Direct amplification of new cellulase genes from woodland soil purified DNA.

Eight genes encoding cellulolytic enzymes were obtained by direct PCR amplification of genomic DNA recovered from woodland soil samples. The direct amplifications were carried out by using primers designed from available online cellulase nucleotide sequences. The isolated genes were all different from each other and homologous to endo-ß-1,4-glucanases of Bacillus subtilis. The cellulases were functionally expressed in Escherichia coli and tested on soluble substrate at 37 and 60 °C, showing different cellulolytic activities. Among these, the enzyme renamed CelWS6 exhibited good activity at higher temperatures. Further analysis of CelWS6 showed a high performance in acid environments (between pH 4.0 and 6.0) and at elevated temperatures with its maximum activity at pH 5.0 and 50 °C. At the optimum pH, it was very stable since more than 80 % of its original activity was maintained after an incubation of 120 min at 60 °C. Because the cellulases had different cellulolytic activities, but similar amino acid sequences, it was possible to assess the relationship between sequence and protein function.

Development of a peptide nucleic acid array platform for the detection of genetically modified organisms in food.

Two previously developed platforms, a multiplex polymerase chain reaction (PCR) and a peptide nucleic acid (PNA) array, the former allowing for the simultaneous detection of five transgenes and two endogenous controls in food and feed matrices and the latter for the assessment of the identity of amplified PCR products, were combined in order to develop a PNA array device for the screening of genetically modified organisms (GMOs) in food. PNA probes were opportunely designed, synthesized, and deposited on commercial slides. The length of the probes as well as the distance of the probes from the surface were evaluated and found to be critical points. The most suitable probes were found to be 15-mer PNAs linked to the slide surface by means of two 2-(2-aminoethoxy)ethoxyacetic acids as spacers. The device was tested on a model system constituted by flour samples containing a mixture of standards at known concentrations of transgenic material, in particular Roundup Ready soybean and Bt11, Bt176, Mon810, and GA21 maize: The DNA was amplified using the specific multiplex PCR method and tested on the PNA array. The method proposed was found to be able to correctly identify every GMO present in the tested samples.

Endonuclease genes up-regulated in tissues undergoing programmed cell death are expressed during male gametogenesis in barley.

In the process of programmed cell death (PCD), a key role has been attributed to endonucleases capable to cleave nuclear DNA at internucleosomal sites. In barley (Hordeum vulgare L.), two such nucleases (Bnuc1 and BEN1) were individually identified in unrelated tissues. In the present work, we demonstrate that their genes are also expressed in immature anthers at different stages of pollen development. Further experiments carried out on RNA extracted from immature barley anthers led to discover a novel endonuclease gene, namely Bnuc2 (AJ311603 in the EMBL/GenBank/DDBJ databases), eventually found up-regulated at the tetrad stage. The protein encoded was found to conserve large sequence portions of Bnuc1 and BEN1 endonucleases, including the domain regions involved in secretion and DNA/RNA binding. A survey conducted on barley EST libraries showed that Bnuc2 and BEN1 mRNAs are jointly present also in the transcriptome of 20 DAP spike and that other endonuclease ESTs are co-expressed with Bnuc1 or BEN1 in tissues where PCD has been recorded. Therefore, it can be concluded that during the PCD process, a set of S1-type endonucleases is synthesised regardless of the tissue considered.

Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods.

The detection of genetically modified organisms (GMOs) in food and feed is an important issue for all the subjects involved in raw material control, food industry, and distribution. Because the number of GMOs authorized in the EU increased during the past few years, there is a need for methods that allow a rapid screening of products. In this paper, we propose a method for the simultaneous detection of four transgenic maize (MON810, Bt11, Bt 176, and GA21) and one transgenic soybean (Roundup Ready), which allows routine control analyses to be sped up. DNA was extracted either from maize and soybean seeds and leaves or reference materials, and the recombinant DNA target sequences were detected with 7 primer pairs, accurately designed to be highly specific for each investigated transgene. Cross and negative controls were performed to ensure the specificity of each primer pair. The method was validated on an interlaboratory ring test and good analytical parameters were obtained (LOD = 0.25%, Repeatability, (r) = 1; Reproducibility, (R) = 0.9). The method was then applied to a model biscuit made of transgenic materials baked for the purpose and to real samples such as feed and foodstuffs. On account of the high recognition specificity and the good detection limits, this multiplex PCR represents a fast and reliable screening method directly applicable in all the laboratories involved in raw material and food control.

Production of a functional human acid maltase in tobacco seeds: biochemical analysis, uptake by human GSDII cells, and in vivo studies in GAA knockout mice.

Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II (GSDII) or Pompe's disease. To investigate whether we could generate a functional recombinant human GAA enzyme (tobrhGAA) in tobacco seeds for future enzyme replacement therapy, we subcloned the human GAA cDNA into the plant expression plasmid-pBI101 under the control of the soybean ß-conglycinin seed-specific promoter and biochemically analyzed the tobrhGAA. Tobacco seeds contain the metabolic machinery that is more compatible with mammalian glycosylation-phosphorylation and processing. We found the tobrhGAA to be enzymatically active was readily taken up by GSDII fibroblasts and in white blood cells from whole blood to reverse the defect. The tobrhGAA corrected the enzyme defect in tissues at 7 days after a single dose following intraperitoneal (IP) administration in GAA knockout (GAA(-/-)) mice. Additionally, we could purify the tobrhGAA since it bound tightly to the matrix of Sephadex G100 and can be eluted by competition with maltose. These data demonstrate indirectly that the tobrhGAA is fully functional, predominantly proteolytically cleaved and contains the minimal phosphorylation and mannose-6-phosphate residues essential for biological activity.

Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model.

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.

Evidence of a sirtuin gene family in grapevine (Vitis vinifera L.).

Sirtuins are known as regulators of age-dependent gene transcription and chromatin modification in yeast and in animals, but information about their occurrence and role in plants is scarce. Sirtuin-like sequences were amplified using two highly degenerate primers designed comparing sirtuin sequences of seven different plant species, and characterised at the sequence level in Vitis vinifera L. cv. Barbera clone R4. The data were confirmed and analysed by searching the published database from two independent grape genome projects with the obtained sequences. Two putative sirtuin genes containing sirtuin-conserved domains were found on chromosome 7 and on chromosome 19, suggesting the occurrence of sirtuins in grapevine.

Evaluation of biodiversity of lactic acid bacteria microbiota in the calf intestinal tracts.

Amplified fragment length polymorphism (AFLPs) were used to analyse the naturally occurring flora of lactic acid bacteria (LAB) in gastrointestinal tracts of two healthy 65-day-old calves. More than 1,000 of presumptive LAB were collected and cultured from the gastrointestinal tracts and, among the isolated colonies, a total of 311 strains were analysed and separated into eight clusters based on AFLP banding patterns. To precisely determine the species inside the clusters, partial sequences of fragments of the 16S ribosomal DNA gene were determined, and sequence homology searches were conducted through GenBank on few strains per cluster. The most representative genera of LAB were Lactobacillus (169 isolates, 54% of total) and Streptococcus (99 isolates, 32% of total), while the most frequent species was identified as L. mucosae with 86 different isolates (51% of the Lactobacillus spp. and 28% of the total). This report gives a first characterization of LAB strain biodiversity recovered directly from calf intestine and is the first account of the presence of the L. mucosae species in calves. Moreover it demonstrates that the AFLP is a robust and useful technique for characterizing the strain level of LAB microflora.

Recombinant human acid beta-glucosidase stored in tobacco seed is stable, active and taken up by human fibroblasts.

Gaucher disease, the most common genetic lysosomal disorder, is caused by the lack of functional acid beta-glucosidase (GCase) and is currently treated at a very high cost by enzyme replacement therapy. In an attempt to provide a safe and cost-effective production system, human placental GCase was produced and purified from transgenic tobacco seeds. Plant-derived recombinant GCase was found to be enzymatically active, uptaken by human fibroblasts and free of immunogenic xylose and fucose residues. This report demonstrates the potential of plant bioreactors in the large-scale production of injectable proteins required for lifelong therapy.