The polypyrimidine tract binding protein (PTB) represses splicing of exon 6B from the beta-tropomyosin pre-mRNA by directly interfering with the binding of the U2AF65 subunit

Splicing of exon 6B from the beta-tropomyosin pre-mRNA is repressed in nonmuscle cells and myoblasts by a complex array of intronic elements surrounding the exon. In this study, we analyzed the proteins that mediate splicing repression of exon 6B through binding to the upstream element. We identified the polypyrimidine tract binding protein (PTB) as a component of complexes isolated from myoblasts that assemble onto the branch point region and the pyrimidine tract. In vitro splicing assays and PTB knockdown experiments by RNA interference demonstrated that PTB acts as a repressor of splicing of exon 6B. Using psoralen experiments, we showed that PTB acts at an early stage of spliceosome assembly by preventing the binding of U2 snRNA on the branch point. Using UV cross-linking and immunoprecipitation experiments with site-specific labeled RNA in PTB-depleted nuclear extracts, we found that the decrease in PTB was correlated with an increase in U2AF65. In addition, competition experiments showed that PTB is able to displace the binding of U2AF65 on the polypyrimidine tract. Our results strongly support a model whereby PTB competes with U2AF65 for binding to the polypyrimidine tract.     

Structural variation and evolution of a defense-gene cluster in natural populations of Aegilops tauschii

Genetic mapping and sequencing of plant genomes have been useful for investigating eukaryotic chromosome structural organization. In many cases, analyses have been limited in the number of representatives sampled from specific groups. The degree of intraspecific genome diversity remains in question. The possibility exists that a single model genome may have limited utility for identifying genes in related members of the species or genus. Crop improvement programs have particular interests in disease resistance genes that are harbored by wild relatives of modern cultivated crops. These genes are evolutionarily dynamic and under selective pressure by a broad range of pathogenic organisms. Using resistance gene analogs as models for gene evolution, intraspecific genome comparisons were made among populations of wild diploid wheat (Aegilops tauschii). We observed that deletion haplotypes are occurring frequently and independently in the genome. Haplotypes are geographically correlated and maintenance of gene complements in localized populations indicates selective advantage. Furthermore, deletion haplotypes are not detrimental to plant health, since genes without adaptive value in alternate environments are eliminated from the genome. Deletion haplotypes appear to be a common form of allelic variation in plants, and we address the consequences on genome restructuring and gene evolution. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Dissection of the nuclear genome of barley by chromosome flow sorting

Isolation of mitotic chromosomes using flow cytometry is an attractive way to dissect nuclear genomes into their individual chromosomal components or portions of them. This approach is especially useful in plants with complex genomes, where it offers a targeted and hence economical approach to genome analysis and gene cloning. In several plant species, DNA of flow-sorted chromosomes has been used for isolation of molecular markers from specific genome regions, for physical mapping using polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), for integration of genetic and physical maps and for construction of chromosome-specific DNA libraries, including those cloned in bacterial artificial chromosome vectors. Until now, chromosome analysis and sorting using flow cytometry (flow cytogenetics) has found little application in barley (2n = 14, 1C ∼ 5,100 Mbp) because of the impossibility of discriminating and sorting individual chromosomes, except for the smallest chromosome 1H and some translocation chromosomes with DNA content significantly different from the remaining chromosomes. In this work, we demonstrate that wheat–barley ditelosomic addition lines can be used to sort any arm of barley chromosomes 2H–7H. Thus, the barley genome can be dissected into fractions representing only about 6–12% of the total genome. This advance makes the flow cytogenetics an attractive tool, which may greatly facilitate genome analysis and gene cloning in barley.     

The urokinase/PAI-2 complex: a new high affinity ligand for the endocytosis receptor low density lipoprotein receptor-related protein

The efficient inactivation of urokinase plasminogen activator (uPA) by plasminogen activator inhibitor type 2 (PAI-2) at the surface of carcinoma cells is followed by rapid endocytosis of the uPA-PAI-2 complex. We now show that one pathway of this receptor-mediated endocytosis is mediated via the low density lipoprotein receptor-related protein (LRP) in prostate cancer cells. Detailed biochemical analyses using ligand binding assays and surface plasmon resonance revealed a novel and distinct interaction mechanism between native, human LRP and uPA-PAI-2. As reported previously for PAI-1, inhibition of uPA by PAI-2 significantly increased the affinity of the complex for LRP (K(D) of 36 nm for uPA-PAI-2 versus 200 nm for uPA). This interaction was maintained in the presence of uPAR, confirming the validity of this interaction at the cell surface. However, unlike PAI-1, no interaction was observed between LRP and PAI-2 in either the stressed or the relaxed conformation. This suggests that the uPA-PAI-2-LRP interaction is mediated by site(s) within the uPA molecule alone. Thus, as inhibition of uPA by PAI-2 resulted in accelerated clearance of uPA from the cell surface possibly via its increased affinity for LRP, this represents a mechanism through which PAI-2 can clear proteolytic activity from the cell surface. Furthermore, lack of a direct interaction between PAI-2 and LRP implies that downstream signaling events initiated by PAI-1 may not be activated by PAI-2.     

Divergence in the plasminogen-binding group a streptococcal M protein family: functional conservation of binding site and potential role for immune selection of variants

Group A streptococci (GAS) display receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M protein (PAM). Characterization of PAM genes from 12 GAS isolates showed significant variation within the plasminogen-binding repeat motifs (a1/a2) of this protein. To determine the impact of sequence variation on protein function, recombinant proteins representing five naturally occurring variants of PAM, together with a recombinant M1 protein, were expressed and purified. Equilibrium dissociation constants for the interaction of PAM variants with biotinylated Glu-plasminogen ranged from 1.58 to 4.99 nm. Effective concentrations of prototype PAM required for 50% inhibition of plasminogen binding to immobilized PAM variants ranged from 0.68 to 22.06 nm. These results suggest that although variation in the a1/a2 region of the PAM protein does affect the comparative affinity of PAM variants, the functional capacity to bind plasminogen is conserved. Additionally, a potential role for the a1 region of PAM in eliciting a protective immune response was investigated by using a mouse model for GAS infection. The a1 region of PAM was found to protect immunized mice challenged with a PAM-positive GAS strain. These data suggest a link between selective immune pressure against the plasminogen-binding repeats and the functional conservation of the binding domain in PAM variants.     

Head-tail patterning of the vertebrate embryo: one, two or many unresolved problems?

When, where and how is the head-tail axis of the embryo set up during development? These are such fundamental and intensely studied questions that one might expect them to have been answered long ago. Not so; we still understand very little about the cellular or molecular mechanisms that lead to the orderly arrangement of body elements along the head-tail axis in vertebrates. In this paper, we outline some of the major outstanding problems and controversies and try to identify some reasons why it has been so difficult to resolve this important issue.     

Cryopreservation of economically valuable marine micro-algae in the classes Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Haptophyceae, Prasinophyceae, and Rhodophyceae

The ability to routinely cryopreserve micro-algal species reduces costs associated with maintaining large culture collections and reduces the risks of losing particular strains or species through contamination and genetic drift. Cryopreservation is also a useful adjunct in aquaculture hatcheries for strains of micro-algae where the nutritional status may change as a result of continuous sub-culture. In this study, cryopreservation of isolates from seven micro-algal classes was investigated. Successful candidates included the marine dinoflagellates Amphidinium carterae, Amphidinium trulla, and Gymnodinium simplex, and the haptophytes Chrysochromulina simplex, Prymnesium parvum, Prymnesium parvum f. patelliferum, Isochrysis galbana, and Pavlova lutheri. Also successfully cryopreserved were the planktonic diatoms Chaetoceros calcitrans, Chaetoceros muelleri, Chaetoceros sp., and the benthic Nitzschia ovalis, the chlorophyte Chlamydomonas coccoides, the rhodophyte Porphyridium purpureum, the prasinophytes Tetraselmis chuii, and Tetraselmis suecica, and the cyanophytes Raphidiopsis sp., and Aphanizomenon flos-aquae. All species were successfully cryopreserved using 15% Me2SO.