P-Element Insertion Alleles of Essential Genes on the Third Chromosome of Drosophila Melanogaster: Correlation of Physical and Cytogenetic Maps in Chromosomal Region 86e-87f

We have established a collection of 2460 lethal or semi-lethal mutant lines using a procedure thought to insert single P elements into vital genes on the third chromosome of Drosophila melanogaster. More than 1200 randomly selected lines were examined by in situ hybridization and 90% found to contain single insertions at sites that mark 89% of all lettered subdivisions of the Bridges' map. A set of chromosomal deficiencies that collectively uncover ~25% of the euchromatin of chromosome 3 reveal lethal mutations in 468 lines corresponding to 145 complementation groups. We undertook a detailed analysis of the cytogenetic interval 86E-87F and identified 87 P-element-induced mutations falling into 38 complementation groups, 16 of which correspond to previously known genes. Twenty-one of these 38 complementation groups have at least one allele that has a P-element insertion at a position consistent with the cytogenetics of the locus. We have rescued P elements and flanking chromosomal sequences from the 86E-87F region in 35 lines with either lethal or genetically silent P insertions, and used these as probes to identify cosmids and P1 clones from the Drosophila genome projects. This has tied together the physical and genetic maps and has linked 44 previously identified cosmid contigs into seven ``supercontigs' that span the interval. STS data for sequences flanking one side of the P-element insertions in 49 lines has identified insertions in the αγ element at 87C, two known transposable elements, and the open reading frames of seven putative single copy genes. These correspond to five known genes in this interval, and two genes identified by the homology of their predicted products to known proteins from other organisms.     

Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo

Reiser, Peter J., William O. Kline, and Pal L. Vaghy.Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo. J. Appl. Physiol. 82(4): 1250-1255, 1997.Fast-twitch skeletal muscles contain more neuronal-type nitricoxide synthase (nNOS) than slow-twitch muscles because nNOS is presentonly in fast (type II) muscle fibers. Chronic in vivo electricalstimulation of tibialis anterior and extensor digitorum longus musclesof rabbits was used as a method of inducing fast-to-slow fiber typetransformation. We have studied whether an increase in musclecontractile activity induced by electrical stimulation alters nNOSexpression, and if so, whether the nNOS expression decreases to thelevels present in slow muscles. Changes in the expression of myosinheavy chain isoforms and maximum velocity of shortening of skinnedfibers indicated characteristic fast-to-slow fiber type transformationafter 3 wk of stimulation. At the same time, activity of NOS doubled inthe stimulated muscles, and this correlated with an increase in theexpression of nNOS shown by immunoblot analysis. These data suggestthat nNOS expression in skeletal muscle is regulated by muscle activityand that this regulation does not necessarily follow the fast-twitchand slow-twitch pattern during the dynamic phase of phenotypetransformation.

     

Direct and efficient plant regeneration from leaf explants of Solanum tuberosum l. cv. Bintje

Summary A two-step procedure was used for plant regeneration from in vitro grown leaf strips (2–3 mm wide) of cv. Bintje. Step I medium was designed with 2,4-dichlorophenoxycetic acid (2,4-D) at 0.0 or 9.0 M, in combination with 2.28 M kinetin (K), benzyl adenine (BA), zeatin (Z) or zeatin riboside (ZR). Step II media were 2,4-D-free media containing 5.78 M gibberellic acid (GA3) and growth regulators similar to those of step I media. Leaf explants cultured in medium I containing zeatin riboside or zeatin for 6 days and then subcultured in medium II containing zeatin riboside produced numerous shoots without callus formation. Zeatin riboside containing step I and II media caused shoot regeneration in a high number (97.5±2.2) of explants. Approximately, 33.7±8.4 shoots were regenerated from each leaf explant.Abbreviations BA benzyladenine - Z zeatin - ZR zeatin riboside (trans isomer) - 2,4-D 2,4-dichlorophenoxyacetic acid     

A new mutation protocol for obtaining auxotrophic mutants of the yeastPhaffia rhodozyma

Summary A new mutation strategy, which involves -irradiation of cells followed by a selective enzymatic enrichment step, was worked out to obtain auxotrophic mutants from the astaxanthin-producing yeast P. rhodozyma. Under the optimized conditions described, different mutants suitable for strain improvement were isolated.     

Solid-state fermentation of sugarcane bagasse with Flammulina velutipes and Trametes versicolor

The mushroom Flammulina velutipes and the white-rot fungus Trametes versicolor were cultivated separately on sugarcane bagasse for 40 days. Trametes versicolor produced laccase and manganese-peroxidase activities, showing a simultaneous degradation of lignin and holocellulose. However, only phenoloxidase activity was found with Flammulina velutipes. A preferential degradation of lignin was detected in F. velutipes, which exhibited a greater reduction in the ratio of weight loss to lignin loss than T. versicolor. A decrease in the syringyl/guaiacyl ratio observed with both fungi indicated the preferential degradation of non-condensed (syringyl-type) lignin units. An increase in the relative abundance of aromatic carboxylic acids suggested that the oxidative transformation of lignin unit side-chains was occurring. This was more noticeable with Flammulina velutipes than with T. versicolor.     

Callus Cultures from Zygotic Embryos of Costus speciosus and Their Morphogenetic Responses

Soft, nodular and hard types of calli were initiated on mature zygotic embryo explants of two tetraploid clones of Costus speciosus, of which, only the hard calli were amenable to morphogenetic responses. The two clones differed in their growth regulator requirements both for the initiation of calli and for shoot regeneration. De novo formation of both shoot bud meristems and somatic embryoids were observed. Latter were encased partially or fUlly by coleoptilar sheath. Embryoids could be isolated as discrete units. On maturity, a stock like appendage developed from the base and finally embryoids got detached from the subtending tissue. Both shoot-bud meristems and somatic embryoids developed into complete plantlets, the former upon sequential transfer of calli on Schenk and Hildebrandt’s (SH) basal medium containing lower levels of growth hormones, while the latter only on basal medium. These culture regenerants were subsequently transferred to the field. The morphogenetic behaviour of these two tetraploid clones reflects their marked genotypic difference inspite of their same ploidy status.     

Localization of 3-phosphoglyceric acid synthesis in the mother cell compartments and forespores ofBacillus megaterium and the effects of manganous ions on its metabolism

Rapidly metabolizable compounds such as glucose or glycerol were not utilized byBacillus megaterium in the absence of manganese when grown in the supplemented nutrient broth medium. Under these conditions, growth ceased at low cell titre, 3-phosphoglyceric acid accumulated inside the cells and normal sporulation process was arrested. Addition of manganese to the medium caused disappearance of 3-phosphoglyceric acid, growth resumed and normal sporulation was observed. Synthesis of 3-phosphoglyceric acid occurred only in the mother cell compartments and it was transported for accumulation inside the forespores ofBacillus megaterium when grown in supplemented nutrient broth medium. Incubation of forespores in the presence of glucose or glycerol had no effect on 3-phosphoglyceric acid synthesis/accumulation, but it was completely utilized when forespores were incubated with manganese plus ionophore (X 537A). No other metal(s) could substitute for manganese suggesting that manganese plays crucial role in 3-phosphoglyceric acid metabolism     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Discrimination of flash-induced fast and slow electric potential components

This work aimed at the resolution of the multi-component electric potential changes induced by single-turnover flash illumination of Photosystem-I-enriched subchloroplast vesicles. If supplemented with ferredoxin and under carefully adjusted redox poising, these vesicles show a pronounced slow-rising and -decaying electric potential component, as monitored by endogenous and exogenous field-sensitive probes, carotenoids and oxonol VI, respectively. The fast and slow potential components can be easily discriminated without the need for computer-assisted deconvolution after selective presaturation of the slow component by preillumination or a transmembrane ΔpH, after selective suppression of the slow component by low valinomycin or uncoupler concentrations or in the absence of ferredoxin. The slow electric potential component, as compared to the fast one, is relatively sensitive to low concentrations of ionophores and uncouplers, detergent, ageing and lower temperatures (4–12°C), is associated with electrogenic proton displacements and is interpreted to respond to a field that is more located on the membrane-bulk interface. Temperature effects show transition temperatures around 20°C for both the rise and decay of the slow potential component. The results provide further evidence that the carotenoids and oxonol VI sense the same (slow) electric field, but may be differently located in the thylakoid membrane.