Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium

Summary The response of oilseed rape cultivars to infection with Agrobacterium tumefaciens and A. rhizogenes and the possibility of regenerating genetically transformed oilseed rape plants were examined. The frequency at which Agrobacterium induced galls or hairy-roots on in vitro cultured plants ranged from 10% to 70%, depending on the cultivar. From galls induced by the tumorigenic strain T37, known to be strongly shoot inducing on tobacco, roots developed frequently. Occasionally, shoots formed and some of these produced tumour cell specific nopaline. Attempts to grow the transformed shoots into plants have so far been unsuccessful. Whole plants transformed with Ri-T-DNA, however, were regenerated. These had crinkled leaves and abundant, frequently branching roots that showed reduced geotropism, similar to previously isolated Ri T-DNA transformed tobacco and potato plants. The transformed oilseed rape plants flowered, but failed to form seeds.     

Localization of inorganic ions by precipitative freeze dissolution

Summary A new method for localization of inorganic diffusible ions in tissue is introduced. It has been applied to localization of Tl⁺ and Rb⁺ in barley roots and is probably also suited for Cs⁺, Ca²⁺, Cl^–, Br^–, PO ₄ ^3– and perhaps K⁺. Its principle consists of dissolution of the ice from frozen tissue in a concentrated aqueous solution of a precipitating agent that is kept at a temperature just above its melting point.     

Genetic relationships among wheat genotypes, as revealed by microsatellite markers and pedigree analysis

Genetic relationships among 20 elite wheat genotypes were studied using microsatellite markers and pedigree analysis. A total of 93 polymorphic bands were obtained with 25 microsatellite primer pairs. Coefficient of parentage (COP) values were calculated using parentage information at the expansion level of 5. The pedigree-based similarity (mean 0.115, range 0.00-0.53) was lower than the similarity assessed using microsatellite markers (mean 0.70, range 0.47-0.91). Similarity estimates were used to construct dendrograms by using the unweighted pair-group method with arithmetic averages (UPGMA). Clustering of genotypes in respect of marker-based similarity revealed two groups. Genotype PBW442 diverged and appeared as distinct from all other genotypes in both marker-based and pedigree-based analysis. The correlation of COP values with genetic similarity values based on microsatellite markers is low (r = 0.285, p < 0.05). The results indicate a need to develop wheat varieties with a diverse genetic background and to incorporate new variability into the existing wheat gene pool.     

Differential effects of somatostatin on exploratory behavior after unilateral injections in to rat neostriatum

The effects of somatostatin (SRIF) microinjected unilaterally (left or right) at a dose of 10, 50, and 100 ng into the neostriatum of male Wistar rats on exploratory behavior were studied. Unilateral injections of SRIF suppressed dose-related the exploratory activity as decreased the number of horizontal and vertical movements compared to the respective controls. The effect was more pronounced when SRIF was microinjected into the right neostriatum as compared to the left neostriatum. These findings suggest some asymmetric effects of SRIF, depending on the dose and the microinjected hemisphere.     

Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73.

The major outer membrane protein (OmpH) of Pasteurella multocida X-73 was purified by selective extraction with detergents, followed by size exclusion chromatography. The planar lipid bilayer assay showed that OmpH has pore-forming function. The average single channel conductance in 1.0 M KCl was 0.62 nS. The gene (ompH) encoding OmpH has been isolated and sequenced by construction of a genomic library and PCR techniques. The coding region of this gene is 1,059 bp long. The predicted primary protein is composed of 353 amino acids, with a 20-amino-acid signal peptide. The mature protein is composed of 333 amino acids with a molecular mass of 36.665 kDa. The ompH gene encoding mature protein has been expressed in Escherichia coli by using a regulatable expression system. The ompH gene was distributed among 15 P. multocida serotypes and strain CU. Protection studies showed that OmpH was able to induce homologous protection in chickens. These findings demonstrate that OmpH is a protective outer membrane porin of strain X-73 and is conserved among P. multocida somatic serotypes.     

Purification and characterization of a multicatalytic proteinase from Atlantic salmon (Salmo salar) muscle

A high molecular mass alkaline proteinase was purified by DEAE-Sepharose and Mono Q chromatography. The mol. wt was estimated to be about 600,000. Under denaturing conditions, the enzyme dissociated into a cluster of subunits with mol. wt ranging from 25,000 to 30,000. The isoelectric point of the enzyme was about pH 7.3. The proteinase was able to hydrolyse N-terminal-blocked 4-methyl-7-coumarylamide substrates for either trypsin- or chymotrypsin-like activity. It was also able to hydrolyse haemoglobin and myosin at temperatures of about 60°C. The activities responded to pH and some chemicals in different ways. The trypsin-like activity was clearly inhibited by several serine protease inhibitors. These results suggest that the enzyme is multicatalytic, having at least two different active sites.     

Identification of a codominant amplified polymorphic DNA marker linked to the verticillium wilt resistance gene in tomato

Resistance to verticillium wilt, a vascular disease causing yield losses in many crops, is conferred in tomato by a single dominant allele, Ve. A population segregating for the Ve allele was generated using near-isogenic tomato lines. Analysis of the parental tomato DNA using the polymerase chain reaction and 400 random primers, each 10 deoxyribonucleotides in length, produced 1,880 amplified DNA fragments. Of the four polymorphisms observed between the resistant and susceptible parental genotypes, only one was linked to the Ve gene. No recombination was observed between this DNA marker and the Ve locus, indicating that the linkage is less than 3.5±2.7 cM. The marker detected both the susceptible and resistant alleles, producing amplified DNA fragments of approximately 1,300 and 1,350 bp, respectively. The sequence of the primer, determined from cloned amplified products, was 5 CTCACATGCA 3 instead of the expected 5 CTCACATGCC 3. The marker will be of value to tomato breeding programs because of the tight linkage, Codominant nature, and analytical procedure utilized.     

Characterization of A11 Neurons Projecting to the Spinal Cord of Mice

The hypothalamic A11 region has been identified in several species including rats, mice, cats, monkeys, zebrafish, and humans as the primary source of descending dopamine (DA) to the spinal cord. It has been implicated in the control of pain, modulation of the spinal locomotor network, restless leg syndrome, and cataplexy, yet the A11 cell group remains an understudied dopaminergic (DAergic) nucleus within the brain. It is unclear whether A11 neurons in the mouse contain the full complement of enzymes consistent with traditional DA neuronal phenotypes. Given the abundance of mouse genetic models and tools available to interrogate specific neural circuits and behavior, it is critical first to fully understand the phenotype of A11 cells. We provide evidence that, in addition to tyrosine hydroxylase (TH) that synthesizes L-DOPA, neurons within the A11 region of the mouse contain aromatic L-amino acid decarboxylase (AADC), the enzyme that converts L-DOPA to dopamine. Furthermore, we show that the A11 neurons contain vesicular monoamine transporter 2 (VMAT2), which is necessary for packaging DA into vesicles. On the contrary, A11 neurons in the mouse lack the dopamine transporter (DAT). In conclusion, our data suggest that A11 neurons are DAergic. The lack of DAT, and therefore the lack of a DA reuptake mechanism, points to a longer time of action compared to typical DA neurons.