Towards coeliac‐safe bread

Gluten‐free foods cannot substitute for products made from wheat flour. When wheat products are digested, the remaining peptides can trigger an autoimmune disease in 1% of the North American and European population, called coeliac disease. Because wheat proteins are encoded by a large gene family, it has been impossible to use conventional breeding to select wheat varieties that are coeliac‐safe. However, one can test the properties of protein variants by expressing single genes in coeliac‐safe cereals like maize. One source of protein that can be considered as coeliac‐safe and has bread‐making properties is teff (Eragrostis tef), a grain consumed in Ethiopia. Here, we show that teff α‐globulin3 (Etglo3) forms storage vacuoles in maize that are morphologically similar to those of wheat. Using transmission electron microscopy, immunogold labelling shows that Etglo3 is almost exclusively deposited in the storage vacuole as electron‐dense aggregates. Of maize seed storage proteins, 27‐kDa γ‐zein is co‐deposited with Etglo3. Etglo3 polymerizes via intermolecular disulphide bonds in maize, similar to wheat HMW glutenins under non‐reducing conditions. Crossing maize Etglo3 transgenic lines with α‐, β‐ and γ‐zein RNA interference (RNAi) lines reveals that Etglo3 accumulation is only dramatically reduced in γ‐zein RNAi background. This suggests that Etglo3 and 27‐kDa γ‐zein together cause storage vacuole formation and behave similar to the interactions of glutenins and gliadins in wheat. Therefore, expression of teff α‐globulins in maize presents a major step in the development of a coeliac‐safe grain with bread‐making properties.     

Development of real-time assays for impedance-based detection of microbial double-stranded DNA targets: optimization and data analysis

A real-time, label free assay was developed for microbial detection, utilizing double-stranded DNA targets and employing the next generation of an impedimetric sensor array platform designed by Sharp Laboratories of America (SLA). Real-time curves of the impedimetric signal response were obtained at fixed frequency and voltage for target binding to oligonucleotide probes attached to the sensor array surface. Kinetic parameters of these curves were analyzed by the integrated data analysis package for signal quantification. Non-specific binding presented a major challenge for assay development, and required assay optimization. For this, differences were maximized between binding curve kinetic parameters for probes binding to complementary targets versus non-target controls. Variables manipulated for assay optimization included target concentration, hybridization temperature, buffer concentration, and the use of surfactants. Our results showed that (i) different target-probe combinations required optimization of specific sets of variables; (ii) for each assay condition, the optimum range was relatively narrow, and had to be determined empirically; and (iii) outside of the optimum range, the assay could not distinguish between specific and non-specific binding. For each target-probe combination evaluated, conditions resulting in good separation between specific and non-specific binding signals were established, generating high confidence in the SLA impedimetric dsDNA assay results.     

Dynamic gene copy number variation in collinear regions of grass genomes

A salient feature of genomes of higher organisms is the birth and death of gene copies. An example is the alpha prolamin genes, which encode seed storage proteins in grasses (Poaceae) and represent a medium-size gene family. To better understand the mechanism, extent, and pace of gene amplification, we compared prolamin gene copies in the genomes of two different tribes in the Panicoideae, the Paniceae and the Andropogoneae. We identified alpha prolamin (setarin) gene copies in the diploid foxtail millet (Paniceae) genome (490 Mb) and compared them with orthologous regions in diploid sorghum (730 Mb) and ancient allotetraploid maize (2,300 Mb) (Andropogoneae). Because sequenced genomes of other subfamilies of Poaceae like rice (389 Mb) (Ehrhartoideae) and Brachypodium (272 Mb) (Pooideae) do not have alpha prolamin genes, their collinear regions can serve as "empty" reference sites. A pattern emerged, where genes were copied and inserted into other chromosomal locations followed by additional tandem duplications (clusters). We observed both recent (species-specific) insertion events and older ones that are shared by these tribes. Many older copies were deleted by unequal crossing over of flanking sequences or damaged by truncations. However, some remain intact with active and inactive alleles. These results indicate that genomes reflect only a snapshot of the gene content of a species and are far less static than conventional genetics has suggested. Nucleotide substitution rates for active alpha prolamins genes were twice as high as for low copy number beta, gamma, and delta prolamin genes, suggesting that gene amplification accelerates the pace of divergence.     

Characterization of three maize bacterial artificial chromosome libraries toward anchoring of the physical map to the genetic map using high-density bacterial artificial chromosome filter hybridization

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the 185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage lambda. The results indicate that the libraries are of high quality with low contamination by organellar and lambda-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6x coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 x Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 +/- 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction.     

Asymmetric localization of seed storage protein RNAs to distinct subdomains of the endoplasmic reticulum in developing maize endosperm cells

Plant storage proteins are synthesized and stored in different compartments of the plant endomembrane system. Developing maize seeds synthesize and accumulate prolamin (zein) and 11S globulin (legumin-1) type proteins, which are sequestered in the endoplasmic reticulum (ER) lumen and storage vacuoles, respectively. Immunofluorescence studies showed that the lumenal chaperone BiP was not randomly distributed within the ER in developing maize endosperm but concentrated within the zein-containing protein bodies. Analysis of the spatial distribution of RNAs in maize endosperm sections by in situ RT-PCR showed that, contrary to the conclusions made in an earlier study [Kim et al. (2002) Plant Cell 14: 655-672], the zein and legumin-1 RNAs are not symmetrically distributed on the ER but, instead, targeted to specific ER subdomains. RNAs coding for 22 kDa alpha-zein, 15 kDa beta-zein, 27 kDa gamma-zein and 10 kDa delta-zein were localized to ER-bounded zein protein bodies, whereas 51 kDa legumin-1 RNAs were distributed on adjacent cisternal ER proximal to the zein protein bodies. These results indicate that the maize storage protein RNAs are targeted to specific ER subdomains in developing maize endosperm and that RNA localization may be a prevalent mechanism to sort proteins within plant cells.     

P(0) glycoprotein overexpression causes congenital hypomyelination of peripheral nerves

We show that normal peripheral nerve myelination depends on strict dosage of the most abundantly expressed myelin gene, myelin protein zero (Mpz). Transgenic mice containing extra copies of Mpz manifested a dose-dependent, dysmyelinating neuropathy, ranging from transient perinatal hypomyelination to arrested myelination and impaired sorting of axons by Schwann cells. Myelination was restored by breeding the transgene into the Mpz-null background, demonstrating that dysmyelination does not result from a structural alteration or Schwann cell-extrinsic effect of the transgenic P(0) glycoprotein. Mpz mRNA overexpression ranged from 30-700%, whereas an increased level of P(0) protein was detected only in nerves of low copy-number animals. Breeding experiments placed the threshold for dysmyelination between 30 and 80% Mpz overexpression. These data reveal new points in nerve development at which Schwann cells are susceptible to increased gene dosage, and suggest a novel basis for hereditary neuropathy.     

Genetic modifiers of the Kv beta2-null phenotype in mice

Shaker-type potassium (K+) channels are composed of pore-forming alpha subunits associated with cytoplasmic beta subunits. Kv beta2 is the predominant Kv beta subunit in the mammalian nervous system, but its functions in vivo are not clear. Kv beta2-null mice have been previously characterized in our laboratory as having reduced lifespans, cold swim-induced tremors and occasional seizures, but no apparent defect in Kv alpha-subunit trafficking. To test whether strain differences might influence the severity of this phenotype, we analyzed Kv beta2-null mice in different strain backgrounds: 129/SvEv (129), C57BL/6J (B6) and two mixed B6/129 backgrounds. We found that strain differences significantly affected survival, body weight and thermoregulation in Kv beta2-null mice. B6 nulls had a more severe phenotype than 129 nulls in these measures; this dramatic difference did not reflect alterations in seizure thresholds but may relate to strain differences we observed in cerebellar Kv1.2 expression. To specifically test whether Kv beta1 is a genetic modifier of the Kv beta2-null phenotype, we generated Kv beta1.1-deficient mice by gene targeting and bred them to Kv beta2-null mice. Kv beta1.1/Kv beta2 double knockouts had significantly increased mortality compared with either single knockout but still maintained surface expression of Kv1.2, indicating that trafficking of this alpha subunit does not require either Kv beta subunit. Our results suggest that genetic differences between 129/SvEv and C57Bl/6J are key determinants of the severity of defects seen in Kv beta2-null mice and that Kv beta1.1 is a specific although not strain-dependent modifier.     

Whole-genome validation of high-information-content fingerprinting

Fluorescent-based high-information-content fingerprinting (HICF) techniques have recently been developed for physical mapping. These techniques make use of automated capillary DNA sequencing instruments to enable both high-resolution and high-throughput fingerprinting. In this article, we report the construction of a whole-genome HICF FPC map for maize (Zea mays subsp. mays cv B73), using a variant of HICF in which a type IIS restriction enzyme is used to generate the fluorescently labeled fragments. The HICF maize map was constructed from the same three maize bacterial artificial chromosome libraries as previously used for the whole-genome agarose FPC map, providing a unique opportunity for direct comparison of the agarose and HICF methods; as a result, it was found that HICF has substantially greater sensitivity in forming contigs. An improved assembly procedure is also described that uses automatic end-merging of contigs to reduce the effects of contamination and repetitive bands. Several new features in FPC v7.2 are presented, including shared-memory multiprocessing, which allows dramatically faster assemblies, and automatic end-merging, which permits more accurate assemblies. It is further shown that sequenced clones may be digested in silico and located accurately on the HICF assembly, despite size deviations that prevent the precise prediction of experimental fingerprints. Finally, repetitive bands are isolated, and their effect on the assembly is studied.     

CARPEL FACTORY,a Dicer homolog,and HEN1, a novel protein,act in microRNA metabolism in Arabidopsis thaliana

BACKGROUND: In metazoans, microRNAs, or miRNAs, constitute a growing family of small regulatory RNAs that are usually 19-25 nucleotides in length. They are processed from longer precursor RNAs that fold into stem-loop structures by the ribonuclease Dicer and are thought to regulate gene expression by base pairing with RNAs of protein-coding genes. In Arabidopsis thaliana, mutations in CARPEL FACTORY (CAF), a Dicer homolog, and those in a novel gene, HEN1, result in similar, multifaceted developmental defects, suggesting a similar function of the two genes, possibly in miRNA metabolism.RESULTS: To investigate the potential functions of CAF and HEN1 in miRNA metabolism, we aimed to isolate miRNAs from Arabidopsis and examine their accumulation during plant development in wild-type plants and in hen1-1 and caf-1 mutant plants. We have isolated 11 miRNAs, some of which have potential homologs in tobacco, rice, and maize. The putative precursors of these miRNAs have the capacity to form stable stem-loop structures. The accumulation of these miRNAs appears to be spatially or temporally controlled in plant development, and their abundance is greatly reduced in caf-1 and hen1-1 mutants. HEN1 homologs are found in bacterial, fungal, and metazoan genomes.CONCLUSIONS: miRNAs are present in both plant and animal kingdoms. An evolutionarily conserved mechanism involving a protein, known as Dicer in animals and CAF in Arabidopsis, operates in miRNA metabolism. HEN1 is a new player in miRNA accumulation in Arabidopsis, and HEN1 homologs in metazoans may have a similar function. The developmental defects associated with caf-1 and hen1-1 mutations and the patterns of miRNA accumulation suggest that miRNAs play fundamental roles in plant development.