Biomedical subjects

Mike J Naldrett

Publications and source records attributed to Mike J Naldrett.

5 recordsLinked to original sources

PubMed2006

Arabidopsis cell wall proteome defined using multidimensional protein identification technology.

With the completion of the sequencing of the Arabidopsis genome and the recent advances in proteomic technology, the identification of proteins from highly complex mixtures is now possible. Rather than using gel electrophoresis and peptide mass fingerprinting, we have used multidimensional protein identification technology (MudPIT) to analyse the "tightly-bound" proteome for purified cell walls from Arabidopsis cell suspension cultures. Using bioinformatics for the prediction of signal peptides for targeting to the secretory pathway and for the absence of ER retention signal, 89 proteins were selected as potential extracellular proteins. Only 33% of these were identified in previous proteomic analyses of Arabidopsis cell walls. A functional classification revealed that a large proportion of the proteins were enzymes, notably carbohydrate active enzymes, peroxidases and proteases. Comparison of all the published proteomic analyses for the Arabidopsis cell wall identified 268 non-redundant genes encoding wall proteins. Sixty of these (22%) were derived from our analysis of tightly-bound wall proteins.

Arabidopsis↗

PubMed2005

Identification of a novel family of 70 kDa microtubule-associated proteins in Arabidopsis cells.

Most plant microtubule-associated proteins (MAPs) have homologues across the phylogenetic spectrum. To find potential plant-specific MAPs that will have evaded bioinformatic searches we devised a low stringency method for isolating proteins from an Arabidopsis cell suspension on endogenous taxol-microtubules. By tryptic peptide mass fingerprinting we identified 55 proteins that were enriched on taxol-microtubules. Amongst a range of known MAPs, such as kinesins, MAP65 isoforms and MOR1, we detected 'unknown' 70 kDa proteins that belong to a family of five closely related Arabidopsis proteins having no known homologues amongst non-plant organisms. To verify that AtMAP70-1 associates with microtubules in vivo, it was expressed as a GFP fusion. This confirmed that the protein decorates all four microtubule arrays in both transiently infected Arabidopsis and stably transformed tobacco BY-2 suspension cells. Microtubule-directed drugs perturbed the localization of AtMAP70-1 but cytochalasin D did not. AtMAP70-1 contains four predicted coiled-coil domains and truncation studies identified a central domain that targets the fusion protein to microtubules in vivo. This study therefore introduces a novel family of plant-specific proteins that interact with microtubules.

Amino Acid Sequence↗

PubMed2005

Concentration and desalting of peptide and protein samples with a newly developed C18 membrane in a microspin column format.

Protein identification plays an important role in today's academic and industrial proteomic research. Commonly used methods for the separation of proteins from complex samples include liquid chromatography (e.g., ion exchange, reversed-phase, hydrophobic interaction), or types of gel electrophoresis (e.g., 1d and 2d PAGE). Relevant proteins separated in the latter way are often cut out, cleaved with trypsin "in gel," and the resulting peptide mixtures combined with matrix and spotted onto a target plate for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-ms) analysis. Subsequently, proteins can be identified by comparison of the resulting peptide mass fingerprints against different databases.(1) since the success of protein identification can be enhanced by the desalting and concentration of the samples, an innovative C18-membrane was incorporated into a microspin column (Vivapure C18 micro spin column, Vivascience AG, Hannover, Germany) to analyze its performance for sample preparation prior to MALDI-ToF-ms. Rapid concentration of single or multiple 200-microl volumes through an available membrane only 2 mm in diameter allowed for analysis of very dilute samples. We observed the successful and rapid desalting of urea-containing protein samples at 100 fmol/mul up to a mass of approximately 70 KDA and the concentration of digest peptides from a solution of 1 fmol/microl using C18-membrane technology.

Animals↗

PubMed2002

The altered pattern of amylose accumulation in the endosperm of low-amylose barley cultivars is attributable to a single mutant allele of granule-bound starch synthase I with a deletion in the 5'-non-coding region.

Reasons for the variable amylose content of endosperm starch from waxy cultivars of barley (Hordeum vulgare) were investigated. The mature grains of most such cultivars contain some amylose, although amounts are much lower than in wild-type cultivars. In these low-amylose cultivars, amylose synthesis starts relatively late in grain development. Starch granules in the outer cell layers of the endosperm contain more amylose than those in the center. This distribution corresponds to that of granule-bound starch synthase I (GBSSI), which is more severely reduced in amount in the center of the endosperm than in the outer cell layers, relative to wild-type cultivars. A second GBSSI in the barley plant, GBSSIb, is not detectable in the endosperm and cannot account for amylose synthesis in the low-amylose cultivars. The change in the expression of GBSSI in the endosperm of the low-amylose cultivars appears to be due to a 413-bp deletion of part of the promoter and 5'-untranslated region of the gene. Although these cultivars are of diverse geographical origin, all carry this same deletion, suggesting that the low-amylose cultivars have a common waxy ancestor. Records suggest a probable source in China, first recorded in the 16th century. Two further families of waxy cultivars have no detectable amylose in the endosperm starch. These amylose-free cultivars were selected in the 20th century from chemically mutagenized populations of wild-type barley. In both cases, 1-bp alterations in the GBSSI gene completely eliminate GBSSI activity.

5' Flanking Region↗

PubMed2002

Characterization of the genes encoding the cytosolic and plastidial forms of ADP-glucose pyrophosphorylase in wheat endosperm.

In most species, the synthesis of ADP-glucose (Glc) by the enzyme ADP-Glc pyrophosphorylase (AGPase) occurs entirely within the plastids in all tissues so far examined. However, in the endosperm of many, if not all grasses, a second form of AGPase synthesizes ADP-Glc outside the plastid, presumably in the cytosol. In this paper, we show that in the endosperm of wheat (Triticum aestivum), the cytosolic form accounts for most of the AGPase activity. Using a combination of molecular and biochemical approaches to identify the cytosolic and plastidial protein components of wheat endosperm AGPase we show that the large and small subunits of the cytosolic enzyme are encoded by genes previously thought to encode plastidial subunits, and that a gene, Ta.AGP.S.1, which encodes the small subunit of the cytosolic form of AGPase, also gives rise to a second transcript by the use of an alternate first exon. This second transcript encodes an AGPase small subunit with a transit peptide. However, we could not find a plastidial small subunit protein corresponding to this transcript. The protein sequence of the purified plastidial small subunit does not match precisely to that encoded by Ta.AGP.S.1 or to the predicted sequences of any other known gene from wheat or barley (Hordeum vulgare). Instead, the protein sequence is most similar to those of the plastidial small subunits from chickpea (Cicer arietinum) and maize (Zea mays) and rice (Oryza sativa) seeds. These data suggest that the gene encoding the major plastidial small subunit of AGPase in wheat endosperm has yet to be identified.

Amino Acid Sequence↗