Molecular cloning of a Brassica napus thiohydroximate S-glucosyltransferase gene and its expression in Escherichia coli

A genomic clone encoding a thiohydroximate S -glucosyltransferase ( S -GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers. Its corresponding cDNA was amplified by rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library screening. The genomic clone was 5 896 bp long and contained a 173-bp intron. At least two copies of the S -GT gene were present in B. napus . The full-length cDNA clone was 1.5 kb long and contained an open reading frame encoding a 51-kDa polypeptide. The deduced amino acid sequence shared a significant degree of homology with other glucosyltransferases characterized in other species, including a highly conserved motif within this family of enzymes corresponding to the glucose-binding domain. The recombinant protein was expressed in Escherichia coli , and the enzyme activity was tested by a biochemical assay based on the measure of glucose incorporation. The high thiohydroximate S -GT activity detected from the recombinant protein confirmed that this clone was indeed a S -glucosyltransferase.     

A desaturase-like protein from white spruce is a Delta(9) desaturase

Gymnospermae seed lipids are characterized by a high degree of desaturation, most having a Delta(9) double bond. By degenerate polymerase chain reaction (PCR) we have isolated a white spruce (Picea glauca) cDNA clone that encodes an amino acid sequence sharing a high degree of homology with other putative plant acyl-coenzyme A (CoA) Des9 desaturases. Both in vivo and in vitro expression studies in a Delta(9) desaturase-deficient yeast strain demonstrated the desaturation functionality of the white spruce clone, and gas chromatography-mass spectrometry (GC-MS) analyses confirmed the regioselectivity of the encoded enzyme. This is the first report of the functional characterization of a plant membrane-bound acyl-CoA-like protein Delta(9) desaturase by heterologous expression in yeast.     

Monitoring ungulates in steep non-forest habitat: a comparison of faecal pellet and helicopter counts

Faecal pellet counts have been widely used to monitor the abundances of introduced ungulates in New Zealand, but ground-based sampling cannot be conducted safely in the steep non-forest habitats that are common in New Zealand's Southern Alps. Helicopter counts may be an effective technique for monitoring ungulates in steep non-forest habitat. We evaluated the relationship between faecal pellet and helicopter counts of ungulates (primarily feral goat Capra hircus) at 12 non-forest sites in the Southern Alps. Within each site we counted the numbers of ungulates from a helicopter on three occasions and the number of intact faecal pellets along 30 transects. Mean observed densities of feral goats derived from helicopter counts ranged from 0.0 to 20.2 km^?2. There was a positive curvilinear (concave down) relationship between faecal pellet and helicopter counts. Compared with faecal pellet counts, helicopter counts were cheaper, could identify ungulate species and provided estimates of absolute density. Helicopter counts are a cost-effective method for monitoring ungulates in the steep non-forest habitats of New Zealand's Southern Alps.     

Sickle cell disease in mice is associated with sensitization of sensory nerve fibers

The pain phenotype in sickle cell disease (SCD) patients is highly variable. A small percentage of SCD patients experience many vaso-occlusive crises/year, 5% of patients account for over 30% of pain episodes, while 39% report few episodes of severe pain. Clearly, a better understanding of the pathobiology of SCD is needed to improve its therapy. Humanized sickle cell mice recapitulate several phenotypes of SCD patients and provide a model for the study of SCD pain. Researchers have shown that one strain of humanized SCD mice, the BERK strain, has abnormal pain phenotype. However, the nociception phenotype of another humanized SCD mouse strain, the Townes strain, has not been described. In a large cross-sectional study of BERK and Townes SCD mice, we examined thermosensory response and sensory nerve fiber function using sine-wave electrical stimulation at 2000, 250, and 5 Hz to stimulate preferentially Aβ, Aδ, and C sensory nerve fibers, respectively. We found that BERK and Townes mice, compared to respective controls, had decreases in 2000, 250, and 5 Hz current vocalization thresholds in patterns that suggest sensitization of a broad spectrum of sensory nerve fibers. In addition, the pattern of sensitization of sensory fibers varied according to strain, sex, age, and mouse genotype. In a similarly variable pattern, Townes and BERKs also had significantly altered sensitivity to noxious thermal stimuli in agreement with what has been shown by others. In summary, the analysis of somatosensory function using sine-wave electrical stimulation in humanized sickle cell mice suggests that in SCD, both myelinated and unmyelinated, fibers are sensitized. The pattern of sensory fiber sensitization is distinct from that observed in pain models of neuropathic and inflammatory pain. These findings raise the possibility that sensitization of a broad spectrum of sensory fibers might contribute to the altered and variable nociception phenotype in SCD.     

Production of fatty acid components of meadowfoam oil in somatic soybean embryos

The seed oil of meadowfoam (Limnanthes alba) and other Limnanthes spp. is enriched in the unusual fatty acid Delta(5)-eicosenoic acid (20:1Delta(5)). This fatty acid has physical and chemical properties that make the seed oil of these plants useful for a number of industrial applications. An expressed sequence tag approach was used to identify cDNAs for enzymes involved in the biosynthesis of 20:1Delta(5)). By random sequencing of a library prepared from developing Limnanthes douglasii seeds, a class of cDNAs was identified that encode a homolog of acyl-coenzyme A (CoA) desaturases found in animals, fungi, and cyanobacteria. Expression of a cDNA for the L. douglasii acyl-CoA desaturase homolog in somatic soybean (Glycine max) embryos behind a strong seed-specific promoter resulted in the accumulation of Delta(5)-hexadecenoic acid to amounts of 2% to 3% (w/w) of the total fatty acids of single embryos. Delta(5)-Octadecenoic acid and 20:1Delta(5) also composed <1% (w/w) each of the total fatty acids of these embryos. In addition, cDNAs were identified from the L. douglasii expressed sequence tags that encode a homolog of fatty acid elongase 1 (FAE1), a beta-ketoacyl-CoA synthase that catalyzes the initial step of very long-chain fatty acid synthesis. Expression of the L. douglassi FAE1 homolog in somatic soybean embryos was accompanied by the accumulation of C(20) and C(22) fatty acids, principally as eicosanoic acid, to amounts of 18% (w/w) of the total fatty acids of single embryos. To partially reconstruct the biosynthetic pathway of 20:1Delta(5) in transgenic plant tissues, cDNAs for the L. douglasii acyl-CoA desaturase and FAE1 were co-expressed in somatic soybean embryos. In the resulting transgenic embryos, 20:1Delta(5) and Delta(5)-docosenoic acid composed up to 12% of the total fatty acids.     

Parasites may help stabilize cooperative relationships

Background  

The persistence of cooperative relationships is an evolutionary paradox; selection should favor those individuals that exploit their partners (cheating), resulting in the breakdown of cooperation over evolutionary time. Our current understanding of the evolutionary stability of mutualisms (cooperation between species) is strongly shaped by the view that they are often maintained by partners having mechanisms to avoid or retaliate against exploitation by cheaters. In contrast, we empirically and theoretically examine how additional symbionts, specifically specialized parasites, potentially influence the stability of bipartite mutualistic associations. In our empirical work we focus on the obligate mutualism between fungus-growing ants and the fungi they cultivate for food. This mutualism is exploited by specialized microfungal parasites (genus Escovopsis) that infect the ant's fungal gardens. Using sub-colonies of fungus-growing ants, we investigate the interactions between the fungus garden parasite and cooperative and experimentally-enforced uncooperative ("cheating") pairs of ants and fungi. To further examine if parasites have the potential to help stabilize some mutualisms we conduct Iterative Prisoner's Dilemma (IPD) simulations, a common framework for predicting the outcomes of cooperative/non-cooperative interactions, which incorporate parasitism as an additional factor.     

Effects of antisense repression of an Arabidopsis thaliana pyruvate dehydrogenase kinase cDNA on plant development⋆

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase (PDH) complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. This report describes the cloning of a pyruvate dehydrogenase kinase cDNA (AtPDHK) from Arabidopsis thaliana and focuses on the effects of antisense down-regulation of its expression on plant growth and development. The deduced amino acid sequence of AtPDHK exhibits extensive similarity to other plant and mammalian PDHKs, containing conserved domains typical of two-component histidine protein kinases. The Escherichia coli expressed AtPDHK specifically phosphorylated mammalian PDH E1 in a time-dependent manner. Antisense expression of the AtPDHK cDNA led to marked elevation of mtPDC activity in transgenic plants with increases ranging from 137% to 330% compared to control plants. Immunoblot analyses performed with a monoclonal antibody to the E1 mtPDH component (the subunit phosphorylated by PDHK) indicated that the increased mtPDC activity was not the result of an increase in the level of PDH protein. MtPDC from transgenic plants showed a reduced sensitivity to ATP-dependent inactivation compared to that observed in wild-type plants. Collectively, these data suggest that the antisense partial silencing of the negative regulator, PDHK, was responsible for the increased mtPDC activity observed in the antisense PDHK plants. Transgenic plants with partially repressed AtPDHK also displayed altered vegetative growth with reduced accumulation of vegetative tissues, early flower development and shorter generation time. The potential role for AtPDHK gene manipulation in crop improvement is discussed.     

Production of 22:2<Superscript>Δ5,Δ13</Superscript> and 20:1<Superscript>Δ5</Superscript> in <Emphasis Type="Italic">Brassica carinata</Emphasis> and soybean breeding lines via introduction of <Emphasis Type="Italic">Limnanthes</Emphasis> genes

Seed oils of meadowfoam (Limnanthes douglasii, L. alba) contain very long-chain fatty acids of strategic importance for a number of industrial applications. These include the monoene 20 1⁵ and the diene 22:2^5,13. Engineering of meadowfoam-type oils in other oilseed crops is desirable for the production of these fatty acids as industrial feedstocks. Accordingly, we have targeted Brassica carinata and soybean (Glycine max) to trangenically engineer the biosynthesis of these unusual fatty acids. An L. douglasii seed-specific cDNA (designated Lim Des5) encoding a homolog of acyl-coenzyme A desaturases found in animals, fungi and cyanobacteria was expressed in B. carinata, which resulted in the accumulation of up to 10% 22:2^5,13 in the seed oil. In soybean, co-expression of Lim Des5 with a cDNA (Lim FAE1) encoding an FAEl (elongase complex condensing enzyme) homolog from L. douglasii resulted in the accumulation of 20:1⁵ to approximately 10% of the total fatty acids of seeds. The content of C₂₀ and C₂₂ fatty acids was also increased from <0.5% in non-transformed soybean seeds to >25% in seeds co-expressing the Lim. douglasii Des5 and FAE1 cDNAs. In contrast, expression of the Lim Des5 in Arabidopsis did not produce the expected 20:2^5,11 in the seed oil. Cumulatively, these results demonstrate the utility of soybean and B. carinata for the production of vegetable oils containing novel C₂₀ and C₂₂ fatty acids, and confirm that the preferred substrates of the Lim Des5 are 20:0 and 22:1³, respectively.     

Phylogenetic position of the acariform mites: sensitivity to homology assessment under total evidence

Background  

Mites (Acari) have traditionally been treated as monophyletic, albeit composed of two major lineages: Acariformes and Parasitiformes. Yet recent studies based on morphology, molecular data, or combinations thereof, have increasingly drawn their monophyly into question. Furthermore, the usually basal (molecular) position of one or both mite lineages among the chelicerates is in conflict to their morphology, and to the widely accepted view that mites are close relatives of Ricinulei.