Comparative genomic mapping between a 754 kb region flanking DREB1A in Arabidopsis thaliana and maize

Comparative mapping between model plant species for which the complete genome sequence is known and crop species has been suggested as a new strategy for the isolation of agronomically valuable genes. In this study, we tested whether comparative mapping between Arabidopsisand maize of a small region (754 kb) surrounding the DREB1A gene in Arabidopsis could lead to the identification of an orthologous region in maize containing the DREB1A homologue. The genomic sequence information available for Arabidopsis allowed for the selection of conserved, low-copy genes that were used for the identification of maize homologues in a large EST database. In total, 17 maize homologues were mapped. A second BLAST comparison of these genes to the recently completed Arabidopsis sequence revealed that 15 homologues are likely to be orthologous as the highest similarity score was obtained either with the original Arabidopsis gene or with a highly similar Arabidopsis gene localized on a duplication of the investigated region on chromosome 5. The map position of these genes showed a significant degree of orthology with the Arabidopsis region. Nevertheless, extensive duplications and rearrangements in the Arabidopsisand maize genomes as well as the evolutionary distance between Arabidopsis and maize make it unlikely that orthology and collinearity between these two species are sufficient to aid gene prediction and cloning in maize.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

A TGFβRII frameshift-mutation-derived CTL epitope recognised by HLA-A2-restricted CD8+ T cells

Microsatellite instability (MSI) is recognised as genome-wide alterations in repetitive DNA sequences caused by defects in the DNA mismatch repair machinery. Such mutation patterns have been found in almost all analysed malignancies from patients with hereditary non-polyposis colorectal cancer, and in approximately 15% of sporadic colorectal cancers. In cancers with the MSI phenotype, microsatellite-like sequences in coding regions of various cancer-related genes, including transforming growth factor beta receptor type II (TGF betaRII), are targets for mutations. The TGF betaRII gene harbours a 10-bp polyadenine tract, and mutations within this region are found in 90% of colorectal cancers with MSI. The frameshift mutations result in new amino acid sequences in the C-terminal part of the proteins, prematurely terminating where a novel stop codon appears. In this study we have defined new cytotoxic T lymphocyte (CTL) epitope (RLSSCVPVA), carrying a good HLA-A*0201 binding motif, and resulting from the most common frameshift mutation in TGF betaRII. A CTL line and several CTL clones were generated from an HLA-A2+ normal donor by repeated stimulation of T cells with dendritic cells pulsed with the peptide. One of the CTL clones was able to kill an HLA-A2+ colon cancer cell line harbouring mutant TGF betaRII. This epitope may be a valuable component in cancer vaccines directed at MSI-positive carcinomas.     

The Sub-Cellular Localization of WRAP53 Has Prognostic Impact in Breast Cancer

WRAP53 protein controls intracellular trafficking of DNA repair proteins, the telomerase enzyme, and splicing factors. Functional loss of the protein has been linked to carcinogenesis, premature aging and neurodegeneration. The aim of this study was to investigate the prognostic significance of WRAP53 protein expression in breast cancer. A tissue microarray was constructed from primary breast tumors and immunostained by a polyclonal WRAP53 antibody to assess the protein expression pattern. Two different patient cohorts with long term follow-up were studied; a test- and a validation set of 154 and 668 breast tumor samples respectively. Breast cancer patients with tumor cells lacking the expression of WRAP53 in the nucleus had a significantly poorer outcome compared to patients with tumor cells expressing this protein in the nuclei (HR = 1.95, 95%CI = 1.09–3.51, p = 0.025). Nuclear localization of WRAP53 was further shown to be an independent marker of prognosis in multivariate analysis (HR = 2.57, 95%CI = 1.27–5.19, p = 0.008), and also significantly associated with better outcome in patients with TP53 mutation. Here we show that the sub-cellular localization of the WRAP53 protein has a significant impact on breast cancer survival, and thus has a potential as a clinical marker in diagnostics and treatment.     

A meta-analysis of seaweed impacts on seagrasses: generalities and knowledge gaps

Seagrasses are important habitat-formers and ecosystem engineers that are under threat from bloom-forming seaweeds. These seaweeds have been suggested to outcompete the seagrasses, particularly when facilitated by eutrophication, causing regime shifts where green meadows and clear waters are replaced with unstable sediments, turbid waters, hypoxia, and poor habitat conditions for fishes and invertebrates. Understanding the situations under which seaweeds impact seagrasses on local patch scales can help proactive management and prevent losses at greater scales. Here, we provide a quantitative review of available published manipulative experiments (all conducted at the patch-scale), to test which attributes of seaweeds and seagrasses (e.g., their abundances, sizes, morphology, taxonomy, attachment type, or origin) influence impacts. Weighted and unweighted meta-analyses (Hedges d metric) of 59 experiments showed generally high variability in attribute-impact relationships. Our main significant findings were that (a) abundant seaweeds had stronger negative impacts on seagrasses than sparse seaweeds, (b) unattached and epiphytic seaweeds had stronger impacts than 'rooted' seaweeds, and (c) small seagrass species were more susceptible than larger species. Findings (a) and (c) were rather intuitive. It was more surprising that 'rooted' seaweeds had comparatively small impacts, particularly given that this category included the infamous invasive Caulerpa species. This result may reflect that seaweed biomass and/or shading and metabolic by-products like anoxia and sulphides could be lower for rooted seaweeds. In conclusion, our results represent simple and robust first-order generalities about seaweed impacts on seagrasses. This review also documented a limited number of primary studies. We therefore identified major knowledge gaps that need to be addressed before general predictive models on seaweed-seagrass interactions can be build, in order to effectively protect seagrass habitats from detrimental competition from seaweeds.     

RhoA guanine exchange factor expression profile in arteries: evidence for a Rho kinase-dependent negative feedback in angiotensin II-dependent hypertension

Sustained overactivation of RhoA is a common component for the pathogenesis of several cardiovascular disorders, including hypertension. Although activity of Rho proteins depends on Rho exchange factors (Rho-GEFs), the identity of Rho-GEFs expressed in vascular smooth muscle cells (VSMC) and participating in the control of Rho protein activity and Rho-dependent functions remains unknown. To address this question, we analyzed by quantitative RT-PCR the expression profile of 28 RhoA-GEFs in arteries of normotensive (saline-treated) and hypertensive (ANG II-treated) rats. Sixteen RhoA-GEFs were downregulated in mesenteric arteries of hypertensive rats, among which nine are also downregulated in cultured VSMC stimulated by ANG II (100 nM, 48 h), suggesting a direct effect of ANG II. Inhibition of type 1 ANG II receptors (losartan, 1 μM) or Rho kinase (fasudil, 10 μM) prevented ANG II-induced RhoA-GEF downregulation. Functionally, ANG II-induced downregulation of RhoA-GEFs is associated with decreased Rho kinase activation in response to endothelin-1, norepinephrine, and U-46619. This work thus identifies a group of RhoA-GEFs that controls RhoA and RhoA-dependent functions in VSMC, and a negative feedback of RhoA/Rho kinase activity on the expression of these RhoA-GEFs that may play an adaptative role to limit RhoA/Rho kinase activation.     

Nitrogen uptake and utilisation as a competition factor between invasive Duchesnea indica and native Fragaria vesca

The Indian mock strawberry [Duchesnea indica (Andrews) Focke] is an invasive plant in several regions of central Europe and Germany. In order to explore its competitive ability, we compared it with the native woodland strawberry (Fragaria vesca L.) by growing it alone as well as in intra- or inter-specific competition in a pot experiment under greenhouse conditions. Nutrient solution was added several times at two nitrogen (N) levels. One addition involved ¹⁵N labelling to determine whether the competition of both plant species depends on their ability to acquire N from soil. Duchesnea had a higher biomass production than Fragaria when grown in nutrient-rich soil, both in competition and as a solitary plant. Under N-poor conditions, root interference could change this superiority due to limited soil space. After 65 days of growth, total plant dry weight, total N content and ¹⁵N content in the plant tissues were determined. The results show that the predominance of Duchesnea in biomass production was confirmed at high, but not at low N availability. The assimilate partitioning strategy of Duchesnea differs from that of Fragaria: the former generally had a higher shoot-to-root ratio. The N content in shoots and roots was affected only by N addition but not by competition or species. Duchesnea allocated more N to the leaves, Fragaria to the roots. The amount of ¹⁵N taken up was nearly equal for both species. In relation to root biomass, Duchesnea had a higher specific uptake rate at low N addition because of the higher root biomass in Fragaria. The roots of Fragaria and Duchesnea did not affect each other when grown together. We conclude that the invasive potential of Duchesnea is only poorly related to the N uptake rate or to better root competition for N. In N-rich environments, however, Duchesnea is highly competitive because of the preferred investment in shoot biomass. Therefore, environments with increased N deposition, i.e. from anthropogenic sources, could promote the invasive potential of Duchesnea.     

MGOUN3: evidence for chromatin-mediated regulation of FLC expression

The MGOUN3(MGO3)/BRUSHY1(BRU1)/TONSOKU(TSK) gene of Arabidopsis thaliana encodes a nuclear leucine-glycine-asparagine (LGN) domain protein that may be implicated in chromatin dynamics and genome maintenance. Mutants with defects in MGO3 display a fasciated stem and disorganized meristem structures. The transition to flowering was examined in mgo3 mutants and it was found that, under short days, the mutants flowered significantly earlier than the wild-type plants. Study of flowering-time associated gene expression showed that the floral transition inhibitor gene FLC was under-expressed in the mutant background. Ectopic expression of the flower-specific genes AGAMOUS (AG), PISTILLATA (PI), and SEPALLATA3 (SEP3) in mgo3 vegetative organs was also detected. Western blot and chromatin immunoprecipitation experiments suggested that histone H3 acetylation may be altered in the mgo3 background. Together, these data suggest that MGO3 is required for the correct transition to flowering and that this may be mediated by histone acetylation and associated changes in FLC expression.