Oat mesophyll protoplasts: their response to various feeder cultures

Oat (Avena sativa L.) mesophyll protoplasts were recently demonstrated to be capable of dedifferentiation, repeated divisions, and colony formation. Since the development of oat mesophyll protoplasts is decisively influenced by the nature of the used feeder culture (species, variety and concentration), we conducted a systematic study of this parameter. Generally, graminaceous feeders promoted protoplast proliferation, while dicot species repressed protoplast divisions. The beneficial effect of those feeders that promote divisions was counterbalanced by a factor that causes necrosis. The correct balance between promotion of divisions or necrosis depended on the nature of the feeder and its plating density.     

The bacteriophage T4 gene for the small subunit of ribonucleotide reductase contains an intron.

The bacteriophage T4 gene nrdB codes for the small subunit of the enzyme ribonucleotide reductase. The T4 nrdB gene was localized between 136.1 kb and 137.8 kb in the T4 genetic map according to the deduced structural homology of the protein to the amino acid sequence of its bacterial counterpart, the B2 subunit of Escherichia coli. This positions the C-terminal end of the T4 nrdB gene approximately 2 kb closer to the T4 gene 63 than earlier anticipated from genetic recombinational analyses. The most surprising feature of the T4 nrdB gene is the presence of an approximately 625 bp intron which divides the structural gene into two parts. This is the second example of a prokaryotic structural gene with an intron. The first prokaryotic intron was reported in the nearby td gene, coding for the bacteriophage T4-specific thymidylate synthase enzyme. The nucleotide sequence at the exon-intron junctions of the T4 nrdB gene is similar to that of the junctions of the T4 td gene: the anticipated exon-intron boundary at the donor site ends with a TAA stop codon and there is an ATG start codon at the putative downstream intron-exon boundary of the acceptor site. In the course of this work the denA gene of T4 (endonuclease II) was also located.     

The relationship between physical and chemical conditions and low microbial diversity in the Blue Lagoon geothermal lake in Iceland

Abstract: The Blue Lagoon in Iceland is a shallow geothermal lake with average temperatures of 37°C, pH 7.5 and about 2.5% salinity. It was formed in 1976 from the effluents of the Svartsengi geothermal power plant and is saturated with silica which constantly precipitates in the lake. It has been colonized by a few types of specialized microorganisms which seem to proliferate in this unusual ecosystem. The average bacterial colony count in the lake was 1.3 × 10⁵ ml⁻¹ on plate count agar made with 50% Blue Lagoon fluid but 2.6 × 10⁶ ml⁻¹ when determined with the MPN method. A total of 99 isolates were purified and characterized by 54 phenotypic tests and then grouped using Numerical Taxonomy. At similarity values of 80%, one major cluster was formed containing 85% of the isolates. Four representative strains from this cluster were further characterized and all shown to be Gram-negative, obligately aerobic, non-motile rods. They were oxidase positive, catalase negative and grew optimally at 45°C and in 3.5% NaCl with doubling time of about 80 min.     

Initial increase and subsequent loss of vasoactive intestinal polypeptide immunoreactivity in acetylcholinesterase-positive neurons of mouse islets transplanted to the kidney

Collagenase-isolated pancreatic islets from C57BL/6J mice were cultured overnight and transplanted under the kidney capsule of non-diabetic syngeneic hosts. Cryostat sections of grafts and fresh islets were stained for acetylcholinesterase (AChE) and vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI). Immediately after isolation, as well as 2-5 days after transplantation, VIP-LI- and AChE-positive nerve cell bodies were clearly seen in the periphery of the islets. Grafts 3-5 days old exhibited a transient and marked increase in VIP-LI nerve cell bodies and fibers. Seven days after transplantation VIP-LI nerve structures began to decrease in number and after 26-52 weeks they were no longer detectable. In contrast, AChE-positive nerve cell bodies and fibers, which showed a relatively constant pattern of distribution, were observed throughout the entire observation period. Restaining experiments demonstrated the coexistence of VIP-LI and AChE activity in the neurons. It is concluded that the grafts were extensively equipped with an intrinsic VIP-ergic and AChE-positive innervation. The initial, transient enhancement of VIP-LI expression probably reflects an adaptation of the neuro-insular complex to the preganglionic denervation, or to the ectopic environment, or both.     

Transformation of sunflower (Helianthus annuus L.): a reliable protocol

Summary A reliable protocol for the transformation of cultivated sunflower (Helianthus annuus L.) has been established, based on microprojectile bombardment of half shoot apices in combination with Agrobacterium tumefaciens coculture. Transgenic shoots have been obtained from 5 inbred lines, although transformation efficiencies varied with the genotype. Plants expressing the transgenes could be recovered from up to 7% of the explants. A minority of plants was shown to be chimaeric for expression of ß-glucuronidase activity while most appeared to be uniformly transformed. Genetic segregation was 31 for both ß-glucuronidase and neomycine phospho transferase in some plants, indicating that the respective mother plants were uniformly transformed. Integration of the foreign genes was also shown by Southern analysis.Abbreviations BAP benzyl amino purine - EDTA ethylene diamine tetraacetic acid - GUS ß-glucuronidase - npt II neomycine phospho-transferase II     

An internal standard improves the reliability of transient expression studies in plant protoplasts

Transient expression of foreign genes introduced on a plasmid into isolated plant protoplasts is widely used to study the control of gene expression. Unfortunately, many experimental variables implicated in this technique are difficult or impossible to control, resulting in a disturbing degree of variability between otherwise identical experiments. We have studied the co-expression of two constitutively expressed genes located on the same plasmid. This has allowed us to identify the lot of plasmid DNA as an important source of variation, along with the protoplast lot. Plasmid DNA concentration was found to be of minor importance. Since the variation of expression level of the two genes was identical for the two genes in all experiments, we propose the use of an internal standard in all comparative transient expression studies, which allows the reduction of the variation between experiments by one order of magnitude.Abbreviations CaMV cauliflower mosaic virus - CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase synthase - MS medium after Murashige and Skoog (1962) - MU methyl umbelliferone - NOS nopaline synthase - NPT II-neomycin phospho transferase - PEG polyethylene glycol - Tris tris-hydroxymethyl aminoethane