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.     

The structure of agarose gels,and the accommodation within them of irregularly shaped particles

This paper shows how the number of cross-linkage points (nodes) in a random reticulum, such as an ideal polysaccharide gel, may be calculated in accordance with mathematical principles. The influence of nodal configurations upon the statistical geometry of the reticulum is discussed, and it is shown from experimental evidence that the nodal configurations in agarose gel are nonrandom. A method is given for calculating the accommodation probability of an irregularly shaped particle in a reticulum, which is relevant to the theory of gel chromatography and to the distribution of cells in tissues permeating a network of capillaries or veins.     

Characterisation in vivo of the reactive thiol groups of the lactose permease from Escherichia coli and a mutant; exposure, reactivity and the effects of substrate binding

The reactivity and accessibility of the reactive thiol groups of the native lactose permease and a mutant have been studied in a number of circumstances and with a number of reagents, in particular using the specific thiol-disulphide exchange reaction. Seven different reactive states of the thiol in the native protein have been characterised by their different second-order rate constants. Interconversion between these states is dependent on the magnitude of the protonmotive force, pH and substrate binding. In the absence of galactoside, reactivity is controlled by an ionisation with apparent pKa 9.3. This pKa is not affected by the protonmotive force, but it is lowered in the presence of external galactoside. The conformation adopted by the permease when in equilibrium with saturating galactoside appears to be different from that of the intermediate that accumulates during net turnover. In the former state, the reactivity of the thiol group is depressed, whereas in the latter state it is enhanced. The thiol group of the native protein is buried in a hydrophobic environment that has a dielectric constant considerably lower than that of water. The environment is not greatly perturbed by changes in the magnitude of the protonmotive force, but it is affected by the binding of galactoside. In a strain which carries the YUN mutation (Wilson, T.H. and Kusch, M. (1972) Biochim. Biophys. Acta 255, 786-797), two reactive thiols were characterised. The more reactive of the two is more exposed than the thiol group of the native molecule and is in an environment that has a dielectric constant close to that of water. The less reactive thiol appears to be more deeply buried than that of the native protein. Thus the mutation appears to produce a conformation change in the central portion of the polypeptide chain that results in greater exposure of the reactive thiol to the aqueous environment.     

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.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : The Purification and Properties of Farnesyl Transferase from Elicited Seedlings

Farnesyl transferase (farnesyl pyrophosphate: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400-fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 ± 3,000 from its behavior on a calibrated G-100 Sephadex molecular sieving column. Mg²⁺ ion at 4 millimolar gave the greatest stimulation of activity; Mn²⁺ ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (K_m = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K_m = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K_m = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes—a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates.     

A probability matrix for the identification of species of Vibrio and related genera

A matrix for the probabilistic identification of species of Vibrio and related genera has been constructed using the data from 1091 strains collected throughout the world and classified. Thirty-eight phenons are included in the matrix, 31 of these represent previously identified species or biovars and seven represent phenons which could not be identified and may represent new species. The identification matrix incorporates 81 characters although a subset of 30 tests can be used to distinguish the 38 phenons from each other. The additional 51 tests were included to assist the identification of some strains for which the initial 30 tests were inadequate. No significant cluster overlap was found at the 5% level and the identification score for the Hypothetical Median Organism of each cluster exceeded 0.9999 in all cases.     

A tentative national reference procedure for isolation and enumeration of Escherichia coli from bivalve molluscan shellfish by most probable number method

In the UK several quantitative methods exist for the examination of bivalve molluscan shellfish for sewage contamination. These methods include roll tubes, pour plates and most probable number (MPN) techniques, but there is no national standard method. A comparative study was made of the most commonly used methods for detection of Escherichia coli in bivalve shellfish. Schemes employing solid media, such as the roll tube and pour plate methods, underestimated faecal contamination in shellfish tissue compared with a liquid MPN multiple test-tube method using minerals-modified-glutamate broth (MMGB) as primary enrichment medium. The composition of MMGB apparently permits repair of sublethally injured cells of E. coli. Incorporation of resuscitation stages into the pour plate technique did not yield higher counts. A standardized MPN technique for examination of bivalve molluscan shellfish for E. coli content is proposed as a possible national reference procedure pending further collaborative assessment.