Whole-embryo culture of Drosophila : development of embryonic tissues in vitro

Summary We have devised techniques to culture whole, dissected embryos of Drosophila melanogaster. We examine multiple aspects of the morphological and physiological development of the epidermis, musculature, nervous system, and internal organs in this cultured preparation, and show that in vitro development closely parallels normal embryogenesis. These techniques permit a wide range of experimental manipulations during embryogenesis and allow us to extend observations through late embryonic stages, after cuticle deposition. Applications of this technique are presented.     

A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae

A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.     

The root-knot nematode resistance gene (Mi) in tomato: construction of a molecular linkage map and identification of dominant cDNA markers in resistant genotypes

A dominant allele at the Mi locus on chromosome 6 of tomato (Lycopersicon esculentum Mill) confers resistance to three species of root-knot nematodes (Meloidogyne). The resistance, which is associated with a localized necrotic response, was originally introduced into tomato from the wild species Lycopersicon peruvianum. As a step towards the molecular cloning of Mi, we have identified closely linked DNA markers from both cDNA and genomic DNA libraries as restriction fragment length polymorphisms (RFLPs). DNA from tomato populations segregating for nematode resistance was analyzed to generate a high-resolution genetic map of this region. Additional information on gene order was obtained by comparing the size of the introgressed L. peruvianum chromosomal segment within a collection of nematode-resistant tomato lines. Among the four cDNA markers that are tightly linked to Mi, three are dominant, i.e. L. peruvianum-specific. One cDNA marker corresponds to a gene family comprising 20-30 members, one of which is diagnostic for all nematode-resistant genotypes tested. The presence of non-homologous sequences around the Mi gene may contribute to the suppression of recombination in this region of the genome in crosses heterozygous for Mi. The potential of 'walking' from closely linked markers to Mi is discussed.     

The cellular and molecular regulation of 1,25(OH)2D3 production

The synthesis of 1,25(OH)₂D₃ is a critical control point in the regulation of calcium metabolism, and possibly in the growth and differentiation of a number of cell types. This paper reviews our current understanding of the regulation of this process at the cellular and molecular levels, with the emphasis on the mechanisms of feedback control 1,25(OH)₂D₃ itself, control of parathyroid hormone, the roles of cyclic AMP dependent protein kinase and protein kinase C, and the interaction between the various intracellular regulators of 1,25(OH)₂D₃ production.     

Nitrogen assimilation in field-grown winter wheat: Direct measurements of nitrate reduction in roots using15N

Summary Nitrate fertiliser labelled with¹⁵N was applied to a field grown crop of winter wheat. Uptake and assimilation of fertiliser nitrate was studied by monitoring the appearance of labelled nitrate and labelled amino acids in the xylem sap. Shortly after applying¹⁵N-nitrate to the soil about 30 per cent of recently absorbed¹⁵N was in the reduced form, indicating that roots of cereal crops can make a substantial contribution in reducing nitrate. Seasonal changes in crop growth andin vivo NRA are also described.     

Analysis of the Drosophila female sterile mutation fs(1) 1304 by pole cell transplantation experiments

The Drosophila melanogaster mutant fs(1)1304 is an ovary autonomous female sterile mutant that causes abnormal morphology of the egg. Vitellogenesis proceeds at an abnormally slow rate in homozygous females. We have used pole cell transplantation to construct germ line mosaics in order to determine whether the 1304 defect depends upon the genotype of the germ line cells (oocyte or nurse cells) or the somatic line (follicle cells). We have found that the germ line is the primary target tissue where the mutant gene is expressed.     

A new,strikingly patterned Calathea (Marantaceae) from Ecuador

Calathea libbyana from Napo Province, Ecuador, is described. It belongs to the dimorphic-bracted species-group ofCalathea seriesComosae. It is in cultivation under the cultivar name ‘Windows’, which refers to its distinctive leaf pattern with pale, semi-translucent markings.     

Differentiation properties of pure populations of human dystrophic muscle cells

The interpretation of the majority of studies of Duchenne muscular dystrophy (DMD) has been complicated by the heterogeneous composition of the cultures used. In addition to muscle cells, muscle tissue contains adipocytes and fibroblasts and the proportion of these cell types varies, especially in disease states. To overcome this problem we developed culture conditions which permitted isolation and characterization of pure populations of clonally derived human muscle cells [1, 2]. Here we report the successful application of these methods to muscle cells from biopsies of individuals with diagnosed DMD. The normal and mutant human muscle cells were used in experiments of muscle differentiation in the same manner as cell lines. Frozen-stored cells were thawed, plated in a series of replicate plates, and allowed to differentiate under similar culture conditions. Yet, in contrast with cell lines, the cells were karyotypically normal, not altered by adaptation to long-term culture, and had a finite lifespan. We have systematically analysed specific properties of the normal and DMD muscle cells which differentiated in culture. The kinetics and extent of myoblast fusion, myotube morphology, and the accumulation and distribution of membrane acetylcholine receptors were monitored. In addition, the isozyme composition of creatine kinase and its intracellular and extracellular distribution were determined. Our results indicate that DMD muscle cells are fully capable of initiating myogenesis in culture and do not differ from normal muscle in several important parameters of differentiation.