Targeted modification of wheat grain protein to reduce the content of celiac causing epitopes.

The prolamin peptides in wheat gluten and in the homologous storage proteins of barley and rye cause painful chronic erasure of microvilli of the small intestine epithelium in celiac patients. If untreated, it can lead to chronic diarrhea, abdominal distension, osteoporosis, weight-loss due to malabsorption of nutrients, and anemia. In addition to congenital cases, life-long exposure to gluten proteins in bread and pasta can also induce development of celiac sprue in adults. To date, the only effective treatment is life-long strict abstinence from the staple food grains. Complete exclusion of dietary gluten is, however, difficult due to use of wheat in many foods, incomplete labeling and social constraints. Thus, finding alternative therapies for this most common foodborne disease remained an active area of research, which has led to many suggestions in last few years. The pros and cons associated with these therapies were reviewed in the present communication. As different celiac patients are immunogenic to different members of the undigestible proline/glutamine rich peptides of ~149 gliadins and low molecular weight glutenin subunits as well as the six high molecular weight glutenin subunits, an exhaustive digestion of the immunogenic peptides in the stomach, duodenum, jejunum, and ileum of celiacs is required. In view of the above, we evaluated the capacity of cereal grains to synthesize and store the enzymes prolyl endopeptidase from Flavobacterium meningosepticum and the barley cysteine endoprotease B2, which in combination are capable of detoxifying immunogenic gluten peptides in a novel treatment of celiac disease.

Heritable alteration in DNA methylation induced by nitrogen-deficiency stress accompanies enhanced tolerance by progenies to the stress in rice (Oryza sativa L.).

Cytosine methylation is responsive to various biotic- and abiotic-stresses, which may produce heritable epialleles. Nitrogen (N)-deficiency is an abiotic stress being repeatedly experienced by plants. To address possible epigenetic consequences of N-deficiency-stress, we investigated the stability of cytosine methylation in rice (Oryza sativa L.) subsequent to a chronic (a whole-generation) N-deficiency at two levels, moderate (20mg/L) and severe (10mg/L), under hydroponic culture. MSAP analysis revealed that locus-specific methylation alteration occurred in leaf-tissue of the stressed plants (S(0)) experiencing either level of N-deficiency, which was validated by gel-blotting. Analysis on three non-stressed self-fed progenies (S(1), S(2) and S(3)) by gel-blotting indicated that ca. 50% of the altered methylation patterns in somatic cells (leaf) of the stressed S(0) plants were recaptured in S(1), which were then stably inherited to S(2) and S(3). Bisulfite sequencing of two variant MSAP loci with homology to low-copy retrotransposons on one stressed plant (S(0)) and its non-stressed progenies (S(1) and S(2)) showed that whereas one locus exhibited limited and non-heritable CHH methylation alteration, the other locus manifested dramatic heritable hypermethylation at nearly all cytosine sites within the assayed region. Intriguingly, when two groups of S(2) plants descended from the same N-deficiency-stressed S(0) plant were re-subjected to the stress, the group inheriting the modified methylation patterns showed enhanced tolerance to the N-deficiency-stress compared with the group bearing the original patterns. Our results thus demonstrate heritability of an acquired adaptive trait in rice, which was accompanied by epigenetic inheritance of modified cytosine methylation patterns, implicating an epigenetic basis underlying the inheritance of an acquired trait in plants.

Childbirth after organ transplantation in Hungary.

BACKGROUND: The first successfully delivered newborn after organ transplantation was reported in 1963; since then, >14,000 women have delivered after transplantation. Patients with an end-stage organ disease develop fertility disturbances. One year after a successful solid organ transplantation with stable graft function, fertile women can give birth to a child from a medical point of view. Pregnant transplant patients do experience a high risk of graft function worsening, a rejection episode, and opportunistic infections. Furthermore, the medical therapy may influence teratogenicity. METHODS: Between 1974 and September 1, 2010, 5 Hungarian centers performed 6802 solid organ transplantation and lungs were grafted in Vienna, Austria. The organ distribution was: 5971 kidney, 454 liver, 187 heart, 90 combined pancreas-kidney, 5 combined islet-kidney, and 95 lung transplantation. There were no pregnancies among heart, lung, and pancreas recipients. RESULTS: In all, 3.9% of the renal and 14.3% of the fertile liver transplanted women gave birth to children. To wit, 23 kidney recipients delivered 27 healthy children (17 boys and 10 girls). In 4 cases, 2 children were born, twice as twins. Among liver recipients, 8 women delivered 8 healthy babies. There was no hepatitis C or B virus-positive patient among the mothers. There was no graft insufficiency, rejection or birth defect. Transplanted mothers often display toxemia or preeclampsia during pregnancy requiring cesarean section. The relatively higher ratio of liver recipients was perhaps due to the rarer occurrence of extrahepatic organ damage, like diabetic nephropathy or cardiomyopathy, and the reversible nature of hepatorenal or hepatopulmonary syndrome. CONCLUSION: Delivery of a child by a transplanted mother carries an high risk, requiring interdisciplinary cooperation. The quality of life of solid organ recipients can be significantly raised by childbirth under appropriate circumstances.

Supplements of transgenic malt or grain containing (1,3-1,4)-beta-glucanase increase the nutritive value of barley-based broiler diets to that of maize.

1. A diet with addition to normal barley of malt from transgenic barley expressing a protein engineered, thermotolerant Bacillus (1,3-1,4)-beta-glucanase during germination has previously been demonstrated to provide a broiler chicken weight gain comparable to maize diets. It also reduced dramatically the number of birds with adhering sticky droppings, but did not entirely eliminate sticky droppings. One of the objectives of the broiler chicken trials reported here was to determine if higher concentrations of transgenic malt could alleviate the sticky droppings. 2. Another aim was to investigate the feasibility of using mature transgenic grain containing the thermotolerant (1,3-1,4)-beta-glucanase as feed addition and to compare diets containing transgenic grain to a diet with the recommended amount of a commercial beta-glucanase-based product. 3. Inclusion of 75 or 151 g/kg transgenic malt containing 4.7 or 98 mg/kg thermotolerant (1,3-1,4)-beta-glucanase with 545 or 469 g/kg non-transgenic barley instead of maize yielded a weight gain in Cornish Cross broiler chickens indistinguishable from presently used maize diets. The gene encoding the enzyme is expressed in the aleurone with a barley alpha-amylase gene promoter and the enzyme is synthesised with a signal peptide for secretion into the endosperm of the malting grain. 4. Equal weight gain was achieved, when the feed included 39 g/kg transgenic barley grain [containing 66 mg/kg thermotolerant (1,3-1,4)-beta-glucanase] and 581 g/kg non-transgenic barley instead of maize. In this case, the gene encoding the enzyme has been expressed with the D-hordein gene (Hor3-1) promoter during grain maturation. The enzyme is synthesised as a precursor with a signal peptide for transport through the endoplasmic reticulum and targeted into the storage vacuoles. Deposition of the enzyme in the prolamin storage protein bodies of the endosperm protects it from degradation during the programmed cell death of the endosperm in the final stages of grain maturation and provides extraordinary heat stability. The large amount of highly active (1,3-1,4)-beta-glucanase in the mature grain allowed the reduction of the transgenic grain ingredient to 0.2 g/kg diet, thus making the ingredient comparable to that of the trace minerals added to standard diets. 5. A direct comparison using transgenic grain supplement at the level of 1 g/kg of feed with the standard recommended addition of the commercial enzyme preparation Avizyme 1100 at 1 g/kg yielded equal weight gain, feed consumption and feed efficiency in birds fed a barley-based diet. 6. The production of sticky droppings characteristic of broilers fed on barley diets was avoided with all 9 experimental diets and reduced to the level observed with a standard maize diet by supplementation with transgenic barley. 7. The excellent growth and normal survival of the 400 broilers tested on barley diets supplemented with transgenic grain or malt showed the grain and malt not to be toxic. 8. The barley feed with added transgenic grain or malt containing thermotolerant (1,3-1,4)-beta-glucanase provides an environmentally friendly alternative to enzyme additives, as it uses photosynthetic energy for production of the enzyme in the grain and thus avoids use of non-renewable energy for fermentation. The deposition of the enzyme in the protein bodies of the grain in the field makes coating procedures for stabilisation of enzyme activity superfluous. 9. Barley feed with the small amount of transgenic grain as additive to normal barley provides an alternative for broiler feed in areas where grain maize cannot be grown for climatic reasons or because of unsuitable soil and thus has to be imported.

Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding.

Like other enveloped viruses, HIV-1 uses cellular machinery to bud from infected cells. We now show that Tsg101 protein, which functions in vacuolar protein sorting (Vps), is required for HIV-1 budding. The UEV domain of Tsg101 binds to an essential tetrapeptide (PTAP) motif within the p6 domain of the structural Gag protein and also to ubiquitin. Depletion of cellular Tsg101 by small interfering RNA arrests HIV-1 budding at a late stage, and budding is rescued by reintroduction of Tsg101. Dominant negative mutant Vps4 proteins that inhibit vacuolar protein sorting also arrest HIV-1 and MLV budding. These observations suggest that retroviruses bud by appropriating cellular machinery normally used in the Vps pathway to form multivesicular bodies.

Nrarp is a novel intracellular component of the Notch signaling pathway.

The Lin12/Notch receptors regulate cell fate during embryogenesis by activating the expression of downstream target genes. These receptors signal via their intracellular domain (ICD), which is released from the plasma membrane by proteolytic processing and associates in the nucleus with the CSL family of DNA-binding proteins to form a transcriptional activator. How the CSL/ICD complex activates transcription and how this complex is regulated during development remains poorly understood. Here we describe Nrarp as a new intracellular component of the Notch signaling pathway in Xenopus embryos. Nrarp is a member of the Delta-Notch synexpression group and encodes a small protein containing two ankyrin repeats. Nrarp expression is activated in Xenopus embryos by the CSL-dependent Notch pathway. Conversely, overexpression of Nrarp in embryos blocks Notch signaling and inhibits the activation of Notch target genes by ICD. We show that Nrarp forms a ternary complex with the ICD of XNotch1 and the CSL protein XSu(H) and that in embryos Nrarp promotes the loss of ICD. By down-regulating ICD levels, Nrarp could function as a negative feedback regulator of Notch signaling that attenuates ICD-mediated transcription.

Improved barley broiler feed with transgenic malt containing heat-stable (1,3-1,4)-beta-glucanase.

The low nutritional value of barley for poultry is because of the absence of an intestinal enzyme for efficient depolymerization of (1, 3-1,4)-beta-glucan, the major polysaccharide of the endosperm cell walls. This leads to high viscosity in the intestine, limited nutrient uptake, decreased growth rate, and unhygienic sticky droppings adhering to chickens and floors of the production cages. Consequently, the 7.5 billion broiler chickens produced annually in the United States are primarily raised on corn-soybean diets. Here we show that addition to normal barley of 6.2% transgenic malt containing a thermotolerant (1,3-1,4)-beta-glucanase (4.28 microg.g(-1) soluble protein) provides a weight gain equivalent to corn diets. The number of birds with adhering sticky droppings is drastically reduced. Intestines and excrements of chickens fed the barley control diet contained large amounts of soluble (1,3-1,4)-beta-glucan, which was reduced by 75 and 50%, respectively, by adding transgenic malt to the diet. The amount of active recombinant enzyme in the small intestine corresponded to that present in the feed, whereas an 11-fold concentration of the enzyme was observed in the ceca, and a 7.5-fold concentration occurred in the excrement. Glycosylation of the beta-glucanase isolated from the ceca testified to its origin from the transgenic barley. Analysis of the data from this trial demonstrates the possibility of introducing individual recombinant enzymes into various parts of the gastrointestinal tract of chickens with transgenic malt and thereby the possibility of evaluating their effect on the metabolism of a given ingredient targeted by the enzyme.

The production of recombinant proteins in transgenic barley grains.

The grain of the self-pollinating diploid barley species offers two modes of producing recombinant enzymes or other proteins. One uses the promoters of genes with aleurone-specific expression during germination and the signal peptide code for export of the protein into the endosperm. The other uses promoters of the structural genes for storage proteins deposited in the developing endosperm. Production of a protein-engineered thermotolerant (1, 3-1, 4)-beta-glucanase with the D hordein gene (Hor3-1) promoter during endosperm development was analyzed in transgenic plants with four different constructs. High expression of the enzyme and its activity in the endosperm of the mature grain required codon optimization to a C+G content of 63% and synthesis as a precursor with a signal peptide for transport through the endoplasmic reticulum and targeting into the storage vacuoles. Synthesis of the recombinant enzyme in the aleurone of germinating transgenic grain with an alpha-amylase promoter and the code for the export signal peptide yielded approximately 1 microgram small middle dotmg(-1) soluble protein, whereas 54 microgram small middle dotmg(-1) soluble protein was produced on average in the maturing grain of 10 transgenic lines with the vector containing the gene for the (1, 3-1, 4)-beta-glucanase under the control of the Hor3-1 promoter.

Activation of Xenopus genes required for lateral inhibition and neuronal differentiation during primary neurogenesis.

XNGN-1, a member of the neurogenin family of basic helix-loop-helix proteins, plays a critical role in promoting neuronal differentiation in Xenopus embryos. When ectopically expressed, XNGN-1 induces the expression of a set of genes required for neuronal differentiation such as XMyT1 and NeuroD. At the same time, however, XNGN-1 induces the expression of genes that antagonize neuronal differentiation by a process called lateral inhibition. Here, we present evidence that XNGN-1 activates the expression of genes required for differentiation and lateral inhibition by recruiting transcriptional coactivators p300/CBP (CREB-binding protein) or PCAF (p3OO/CBP-associated protein), both of which contain histone acetyltransferase (HAT) activity. Significantly, transcriptional activation of the genes in the lateral inhibitory pathway is less dependent on the HAT activity than is the activation of the genes that mediate differentiation. We propose that this difference enables the genes in the lateral inhibition pathway to be induced prior to the genes that promote differentiation, thus enabling lateral inhibition to establish a negative feedback loop and restrict the number of cells undergoing neuronal differentiation.

A two-step mechanism generates the spacing pattern of the ciliated cells in the skin of Xenopus embryos.

The skin of Xenopus embryos contains a population of specialized ciliated cells that are distributed in an evenly spaced pattern. Here we describe two successive steps that govern the differentiation and the generation of the spacing pattern of these ciliated cells. The first step occurs in the inner or sensorial layer of the non-neural ectoderm where a subset of cells are chosen to differentiate into ciliated-cell precursors. This choice is under the control of lateral inhibition mediated by a Suppressor of Hairless-dependent Notch signaling pathway, in which X-Delta-1 is the putative ligand driving the selection process, and a new Enhancer-of-Split-related gene is an epidermal target of Notch signaling. Because nascent ciliated-cell precursors prevent neighboring cells from taking on the same fate, a scattered pattern of these precursors is generated within the deep layer of the non-neural ectoderm. Ciliated-cell precursors then intercalate into the outer layer of cells in the epidermis. We show that the intercalation event acts as a second step to regulate the spacing of the mature ciliated cells. We propose that the differentiation of the ciliated cells is not only regulated by Notch-mediated lateral inhibition, but is also an example where differentiation is coupled to the movement of cells from one cell layer to another.

Molecular basis for semidominance of missense mutations in the XANTHA-H (42-kDa) subunit of magnesium chelatase.

During biosynthesis of bacteriochlorophyll or chlorophyll, three protein subunits of 140, 70, and 42 kDa interact to insert Mg2+ into protoporphyrin IX. The semidominant Chlorina-125, -157, and -161 mutants in barley are deficient in this step and accumulate protoporphyrin IX after feeding on 5-aminolevulinate. Chlorina-125, -157, and -161 are allelic to the recessive xantha-h mutants and contain G559A, G806A, and C271T mutations, respectively. These mutations cause single amino acid substitutions in residues that are conserved in all known primary structures of the 42-kDa subunit. In vitro complementation and reconstitution of Mg-chelatase activity show that the 42-kDa subunits are defective in the semidominant Chlorina mutants. A mutated protein is maintained in the Chlorina plastids, unlike in the xantha-h plastids. Heterozygous Chlorina seedlings have 25-50% of the Mg-chelatase activity of wild-type seedlings. Codominant expression of active and inactive 42-kDa subunits in heterozygous Chlorina seedlings is likely to produce two types of heterodimers between the strongly interacting 42-kDa and 70-kDa subunits. Reduced Mg-chelatase activity is explained by the capacity of heterodimers consisting of mutated 42-kDa and wild-type 70-kDa protein to bind to the 140-kDa subunit. The 42-kDa subunit is similar to chaperones that refold denatured polypeptides with respect to its ATP-to-ADP exchange activity and its ability to generate ATPase activity with the 70-kDa subunit. We hypothesize that the association of the 42-kDa subunit with the 70-kDa subunit allows them to form a specific complex with the 140-kDa subunit and that this complex inserts Mg2+ into protoporphyrin IX.

Barley glutamyl tRNAGlu reductase: mutations affecting haem inhibition and enzyme activity.

Glutamyl tRNA(Glu) reductase converts glutamate molecules that are ligated at their alpha-carboxyl groups to tRNA(Glu) into glutamate 1-semialdehyde, an intermediate in the synthesis of 5-aminolevulinate, chlorophyll and haem. The mature plant enzymes contain a highly conserved extension of 31-34 amino acids at the N-terminus not present in bacterial enzymes. It is shown that barley glutamyl tRNAGlu reductases with a deletion of the 30 N-terminal amino acids have the same high specific activity as the untruncated enzymes, but are highly resistant to feed-back inhibition by haem. This peptide domain thus interacts directly or indirectly with haem and the toxicity of the 30 amino acid peptide for Escherichia coli experienced in mutant rescue and overexpression experiments can be explained by extensive haem removal from the metabolic pools that cannot be tolerated by the cell. Induced missense mutations identify nine amino acids in the 451 residue long C-terminal part of the barley glutamyl tRNA(Glu) reductase which upon substitution curtail drastically, but do not eliminate entirely the catalytic activity of the enzyme. These amino acids are thus important for the catalytic reaction or tRNA binding.

Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD.

Magnesium chelatase catalyses the insertion of Mg2+ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts, inserted Mg2+ into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley extracts optimal activity was observed with 40 mM Mg2+. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid stroma protein preparations from non-leaky xantha-f -g and -h mutants. This establishes that, as in Rhodobacter, three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272,000 x g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored. Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays using stroma protein from the xantha-f, -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A260:A280 ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the plastid and bacterial ribosomal RNA, respectively.

The Notch ligand, X-Delta-2, mediates segmentation of the paraxial mesoderm in Xenopus embryos.

Segmentation of the vertebrate embryo begins when the paraxial mesoderm is subdivided into somites, through a process that remains poorly understood. To study this process, we have characterized X-Delta-2, which encodes the second Xenopus homolog of Drosophila Delta. Strikingly, X-Delta-2 is expressed within the presomitic mesoderm in a set of stripes that corresponds to prospective somitic boundaries, suggesting that Notch signaling within this region establishes a segmental prepattern prior to somitogenesis. To test this idea, we introduced antimorphic forms of X-Delta-2 and Xenopus Suppressor of Hairless (X-Su(H)) into embryos, and assayed the effects of these antimorphs on somite formation. In embryos expressing these antimorphs, the paraxial mesoderm differentiated normally into somitic tissue, but failed to segment properly. Both antimorphs also disrupted the segmental expression of X-Delta-2 and Hairy2A, a basic helix-loop-helix (bHLH) gene, within the presomitic mesoderm. These observations suggest that X-Delta-2, via X-Notch-1, plays a role in segmentation, by mediating cell-cell interactions that underlie the formation of a segmental prepattern prior to somitogenesis.

The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis.

The X-Notch-1 receptor, and its putative ligand, X-Delta-1, are thought to mediate an inhibitory cell-cell interaction, called lateral inhibition, that limits the number of primary neurons that form in Xenopus embryos. The expression of Xenopus ESR-1, a gene related to Drosophila Enhancer of split, appears to be induced by Notch signaling during this process. To determine how the activation of X-Notch-1 induces ESR-1 expression and regulates primary neurogenesis, we isolated the Xenopus homolog of Suppressor of Hairless (X-Su(H)), a component of the Notch signaling pathway in Drosophila. Using animal cap assays, we show that X-Su(H) induces ESR-1 expression, perhaps directly, when modified by the addition of ankyrin repeats. Using a DNA binding mutant of X-Su(H), we show that X-Su(H) activity is required for induction of ESR-1. Finally, expression of the DNA binding mutant in embryos leads to a neurogenic phenotype as well as increased expression of both X-Delta-1 and XNGNR1, a proneural gene expressed during primary neurogenesis. These results suggest that activation of X-Su(H) is a key step in the Notch signaling pathway during primary neurogenesis in Xenopus embryos.

Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.

delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway. The enzyme glutamyl tRNA(Glu) reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNA(Glu) reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1. The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E. coli tRNA(Glu) and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an approximately equal to 60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.

Expression of an Erwinia pectate lyase in three species of Aspergillus.

Transgenic filamentous fungi of the species Aspergillus niger, A. nidulans and A. awamori expressing and secreting Erwinia carotovora subsp. atroseptica pectate lyase 3 (PL3) were generated. Correct processing of the pre-enzyme was achieved using the A. niger pectin lyase A (PEL A) signal peptide. With the prepro-peptide of A. niger polygalacturonase II, secreted enzymes still possessed the 6- aa pro-sequence, indicating the importance of the conformation of the precursor protein for correct cleavage of the signal sequence. PL3 expression was markedly increased in media optimized for limited protease activity, and reached 0.4, 0.8 and 2.0 mg/l for expression in A. niger, A. awamori and A. nidulans, respectively. Glycans attached to the PL3 enzymes exhibited species-specific differences, and an increase of molecular mass coincided with reduced specific activities of the enzymes.

Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-beta-glucanase during germination.

The codon usage of a hybrid bacterial gene encoding a thermostable (1,3-1,4)-beta-glucanase was modified to match that of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene. Both the modified and unmodified bacterial genes were fused to a DNA segment encoding the barley high-pI alpha-amylase signal peptide downstream of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene promoter. When introduced into barley aleurone protoplasts, the bacterial gene with adapted codon usage directed synthesis of heat stable (1,3-1,4)-beta-glucanase, whereas activity of the heterologous enzyme was not detectable when protoplasts were transfected with the unmodified gene. In a different expression plasmid, the codon modified bacterial gene was cloned downstream of the barley high-pI alpha-amylase gene promoter and signal peptide coding region. This expression cassette was introduced into immature barley embryos together with plasmids carrying the bar and the uidA genes. Green, fertile plants were regenerated and approximately 75% of grains harvested from primary transformants synthesized thermostable (1,3-1,4)-beta-glucanase during germination. All three trans genes were detected in 17 progenies from a homozygous T1 plant.

Structural genes for Mg-chelatase subunits in barley: Xantha-f, -g and -h.

Barley mutants in the loci Xantha-f, Xantha-g and Xantha-h, when fed with 5-aminolevulinate in the dark, accumulate protoporphyrin IX. Mutant alleles at these loci that are completely blocked in protochlorophyllide synthesis are also blocked in development of prolamellar bodies in etioplasts. In contrast to wild type, the xan-f, -g and -h mutants had no detectable Mg-chelatase activity, whereas they all had methyltransferase activity for synthesis of Mg-protoporphyrin monomethyl ester. Antibodies recognising the CH42 protein of Arabidopsis thaliana and the OLIVE (OLI) protein of Antirrhinum majus immunoreacted in wild-type barley with 42 and 150 kDa proteins, respectively. The xan-h mutants lacked the protein reacting with antibodies raised against the CH42 protein. Two xan-f mutants lacked the 150 kDa protein recognised by the anti-OLI antibody. Barley genes homologous to the A. majus olive and the A. thaliana Ch-42 genes were cloned using PCR and screening of cDNA and genomic libraries. Probes for these genes were applied to Northern blots of RNA from the xantha mutants and confirmed the results of the Western analysis. The mutants xan-f27, -f40, -h56 and -h57 are defective in transcript accumulation while -h38 is defective in translation. Southern blot analysis established that h38 has a deletion of part of the gene. Mutants xan-f10 and -f41 produce both transcript and protein and it is suggested that these mutations are in the catalytic sites of the protein. It is concluded that X an-f -h genes encode two subunits of the barley Mg-chelatase and that X an-g is likely to encode a third subunit. The XAN-F protein displays 82% amino acid sequence identity to the OLI protein of Antirrhinum, 66% to the Synechocystis homologue and 34% identity to the Rhodobacter BchH subunit of Mg-chelatase. The XAN-H protein has 85% amino acid sequence identity to the Arabidopsis CH42 protein, 69% identity to the Euglena CCS protein, 70% identity to the Cryptomonas BchA and Olisthodiscus CssA proteins, as well as 49% identity to the Rhodobacter BchI subunit of Mg-chelatase. Identification of the barley X an-f and X an-h encoded proteins as subunits required for Mg-chelatase activity supports the notion that the Antirrhinum OLI protein and the Arabidopsis Ch42 protein are subunits of Mg-chelatase in these plants. The expression of both thet X an-f and -h genes in wild-type barley is light induced in leaves of greening seedlings, and in green tissue the genes are under the control of a circadian clock.