Molecular evolution of the MAGUK family in metazoan genomes

Background  

Development, differentiation and physiology of metazoans all depend on cell to cell communication and subsequent intracellular signal transduction. Often, these processes are orchestrated via sites of specialized cell-cell contact and involve receptors, adhesion molecules and scaffolding proteins. Several of these scaffolding proteins important for synaptic and cellular junctions belong to the large family of membrane-associated guanylate kinases (MAGUK). In order to elucidate the origin and the evolutionary history of the MAGUKs we investigated full-length cDNA, EST and genomic sequences of species in major phyla.     

Salinity tolerance of Great Lakes invaders

1. The Laurentian Great Lakes are among the most invaded freshwater ecosystems in the world. Historically, the major vector for the introduction of non‐indigenous species (NIS) has been the release of contaminated ballast water via transoceanic ships. Despite regulations implemented in 1993, requiring vessels carrying fresh ballast water to exchange this water with saline ocean water, new reports of invasions have continued. 2. NIS often have a wide environmental tolerance allowing them to adapt to and invade a variety of habitats. It has been hypothesized that NIS with broad salinity tolerance may be able to survive ballast water exchange (BWE) and continue to pose an invasion risk to the Great Lakes. 3. We tested the short‐term salinity tolerance of eight recent invaders to the Great Lakes, specifically three cladocera (Bosmina coregoni, Bythotrephes longimanus, Cercopagis pengoi), two molluscs (Dreissena polymorpha, Dreissena rostriformis bugensis), and one species each of the families Gammaridae, Mysidae and Gobidae (Echinogammarus ischnus, Hemimysis anomala, Neogobius melanostomus) to determine if they could have survived salinities associated with BWE. 4. Overall, short‐term exposure to highly saline water dramatically reduced survival of all species. Two different methods of BWE tested, simultaneous and sequential, were equally effective in reducing survival. Species that survived the longest in highly saline water either possess behavioural characteristics that reduce exposure to adverse environments (valve closure; both Dreissena species) or are reported to have some degree of salinity tolerance in their native region (Echinogammarus). Given that exposure in our trials lasted a maximum of 48 h, and that species in ballast tanks would typically be exposed to saline water for c. 5 days, it appears that BWE is an effective method to reduce the survival of these NIS. These results provide impetus for tightening policy and monitoring of BWE, in particular for ships entering the Great Lakes from freshwater ports.     

Relationships between rust resistance genes at the M locus in flax

Genes at the M locus in flax ( Linum usitatissimum ) that confer resistance to flax rust ( Melampsora lini ) occur in complex haplotypes containing up to 15 related genes or gene fragments. We have cloned two additional functional resistance genes at this locus, M1 and M3 , by transposon tagging and candidate gene approaches, and investigated the genetic relationships between four genes ( M , M1 , M3 and M4 ) by recombination analysis. M1 and M3 , like M , are members of the nucleotide binding site, leucine-rich repeat (NBS-LRR) family. Comparisons of the predicted M1 and M3 amino acid sequences with M and L6 reveal that: (i) M1 contains four additional LRRs, probably as a result of an unequal crossover event between duplicated regions; (ii) M1 shares large segments of exact identity with M and M3, indicative of intragenic recombination events; and (iii) a large number of amino acid differences are scattered throughout the M, M1 and M3 proteins. Recombination analysis (here and in previous studies) has revealed that M readily recombines with M1 , M3 and M4 , whereas these three genes fail to recombine despite large family sizes (>5800) in two test-cross families, suggesting that they may occupy allelic positions in the gene cluster. Several restriction fragment length polymorphism markers within or near the M locus were mapped with respect to seven crossover events between M and M1 . The results of this and previous studies provide evidence of structural differences between: (i) homoeologous loci in the different genomes of flax; (ii) different haplotypes at the M locus; (iii) different resistance genes in the M group; and (iv) the flanking regions downstream of M locus resistance genes.     

Canopy development and tillering of field-grown crops of two contrasting cultivars of winter wheat (Triticum aestivum) in response to CO2 and temperature

Elevated CO₂ (691 cf. 371 /miol CO₂ mol^-1 air) and warmer temperatures (over the range 1.0^UC below to 1.6^oC above ambient) increased light interception by crops of two contrasting cultivars (Hereward and Soissons) of winter wheat (Triticum aestivum L.) during winter growth in the field. The fractional interception of light by the canopy increased more rapidly initially in Soissons than in Hereward, but Hereward showed a much greater response to CO₂ (35% increase in Hereward but only 7% in Soissons) at 500^oCd after sowing. By terminal spikelet formation, in contrast, fractional interception was greater in Hereward than in Soissons, while the effect of CO₂ was the same in both cultivars (9%). Thus, although differences in the relative response of canopy development to CO₂ were detected between cultivars initially, differences were negligible during later development. The greater interception of light by the canopy in elevated CO₂, at any one temperature, resulted from increased tillering. The number of tillers plant“‘ at terminal spikelet was a linear function of main stem dry mass at this developmental stage but with a greater response in elevated CO₂, viz 2.3 and 3.8 tillers g^-1 main stem dry mass at 371 and 691 /μmol CO₂ mol^-1 air, respectively; these relations were unaffected by cultivar.     

The development of an action threshold for cabbage aphid (Brevicoryne brassicae) in oilseed rape in the UK

Replicated small plot field experiments were done at two sites growing winter oilseed rape (ADAS Boxworth, Cambridgeshire and ADAS High Mowthorpe, North Yorkshire) and two sites growing spring oilseed rape (ADAS Bridgets, Hampshire and ADAS Rosemaund, Herefordshire) to investigate the effect of cabbage aphid (Brevicoryne brassicae) on crop yield and quality. All four sites were included in the first 2 yr of the experiment in 1994 and 1995 but only those with winter oilseed rape were continued into the final year in 1996. Plots were artificially inoculated with cabbage aphids at either five aphid 4 m^-2 or 5 aphids 16 m^-2 or left uninoculated to become naturally infested. In 1995 and 1996 the naturally infested treatment was omitted. Sprays of the aphicide pirimicarb at GS 3.3, 3.7, 4.5, 4.9 and 5.5 were used to manipulate aphid populations. Once a plot had been treated at a target growth stage it was sprayed on all subsequent occasions to prevent recolonisation. Aphid numbers were assessed prior to each spray date and their effect on the crop measured in terms of yield of seed and oil and glucosinolate content. Artificial inoculation of aphids was often successful in establishing different populations of the pest at a range of growth stages. Results showed that cabbage aphid sometimes reduced both crop yield and quality. Yield responses to insecticide treatment tended to be larger in spring oilseed rape than in winter oilseed rape mainly because it became more heavily infested at an early growth stage. Tentative thresholds are proposed for control of the pest in both winter and spring oilseed rape. It is stressed that cabbage aphid is a sporadic pest and rarely likely to reach these threshold levels in field crops.     

ASSESSMENT OF AN ESCHERICHIA COLI METHIONINE AUXOTROPH GROWTH ASSAY FOR QUANTIFYING CRYSTALLINE METHIONINE SUPPLEMENTED IN POULTRY FEEDS

Estimating availability of methionine is relevant to feed formulation since diets can be supplemented with crystalline methionine to meet the minimum requirements of rapidly growing birds. Bacterial assays have been developed to measure the bioavailable levels of several essential amino acids in feeds, including methionine. The E. coli methionine auxotroph strain used in this study exhibited a linear extent of growth response to increasing concentrations of methionine added to the minimal test media, in the range of 0 to 4 μg/mL. In addition the growth rates of the E. coli auxotroph were significantly (P < 0.01) different when the methionine concentrations were varied (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 μg/mL) in minimal media. To assay feeds, feed grade methionine was added to poultry feed mixtures and samples were diluted with M9 media. Using this assay for estimating crystalline methionine added to feed, the extent of growth of the methionine auxotroph was correlated with the levels of crystalline methionine supplemented in the feed (R²= 0.9873). For all supplementation levels methionine recovery percentages ranged from 71 to 80% indicating that the bacterial assay response to crystalline methionine was relatively constant in the presence of the feed matrix. The overall results indicate that the rapid detection of crystalline methionine added to feeds is possible using this E. coli methionine auxotroph growth-based assay.     

Development of pepper (Capsicum annuum) seed quality

Changes in seed quality in pepper (Capsicum annuum L.) were monitored during seed development and maturation in two seasons. Seed quality was assessed by a number of different tests, but principally by determining seed storage longevity in laboratory tests and seedling growth in glasshouse tests. Mass maturity (defined as the end of the seed-filling phase) occurred 49–53 days after anthesis (DAA) in 1989 (varying among fruit layers) and 53 DAA in 1990 when seed moisture contents were 51–53%. The onset of both germinability and desiccation tolerance occurred either just before or at mass maturity. Maximum potential longevity (assessed by the value of the seed lot constant K_i) was achieved 63–65 DAA, i.e. not until 10–12 days after mass maturity (DAMM), in both years. Seedling dry weights in the glasshouse growth tests were maximal later still - for seeds harvested 17–21 DAMM in 1989 and 17 DAMM in 1990; the effects on seedling weight arose from differences in times from sowing to emergence (P < 0.005) among different seed harvests, with no significant differences in subsequent relative growth rates (P > 0.25). Seed priming reduced mean germination times for seeds harvested at all stages of development, but had little effect on germination capacity and potential longevity, and did not affect the pattern of changes in potential longevity during seed development and maturation. The results contradict the hypothesis that seed quality is maximal at the end of the seed-filling phase and that viability and vigour begin to decline immediately thereafter.     

Exploitation of the resistance to carrot fly in the wild carrot species Daucus capillifolius

Under field conditions a wild Daucus species from Libya, D. capillifolius, supported less than one tenth as many carrot flies (Psila rosae) as the susceptible carrot cultivar Danvers Half Long 126. Breeding lines developed from crosses between D. capillifolius and three different carrot types were grown in a series of field experiments at Wellesbourne between 1980 and 1989. Each year selections were made for agronomic quality and/or for increased resistance to carrot fly. The programme produced lines which for size, shape and colour represented most of the commercially-important carrot types. Some of these lines were also significantly more resistant to carrot fly than selections from the partially-resistant cv. Sytan. However, the best lines were not as resistant as the wild parent. The highest quality resistant lines were sold to seed companies for variety production.