Can we stop transgenes from taking a walk on the wild side?

Whether the potential costs associated with broad‐scale use of genetically modified organisms (GMOs) outweigh possible benefits is highly contentious, including within the scientific community. Even among those generally in favour of commercialization of GM crops, there is nonetheless broad recognition that transgene escape into the wild should be minimized. But is it possible to achieve containment of engineered genetic elements in the context of large scale agricultural production? In a previous study, Warwick et al. (2003) documented transgene escape via gene flow from herbicide resistant (HR) canola (Brassica napus) into neighbouring weedy B. rapa populations ( Fig. 1 ) in two agricultural fields in Quebec, Canada. In a follow‐up study in this issue of Molecular Ecology, Warwick et al. (2008) show that the transgene has persisted and spread within the weedy population in the absence of selection for herbicide resistance. Certainly a trait like herbicide resistance is expected to spread when selected through the use of the herbicide, despite potentially negative epistatic effects on fitness. However, Warwick et al.'s findings suggest that direct selection favouring the transgene is not required for its persistence. So is there any hope of preventing transgene escape into the wild?

Figure 1 Open in figure viewer PowerPoint Weedy Brassica rapa (orange flags) growing in a B. napus field. (Photo: MJ Simard)     

Familial Chordoma, a Tumor of Notochordal Remnants, Is Linked to Chromosome 7q33

Chordoma is a rare tumor originating from notochordal remnants that is usually diagnosed during midlife. We performed a genomewide analysis for linkage in a family with 10 individuals affected by chordoma. The maximum two-point LOD score based on only the affected individuals was 2.21, at recombination fraction 0, at marker D7S2195 on chromosome 7q. Combined analysis of additional members of this family (11 affected individuals) and of two unrelated families (one with 2 affected individuals and the other with 3 affected individuals), with 20 markers on 7q, showed a maximum two-point LOD score of 4.05 at marker D7S500. Multipoint analysis based on only the affected individuals gave a maximum LOD score of 4.78, with an approximate 2-LOD support interval from marker D7S512 to marker D7S684. Haplotype analysis of the three families showed a minimal disease-gene region from D7S512 to D7S684, a distance of 11.1 cM and approximately 7.1 Mb. No loss of heterozygosity was found at markers D7S1804, D7S1824, and D7S2195 in four tumor samples from affected family members. These results map a locus for familial chordoma to 7q33. Further analysis of this region, to identify this gene, is ongoing.     

Neisseria meningitidis App,a new adhesin with autocatalytic serine protease activity

Neisseria meningitidis is a Gram-negative bacterium which colonizes the human upper respiratory tract. Occasionally, it translocates to the bloodstream causing sepsis and from there it can cross the blood-brain barrier and cause meningitis. Many of the molecules, which mediate the interaction of N. meningitidis to host cells, are still unknown. Recently, App (Adhesion and penetration protein) was described as a member of the autotransporter family and a homologue to the Hap (Haemophilus adhesion and penetration) protein of Haemophilus influenzae, a molecule that plays a role in the interaction with human epithelial cells. In this study we expressed app in Escherichia coli in order to analyse the functional properties of the protein. We show that the protein is exported to the E. coli surface, processed by an endogenous serine-protease activity and released in the culture supernatant. Escherichia coli expressing app adhere to Chang epithelial cells, showing that App is able to mediate bacterial adhesion to host cells. The serine protease activity is localized at the amino-terminal domain, whereas the binding domain is in the carboxy-terminal region. The role of App in adhesion was confirmed also in N. meningitidis.     

HPLC isolation of the anti-plasmodially active bisbenzylisoquinone alkaloids present in roots of Cissampelos mucronata

The methanolic extract of dried, powdered Cissampelos mucronata roots possesses significant in vitro activity against Plasmodium falciparum. In order to enable further pharmacological testing, the substances responsible for the observed activity were purified, mainly by HPLC, using various stationary and mobile phases. The active principles were determined to be a series of bisbenzylisoquinoline alkaloids, a group of natural products for which one of the first routine preparative HPLC separation methods is described.     

The avian chB6 alloantigen induces apoptosis in DT40 B cells

In avian species, B-lymphocytes develop in the bursa of Fabricius. Cells developing in the bursa are subject to signals regulating their survival, with the majority of cells dying by apoptosis within the bursa. However, the molecules delivering the signals influencing this life and death decision remain enigmatic. We have previously shown that antibodies against the chB6 alloantigen present on avian B-lymphocytes can induce a rapid form of cell death. Here we extend this finding by showing that anti-chB6 antibodies induce true apoptosis in DT40 cells without visible membrane damage. This apoptosis results in DNA degradation and morphologic changes characteristic of apoptosis. Furthermore, this apoptosis is coincident with a loss of mitochondrial membrane potential and is inhibited by either overexpression of bcl-x(L) or the presence of inhibitors of caspase 8, 9, or 3 activity. Collectively these data argue that chB6 may function as a novel death receptor on avian B-lymphocytes and support the use of DT40 as an amenable model to study the signaling involved in chB6-induced apoptosis.     

Involvement of a sialic acid-binding lectin with hemagglutination and hydrophobicity of Flavobacterium psychrophilum

Strains of Flavobacterium psychrophilum were studied for their ability to adhere and cause agglutination of erythrocytes and yeast cells. Strains of the serotype Th showed low or no hemagglutinating (HA) properties toward human, avian, bovine, and rainbow trout erythrocytes, whereas strains of serotype Fd and Fp(T) exhibited distinct HA properties. None of the strains was able to cause agglutination of yeast cells. Greater adherence specificity toward rainbow trout blood cells was seen for the HA-positive strains. Growth at 5 degrees C, compared to that at 15 degrees C, induced an increase in the hemagglutination of some strains. HA activities of F. psychrophilum were inhibited only by sialic acid (N-acetyl-neuraminic acid), heat treatment at 65 degrees C, and proteinase K treatment and not by any of seven other carbohydrates, periodate oxidation, or treatment with trypsin. The supernatant from washed bacterial cells also showed some HA properties. All strains were shown to be highly hydrophobic by the hydrophobic interaction chromatography test, although some contradictions to the results of the salt aggregation test (showing some strains as less hydrophobic) were seen. These results indicate that the aggregation of F. psychrophilum and erythrocytes depend on a lectin present on the surface of HA-positive F. psychrophilum strains and absent on HA-negative strains. This lectin reacts specifically with sialic acid. The adhesion differences observed for F. psychrophilum strains do not appear to correlate with the virulence but still provide insights into the interaction of F. psychrophilum and rainbow trout.     

Proteomic analysis of the effect of heat stress on hexaploid wheat grain: Characterization of heat-responsive proteins from total endosperm

High temperatures during grain filling have been reported to be one of the factors that can affect the dough properties and quality characteristics of wheat. Responses to high temperature have been related to changes in protein composition at both quantitative and qualitative levels. The present study was conducted to determine the influence of high temperature during grain filling on the protein composition of bread wheat evaluated by proteomic tools. Plants were grown in the field and transferred to cabinets soon after flowering. They were subjected to two thermal regimes 18 degrees C/10 degrees C (day/night) and 34 degrees C/10 degrees C. Total proteins were extracted from control grains and treated plants at three different post-anthesis stages. The proteins were separated by two-dimensional gel electrophoresis and analysed by Melanie 3 software. Of the total number of mature wheat grain proteins, 37 were identified as significantly changed by heat treatment. Analysis by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry coupled with database searching allowed the characterization of 25 heat-induced proteins and only one heat-decreased protein spot. To learn more about the function of the identified proteins, we examined their expression during treatment.     

A specific enzymatic high-performance liquid chromatography method to determine N-methyl-D-aspartic acid in biological tissues

Recently we demonstrated that N-methyl-D-aspartic acid (NMDA) is present as an endogenous compound in the nervous tissues and endocrine glands of the rat where it plays a role in the regulation of the luteinizing hormone, growth hormone, and prolactin (FASEB J. 14 (2000) 699; Endocrinology 141 (2000) 3861). Based on the prediction that NMDA could have future importance in neuroendocrinology, we have devised an improved method for the specific and routine determination of NMDA in biological tissue. This method is based on the detection by HPLC of methylamine (CH(3)NH(2)) which comes from the oxidation of NMDA by D-aspartate oxidase, an enzyme which specifically oxidizes NMDA, yielding CH(3)NH(2) as one of the oxidative products of the reaction. The sensitivity of the method permits the accurate determination of NMDA in the supernatant of a tissue homogenate at levels of about 5-10 picomol/assay. However, for those tissues in which the concentration of NMDA is less than 1nmol/g, the sample must be further purified by treatment with o-phthaldialdehyde in order to separate the NMDA from the other amino acids and amino compounds and then concentrated and analyzed by HPLC. Using this method we have conducted a comparative study in order to measure the amount of NMDA in neuroendocrine and other tissues of various animal phyla from mollusks to mammals.     

The projection domain of MAP4 suppresses the microtubule-bundling activity of the microtubule-binding domain

Microtubule-associated protein 4 (MAP4), a major MAP expressed in proliferating non-neuronal cells, consists of an N-terminal projection (PJ) domain and a C-terminal microtubule-binding (MTB) domain. The PJ domain of MAP4 is divided into three regions; the N-terminal acidic region (the Na-region), the multiple KDM-repeated sequence region (the KDM-region), and the b-region followed by the MTB domain. To investigate roles of the PJ domain, we prepared three truncated forms of human MAP4 with different PJ domain lengths; PJ1, PJ2 and MTB with deletion of about one-third, two-third and all of the PJ domain, respectively, and examined their effects on bundle formation of microtubules (MTs). MTs polymerized by full length MAP4 were singly distributed as observed by both negative staining electron microscopy and dark field microscopy. MTs with PJ1 were also separated in solution but became pairs when pelleted by centrifugation. PJ2 formed planar two-dimensional bundles consisting of several MTs (the 2D-bundle). MTB induced large bundles of many MTs, tightly packed without space in between (termed the 3D-bundle). To study how the PJ domain decreases the bundle-forming activity of the MTB domain of MAP4, we made three additional deletion-mutants of MAP4, called Na-MTB, KDM-MTB and Na-PJ2. Na-MTB and KDM-MTB, in which the KDM/b-region and both of Na- and b-regions were deleted respectively, were prepared by fusing the Na-region or KDM-region to MTB. Both of Na-MTB and KDM-MTB suppressed the 3D-bundle formation as effectively as PJ2. MTs polymerized with Na-PJ2, the KDM-deletion mutant made by adding the Na-region to PJ2, were singular and did not become bundles. These results indicated that the PJ domain kept individual MTs separated by suppressing the bundle-forming ability of the MTB domain. The suppressive activity of the PJ domain was correlated with the length, but not the amino acid sequence, of the PJ.