aggregates. Here we ask whether expanded polyQ is sufficient to cause pathology and aggregate formation. By addressing the sufficiency question, one can identify cellular processes and structural parameters that influence HD pathology and HTT subcellular behavior (i.e. aggregation state and subcellular location). Using Drosophila, we compare the effects of expressing mutant full-length human HTT (fl-mHTT) to the effects of mutant human HTTexon1 and to two commonly used synthetic fragments, HTT171 and shortstop (HTT118). Expanded polyQ alone is not sufficient to cause inclusion formation since full-length HTT and HTTex1 with expanded polyQ are both toxic although full-length HTT remains diffuse while HTTex1 forms inclusions. Further, inclusions are not sufficient to cause pathology since HTT171-120Q forms inclusions but is benign and co-expression of HTT171-120Q with non-aggregating pathogenic fl-mHTT recruits fl-mHTT to aggregates and rescues its pathogenicity. Additionally, the influence of sequences outside the expanded polyQ domain is revealed by finding that small modifications to the HTT118 or HTT171 fragments can dramatically alter their subcellular behavior and pathogenicity. Finally, mutant HTT subcellular behavior is strongly modified by different cell and tissue environments (e.g. fl-mHTT appears as diffuse nuclear in one tissue and diffuse cytoplasmic in another but toxic in both). These observations underscore the importance of cellular and structural context for the interpretation and comparison of experiments using different fragments and tissues to report the effects of expanded polyQ.

Lipin/Pah phosphatidic acid phosphatases (PAPs) generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Inactivating mutations cause rhabdomyolysis, autoinflammatory disease, and aberrant fat storage. Disease-mutations cluster within the conserved N-Lip and C-Lip regions that are separated by 500-residues in humans. To understand how the N-Lip and C-Lip combine for PAP function, we determined crystal structures of Tetrahymena thermophila Pah2 (Tt Pah2) that directly fuses the N-Lip and C-Lip. Tt Pah2 adopts a two-domain architecture where the N-Lip combines with part of the C-Lip to form an immunoglobulin-like domain and the remaining C-Lip forms a HAD-like catalytic domain. An N-Lip C-Lip fusion of mouse lipin-2 is catalytically active, which suggests mammalian lipins function with the same domain architecture as Tt Pah2. HDX-MS identifies an N-terminal amphipathic helix essential for membrane association. Disease-mutations disrupt catalysis or destabilize the protein fold. This illustrates mechanisms for lipin/Pah PAP function, membrane association, and lipin-related pathologies.

Covalent inhibitors of K-Ras(G12C) have been reported that exclusively recognize the GDP state. Here, we utilize disulfide tethering of a non-natural cysteine (K-Ras(M72C)) to identify a new switch-II pocket (S-IIP) binding ligand (2C07) that engages the active GTP state. Co-crystal structures of 2C07 bound to H-Ras(M72C) reveal binding in a cryptic groove we term S-IIG. In the GppNHp state, 2C07 binding to a modified S-IIP pushes switch I away from the nucleotide, breaking the network of polar contacts essential for adopting the canonical GTP state. Biochemical studies show that 2C07 alters nucleotide preference and inhibits SOS binding and catalyzed nucleotide exchange. 2C07 was converted to irreversible covalent analogs, which target both nucleotide states, inhibit PI3K activation in vitro, and function as occupancy probes to detect reversible engagement in competition assays. Targeting both nucleotide states opens the possibility of inhibiting oncogenic mutants of Ras, which exist predominantly in the GTP state in cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to spread relentlessly, associated with a high frequency of respiratory failure and mortality. Children experience largely asymptomatic disease, with rare reports of multisystem inflammatory syndrome in children (MIS-C). Identifying immune mechanisms that result in these disparate clinical phenotypes in children could provide critical insights into coronavirus disease 2019 (COVID-19) pathogenesis. Using systems serology, in this study we observed in 25 children with acute mild COVID-19 a functional phagocyte and complement-activating IgG response to SARS-CoV-2, similar to the acute responses generated in adults with mild disease. Conversely, IgA and neutrophil responses were significantly expanded in adults with severe disease. Moreover, weeks after the resolution of SARS-CoV-2 infection, children who develop MIS-C maintained highly inflammatory monocyte-activating SARS-CoV-2 IgG antibodies, distinguishable from acute disease in children but with antibody levels similar to those in convalescent adults. Collectively, these data provide unique insights into the potential mechanisms of IgG and IgA that might underlie differential disease severity as well as unexpected complications in children infected with SARS-CoV-2. A study of multisystem inflammatory syndrome in children (MIS-C) shows maintenance of elevated levels of monocyte-activating pathogen-specific IgG not seen in children infected with SARS-CoV-2 who do not develop MIS-C., Author(s): Yannic C. Bartsch [sup.1] , Chuangqi Wang [sup.2] , Tomer Zohar [sup.1] [sup.2] , Stephanie Fischinger [sup.1] , Caroline Atyeo [sup.1] , John S. Burke [sup.1] , Jaewon Kang [...]

BackgroundShoot branching is an important determinant of plant architecture and influences various aspects of growth and development. Selection on branching has also played an important role in the domestication of crop plants, including sunflower (Helianthus annuus L.). Here, we describe an investigation of the genetic basis of variation in branching in sunflower via association mapping in a diverse collection of cultivated sunflower lines.ResultsDetailed phenotypic analyses revealed extensive variation in the extent and type of branching within the focal population. After correcting for population structure and kinship, association analyses were performed using a genome-wide collection of SNPs to identify genomic regions that influence a variety of branching-related traits. This work resulted in the identification of multiple previously unidentified genomic regions that contribute to variation in branching. Genomic regions that were associated with apical and mid-apical branching were generally distinct from those associated with basal and mid-basal branching. Homologs of known branching genes from other study systems (i.e., Arabidopsis, rice, pea, and petunia) were also identified from the draft assembly of the sunflower genome and their map positions were compared to those of associations identified herein. Numerous candidate branching genes were found to map in close proximity to significant branching associations.ConclusionsIn sunflower, variation in branching is genetically complex and overall branching patterns (i.e., apical vs. basal) were found to be influenced by distinct genomic regions. Moreover, numerous candidate branching genes mapped in close proximity to significant branching associations. Although the sunflower genome exhibits localized islands of elevated linkage disequilibrium (LD), these non-random associations are known to decay rapidly elsewhere. The subset of candidate genes that co-localized with significant associations in regions of low LD represents the most promising target for future functional analyses.

Although Huntington's disease is caused by the expansion of a CAG triplet repeat within the context of the 3144-amino acid huntingtin protein (HTT), studies reveal that N-terminal fragments of HTT containing the expanded PolyQ region can be produced by proteolytic processing and/or aberrant splicing. N-terminal HTT fragments are also prevalent in postmortem tissue, and expression of some of these fragments in model organisms can cause pathology. This has led to the hypothesis that N-terminal peptides may be critical modulators of disease pathology, raising the possibility that targeting aberrant splicing or proteolytic processing may present attractive therapeutic targets. However, many factors can contribute to pathology, including genetic background and differential expression of transgenes, in addition to intrinsic differences between fragments and their cellular effects. We have used Drosophila as a model system to determine the relative toxicities of different naturally occurring huntingtin fragments in a system in which genetic background, transgene expression levels and post-translational proteolytic processing can be controlled. These studies reveal that among the naturally occurring N-terminal HTT peptides, the exon 1 peptide is exceptionally pathogenic and exhibits unique structural and biophysical behaviors that do not appear to be incremental changes compared with other fragments. If this proves correct, efforts to specifically reduce the levels of exon 1 peptides or to target toxicity-influencing post-translational modifications that occur with the exon 1 context are likely to have the greatest impact on pathology.

Knowledge of the nature and extent of karyotypic differences between species provides insight into the evolutionary history of the genomes in question and, in the case of closely related species, the potential for genetic exchange between taxa. We constructed high-density genetic maps of the silverleaf sunflower (Helianthus argophyllus) and Algodones Dune sunflower (H. niveus ssp. tephrodes) genomes and compared them to a consensus map of cultivated sunflower (H. annuus) to identify chromosomal rearrangements between species. The genetic maps of H. argophyllus and H. niveus ssp. tephrodes included 17 linkage groups each and spanned 1337 and 1478 cM, respectively. Comparative analyses revealed greater divergence between H. annuus and H. niveus ssp. tephrodes (13 inverted segments, 18 translocated segments) than between H. annuus and H. argophyllus (10 inverted segments, 8 translocated segments), consistent with their known phylogenetic relationships. Marker order was conserved across much of the genome, with 83 and 64% of the H. argophyllus and H. niveus ssp. tephrodes genomes, respectively, being syntenic with H. annuus. Population genomic analyses between H. annuus and H. argophyllus, which are sympatric across a portion of the natural range of H. annuus, revealed significantly elevated genetic structure in rearranged portions of the genome, indicating that such rearrangements are associated with restricted gene flow between these two species.

Phosphatidylinositol 4-kinases (PI4Ks) and small guanosine triphosphatases (GTPases) are essential for processes that require expansion and remodeling of phosphatidylinositol 4-phosphate (PI4P)-containing membranes, including cytokinesis, intracellular development of malarial pathogens, and replication of a wide range of RNA viruses. However, the structural basis for coordination of PI4K, GTPases, and their effectors is unknown. Here, we describe structures of PI4Kβ (PI4KIIIβ) bound to the small GTPase Rab11a without and with the Rab11 effector protein FIP3. The Rab11-PI4KIIIβ interface is distinct compared with known structures of Rab complexes and does not involve switch regions used by GTPase effectors. Our data provide a mechanism for how PI4KIIIβ coordinates Rab11 and its effectors on PI4P-enriched membranes and also provide strategies for the design of specific inhibitors that could potentially target plasmodial PI4KIIIβ to combat malaria.

The phospholipase A(2) (PLA(2)) superfamily consists of 16 groups and many subgroups and constitutes a diverse set of enzymes that have a common catalytic activity due to convergent evolution. However, different PLA(2) types have unique three-dimensional structures and catalytic residues as well as specific tissue localization and distinct biological functions. Understanding how the different PLA(2) enzymes associate with phospholipid membranes, specific phospholipid substrate molecules, and inhibitors on a molecular basis has advanced in recent years due to the introduction of hydrogen/deuterium exchange mass spectrometry. Its theory, practical considerations, and application to understanding PLA(2)/membrane interactions are addressed.

There is growing recognition of the ways in which maternal effects can influence offspring size, physiological performance, and survival. Additionally, environmental contaminants increasingly act as stressors in maternal environments, possibly leading to maternal effects on subsequent offspring. Thus, it is important to determine whether contaminants and other stressors can contribute to maternal effects, particularly under varied ecological conditions that encompass the range under which offspring develop. We used aquatic mesocosms to determine whether maternal effects of mercury (Hg) exposure shape offspring phenotype in the American toad (Bufo americanus) in the presence or absence of larval predators (dragonfly naiads). We found significant maternal effects of Hg exposure and significant effects of predators on several offspring traits, but there was little evidence that maternal effects altered offspring interactions with predators. Offspring from Hg-exposed mothers were 18% smaller than those of reference mothers. Offspring reared with predators were 23% smaller at metamorphosis than those reared without predators. There was also evidence of reduced larval survival when larvae were reared with predators, but this was independent of maternal effects. Additionally, 5 times more larvae had spinal malformations when reared without predators, suggesting selective predation of malformed larvae by predators. Lastly, we found a significant negative correlation between offspring survival and algal density in mesocosms, indicating a role for top-down effects of predators on periphyton communities. Our results demonstrate that maternal exposure to an environmental stressor can induce phenotypic responses in offspring in a direction similar to that produced by direct exposure of offspring to predators.

Centromeres are the most dynamic regions of the genome, yet they are typified by little or no crossing over, making it difficult to explain the origin of this diversity. To address this question, we developed a novel CENH3 ChIP display method that maps kinetochore footprints over transposon-rich areas of centromere cores. A high level of polymorphism made it possible to map a total of 238 within-centromere markers using maize recombinant inbred lines. Over half of the markers were shown to interact directly with kinetochores (CENH3) by chromatin immunoprecipitation. Although classical crossing over is fully suppressed across CENH3 domains, two gene conversion events (i.e., non-crossover marker exchanges) were identified in a mapping population. A population genetic analysis of 53 diverse inbreds suggests that historical gene conversion is widespread in maize centromeres, occurring at a rate >1x10(-5)/marker/generation. We conclude that gene conversion accelerates centromere evolution by facilitating sequence exchange among chromosomes.

The Group VIA-2 Ca(2+)-independent phospholipase A(2) (GVIA-2 iPLA(2)) is composed of seven consecutive N-terminal ankyrin repeats, a linker region, and a C-terminal phospholipase catalytic domain. No structural information exists for this enzyme, and no information is known about the membrane binding surface. We carried out deuterium exchange experiments with the GVIA-2 iPLA(2) in the presence of both phospholipid substrate and the covalent inhibitor methyl arachidonoyl fluorophosphonate and located regions in the protein that change upon lipid binding. No changes were seen in the presence of only methyl arachidonoyl fluorophosphonate. The region with the greatest change upon lipid binding was region 708-730, which showed a >70% decrease in deuteration levels at numerous time points. No decreases in exchange due to phospholipid binding were seen in the ankyrin repeat domain of the protein. To locate regions with changes in exchange on the enzyme, we constructed a computational homology model based on homologous structures. This model was validated by comparing the deuterium exchange results with the predicted structure. Our model combined with the deuterium exchange results in the presence of lipid substrate have allowed us to propose the first structural model of GVIA-2 iPLA(2) as well as the interfacial lipid binding region.

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) catalyzes release of arachidonic acid from glycerophospholipids, leading to thromboxane A(2) (TxA(2)) production. Some platelet agonists stimulate cPLA(2)alpha, but others require fibrinogen binding to alphaIIbbeta3 to elicit TxA(2). Therefore, relationships between cPLA(2)alpha and alphaIIbbeta3 were examined. cPLA(2)alpha and a cPLA(2)alpha binding partner, vimentin, coimmunoprecipitated with alphaIIbbeta3 from platelets, independent of fibrinogen binding. Studies with purified proteins and with recombinant proteins expressed in CHO cells determined that the interaction between cPLA(2)alpha and alphaIIbbeta3 was indirect and was dependent on the alphaIIb and beta3 cytoplasmic tails. Fibrinogen binding to alphaIIbbeta3 caused an increase in integrin-associated cPLA(2)alpha activity in normal platelets, but not in cPLA(2)alpha-deficient mouse platelets or in human platelets treated with pyrrophenone, a cPLA(2)alpha inhibitor. cPLA(2)alpha activation downstream of alphaIIbbeta3 had functional consequences for platelets in that it was required for fibrinogen-dependent recruitment of activated protein kinase Cbeta to the alphaIIbbeta3 complex and for platelet spreading. Thus, cPLA(2)alpha and alphaIIbbeta3 interact to reinforce each other's functions during alphaIIbbeta3 signaling. This provides a plausible explanation for the role of alphaIIbbeta3 in TxA(2) formation and in the defective hemostatic function of mouse or human platelets deficient in cPLA(2)alpha.

The Group IVA (GIVA) phospholipase A(2) associates with natural membranes in response to an increase in intracellular Ca(2+) along with increases in certain lipid mediators. This enzyme associates with the membrane surface as well as binding a single phospholipid molecule in the active site for catalysis. Employing deuterium exchange mass spectrometry, we have identified the regions of the protein binding the lipid surface and conformational changes upon a single phospholipid binding in the absence of a lipid surface. Experiments were carried out using natural palmitoyl arachidonyl phosphatidylcholine vesicles with the intact GIVA enzyme as well as the isolated C2 and catalytic domains. Lipid binding produced changes in deuterium exchange in eight different regions of the protein. The regions with decreased exchange included Ca(2+) binding loop one, which has been proposed to penetrate the membrane surface, and a charged patch of residues, which may be important in interacting with the polar head groups of phospholipids. The regions with an increase in exchange are all located either in the hydrophobic core underneath the lid region or near the lid and hinge regions from 403 to 457. Using the GIVA phospholipase A(2) irreversible inhibitor methyl-arachidonyl fluorophosphonate, we were able to isolate structural changes caused only by pseudo-substrate binding. This produced results that were very similar to natural lipid binding in the presence of a lipid interface with the exception of the C2 domain and region 466-470. This implies that most of the changes seen in the catalytic domain are due to a substrate-mediated, not interface-mediated, lid opening, which exposes the active site to water. Finally experiments carried out with inhibitor plus phospholipid vesicles showed decreases at the C2 domain as well as charged residues on the putative membrane binding surface of the catalytic domain revealing the binding sites of the enzyme to the lipid surface.

The GIVA phospholipase A(2) (PLA(2)) contains two domains: a calcium-binding domain (C2) and a catalytic domain. These domains are linked via a flexible tether. GIVA PLA(2) activity is Ca(2+)-dependent in that calcium binding promotes protein docking to the phospholipid membrane. In addition, the catalytic domain has a lid that covers the active site, presumably regulating GIVA PLA(2) activity. We now present studies that explore the dynamics and conformational changes of this enzyme in solution utilizing peptide amide hydrogen/deuterium (H/D) exchange coupled with liquid chromatography-mass spectrometry (DXMS) to probe the solvent accessibility and backbone flexibility of the C2 domain, the catalytic domain, and the intact GIVA PLA(2). We also analyzed the changes in H/D exchange of the intact GIVA PLA(2) upon Ca(2+) binding. The DXMS results showed a fast H/D-exchanging lid and a slow exchanging central core. The C2 domain showed two distinct regions: a fast exchanging region facing away from the catalytic domain and a slow exchanging region present in the "cleft" region between the C2 and catalytic domains. The slow exchanging region of the C2 domain is in tight proximity to the catalytic domain. The effects of Ca(2+) binding on GIVA PLA(2) are localized in the C2 domain and suggest that binding of two distinct Ca(2+) ions causes tightening up of the regions that surround the anion hole at the tip of the C2 domain. This conformational change may be the initial step in GIVA PLA(2) activation.

Fibroblasts represent the key cell type in fibrostenosing Crohn's disease (FCD) pathogenesis. S 100A4 is an EF-hand calcium-binding protein family member, implicated in epithelial-mesenchymal transition and as a marker of activated T lymphocytes and fibroblasts in chronic tissue remodeling. The aim of this study was to examine the expression profile of S100A4 in the resected ileum of patients with FCD. Mucosa, seromuscular explants, and transmural biopsies were harvested from diseased and proximal, macroscopically normal margins of ileocecal resections from patients with FCD. Samples were processed for histochemistry, immunohistochemistry, real-time RTPCR, Western blotting, and transmission electron microscopy. Primary explant cultures of seromuscular fibroblasts were exposed to transforming growth factor (TGF)-[beta]1 (1 ng/ml), and S I00A4 expression and scratch wound-healing activity were assessed at 24 h. CCD-18Co fibroblasts were transfected with S 100A4 small interfering RNA, treated with TGF-[beta]1 (l ng/ml) for 30 min or 24 h, and then assessed for S100A4 and Smad3 expression and scratch woundhealing activity. S100A4 expression was increased in stricture mucosa, in the lamina propria, and in CD3-positive intraepithelial CD3positive T lymphocytes. Fibroblastic S100A4 staining was observed in seromuscular scar tissue. Stricture fibroblast explant culture showed significant upregulation of S100A4 expression. TGF-[beta]1 increased S100A4 expression in cultured ileal fibroblasts. In CCD-18Co fibroblasts, S100A4 small interfering RNA inhibited scratch wound healing and modestly inhibited Smad3 activation. S100A4 expression is increased in fibroblasts, as well as immune cells, in Crohn's disease stricture and induced by TGF-[beta]1. Results from knockdown experiments indicate a potential role for S100A4 in mediating intestinal fibroblast migration. fibrosis; epithelial-mesenchymal transition doi: 10.1152/ajpgi.00351.2009.

Analyses of genetic variation in crop gene pools are a powerful tool for investigating the origin and early evolution of crop lineages. Such analyses also have the potential to identify unique genetic resources for continued crop improvement. The oilseed crop safflower (Carthamus tinctorius) is believed to have been domesticated in the Fertile Crescent region, but up to 10 geographic centers of similarity throughout the world have been proposed based on morphology. Nuclear microsatellite analysis of accessions from each of the 10 proposed centers of similarity, as well as individuals of the progenitor species, suggested the presence of five genetic clusters (1, Europe; 2, Turkey--Iran--Iraq--Afghanistan; 3, Israel--Jordan--Syria; 4, Egypt--Ethiopia; and 5, the Far East-India--Pakistan). North American accessions, products of a secondary introduction from the native range, suggest that a subset of the native accessions harbor unique genetic diversity that could be useful in future breeding efforts. Overall, a Near Eastern origin of safflower was confirmed based on the genetic similarity between the progenitor and the Near Eastern safflower accessions, as well as previous archaeological finds. Genetic differentiation between geographical clusters of accessions is evident, although not to the degree proposed based on morphology. Key words: Asteraceae; Carthamus; crop evolution; domestication; genetic variation; population structure; safflower. doi: 10.3732/ajb.0900137

The domestication and improvement of crop plants have long fascinated evolutionary biologists, geneticists, and anthropologists. In recent years, the development of increasingly powerful molecular and statistical tools has reinvigorated this now fast-paced field of research. In this paper, we provide an overview of how such tools have been applied to the study of crop evolution. We also highlight lessons that have been learned in light of a few long-standing and interrelated hypotheses concerning the origins of crop plants and the nature of the genetic changes underlying their evolution. We conclude by discussing compelling evolutionary genomic approaches that make possible the efficient and unbiased identification of genes controlling crop-related traits and provide further insight into the actual timing of selection on particular genomic regions. Key words: association mapping; crop improvement; crop origins; domestication genes; genetic architecture; genome scans; QTL mapping.

Genetic diversity in modern sunflower (Helianthus annuus L.) cultivars (elite oilseed inbred lines) has been shaped by domestication and breeding bottlenecks and wild and exotic allele introgression--the former narrowing and the latter broadening genetic diversity. To assess single nucleotide polymorphism (SNP) frequencies, nucleotide diversity, and linkage disequilibrium (LD) in modern cultivars, alleles were resequenced from 81 genic loci distributed throughout the sunflower genome. DNA polymorphisms were abundant; 1078 SNPs (1/45.7 bp) and 178 insertions-deletions (INDELs) (1/277.0 bp) were identified in 49.4 kbp of DNA/genotype. SNPs were twofold more frequent in noncoding (1/32.1 bp) than coding (1/ 62.8 bp) sequences. Nucleotide diversity was only slightly lower in inbred lines ([theta] = 0.0094) than wild populations ([theta] = 0.0128). Mean haplotype diversity was 0.74. When extraploted across the genome (~3500 Mbp), sunflower was predicted to harbor at least 76.4 million common SNPs among modern cultivar alleles. LD decayed more slowly in inbred lines than wild populations (mean LD declined to 0.32 by 5.5 kbp in the former, the maximum physical distance surveyed), a difference attributed to domestication and breeding bottlenecks. SNP frequencies and LD decay are sufficient in modern sunflower cultivars for very high-density genetic mapping and high-resolution association mapping.

Genetic analyses of the domestication syndrome have revealed that domestication-related traits typically have a very similar genetic architecture across most crops, being conditioned by a small number of quantitative trait loci (QTL), each with a relatively large effect on the phenotype. To date, the domestication of sunflower (Helianthus annuus L.) stands as the only counterexample to this pattern. In previous work involving a cross between wild sunflower (also H. annuus) and a highly improved oilseed cultivar, we found that domestication-related traits in sunflower are controlled by numerous QTL, typically of small effect. To provide insight into the minimum genetic changes required to transform the weedy common sunflower into a useful crop plant, we mapped QTL underlying domestication-related traits in a cross between a wild sunflower and a primitive Native American landrace that has not been the target of modern breeding programs. Consistent with the results of the previous study, our data indicate that the domestication of sunflower was driven by selection on a large number of loci, most of which had small to moderate phenotypic effects. Unlike the results of the previous study, however, nearly all of the QTL identified herein had phenotypic effects in the expected direction, with the domesticated allele producing a more crop-like phenotype and the wild allele producing a more wild-like phenotype. Taken together, these results are consistent with the hypothesis that selection during the post-domestication era has resulted in the introduction of apparently maladaptive alleles into the modern sunflower gene pool.

The result of photocross-linking analysis of native and minimal hammerheads containing photoreactive nucleobases, introduced at specific sites within catalytic core is described. The results obtained support a model in which both hammerheads partition between 2-folds, an inactive ground state similar to the crystallographic structures and an active fold characterized by interactions between putative catalytic nucleobases G8 and G12 and the substrate cleavage site.

Interest in the level and organization of nucleotide diversity in domesticated plant lineages has recently been motivated by the potential for using association-based mapping techniques as a means for identifying the genes underlying complex traits. To date, however, such data have been available only for a relatively small number of well-characterized plant taxa. Here we provide the first detailed description of patterns of nucleotide polymorphism in wild and cultivated sunflower (Helianthus annuus), using sequence data from nine nuclear genes. The resuflts of this study indicate that wild sunflower harbors at least as much nucleotide diversity as has been reported in other wild plant taxa, with randomly selected sequence pairs being expected to differ at 1 of every 70 bp. In contrast, cultivated sunflower has retained only 40-50% of the diversity present in the wild. Consistent with this dramatic reduction in polymorphism, a phylogenetic analysis of our data revealed that the cultivars form a monophyletic clade, adding to the growing body of evidence that sunflower is the product of a single domestication. Eight of the nine loci surveyed appeared to be evolving primarily under purifying selection, while the remaining locus may have been the subject of positive selection. Linkage disequilibrium (LD) decayed very rapidly in the self-incompatible wild sunflower, with the expected LD falling to negligible levels within 200 bp. The cultivars, on the other hand, exhibited somewhat higher levels of LD, with nonrandom associations persisting up to ~1100 bp. Taken together, these results suggest that association-based approaches will provide a high degree of resolution for the mapping of functional variation in sunflower.

We mapped quantitative trait loci (QTL) controlling differences in seed oil content and composition between cultivated and wild sunflower and used the results, along with those of a previous study of domestication-related QTL, to guide a genome-wide analysis of genetic variation for evidence of past selection. The effects of the seed oil QTL were almost exclusively in the expected direction with respect to the parental phenotypes. A major, oil-related QTL cluster mapped near a cluster of domestication-related QTL on linkage group six (LG06), the majority of which have previously been shown to have effects that are inconsistent with the parental phenotypes. To test the hypothesis that this region was the target of a past selective sweep, perhaps resulting in the fixation of the antagonistic domestication-related QTL, we analyzed simple sequence repeat (SSR) diversity from 102 markers dispersed throughout the sunflower genome. Our results indicate that LG06 was most likely the target of multiple selective sweeps during the postdomestication era. Strong directional selection in concert with genetic hitchhiking therefore offers a possible explanation for the occurrence of numerous domestication-related QTL with apparently maladaptive phenotypic effects.

New species may arise via hybridization and without a change in ploidy. This process, termed homoploid hybrid speciation, is theoretically difficult because it requires the development of reproductive barriers in sympatry or parapatry. Theory suggests that isolation may arise through rapid karyotypic evolution and/or ecological divergence of hybrid neospecies. Here, we investigate the role of karyotypic change in homoploid hybrid speciation by generating detailed genetic linkage maps for three hybrid sunflower species, Helianthus anomalus, H. deserticola, and H. paradoxus, and comparing these maps to those previously generated for the parental species, H. annuus and H. petiolaris. We also conduct a quantitative trait locus (QTL) analysis of pollen fertility in a B[C.sub.2] population between the parental species and assess levels of pollen and seed fertility in all cross-combinations of the hybrid and parental species. The three hybrid species are massively divergent from their parental species in karyotype; gene order differences were observed for between 9 and 11 linkage groups (of 17 total), depending on the comparison. About one-third of the karyoypic differences arose through the sorting of chromosomal rearrangements that differentiate the parental species, but the remainder appear to have arisen de novo (six breakages/six fusions in H. anomalus, four breakages/three fusions in H. deserticola, and five breakages/five fusions in H. paradoxus). QTL analyses indicate that the karyotypic differences contribute to reproductive isolation. Nine of 11 pollen viability QTL occur on rearranged chromosomes and all but one map close to a rearrangement breakpoint. Finally, pollen and seed fertility estimates for [F.sub.1]'s between the hybrid and parental species fall below 11%, which is sufficient for evolutionary independence of the hybrid neospecies.

The acquisition and analysis of a large set of photocrosslinks, primarily those covalently linking ribozyme residues to the substrate in minimal hammerhead ribozyme constructs HH16 are described. The analysis shows that residues involved in the chemistry of hammerhead catalysis are likely located in that region containing two guanosines (G8 and G12).

Comparative genetic linkage maps provide a powerful tool for the study of karyotypic evolution. We constructed a joint SSR/RAPD genetic linkage map of the Helianthus petiolaris genuine and used it, along with an integrated SSR genetic linkage map derived from four independent H. annuus mapping populations, to examine the evolution of genuine structure between these two annual sunflower species. The results of this work indicate the presence of 27 colinear segments resulting from a minimum of eight translocations and three inversions. These 11 rearrangements are more than previously suspected on the basis of either cytological or genetic map-based analyses. Taken together, these rearrangements required a minimum of 20 chromosomal breakages/fusions. On the basis of estimates of the time since divergence of these two species (750,000-1,000,000 years), this translates into an estimated rate of 5.5-7.3 chromosomal rearrangements per million years of evolution, the highest rate reported tot any taxonomic group to date.

Quantitative trait loci (QTL) controlling phenotypic differences between cultivated sunflower and its wild progenitor were investigated in an [F.sub.3] mapping population. Composite interval mapping revealed the presence of 78 QTL affecting the 18 quantitative traits of interest, with 2--10 QTL per trait. Each QTL explained 3.0-68.0% of the phenotypic variance, although only 4 (corresponding to 3 of 18 traits) had effects >25%. Overall, 51 of the 78 QTL produced phenotypic effects in the expected direction, and for 13 of 18 traits the majority of QTL had the expected effect. Despite being distributed across 15 of the 17 linkage groups, there was a substantial amount of clustering among QTL controlling different traits. In several cases, regions influencing multiple traits harbored QTL with antagonistic effects, producing a cultivar-like phenotype for some traits and a wild-like phenotype for others. On the basis of the directionality of QTL, strong directional selection for increased achene size appears to have played a central role in sunflower domestication. None of the other traits show similar evidence of selection. The occurrence of numerous wild alleles with cultivar-like effects, combined with the lack of major QTL, suggests that sunflower was readily domesticated.

Research has been conducted on the catalysis of the site-specific RNA cleavage and ligation by hairpin ribozyme that requires tertiary interaction between two folded internal loop domains. Results indicate that the docking of these two domains correlates with the loss of the UV reactivity of the G21 and U42 bases.

A single base mutation can trap the hairpin ribozyme-RNA substrate complex in a native-like fold, which precedes the transition state. This ribozyme has been studied as gene therapy for inactivating RNA.

John M. Burke (1) Michael L. Arnold (2) Key Words adaptation, epistasis, hybridization, speciation, transgressive segregation * Abstract Over the years, the evolutionary importance of natural hybridization has been a [...]

Frequency and spatial patterning of clonal reproduction were studied in Louisiana iris hybrid populations. Much clonality was seen, however. Clonal reproduction in hybrid populations might facilitate introgression through increase in the number of flowering ramets per genet and/or the survivorship of early generation hybrids. However, the clonal reproduction might also influence the mating system in such populations. Any potential increase in the selfing rate caused by cross-pollination among ramets of the same genet may increase the likelihood of homoploid hybrid speciation

In the hairpin ribozyme, an interaction between the guanosine downstream from the reactive phosphodiester and C(sub)25 is essential for activity. The hairpin ribozyme-substrate complex has two folding domains that must interact with one another to form the catalytically active complex.

Hydroxyl radical footprinting was used to define the interface between the two domains of the hairpin ribozyme responsible for the interaction with the substrate. Results reveal the presence of a solvent-protected core associated with the internal loops of the enzyme-substrate complex. The experimental protocol used in the investigation can be utilized in the investigation of the requirements in the interaction between the two domains and the analysis of the interactions between them.

A study was conducted to determine whether genetic interactions lead to hybrid offsprings with lower levels of viability and fertility. The nuclear and cytonuclear epistatic interactions in two Louisiana iris species, Iris fulva and I. brevicaulis, were investigated using cpDNA and randomly amplified polymorphic analyses. Results show that genetic interactions do not affect the fitness of hybrid genotypes.

The evolutionary importance of natural hybridization is a topic over which there has been considerable debate. The predominant viewpoint is that hybridization is evolutionarily unimportant because crosses between genetically divergent [...]

The relative flow rates of the tricarboxylic acid cycle was analyzed based on the quantitative determination of isotopomers in the liver via nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). The accuracy of the 13C-NMR method in the quantitative analysis of tricarboxylic acid cycle flow rates was enhanced by the utilization of GC-MS data which provided realiable estimates of the flow rates in the metabolic system. Furthermore, the technique can be utilized for the analysis of relatively low flow rates.

A stable isotope tracer arteriovenous difference protocol was utilized to determine the kinetic mechanisms of whole body arginine and citrulline metabolism in the kidneys and splanchnic region. Analysis of arginine and citrulline metabolism in chronic multicatheritized dog models indicated a higher rate of arginine uptake in the liver compared to the intestines. Furthermore, citrulline from the liver and intestines are converted into arginine in the kidneys and intestines.

Studies of animal and plant hybrid zones have been used to examine the early stages of speciation and mechanisms of reproductive isolation, as well as the interactions between genetic and [...]

The hairpin ribozyme can be manipulated to function properly in the form of a ribonucleoprotein in vitro and provides scope for future research of the mechanism of protein modulation of catalytic RNA activity. The incorporation of the protein-binding domain in the absence of protein increases the catalytic efficiency of the hairpin ribozyme by two times for the cleavage reaction and by sixteen times for the ligation reaction.

Rectal temperature measurements after endotoxin injection in rats reveals that injection of lipopolysaccharide (LPS) leads to different rates of transcription in tumor necrosis factor (TNF) and interleukin-1 beta (IL-1 beta). IL-1 beta and TNF have independent regulatory mechanisms for their synthesis and secretion. The differences in the rate of synthesis of TNF and IL-1beta agree with the results obtained from other studies.

A model comprises equations expressing the intensity of 13C NMR patterns as a relation of the mole fraction of specific isotopomers of oxaloacetate and acetyl CoA (ACC). The model is useful in 13C NMR studies that deal with high concentration of labeled substrates and to estimate normalized pyruvates carboxylase activity. The model provides information on mass spectroscopy and proton NMR, which are used to obtain absolute fluxes for the pyruvate and ACC components.

The schizosaccharomyces pombe cdc 3 (+) gene encodes profilin, an actin-monomer-binding protein, which is involved in cytokinesis. Cdc 3-profilin, which is situated in the medial domain of the cell, catalyzes the production of the F-actin contractile ring. Cells which generate cdc3-profilin in excess acquire an elongated dumbbell shape.

Hairpin ribozyme species have an ultraviolet-sensitive tertiary structure domain. Two cross-linked RNA species which are isolated from unmodified structure are produced by irradiation at 254 nanometers during gel electrophoresis. The presence of high efficiency and mapping between oligonucleotides produce the cross links. The intermolecular crosslinks are independent of magnesium ion concentration.

Two studies were conducted on four healthy young adult men who were administered a diet consisting of 1g protein.kg-1 day-1 for 7 and 10 days before performing a primed constant tracer infusion procedure, to examine the assimilation of dietary arginine and leucine by the splanchnic area. Plasma arginine fluxes were almost identical for the two arginine traces, but varied when the type of infusion was different. The subjects in study 2 were given sustained intravenous infusion of L-(guanidino-13C)arginine and L-(5,5,5-2H3)leucine and an intragastric infusion of L-(5,5,-2H2;guanidino-15N2)arginine and (1-13C)leucine.

The 3' end of a group I ribozyme has the capacity to accept altered nucleotides through a new technique which places them at particular locations. The deoxyribonucleotides are substituted with particular ribonucleotides by this method. 3' splice site reactions which require four 2'-hydroxyl clusters are observed in the 3' end of the Azoarcus group ribozyme. The exon ligation reactions occur forward and backward, and necessitate the presence of three 2'-hydroxyl groups in exon 1 series. Catalysis of the 3' splice site reactions by the group I ribozymes help in determining their geometry and association with the working.