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.     

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.     

Metabolism of Imidazole by a Pseudomonad

Intermediates formed during the microbial degradation of imidazole, namely 4(5)-imidazolone, formiminoglycine, and possibly glycine, are similar to those formed during metabolism of imidazole derivatives.     

Characterization of protein–polysaccharides of articular cartilage from mature and immature pigs

Protein-polysaccharides of femoral articular cartilage from pigs of ages 9 months and 5 weeks were compared after extraction at pH6.8 with iso-osmotic sodium acetate followed by 0.63m-calcium acetate. The cartilage from the younger animals had a higher moisture content and contained considerably larger amounts of protein-polysaccharide, but less than half as much collagen/g. dry weight, than cartilage from the older pigs. There was notably less keratan sulphate in the fractions from the less mature animals. After gel filtration on 6% agarose, elution profiles of the calcium acetate extracts were similar to those of the sodium acetate extracts of the same tissue. Chemical analyses, however, showed that in both age-groups the extraction procedure had achieved a sequential solubilization of protein-polysaccharides in that the initial extracts contained a higher proportion of keratan sulphate than those that were extracted subsequently. Both extracts from the older animals contained up to 25% of a relatively small protein-polysaccharide that was retarded on 6% agarose and that had a lower protein content and less keratan sulphate than the larger protein-polysaccharides. In contrast, in extracts from the less mature cartilage only about 5% of the protein-polysaccharides were small enough to be retarded by 6% agarose, suggesting that the small components may not be precursors of the larger. The average length of chondroitin sulphate chains, as calculated from the analytical data, was the same in the smaller protein-polysaccharides as in the larger.     

CHORIZOCOCCUS HERBICOLA (MASKELL) (COMB. N): (HOMOPTERA: PSEUDOCOCCIDAE) A GRASS-INFESTING SPECIES IN AUSTRALIA

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The Maskell species Dactylopius herbicola , a mealybug infesting grass in Australia, is redescribed and designated as a new combination, Chorizococcus herbicola (Maskell).