Identification of SNPs and development of allele-specific PCR markers for γ-gliadin alleles in Triticum aestivum

The coding regions of 28 entries of hexaploid wheat gamma-gliadin genes, gene fragments or pseudogenes in GenBank were used for nucleotide alignment. These sequences could be divided into nine subgroups based on nucleotide variation. The chromosomal locations of five of the seven unassigned subgroups were identified through subgroup-specific polymerase chain reactions (PCR) using Chinese Spring group-1 nulli-tetrasomic lines. Multiple single nucleotide polymorphisms (SNPs) and small insertions/deletions were identified in each subgroup. With further mining from wheat expressed sequence tag databases and targeted DNA sequencing, two SNPs were confirmed and one SNP was discovered for genes at the Gli-A1, Gli-B1 and Gli-D1 loci. A modified allele-specific PCR procedure for assaying SNPs was used to generate dominant DNA markers based on these three SNPs. For each of these three SNPs, two allele-specific primer sets were used to test Chinese Spring and 52 commercial Australian wheat varieties representing a range of low-molecular-weight (LMW) alleles. PCR results indicated that all were positive with one of the primer sets and negative with the other, with the exception of three varieties containing the 1BL/1RS chromosomal translocation that were negative for both. Furthermore, markers GliA1.1, GliB1.1 and GliD1.1 were found to be correlated with Glu-A3 a, b or c, Glu-B3 b, c, d or e and Glu-D3 a, b or e LMW glutenin alleles, respectively. Markers GliA1.2, GliB1.2 and GliD1.2 were found to be correlated with the Glu-A3 d or e, Glu-B3 a, g or h and Glu-D3 c alleles, respectively. These results indicated that the gamma-gliadin SNP markers could be used for detecting linked LMW glutenin subunit alleles that are important in determining the quality attributes of wheat products.     

Characterization of a subfamily of beetle odorant-binding proteins found in hemolymph

In insects, hydrophobic odorants are transported through the sensillar lymph to receptors on sensory neurons by odorant-binding proteins (OBPs). The beetle Tenebrio molitor, which is a pest of stored grain products, produces a set of 12-14-kDa OBP-like proteins in its hemolymph. The structure of one of these proteins and that of a moth pheromone-binding protein have been solved. Both proteins have at least six alpha-helices with an internal, hydrophobic, ligand-binding pocket, but the beetle OBP lacks one of the disulfide bonds immediately adjacent to this pocket. To explore this difference and to sample isoform diversity, T. molitor hemolymph OBPs were fractionated by size-exclusion chromatography and reversed-phase high performance liquid chromatography. Selected fractions were reduced and alkylated, and tryptic peptides were sequenced by tandem mass spectrometry. Partial sequences of 7 different isoforms were obtained and used to clone 9 new cDNAs encoding OBPs with identities from 32 to 99%. The more divergent isoforms have numerous substitutions of hydrophobic residues that presumably alter the shape and specificity of the ligand-binding pocket. These isoforms all lack the same third disulfide bridge and are more similar to one another than to any of the 38 OBPs in Drosophila melanogaster. They have presumably arisen via gene duplication following separation of the major insect orders.     

Comprehensive identification of post-translational modifications of rat bone osteopontin by mass spectrometry

Osteopontin (OPN) is a highly modified protein that is found in many tissues and has been associated with a variety of physiological and pathological processes. Bone OPN is a potent inhibitor of hydroxyapatite crystal formation and stimulates bone resorption by osteoclasts; these activities, as well as others, are dependent upon phosphorylation of the protein. We have used mass spectrometry (MS) to perform a comprehensive analysis of the post-translational modification of OPN purified from rat bone. Matrix-assisted laser desorption time-of-flight (MALDI-TOF) MS showed masses of 37.6 and 36.8 kDa before and after enzymatic dephosphorylation, respectively, corresponding to a content of approximately 10.4 phosphate groups. Using proteolytic digestion and tandem MS, we localized 29 sites of phosphorylation: S10, S11, S46, S47, T50, S60, S62, S65, S146, T154, S160, S164, S167, S193, S196, S203, S220, S223, S232, S241, S245, S257, S262, S267, S278, S290, S295, S296, and S297. In addition, Y150 was shown to be sulfated and T107, T110, T116, and T121 are O-glycosylated. No glycan was detected at the potential N-glycosylation site. Other modifications, including deamidation, oxidation, and carbamylation, are also present. A 36-amino acid sequence from residues 67-102 could not be analyzed in detail, even after sialidase treatment, presumably because of the presence of a large number of acidic residues. In comparison to the previously characterized cow milk isoform, rat bone OPN is sulfated and has an additional site of glycosylation, many different sites of phosphorylation, and a lower overall phosphate content.     

Fast neutron treatment as an alternative to radical surgery for malignant tumours of the facial area.

Thirty one patients with very advanced tumours of the maxillary sinus were treated with fast neutrons. Tumour regressed completely in 29 (94%) and subsequently recurred in four (14%). No surgical excision of bone, skin, or nerve was required, and an artificial eye was well tolerated in cases where the eye received the tumour dose and had to be removed. Complications occurred in 10 patients, two of whom had already received radical x ray treatment. The overall duration of neutron treatment was four weeks, and admission to hospital was usually unnecessary. These results compared well with those obtained with surgery. Surgery with curative intent for even moderately advanced tumours of the facial region, particularly the paranasal sinuses, results in deformity, which is often severe and always irreversible. Even so, the cure rate is only about 35%. The high rates of tumour control and the avoidance of severe cosmetic and functional defects after fast neutron treatment make it an alternative to radical surgery in the management of malignant tumours of the facial area.     

Effect of cyclodextrins on the reactivity of fenitrothion

The hydrolysis reaction of fenitrothion was studied in water containing 2% dioxane and in the presence of native cyclodextrins (α-, β- and γ-CD) and two commercially available modified derivatives, namely, permethylated β- and α-cyclodextrin (TRIMEB and TRIMEA, respectively). The kinetics of the reaction in the presence of TRIMEA could not be measured because the complex formed is insoluble and precipitated even at low concentration. On the other hand, the reaction is only weakly affected by the presence of α-CD. The hydrolysis reaction is inhibited by all the other cyclodextrins. From the kinetic data the association equilibrium constants for the formation of the 1:1 inclusion complexes were determined as 417, 511 and 99 M⁻¹ for β-CD, TRIMEB and γ-CD, respectively. Despite the differences in the association constants for β- and γ-CD, the observed inhibition effect is about the same and this is due to the fact that the rate of hydrolysis in the cavity of γ-CD is smaller than that in the cavity of β-CD. The strongest inhibitor is TRIMEB and this result is consistent with the known structure of the complex in the solid state.     

iDArTs: increasing the value of genomic resources at no cost

For many years, genetic markers have been the building blocks in assembling genomic knowledge. Improved technology and methods for collecting marker data have increased accuracy, increased throughput, and reduced cost. However, common genotyping technology still produces far fewer markers in plant species than in animals and humans. We propose a new type of genetic marker based on the Diversity Arrays Technology (DArT) genotyping system for organisms lacking a reference genetic sequence. These markers are based directly on microarray probe intensity profiles and hence are called iDArTs. They require no additional genotyping beyond screening with a DArT array. Since standard methods of genetic analysis cannot be used with these continuous markers, we develop novel methods for the common bi-parental experimental designs doubled haploids, recombinant inbred lines, and backcrosses. These enable the augmentation of genetic maps with iDArTs and permit quantitative trait locus mapping with both discrete and continuous markers. We use simulation to demonstrate the power of this approach for marker mapping. In addition, we construct maps and perform linkage analysis for these DArT genotypes using the doubled haploid progeny lines from a cross between the wheat cultivars Chara and Glenlea. These methods allow access to a previously untapped genetic resource by extracting additional information from the raw data. With no additional genotyping cost, we are able to double the number of markers mapped and thereby increase genome coverage.     

Protein kinase C isoforms differentially phosphorylate human choline acetyltransferase regulating its catalytic activity

Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons; regulation of its activity or response to physiological stimuli is poorly understood. We show that ChAT is differentially phosphorylated by protein kinase C (PKC) isoforms on four serines (Ser-440, Ser-346, Ser-347, and Ser-476) and one threonine (Thr-255). This phosphorylation is hierarchical, with phosphorylation at Ser-476 required for phosphorylation at other serines. Phosphorylation at some, but not all, sites regulates basal catalysis and activation. Ser-476 with Ser-440 and Ser-346/347 maintains basal ChAT activity. Ser-440 is targeted by Arg-442 for phosphorylation by PKC. Arg-442 is mutated spontaneously (R442H) in congenital myasthenic syndrome, rendering ChAT inactive and causing neuromuscular failure. This mutation eliminates phosphorylation of Ser-440, and Arg-442, not phosphorylation of Ser-440, appears primarily responsible for ChAT activity, with Ser-440 phosphorylation modulating catalysis. Finally, basal ChAT phosphorylation in neurons is mediated predominantly by PKC at Ser-476, with PKC activation increasing phosphorylation at Ser-440 and enhancing ChAT activity.