Inter‐ and intraspecific variation in mycotoxin tolerance: A study of four Drosophila species

Many mycophagous Drosophila species have adapted to tolerate high concentrations of mycotoxins, an ability not reported in any other eukaryotes. Although an association between mycophagy and mycotoxin tolerance has been established in many Drosophila species, the genetic mechanisms of the tolerance are unknown. This study presents the inter‐ and intraspecific variation in the mycotoxin tolerance trait. We studied the mycotoxin tolerance in four Drosophila species from four separate clades within the immigrans‐tripunctata radiation from two distinct locations. The effect of mycotoxin treatment on 20 isofemale lines per species was studied using seven gross phenotypes: survival to pupation, survival to eclosion, development time to pupation and eclosion, thorax length, fecundity, and longevity. We observed interspecific variation among four species, with D. falleni being the most tolerant, followed by D. recens, D. neotestacea, and D. tripunctata, in that order. The results also revealed geographical variation and intraspecific genetic variation in mycotoxin tolerance. This report provides the foundation for further delineating the genetic mechanisms of the mycotoxin tolerance trait.     

Immunopathogenesis of coronavirus infections: implications for SARS

At the end of 2002, the first cases of severe acute respiratory syndrome (SARS) were reported, and in the following year, SARS resulted in considerable mortality and morbidity worldwide. SARS is caused by a novel species of coronavirus (SARS-CoV) and is the most severe coronavirus-mediated human disease that has been described so far. On the basis of similarities with other coronavirus infections, SARS might, in part, be immune mediated. As discussed in this Review, studies of animals that are infected with other coronaviruses indicate that excessive and sometimes dysregulated responses by macrophages and other pro-inflammatory cells might be particularly important in the pathogenesis of disease that is caused by infection with these viruses. It is hoped that lessons from such studies will help us to understand more about the pathogenesis of SARS in humans and to prevent or control outbreaks of SARS in the future.     

Localization of an Ataxia-Telangiectasia Gene to an −500-kb Interval on Chromosome 11q23.1: Linkage Analysis of 176 Families by an International Consortium

We describe a 20-point linkage analysis map of chromosome 11q22-23 that is based on genotyping 249 families (59 CEPH and 190 A-T). Monte Carlo linkage analyses of 176 ataxia-telangiectasia (A-T) families localizes the major A-T locus to the region between S1819(A4) and S1818(A2). When seven nonlinking families were excluded from subsequent analyses, a 2-lod support interval of ~500 kb was identified between S1819(A4) and S1294. No recombinants were observed between A-T and markers S384, B7, S535, or S1294. Only 17 of the international consortium families have been assigned to complementation groups. The available evidence favors either a cluster of A-T genes on chromosome 11 or intragenic defects in a single gene.     

Identification of Stylar RNases Associated with Gametophytic Self-Incompatibility in Almond (Prunus dulcis)

Stylar proteins of 13 almond (Prunus dulcis) cultivars withknown S-genotypes were surveyed by IEF and 2D-PAGE combinedwith immunoblot and N-terminal amino acid sequence analysesto identify S-RNases associated with gametophytic self-incompatibility(SI) in this plant species. RNase activities corresponding toS_a and S_b, two of the four S-alleles tested, were identifiedby IEF and RNase activity staining. The S_a-RNase band reactedwith the anti-S₄serum prepared from Japanese pear (Pyrus serotina);no reaction with the antiserum was observed with the s_bRNaseband. When the s_a-RNase band was excised from an IEF gel stainedfor RNase activity, subjected to SDS-PAGE, and detected by immunoblotting,it appeared that this band consisted of a single protein thatreacted with the anti-s₄serum with M_r of about 28 kDa. With2D-PAGE and silver staining of the stylar extracts, all fourS-proteins could be successfully distinguished from each otherin the highly basic zone of the gel. Although S_b-, S_c-, andS_dproteins had roughly the same M_r of about 30 kDa, the S_c-proteinseemed to be slightly smaller than the S_b-protein and slightlylarger than the S_aprotein. In 2D-PAGE profiles as well, theS_a-protein had M_r of about 28 kDa, apparently smaller than theother three proteins. A bud sport, in which one of the two S-allelesof the original cultivar is impaired, was visualized as a lossof S_cprotein, which is consistent with the previous pollinationstudy. All four S-proteins reacted with the anti-S₄serum, probablybecause of the differing conformations of these S-proteins inthe IEF and 2D-PAGE gels. The S_a-protein in 2D-PAGE appearedto be identical to S_a-RNase in IEF; both bad the same M_r andwere reactive with the anti-S₄-serum. N-terminal amino acidsequence analysis of the four 5-proteins revealed that theywere highly homologous to each other and similar to the 5-RNasesof Malus, Pyrus, Scrophulariaceae, and Solanaceae. Taken together,RNases in the style are strongly suggested to be associatedwith the gametophytic SI of al- mond. This is the first reportidentofiying and characterizing S-RNase in almond. (Received July 11, 1996; Accepted December 26, 1996)     

3-Ketoglycoside-mediated metabolism of sucrose in E. coli as conferred by genes from Agrobacterium tumefaciens

Escherichia coli strains that did not have the ability to use sucrose as a sole carbon source gained this ability after receiving a cloned fragment of DNA from Agrobacterium tumefaciens. No invertase was detected in the sucrose-metabolizing E. coli, but evidence for the activity of certain enzymes, known to be produced by biotype 1 strains of Agrobacterium, were found. Evidence was found for the presence of d-glucoside 3-dehydrogenase (G3DH) and α-3-ketoglucosidase. The activity of enzyme extracts on 3-ketosucrose also indicated that 3-ketoglucose reductase, or some enzyme that acts on 3-ketoglucose, was present in the Suc⁺ E. coli as well. The fragment was found to complement a G3DH mutant of A. tumefaciens and was also found to confer chemotaxis towards sucrose in E. coli. Received: 13 September 1996 / Received revision: 15 January 1997 / Accepted: 24 January 1997     

Translational Coupling Controls Expression and Function of the DrrAB Drug Efflux Pump

This study investigates the role of translational coupling in the expression and function of DrrA and DrrB proteins, which form an efflux pump for the export of anticancer drugs doxorubicin and daunorubicin in the producer organism Streptomyces peucetius. Interest in studying the role of translational coupling came from the initial observation that DrrA and DrrB proteins confer doxorubicin resistance only when they are expressed in cis. Because of the presence of overlapping stop and start codons in the intergenic region between drrA and drrB, it has been assumed that the translation of drrB is coupled to the translation of the upstream gene drrA even though direct evidence for coupling has been lacking. In this study, we show that the expression of drrB is indeed coupled to translation of drrA. We also show that the introduction of non-coding sequences between the stop codon of drrA and the start of drrB prevents formation of a functional complex, although both proteins are still produced at normal levels, thus suggesting that translational coupling also plays a crucial role in proper assembly. Interestingly, replacement of drrA with an unrelated gene was found to result in very high drrB expression, which becomes severely growth inhibitory. This indicates that an additional mechanism within drrA may optimize expression of drrB. Based on the observations reported here, it is proposed that the production and assembly of DrrA and DrrB are tightly linked. Furthermore, we propose that the key to assembly of the DrrAB complex lies in co-folding of the two proteins, which requires that the genes be maintained in cis in a translationally coupled manner.     

Trans-splicing of pre-mRNA is predicted to occur in a wide range of organisms including vertebrates.

Several known trans-splicing RNA structures were used to define a canonical trans-splicing structure which was then used to perform a computer search of the EMBL nucleotide database. In addition to most known trans-splicing structures, many putative new trans-splicing sites were detected. These were found in a broad range of organisms including the vertebrates. Control experiments indicate that the search predicts known false positives at a rate of only 20%. Trans-splicing may therefore be a very wide-spread phenomenon.     

High-level transient production of a heterologous protein in plants by optimizing induction of a chemically inducible viral amplicon expression system

We have demonstrated that the method of chemical induction using a chemically inducible viral amplicon expression system can be optimized to increase expression of a heterologous protein in plants. A cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce a recombinant human blood protein, alpha-1-antitrypsin (AAT), by co-infiltrating intact and detached Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Infiltrated plants were induced by either topical applications or pressure injections and inducer was applied at either a single or multiple time points. Applying induction solution every 2 days via topical application resulted in increasing maximum levels of biologically functional rAAT from 0.71% to 1.3% of the total soluble protein (TSP) in detached plant leaves, a 1.8-fold improvement. Multiple applications of induction solution via pressure injection into intact leaves resulted in maximum levels of biologically functional rAAT being elevated 3-fold up to 2.4% of TSP compared to 0.8% of TSP when using the conventional method of a single topical application, and expression levels remained high 6 days post-induction. Overall production of rAAT in intact leaves was found to have a maximum level of 5.8% of TSP or 390 mg rAAT per kg leaf tissue when applying multiple injections of chemical induction solution.     

Deep metazoan phylogeny

We reconstructed a robust phylogenetic tree of the Metazoa, consisting of almost 1,500 taxa, by profile neighbor joining (PNJ), an automated computational method that inherits the efficiency of the neighbor joining algorithm. This tree supports the one proposed in the latest review on metazoan phylogeny. Our main goal is not to discuss aspects of the phylogeny itself, but rather to point out that PNJ can be a valuable tool when the basal branching pattern of a large phylogenetic tree must be estimated, whereas traditional methods would be computationally impractical.