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Background The objective of this research was to investigate the change in dimensional stability of 3D printed dental model construction types with time. By comparing the models to each other, we sought to find the most suitable building technique based on causing the least deformation of the 3D printed model with time.Methods Four dental casts were printed at an angle of 0°, polymerizing every 50 µm using UV light with a wavelength of 405 nm. The four types of models were as follows: 2.5 mm wall thickness hollow (2,5 mm.H), 2 mm wall thickness hollow (2 mm.H), 2 mm wall thickness hollow with stabilization bars (2 mm.B), and 2 mm wall thickness with hollow with gypsum base (2 mm.G). Casts were digitized with a laboratory scanner (3Shape E3 Red E Scanner) after printing, to create the reference Standard Tessellation Language (STL) files. All models were stored under constant conditions (25°C and 50% humidity) and scanned again after 1 day and 1, 2 and 10 weeks. These scan data were compared to the reference STL file and were analysed by comparing the deformation in a surface fitting software (Geomagic Control X, 3D Systems). The results were statistically evaluated using paired Student’s t tests with the SPSS 23.0 software program. The significance level was set at p Results There were significant differences in dimensional stability after 10 weeks between the 2.5 mm.H(18,45 µm ± 7,661) and 2 mm.B(11,49 µm ± 2,648), p Conclusions For of all casts, the deformation stayed within the clinical acceptance range, but there were significant differences between the different building structures. Of this four structures, the 2 mm cast with a stabilization bar (2 mm.B) showed the best results.

There are worrying signs that arthropods are in decline both in density and diversity. This threatens global biodiversity as well as the ecosystem services provided by arthropods. Nonetheless, entomological research, even when studying arthropods with a conservation focus, frequently uses lethal methods. We analysed 1029 articles published in the major biological conservation journals between 2014 and 2020 and found that, while single-species-focused studies used more non-lethal than lethal methods (76.3 % vs. 23.7 %, respectively), the opposite was true for multiple-species ones (24.0 % vs. 76.0 %). In tropical regions, 74.6 % of studies used lethal methods vs. 18.5 % non-lethal ones. Of the major orders, Odonata, Lepidoptera and Orthoptera were generally studied using non-lethal methods (88.1 %, 80.7 %, and 70.8 %, respectively) in non-tropical regions, while in the tropics, only Lepidoptera were frequently (51.9 %) studied by such methods. We argue that even if the evidence for arthropod decline were uncertain, and even if research would not add much to the overall level of mortality, entomologists should be showing an example. If research on invertebrates continues to be ethically blind, entomologists risk losing public support for conserving arthropod diversity.

The experiment has been set up in the University of Debrecen Látókép Experimental Station in three different years (2014, 2015 and 2016), three different plant densities 200, 350 and 500 thousand ha-1, four replications of the same nutrient supply with using a line spacing of 45 cm. In the experiment, the fore crop was winter wheat in each year. The amount of weeds was observed five times in the last experimental year (2016/2017). In the three experimental years, the highest yield was harvested from the early sowing plot with the highest plant density. On the basis of the Pearson’s correlation analysis there was significant negative correlation (r=-0.583) between the effect of the annual year and yield of the hybrid.

We made plant physiology examinations in Arkaso winter oilseed rape hybrid substance: relative chlorophyll content (SPAD) and leaf area index (LAI) measurements. The experiment was set in University of Debrecen Agricultural Sciences Center at Látóképi Experimental Station in four replications, in two different sowing times (I. sowing date on 08/22/2014 and II. sowing date on 09/09/2014 sowing againhappened because of the incomplete germination in the second subtance 01/10/2014) Three different plant density 200, 350 and 500 thousand ha-1, under the same nutrient supply, 45 cm row spacing. The experiment was green crop of winter wheat. The relative chlorophyll content (SPAD) and leaf area index (LAI) measurements were made in seven different times. We measured the maximum value of chlorophyll content in the first sowing time at 500, and the second sowing time at 350 thousand ha-1 plant density. The measurement results proved that there was a linear relationship between the number of plants and the LAI. The maximum leaf area index values we measured in both the sowing time at 500 thousand ha-1 reached.

Upon the establishment of an effective nitrogen-fixing symbiosis in amide-transporting plants the enzymatic activity and transcript levels of L-asparaginase are dramatically decreased. This decrease in L-asparaginase activity is essential for the correct functioning of the Rhizobium-legume symbiosis in lupin in which asparagine, synthesized from recently fixed nitrogen, is exported to aerial parts of the plant for use in growth and development. Concomitant with this decrease in L-asparaginase transcript a DNA-binding protein was detected in the nodules. This binding protein was not detectable in ineffective nodules, in nodules treated with nitrate, or in root tips, mature roots, developing flowers or developing seeds. The DNA-binding activity was shown to interact with a 59 bp sequence proximal to the transcription start site. Within this sequence a CTAAAAT direct repeat and a ACTGT/TGTCA incomplete inverted repeat were implicated in the binding of protein to the DNA by DNase I protection experiments. Competitive binding studies with synthesized binding sites were consistent with the CTAAAAT/TGTCA sequence pair proximal to the transcription start site having the highest affinity for the DNA-binding protein. We postulate that this DNA-binding protein is associated with repression of L-asparaginase gene expression in mature lupin root nodules.

The cisgenesis concept implies that plants are transformed only with their own genetic materials or genetic materials from closely related species capable of sexual hybridization. Furthermore, foreign sequences such as selection genes and vector-backbone sequences should be absent. We used a barley phytase gene (HvPAPhy_a) expressed during grain filling to evaluate the cisgenesis concept in barley. The marker gene elimination method was used to obtain marker-free plant lines. Here, the gene of interest and the selection gene are flanked by their own T-DNA borders to allow unlinked integration of the two genes. We analysed the transformants for co-transformation efficiency, increased phytase activities in the grain, integration of the kanamycin resistance gene of the vector-backbone and segregation between the HvPAPhy_a insert and the hygromycin resistance gene. The frequencies of the four parameters imply that it should be possible to select 11 potentially cisgenic T1-lines out of the 72 T0-lines obtained, indicating that the generation of cisgenic barley is possible at reasonable frequencies with present methods. We selected two potential cisgenic lines with a single extra copy of the HvPAPhy_a insert for further analysis. Seeds from plants homozygous for the insert showed 2.6- and 2.8-fold increases in phytase activities and the activity levels were stable over the three generations analysed. In one of the selected lines, the flanking sequences from both the left and right T-DNA borders were analysed. These sequences confirmed the absence of truncated vector-backbone sequences linked to the borders. The described line should therefore be classified as cisgenic.

Ecological Risk Assessment (ERA) of GMO is basically identical to ERA of chemical substances, when it comes to assessing specific effects of the GMO plant material on the soil ecosystem. The tiered approach always includes the option of studying more complex but still realistic ecosystem level effects in 2nd tier caged experimental systems, cf. the new GMO ERA guidance: EFSA Journal 2010; 8(11):1879. We propose to perform a trophic structure analysis, TSA, and include the trophic structure as an ecological endpoint to gain more direct insight into the change in interactions between species, i.e. the food-web structure, instead of relying only on the indirect evidence from population abundances. The approach was applied for effect assessment in the agro-ecosystem where we combined factors of elevated CO2, viz. global climate change, and GMO plant effects. A multi-species (Collembola, Acari and Enchytraeidae) mesocosm factorial experiment was set up in a greenhouse at ambient CO2 and 450 ppm CO2 with a GM barley variety and conventional varieties. The GM barley differed concerning the composition of amino acids in the grain (antisense C-hordein line). The fungicide carbendazim acted as a positive control. After 5 and 11 weeks, data on populations, plants and soil organic matter decomposition were evaluated. Natural abundances of stable isotopes, 13C and 15N, of animals, soil, plants and added organic matter (crushed maize leaves) were used to describe the soil food web structure.

Genetic transformation is currently met with substantial scepticism among the general public. One major concern is the mingling of genetic material between species. We have initiated a collaborating project with different groups from University of Copenhagen, the Danish Agricultural Advisory Service and Sejet Plant Breeding based on the Cisgenesis concept. Cisgenesis implies that the plants are transformed only with its own or very closely related genetic material and that the final Cisgenic plants have to be free of any foreign genes. The Cisgenesis concept allows for the introduction of extra gene copies of a particular gene whereby a particular trait can be accentuated. Transgenic crops generated by the Cisgenesis concept are accordingly very similar to those generated by conventional breeding. In our part of the project we are focusing on barley phytases as candidate genes for Cisgenesis. Recently, Dionisio et al. (2011) have cloned and characterized phytases belonging to the purple acid phosphatases (PAPs) in barley. We have isolated the genomic PAP-clone of the isoform expressed during grain filling including 2.3 kb of the promoter region and 600 bp of the terminator region using a genomic barley lambda library. The clone has been inserted into a Cisgenic Agrobacterium vector where both the gene of interest and the selection gene are flanked by their own T-DNA borders in order to promote integration of the two genes at unlinked places in the plant genome. T0-plants show increases in the phytase activity of mature seeds from 1350 in wild type to 7500 FTU/kg in T0-plants. We have identified two Cisgenic T1-lines without selection gene and vector backbone but with one additional genomic clone of the phytase gene. Lines homozygous for the additional cisgene show 2-3 fold increases in phytase activity. The integration pattern of the cisgenes are currently investigated. Genetic transformation is currently met with substantial scepticism among the general public. One major concern is the mingling of genetic material between species. We have initiated a collaborating project with different groups from University of Copenhagen, the Danish Agricultural Advisory Service and Sejet Plant Breeding based on the Cisgenesis concept. Cisgenesis implies that the plants are transformed only with its own or very closely related genetic material and that the final Cisgenic plants have to be free of any foreign genes. The Cisgenesis concept allows for the introduction of extra gene copies of a particular gene whereby a particular trait can be accentuated. Transgenic crops generated by the Cisgenesis concept are accordingly very similar to those generated by conventional breeding. In our part of the project we are focusing on barley phytases as candidate genes for Cisgenesis. Recently, Dionisio et al. (2011) have cloned and characterized phytases belonging to the purple acid phosphatases (PAPs) in barley. We have isolated the genomic PAP-clone of the isoform expressed during grain filling including 2.3 kb of the promoter region and 600 bp of the terminator region using a genomic barley lambda library. The clone has been inserted into a Cisgenic Agrobacterium vector where both the gene of interest and the selection gene are flanked by their own T-DNA borders in order to promote integration of the two genes at unlinked places in the plant genome. T0-plants show increases in the phytase activity of mature seeds from 1350 in wild type to 7500 FTU/kg in T0-plants. We have identified two Cisgenic T1-lines without selection gene and vector backbone but with one additional genomic clone of the phytase gene. Lines homozygous for the additional cisgene show 2-3 fold increases in phytase activity. The integration pattern of the cisgenes are currently investigated.

Genetic transformation is currently met with substantial skepticism among the general public in Europe and consequently also by the growers, the agro-industry, and the retailers. One major concern is the mingling of genetic material between species. In the light of this, we have initiated a project based on the cisgenesis concept. In contrast to transgenesis, cisgenesis implies that the plant is transformed only with its own or very closely related genetic material. Furthermore, all “helper” genes and gene sequences of foreign nature are removed from the transformed plant lines. Cisgenic crops are accordingly very similar to those generated by conventional breeding. The cisgenesis concept allows for the introduction of extra gene copies of a particular gene to accentuate the trait. We are using a barley purple acid phosphatase expressed during grain filling as candidate gene for cisgenesis. A genomic barley lambda library has been used to isolate the genomic clone of this phytase including 2.3 kb of the promoter region and 600 bp of the terminator region. The clone has been inserted into a cisgenic Agrobacterium vector where both the gene of interest and the selection gene are flanked by their own T-DNA borders in order to promote integration of the two genes at unlinked places in the plant genome. Transformed T0 plants show increases in the phytase activity of mature seeds from 1,400 in wild type to 8,950 FTU/kg in T0 plants. T1 plants of each transformant are currently screened with PCR for extra copies of the genomic phytase gene and the selection gene to identify segregation between the two genes. Presently, we have identified two cisgenic T1 plants without vector backbone and selection gene but with an extra copy of the genomic phytase gene. Genetic transformation is currently met with substantial skepticism among the general public in Europe and consequently also by the growers, the agro-industry, and the retailers. One major concern is the mingling of genetic material between species. In the light of this, we have initiated a project based on the cisgenesis concept. In contrast to transgenesis, cisgenesis implies that the plant is transformed only with its own or very closely related genetic material. Furthermore, all “helper” genes and gene sequences of foreign nature are removed from the transformed plant lines. Cisgenic crops are accordingly very similar to those generated by conventional breeding. The cisgenesis concept allows for the introduction of extra gene copies of a particular gene to accentuate the trait. We are using a barley purple acid phosphatase expressed during grain filling as candidate gene for cisgenesis. A genomic barley lambda library has been used to isolate the genomic clone of this phytase including 2.3 kb of the promoter region and 600 bp of the terminator region. The clone has been inserted into a cisgenic Agrobacterium vector where both the gene of interest and the selection gene are flanked by their own T-DNA borders in order to promote integration of the two genes at unlinked places in the plant genome. Transformed T0 plants show increases in the phytase activity of mature seeds from 1,400 in wild type to 8,950 FTU/kg in T0 plants. T1 plants of each transformant are currently screened with PCR for extra copies of the genomic phytase gene and the selection gene to identify segregation between the two genes. Presently, we have identified two cisgenic T1 plants without vector backbone and selection gene but with an extra copy of the genomic phytase gene.

A genomic sequence encoding Lupinus angustifolius L-asparaginase has been obtained, and is the first report of this gene from a plant source. The 3.2 kb of DNA sequenced contains a 1136 bp 5' flanking sequence, four exons and three introns. Intron-exon borders were mapped by comparing the genomic sequence with that of a L. arboreus cDNA. Primer extension analysis revealed transcription start sites 16 bp and 13 bp 5' of the initiating ATG for L. angustifolius and L. arboreus, respectively. The 5' flanking region contained sequences associated with seed-specific expression.

We present the primary structure of a nudule-specific gene, Nms-25 from Medicago sativa L. cultivar Nagyszenasi. Analysis of the nucleotide sequence of Nms-25 revealed that this gene shows all the characteristics of an interrupted plant gene consisting of 13 exons and 12 introns. The promoter region of Nms-25 contains the common promoter elements of plant genes as well as motifs which are supposed to be involved in nodule-specific expression. There are two exon-like sequences in the gene named PE1 and PE2 which are not present in the cDNA clones of Medicago sativa cultivar Cardinal. Intron 9 carries a retrotransposon-like element, Tms1, which might be responsible for downstream deletion events in which a heptanucleotide, ATTAGCT, might have been involved. Most of the exons, exept 1, 12 and 13, are similar to each other both in length (54 bp) and sequence (up to 94% sequence similarity). All exons are interrupted by introns in the same phase (type I). It is suggested that exon-shuffling based on illegitimate recombination in which the ATTAGCT motif might have played an active role, and retrotransposon-mediated DNA rearrangements were the primary events in the molecular evolution of the Nms-25 gene. The nucleotide sequence of the genomic clone of Nms-25 from Medicago sativa is available from the EMBL/GenBank/DDBJ Nucleotide Sequence Databases under the accession number X13287.

A trans -acting protein interacting with a specific sequence motif proximal to the transcriptional start site of the l -asparaginase promoter has been observed previously (E. Vincze, J. M. Reeves, E. Lamping, K. J. F. Farnden, and P. H. S. Reynolds, Plant Mol. Biol. 26:303-311, 1994). Gel retardation experiments in which protein extracts of Mesorhizobium loti and developing nodules were used suggested a bacterial origin for the repressor binding protein ( rep2037 ). Nodulation tests were performed by using different Fix − Tn 5 mutants of M. loti. Analyses of these mutants revealed a correlation between the presence of Mesorhizobium in the nodule-like structures and the ability of nodule protein extracts to bind the repressor binding domain (RBD). Through the use of mutated RBD sequences, the RBD sequence was identified as CTAAAAT. The repressor protein was isolated from M. loti NZP2037 by multiple chromatographic procedures and affinity separation by using concatemers of RBD attached to magnetic beads. Sequencing of the recovered protein resulted in identification of the repressor protein as the sarcosine oxidase α subunit. This was confirmed by expression of the gene encoding the M. loti α subunit of sarcosine oxidase in Escherichia coli. When the expressed peptide was bound to RBD, the gel retardation result was identical to the result obtained with rep2037 from M. loti strain NZP2037.

It is shown here that the phosphate groups at the cos ends of phage lambda DNA are not a prerequisite for in vitro packaging. Molecules with phosphatase-treated cos ends are packaged in vitro as efficiently as native lambda DNA. This observation can be used for an alternative strategy to improve the efficiency of gene library construction, since cos-cos ligation decreases in vitro encapsidation and infectivity. Dephosphorylated cos ends and a new phasmid vector lambda pGY97 have been used to construct a representative gene bank of alfalfa in a Mcr- (5-methylcytosine restriction deficient) Escherichia coli host strain. These recombinant clones can be propagated as phages or more conveniently as plasmids in recA- E. coli, to prevent possible homologous recombination events between repetitive sequences of the insert that would otherwise interfere with clone stability. The 5-19-kb inserts can be easily recloned as plasmids from the recombinant phasmids with simple EcoRI digestion and re-ligation. This observation also implies that the construction of gene libraries in cosmid vectors can be made more efficient if cos-cos ligates were cleaved by lambda terminase just before in vitro packaging.

A new nodule-specific gene, Nms-25 (nodulin-25), was identified in cDNA clones isolated from a nodule-specific cDNA library of Medicago sativa. The first transcript of this gene appeared 9 days after inoculation of the roots with Rhizobium meliloti. The time of expression and the quantity of the transcripts of the Nms-25 gene was similar to that of leghemoglobin genes suggesting a similar regulation. A protein of 246 amino acids could be deduced from a full-length cDNA clone. The first 24 amino acids at the N-terminal end of this protein formed a signal sequence which might direct membrane transport into the peribacteroid space. Using different predictive methods the signal sequence cleaved protein was tentatively predicted to be a water-soluble enzyme, but not hydrolase.

In order to investigate the role of potential genotypic differences in three economically important barley cultivars, experiments were carried out to determine the influence of supplemental ultraviolet-B (UV-B, 280–320 nm) radiation on reactive oxygen species (ROS), antioxidant activity and photosynthesis. Greenhouse-grown barley (Hordeum vulgare L.) cultivars ‘Cork’, ‘Prestige’ and ‘Golden Promise’ showed different responses to supplemental 280–320 nm (UV-B) representing 100, 138 and 238% levels of ambient biologically active UV-B radiation, respectively. Among the three cultivars studied, cv. Golden Promise was the most tolerant to UV-B, cv. Prestige was slightly more sensitive than cv. Cork. A comparison with the other two cultivars showed that under supplemental UV-B, Golden Promise leaves (i) retained a higher quantum yield of photosynthesis under photosynthetically active radiation (PAR, 400–700 nm) corresponding to growth conditions; (ii) had the smallest decrease in both electron transport rate and non-photochemical quenching under high PAR; (iii) contained less oxidized ascorbate [measured as dehydroascorbate or electron paramagnetic resonance (EPR) detectable monodehydroascorbate radicals] than either Cork or Prestige. Under the highest UV-B level applied, Golden Promise leaves maintained the same activity of both monodehydroascorbate-reductase (MDAR) and ascorbate-peroxidase (APX) enzymes, as untreated controls, while MDAR markedly decreased in the other two cultivars and APX slightly increased in cv. Prestige. These features, together with the observation of directly EPR-trappable free radicals and the light-independent accumulation of monodehydroascorbate radicals in Cork and Prestige but not in Golden Promise leaves under high UV-B suggest that Golden Promise plants suffered less oxidative stress than the two other cultivars.

Twenty-five mutants unable to utilize nitrate as sole nitrogen source were isolated from Rhizobium meliloti 41. These mutations mapped at four different sites, narA, narB, narC and narD; narB, C and D were located between trp-15 and ade-15 on the chromosome. NarA mutants were affected in assimilatory nitrate reduction but not in‘respiratory’ nitrate reduction and had methyl viologen-coupled nitrate reductase activity. NarB mutants were affected in both assimilatory and ‘respiratory’ nitrate reduction and lacked methyl viologen-coupled nitrate reductase activity. NarC and narD mutants were impaired not only in assimilatory and ‘respiratory’ nitrate reduction but lacked xanthine dehydrogenase activity as well. Acid-treated crude extracts of these two mutant classes were unable to restore NADPH-coupled nitrate reductase activity to the nit-1 mutant of Neurospora crassa, indicating the lack of active molybdenum cofactor. All mutants tested were effective in symbiotic plant tests and had normal nitrogenase activity, indicating that nitrogenase and nitrate reductase do not share the same molybdenum cofactor.

We have demonstrated that Rhizobium leguminosarum strain LPR1105 contains a heat stable and a heat labile glutamine synthetase (EC 6.3.1.2) activity similar to those described for other Rhizobiaceae. Most of the activity is heat stable when this strain is grown on glutamine as sole nitrogen source, but most is heat labile when grown on nitrate. Using a gene bank of R. leguminosarum DNA we have isolated two clones, which code for heat stable (p7D9) and heat labile (p4F7) glutamine synthetase activity, by complementing the glutamine auxotrophy of Klebsiella pneumoniae glnA mutants. Cross-hybridization of p7D9 with a fragment of the glnA gene of K. pneumoniae was observed, but no cross-hybridization between p7D9 and p4F7 was found. Since these two regions hybridize to genomic DNA of R. leguminosarum they are probably the structural genes for GSI and GSII, and the availability of these genes will make it possible to test this hypothesis. Clone p4F7 complements an ntrC+ but not an ntrC K. pneumoniae glnA mutant, suggesting that the ntrC gene is required for the complementation of the glutamine auxotrophy by this plasmid.

Linkage maps of R. meliloti 2011 (Rm2011), R. meliloti 41 (Rm41) and R. leguminosarum 300 (R1300), all constructed by means of P1 plasmid-mediated recombination, were compared. Recombination between the two R. meliloti strains occurred at high frequency but was barely detectable in matings between R1300 and Rm41. When co-inheritance data for the three strains were transformed into additive map distances the arrangement of markers showed striking similarities. Each of eight R68.45-primes, carrying different sections of the Rm2011 chromosome, suppressed only those markers of both R1300 and Rm41 which had the same phenotype and map location. Each of these R-primes promoted polarized chromosome transfer in an anticlockwise direction in Rm41, starting from the region corresponding to that carried on the plasmid.

GENETIC studies on symbiotic nitrogen-fixing rhizobia are greatly hampered by the lack of an efficient system for genetic mapping in rhizobia. Although there have been reports on transformation1–3 and transduction4 in nodulating rhizobia as well as conjugation5 in a non-nodulating strain of Rhizobium lupini, the genetic map of a nodulating and nitrogen-fixing Rhizobium strain has not been established yet. Plasmids of the P-1 incompatibility group have a remarkably wide host range and can be transferred to many Rhizobium species6,7. Some of these plasmids can promote chromosome transfer in Pseudomonas aeruginosa strain PAT but show little chromosome donor ability (Cda) in strain PAO8,9. Recently a variant (R68.45) of the P group plasmid R68, with high Cda property in strain PAO, was isolated by Haas and Holloway10. Plasmid R68.45, unlike most of the sex factors, promotes chromosome transfer for a range of chromosomal genes in Rhodopseudomonas, P. putida, Escherichia coil (B. W. Holloway, personal communication) and in Rhizobium leguminosarum11. We have found that this plasmid can promote chromosome transfer also in Rhizobium meliloti and can be used for mapping of the chromosome. We present here a circular genetic map of the R. meliloti 41 chromosome and describe a new approach in the evaluation of conjugational mapping data which may be applicable to other Gram-negative bacteria.

A total of 5 Nod- and 57 Fix- symbiotic mutants of Rhizobium meliloti strain 41 have been isolated after either nitrosoguanidine or Tn5 transposition mutagenesis. Chromosomal locations of mutations in 1 Nod- and 11 Fix- derivatives were ascertained by transferring the chromosome (mobilized by plasmid R68.45), in eight fragments, into symbiotically effective recipients and testing the recombinants for symbiotic phenotype. Alternatively, the kanamycin resistance marker of Tn5 was mapped. In five mutants the fix alleles were localized on different chromosomal regions, but six other fix mutations and one nod mutation tested did not map onto the chromosome. It was shown that the chromosome-mobilizing ability (Cma+) of R68.45 was not involved in the mobilization of genes located extrachromosomally. Moreover, Cma- derivatives of R68.45 could mobilize regions of the indigenous plasmid pRme41b but not chromosomal genes. Thus, mobilization of a marker by Cma- R68.45 indicates its extrachromosomal location. With a 32P-labeled DNA fragment carrying Tn5 as a hybridization probe, it was shown that in five extrachromosomally located Tn5-induced fix mutants and one nod mutant Tn5 was localized on plasmid pRme41b. This is in agreement with the genetic mapping data.

Using a simple enrichment procedure, we isolated an R-prime derivative of plasmid R68.45 carrying a 17.8-megadalton segment of the Rhizobium meliloti 41 chromosome. The chromosomal segment carried on this plasmid (pGY1) includes the markers cys-24+, cys-46+, and att16-3. Plasmid pGY1 mobilized the chromosome in a polarized way starting from the region of homology, but cannot promote chromosome transfer from other sites. The att16-3 site on pGY1 allowed the integration of phage 16-3 into pGY1, and a composite plasmid of 91.8 megadaltons was formed. This vector (pGY2) is suitable for the introduction of Rhizobium bacteriophage 16-3 into other gram-negative bacteria.

A histone H3 gene was isolated from a dicotyledonous plant, alfalfa (Medicago sativa). The sequence analysis of this gene revealed no obvious GC preference in its codon usage. Apart from containing most of the typical consensus sequences found in both animal and plant histone genes, the alfalfa H3 gene exhibits distinct structural features such as (1) the unusual location of two GATCC motifs in its 5′ flanking sequence, (2) the existence of a CGCGGATC on the nonsense strand at position −232, (3) the existence of a long palindromic structure, and (4) several polyadenylation signal-like sequences in the 3′ flanking region. There are about 160 copies of histone H3 gene in alfalfa tetraploid genome. Using the alfalfa H3 gene as a probe to study the pattern of histone H3 transcripts in the alfalfa, we found that the H3 RNAs are undetectable in leaves, more in stems than in roots, and highest in somatic embryos. Moreover, the RNA products of H3 genes in all alfalfa tissues tested show unusually long nontranslated region compared to those of animal histone genes. An additional high molecular weight species of H3 transcript was detected only in somatic embryos.

Two nodule specific genes have been isolated from nodule c-DNA clone bank of Medicago sativa using differential hybridization. One of the two genes was leghemoglobin which showed high level of sequence homology to leghemoglobin from other legumes. The second c-DNA gene, nodulin-1 was also sequenced and revealed two direct repeats RA and RB. Repeat RB consists of three tandem copies of 18 amino acids. From the genomic library of Medicago sativa constructed in our lab, nodulin-1 was isolated and partially characterised. The nucleotide sequence showed that this gene contains several introns. The function of nodulin-1 in symbiotic nitrogen fixation is not known at present.

The soil bacterium Rhizobium meliloti fixes molecular nitrogen after establishment of a symbiotic association with the plant host Medigaco sativa. Certain genes coding for the development of this symbiosis reside in the bacterium, others are carried by the plant partner. This study was devoted to localize and identify symbiotic nitrogen fixation genes in the bacterium Rhizobium meliloti strain 41.

The aim of the study was to understand the molecular and biochemical mechanisms underpinning the effect of nitrogen (N) on barley (Hordeum vulgare) storage protein production (hordeins) during grain filling. Using a combination of advanced biochemistry methods, we could comprehensively describe changes in transcripts (using DNA microarray and qRT-PCR), amino acids (Ultra Performance Liquid Chromatography) and protein abundance (2D-DIGE combined with MS/MS). Examining all known hordein homologues revealed markedly different temporal profiles in their expression patterns. Furthermore, different N-regimes caused significant differences in both quantity and quality of the storage proteins transcripts. Principal Component Analysis of the amino acid (AA) profiles also indicated dissimilarity in individual AA percentages, correlated to hordein content. The abundance values of proteins of interest confirmed the various hordein patterns during grain development and the differential response to environmental stimuli.

Barley (Hordeum vulgare) is mostly used in feed and malt production but has the ability to provide humans nutritional benefits. The current wheat based “barley” breads can unfortunately not exceed more than 20% barley flour mixed into the dough due to poor leavening properties. Therefore the objective is to develop barley varieties with good baking properties. The poor leavening properties of barley can be attributed, at least partially, to the physical properties of the storage proteins. Studies based on small scaled baking trials and protein pattern analysed by SDS-PAGE on a large number of high protein barley cultivars suggested differences in baking quality and correlation between baking quality and D-hordeins. The amino acid composition was measures using the newly developed AccQ Tag Ultra Amino acid (AA) derivatisation system designed for the Acquity UPLC. Obtained results from the total AA composition and the hordein pattern indicated that there are genetic variations not only in the distributions of the hordein polypeptides but also in the relative proportions of the storage proteins affecting the AA compositions. The free AA composition in the grain may also provide us the opportunity to give a forecast of the taste of the bread, as the AA composition is known to control certain aspects of the taste. We uses a MSE approach on a time of flight instrument coupled to a UPLC and in gel digestion to identify and characterize the different D-hordeins responsible for baking quality. Digesting the proteins with chymotrypsin results in approx 60% sequence coverage in Biopharmalynx but higher sequence coverage is expected using both chymotrypsin and trypsin as overlapping peptides are produced in the optimal mass range. Sequencing the digested peptides also revealed post translational modifications of the identified hordein which needs further investigation. Our results strongly indicate genetic variations in the relative proportions of the storage proteins affecting the amino acid compositions and baking quality. Barley (Hordeum vulgare) is mostly used in feed and malt production but has the ability to provide humans nutritional benefits. The current wheat based “barley” breads can unfortunately not exceed more than 20% barley flour mixed into the dough due to poor leavening properties. Therefore the objective is to develop barley varieties with good baking properties. The poor leavening properties of barley can be attributed, at least partially, to the physical properties of the storage proteins. Studies based on small scaled baking trials and protein pattern analysed by SDS-PAGE on a large number of high protein barley cultivars suggested differences in baking quality and correlation between baking quality and D-hordeins. The amino acid composition was measures using the newly developed AccQ Tag Ultra Amino acid (AA) derivatisation system designed for the Acquity UPLC. Obtained results from the total AA composition and the hordein pattern indicated that there are genetic variations not only in the distributions of the hordein polypeptides but also in the relative proportions of the storage proteins affecting the AA compositions. The free AA composition in the grain may also provide us the opportunity to give a forecast of the taste of the bread, as the AA composition is known to control certain aspects of the taste. We uses a MSE approach on a time of flight instrument coupled to a UPLC and in gel digestion to identify and characterize the different D-hordeins responsible for baking quality. Digesting the proteins with chymotrypsin results in approx 60% sequence coverage in Biopharmalynx but higher sequence coverage is expected using both chymotrypsin and trypsin as overlapping peptides are produced in the optimal mass range. Sequencing the digested peptides also revealed post translational modifications of the identified hordein which needs further investigation. Our results strongly indicate genetic variations in the relative proportions of the storage proteins affecting the amino acid compositions and baking quality.

Barley (Hordeum vulgare) has the potential to offer considerable human nutritional benefits, especially as supplement to wheat-based breads. Under current commercial baking conditions it is not possible to introduce more that 20% barley flour to the wheat bread without negative impact on the physical chemical properties of the bread products due to the poor baking properties of barley flour. As a consequence, the nutritional advantages of barley are not fully exploited. The inferior leavening and baking properties of barley can, in part, be attributed to the physical properties of the storage proteins. Changing the storage protein composition can lessen this problem. Our working hypothesis was that exploiting the substantial genetic variation within the gene pool for storage proteins could enable improving the baking qualities of barley flour. We characterised forty-nine barley cultivars for variations in storage protein and AA composition. These cultivars were selected based on their higher protein contents (11.8–17.6%). The results obtained indicated that substantial variation not only in the distribution of the hordein polypeptides but also in the relative proportions of the storage proteins affecting the AA compositions as well. Studies based on small scaled baking trials and protein pattern analysis by SDS-PAGE on the high protein barley cultivars suggested differences in baking quality and a correlation between baking quality and the high molecular weight (HMW)-storage proteins. Mass spectrometry (Q-TOF) proved the identity of the HMW proteins and sequencing the digested peptides also revealed possible post translational modifications of the identified HMW hordeins. Our results strongly indicate that the variations in the relative proportions of the storage proteins of barley affect both the amino acid compositions and baking quality. Barley (Hordeum vulgare) has the potential to offer considerable human nutritional benefits, especially as supplement to wheat-based breads. Under current commercial baking conditions it is not possible to introduce more that 20% barley flour to the wheat bread without negative impact on the physical chemical properties of the bread products due to the poor baking properties of barley flour. As a consequence, the nutritional advantages of barley are not fully exploited. The inferior leavening and baking properties of barley can, in part, be attributed to the physical properties of the storage proteins. Changing the storage protein composition can lessen this problem. Our working hypothesis was that exploiting the substantial genetic variation within the gene pool for storage proteins could enable improving the baking qualities of barley flour. We characterised forty-nine barley cultivars for variations in storage protein and AA composition. These cultivars were selected based on their higher protein contents (11.8–17.6%). The results obtained indicated that substantial variation not only in the distribution of the hordein polypeptides but also in the relative proportions of the storage proteins affecting the AA compositions as well. Studies based on small scaled baking trials and protein pattern analysis by SDS-PAGE on the high protein barley cultivars suggested differences in baking quality and a correlation between baking quality and the high molecular weight (HMW)-storage proteins. Mass spectrometry (Q-TOF) proved the identity of the HMW proteins and sequencing the digested peptides also revealed possible post translational modifications of the identified HMW hordeins. Our results strongly indicate that the variations in the relative proportions of the storage proteins of barley affect both the amino acid compositions and baking quality.