Genome‐wide Linkage and Association Analyses to Identify Genes Influencing Adiponectin Levels: The GEMS Stud

Adiponectin has a variety of metabolic effects on obesity, insulin sensitivity, and atherosclerosis. To identify genes influencing variation in plasma adiponectin levels, we performed genome‐wide linkage and association scans of adiponectin in two cohorts of subjects recruited in the Genetic Epidemiology of Metabolic Syndrome Study. The genome‐wide linkage scan was conducted in families of Turkish and southern European (TSE, n = 789) and Northern and Western European (NWE, N = 2,280) origin. A whole genome association (WGA) analysis (500K Affymetrix platform) was carried out in a set of unrelated NWE subjects consisting of approximately 1,000 subjects with dyslipidemia and 1,000 overweight subjects with normal lipids. Peak evidence for linkage occurred at chromosome 8p23 in NWE subjects (lod = 3.10) and at chromosome 3q28 near ADIPOQ, the adiponectin structural gene, in TSE subjects (lod = 1.70). In the WGA analysis, the single‐nucleotide polymorphisms (SNPs) most strongly associated with adiponectin were rs3774261 and rs6773957 (P < 10^?7). These two SNPs were in high linkage disequilibrium (r² = 0.98) and located within ADIPOQ. Interestingly, our fourth strongest region of association (P < 2 × 10^?5) was to an SNP within CDH13, whose protein product is a newly identified receptor for high‐molecular‐weight species of adiponectin. Through WGA analysis, we confirmed previous studies showing SNPs within ADIPOQ to be strongly associated with variation in adiponectin levels and further observed these to have the strongest effects on adiponectin levels throughout the genome. We additionally identified a second gene (CDH13) possibly influencing variation in adiponectin levels. The impact of these SNPs on health and disease has yet to be determined.     

Gibberellin-photoaffinity labeling of wild oat (Avena fatua L.) aleurone protoplasts

Aleurone protoplasts of wild oat (Avena fatua L.), and subcellular fractions isolated from them, were photoaffinity labeled using the synthetic gibberellin (GA) derivative GA₄-17-yl-1-(1-thia)propan-3-ol-4-azido-5-[¹²⁵I]iodosalicylate. Labeled polypeptides were identified by electrophoresis under denaturing conditions followed by autoradiography. GA-photoaffinity labeling of both intact protoplasts and isolated subcellular fractions led to the covalent attachment of the reagent to many polypeptides. A 50 kD polypeptide in the soluble fraction of homogenates of aleurone protoplasts GA-photoaffinity labeled in vivo showed specific binding. The biologically active GA₁, GA₄ and GA₄-17-yl-1(1-thia)propan-3-ol-4-azidosalicylate completed for binding whereas the biologically inactive GA₈ and GA₃₄ did not. The GA-photoaffinity labeling characteristics of this polypeptide suggested that it might interact specifically with biologically active GAs in vivo. Attempts to detect specific GA-binding in in vitro GA-photoaffinity labeling experiments met with only limited success perhaps indicating the labile nature of specific binding observed in vivo. The potential of GA-photoaffinity labeling for identifying GA-binding proteins in aleurone and other GA-responsive tissues is discussed.Abbreviations azido IAA = 5-azido-7-[³H]indole-3-acetic acid - azido NPA = 5-azido-[3,6-³H]1-N-napthylpthalamic acid - BTP = 1,3-bis(Tris(hydroxymethyl)methylamino)-propane - GA₄-O-ASA = GA₄-17-yl-1-(1-thia)propane-3-ol-4-azidosalicylate - [¹²⁵I]GA₄-O-ASA = GA₄-17-yl-1-(1-thia)propan-3-ol-4-azido-5-[¹²⁵I]iodosalicylate - NPA = 1-Naphthylphthalmic acid - PAGE = Polyacrylamide gel electrophoresis - PMSF = phenylmethylsulfonyl fluoride - SDS = Sodium dodecyl sulphate - TLCK = L-1-Chloro-3-(4-tosylamido)-7-amino-2-heptanone-HCl     

Preparation and polypeptide composition of plasma membrane and other subcellular fractions from wild oat (Avena fatua) aleurone

Plasma membranes can be isolated from a variety of plant tissues by first preparing a post-mitochondrial membrane fraction enriched in plasma membranes, by differential centrifugation, and partitioning this on a dextran-polyethylene glycol two-phase system. With wild oat aleurone, however, we observed that differential centrifugation could not be used to produce a microsomal fraction enriched in plasma membrane. Approximately 70% of the plasma membrane in aleurone homogenates was pelleted by sequential centrifugation at 100 g× 10 min and 1000 g× 10 min. The remainder sedimented at 112 000 g× 1 h. All the material that was pelletable by centrifugation was, therefore, subjected to dextran-polyethylene glycol two-phase partitioning. The plasma membrane marker enzymes glucan synthase II (GSII, EC 2. 4. 1. 34) and UDP-glucose:sterol glucosyltransferase (SGT, EC 2. 4. 1.) were enriched in the upper phase, whereas cytochrome c oxidase activity (EC 1. 9. 3. 1), a mitochondrial marker enzyme, was depleted. The presence of endoplasmic reticulum (ER) and protein body membranes in the phase system was assessed by probing western blots, of SDS-PAGE separated proteins, with polyclonal antiserum either to binding protein (BiP, an ER marker) or to tonoplast intrinsic protein (TIP, a protein body membrane marker). BiP and TIP were present in the lower phase, but were not detected in the upper phase. In addition, the polypeptide patterns of material in the upper and lower phases were very different. These observations suggested that high purity aleurone plasma membrane had been isolated. Although the procedure for isolating plasma membranes was applicable to both aleurone protoplasts and layers, the polypeptide patterns of plasma membranes prepared from these sources were very different. The major protein components of wild oat aleurone were 7 S and 12 S storage globulins. These proteins were present in the lower phase, but not in the plasma membrane enriched upper phase, after aqueous two-phase partitioning. Differential centrifugation studies showed that it was necessary to homogenise aleurone in a buffer of pH 6. 0 or less if a soluble protein fraction, essentially devoid of storage globulins, was to be obtained. The use of these fractionation techniques is discussed in relation to photoaffinity labelling of gibberellin (GA)-binding proteins in aleurone.     

Loss of meiosis in Aspergillus

If strictly mitotic asexual fungi lack recombination, the conventional view predicts that they are recent derivatives from older meiotic lineages. We tested this by inferring phylogenetic relationships among closely related meiotic and strictly mitotic taxa with Aspergillus conidial (mitotic) states. Phylogenies were constructed by using DNA sequences from the mitochondrial small ribosomal subunit, the nuclear ribosomal internal transcribed spacers, and the nuclear 5.8S ribosomal gene. Over 920 bp of sequence was analyzed for each taxon. Phylogenetic analysis of both the mitochondrial and nuclear data sets showed at least four clades that possess both meiotic and strictly mitotic taxa. These results support the hypothesis that strictly mitotic lineages arise frequently from more ancient meiotic lineages with Aspergillus conidial states. Many of the strictly mitotic species examined retained characters that may be vestiges of a meiotic state, including the production of sclerotia, sclerotium-like structures, and hulle cells.      

The phylogenetic status of arthropods, as inferred from 18S rRNA sequences

Partial 18S rRNA sequences of five chelicerate arthropods plus a crustacean, myriapod, insect, chordate, echinoderm, annelid, and platyhelminth were compared. The sequence data were used to infer phylogeny by using a maximum-parsimony method, an evolutionary-distance method, and the evolutionary-parsimony method. The phylogenetic inferences generated by maximum-parsimony and distance methods support both monophyly of the Arthropoda and monophyly of the Chelicerata within the Arthropoda. These results are congruent with phylogenies based on rigorous cladistic analyses of morphological characters. Results support the inclusion of the Arthropoda within a spiralian or protostome coelomate clade that is the sister group of a deuterostome clade, refuting the hypothesis that the arthropods represent the "primitive" sister group of a protostome coelomate clade. Bootstrap analyses and consideration of all trees within 1% of the length of the most parsimonious tree suggest that relationships between the nonchelicerate arthropods and relationships within the chelicerate clade cannot be reliably inferred with the partial 18S rRNA sequence data. With the evolutionary-parsimony method, support for monophyly of the Arthropoda is found in the majority of the combinations analyzed if the coelomates are used as "outgroups." Monophyly of the Chelicerata is supported in most combinations assessed. Our analyses also indicate that the evolutionary-parsimony method, like distance and parsimony, may be biased by taxa with long branches. We suggest that a previous study's inference of the Arthropoda as paraphyletic may be the result of (a) having two few arthropod taxa available for analysis and (b) including long-branched taxa.      

Enigma variations for peptides and their transporters in higher plants

BACKGROUND: Two families of proteins that transport small peptides, the oligopeptide transporters (OPTs) and the peptide transporters (PTRs), have been recognized in eukaryotes. Higher plants contain a far greater number of genes for these transporters than do other eukaryotes. This may be indicative of the relative importance of (oligo)peptides and their transport to plant growth and metabolism. RECENT PROGRESS: Recent studies are now allowing us to assign functions to these transporters and are starting to identify their in-planta substrates, revealing unexpected and important contributions of the transporters to plant growth and developmental processes. This Botanical Briefing appraises recent findings that PTRs and OPTs have key roles to play in the control of plant cell growth and development. Evidence is presented that some of these transporters have functions outside that of nitrogen nutrition and that these carriers can also surprise us with their totally unexpected choice of substrates.