Characterization of Monoclonal Antibodies Specific for Erwinia carotovora subsp. atroseptica and Comparison of Serological Methods for Its Sensitive Detection on Potato Tubers

Seven monoclonal antibodies (MAbs) to Erwinia carotovora subsp. atroseptica have been produced. One, called 4G4, reacted with high specificity for serogroup I of E. carotovora subsp. atroseptica, the most common serogroup on potato tubers in different serological assays. Eighty-six strains belonging to different E. carotovora subsp. atroseptica serogroups were assayed. Some strains of serogroup XXII also reacted positively. No cross-reactions were observed against other species of plant pathogenic bacteria or 162 saprophytic bacteria from potato tubers. Only one strain of E. chrysanthemi from potato cross-reacted. A comparison of several serological techniques to detect E. carotovora subsp. atroseptica on potato tubers was performed with MAb 4G4 or polyclonal antibodies. The organism was extracted directly from potato peels of artificially inoculated tubers by soaking or selective enrichment under anaerobiosis in a medium with polypectate. MAb 4G4 was able to detect specifically 240 E. carotovora subsp. atroseptica cells per ml by indirect immunofluorescence and immunofluorescence colony staining and after soaking by ELISA-DAS (double-antibody sandwich enzyme-linked immunosorbent assay) after enrichment. The same amount of cells was detected by using immunolectrotransfer with polyclonal antibodies, and E. carotovora subsp. atroseptica and subsp. carotovora were distinguished by the latter technique. ELISA-DAS using MAb 4G4 with an enrichment step also efficiently detected E. carotovora subsp. atroseptica in naturally infected tubers and plants.     

Interactions between panicle size, insect density, and environment for genotypic resistance in sorghum to head bug,Calocoris angustatus

Studies were undertaken on interactions between panicle size, insect density, host plant, and the environment for sorghum head bug,Calocoris angustatus Lethiery on five sorghum genotypes in terms of bug population increase, grain damage and loss in grain mass across four panicle sizes (5, 10 or 20 branches/panicle and whole panicle), and three infestation levels (5, 10 and 15 pairs of bugs/panicle). Head bug numbers increased and grain damage decreased with an increase in panicle size in the head bug susceptible cultivars, CSH 1 and CSH 5. However, the increase in bug numbers or decrease in grain damage was not significant in head bug resistant genotypes, IS 17610 and IS 17645. Head bug numbers increased with an increase in infestation level in CSH 1 and CSH 5, however, such an increase was not substantial in IS 17610 and IS 17645. Grain damage was significantly lower in IS 17610 and IS 17645 compared with CSH 1 and CSH 5 across infestation levels. Head bug population increased at a greater rate during the rainy season compared with the dry season. Panicle size and infestation levels accounted for greater variation in grain damage and percentage loss in grain mass during the rainy season than in the dry season. To identify reliable sources of resistance to insects, it is important to study insect host plant-interactions across panicle sizes (levels of food availability), infestation levels and seasons.     

Nucleocapsid-glycoprotein interactions required for assembly of alphaviruses.

We have studied interactions between nucleocapsids and glycoproteins required for budding of alphaviruses, using Ross River virus-Sindbis virus chimeras in which the nucleocapsid protein is derived from one virus and the envelope glycoproteins are derived from the second virus. A virus containing the Ross River virus genome in which the capsid protein had been replaced with that from Sindbis virus was almost nonviable. Nucleocapsids formed in normal numbers in the infected cell, but very little virus was released from the cell. There are 11 amino acid differences between Ross River virus and Sindbis virus in their 33-residue E2 cytoplasmic domains. Site-specific mutagenesis was used to change 9 of these 11 amino acids in the chimera from the Ross River virus to the Sindbis virus sequence in an attempt to adapt the E2 of the chimera to the nucleocapsid. The resulting mutant chimera grew 4 orders of magnitude better than the parental chimeric virus. This finding provides direct evidence for a sequence-specific interaction between the nucleocapsid and the E2 cytoplasmic domain during virus budding. The mutated chimeric virus readily gave rise to large-plaque variants that grew almost as well as Ross River virus, suggesting that additional single amino acid substitutions in the structural proteins can further enhance the interactions between the disparate capsid and the glycoproteins. Unexpectedly, change of E2 residue 394 from lysine (Ross River virus) to glutamic acid (Sindbis virus) was deleterious for the chimera, suggesting that in addition to its role in nucleocapsid-E2 interactions, the N-terminal part of the E2 cytoplasmic domain may be involved in glycoprotein-glycoprotein interactions required to assemble the glycoprotein spikes. The reciprocal chimera, Sindbis virus containing the Ross River virus capsid, also grew poorly. Suppressor mutations arose readily in this chimera, producing a virus that grew moderately well and that formed larger plaques.     

Molecular cytogenetic analysis of a duplication Xp in a male: further delineation of a possible sex influencing region on the X chromosome

We describe a male infant with severe mental retardation and autism with a duplication of the short arm of the X chromosome. Chromosome painting confirmed the origin of this X duplication. Molecular cytogenetic analysis with fluorescence in situ hybridization (FISH) identified one copy of the zinc finger protein on the X chromosome (ZFX) and two copies of the steroid sulfatase gene (STS), further delineating the breakpoints. Based on cytogenetic and molecular comparisons of cases from the literature of sex-reversal in dup(X),Y patients and our patient, we suggest that a possible secondary sexinfluencing gene involved in the regulation of sex determination or testis morphogenesis is present at the distal Xp21.1 to p21.2 region.     

A bifunctional protein in the folate biosynthetic pathway of Streptococcus pneumoniae with dihydroneopterin aldolase and hydroxymethyldihydropterin pyrophosphokinase activities.

A protein encoded by sulD, one of four genes in a previously cloned folate biosynthetic operon of Streptococcus pneumoniae, had been shown to harbor 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase activity. This SulD protein was purified and shown now to harbor also dihydroneopterin aldolase activity. The bifunctional protein therefore catalyzes two successive steps in folate biosynthesis. The aldolase activity can be ascribed to the N-terminal domain of the SulD polypeptide, and the pyrophosphokinase activity can be ascribed to the C-terminal domain. Homologs of the dihydroneopterin aldolase domain were identified in other species, in one of which the domain was encoded as a separate polypeptide. The native SulD protein is a trimer or tetramer of a 31-kDa subunit, and it dissociated reversibly after purification. Dihydroneopterin aldolase activity required the multimeric protein, whereas pyrophosphokinase was expressed by the monomeric form. With purified SulD, the amount of 6-hydroxymethyl-7,8-dihydropterin product formed by the aldolase was proportional to the fourth power of the enzyme concentration, as expected for a reversibly dissociating tetramer. By identifying the gene encoding dihydroneopterin aldolase, this work extends our understanding of the molecular basis of the folate biosynthetic system common to many organisms.     

Physical Mapping of Meiotic Crossover Events in a 200-Kb Region of Neurospora Crassa Linkage Group I

We propose a general restriction fragment length polymorphism-based strategy to analyze the distribution of meiotic crossover events throughout specific genetic intervals. We have isolated 64 recombinant chromosomes carrying independent meiotic crossover events in the genetic interval eth-1-un-2 on linkage group I of Neurospora crassa. Thirty-eight crossover events were physically mapped with reference to a 200-kb region cloned by chromosome walking, using N. crassa λ and cosmid libraries. Crossovers were homogeneously distributed at intervals of 5.0 +/- 2.3 kb along the entire cloned interval. The ratio of physical to genetic distance appears to be higher in the region than in the overall N. crassa genome, suggesting that recombinational activity is less in large chromosomes than in small ones. The present work provides a method for defining the centromeric-telomeric orientation of single cloned DNA fragments. Their physical distance can also be estimated with respect to linked loci, provided that crossover events are distributed homogeneously in the interval. This strategy overcomes typical difficulties in defining the position and direction of chromosome walking steps on conventional linkage maps.     

Vertical distribution of planktonic rotifers in a karstic meromictic lake

Vertical distribution of planktonic rotifers is described in relation to temperature and oxygen in Lake La Cruz, a single-doline, closed karstic lake (121 m diameter and 25 m maximum depth) which shows iron meromixis. Samples were taken by peristaltic pumping at 10 cm depth intervals in the oxycline zone from June 1987 to September 1988. A model of rotifer vertical structure in stratified lakes is proposed. Rotifers concentrate their populations at the depths with intense gradients. As stratification develops some rotifer populations show a downward migration following the thermocline and some others show an upward migration following the oxycline. The production-respiration balance in the lake, and so the position of the oxycline with respect to the thermocline and the layer of maximum production, depends on meteorological conditions. A shift in the dominance of congeneric or related species can occur in consecutive years. In Lake La Cruz, mixing conditions and subterranean inflow in spring were much more intense in 1988 than 1987, and the distance between production and decomposition depths was smaller in 1988. Anuraeopsis miraclei, an oxycline-bound species with high abundance in 1987, was displaced by A. fissa in 1988. A. fissa, which was a metalimnetic species during early summer, reached peak densities (3 × 10⁴ ind l^–1) at the oxycline, equaling the abundance of A. miraclei the preceeding year.