Expression of RPS4 in tobacco induces an AvrRps4-independent HR that requires EDS1, SGT1 and HSP90

The Arabidopsis RPS4 gene belongs to the Toll/interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) class of plant resistance (R) genes. It confers resistance to Pseudomonas syringae carrying the avirulence gene avrRps4. Transient expression of genomic RPS4 driven by the 35S promoter in tobacco leaves induces an AvrRps4-independent hypersensitive response (HR). The same phenotype is seen after expression of a full-length RPS4 cDNA. This indicates that alternative splicing of RPS4 is not involved in this HR. The extent of HR is correlated with RPS4 protein levels. Deletion analyses of RPS4 domains show the TIR domain is required for the HR phenotype. Mutations in the P-loop motif of the NB domain abolish the HR. Using virus-induced gene silencing, we found that the cell death resulting from RPS4 expression is dependent on the three plant signalling components EDS1, SGT1 and HSP90. All these data suggest that heterologous expression of an R gene can result in activation of cell death even in the absence of its cognate avirulence product, and provides a system for studying the RPS4 domains required for HR.     

The tomato homolog of the gene encoding UV-damaged DNA binding protein 1 (DDB1) underlined as the gene that causes the<Emphasis Type="Italic"> high pigment-1</Emphasis> mutant phenotype

A tomato EST sequence, highly homologous to the human and Arabidopsis thaliana UV-damaged DNA binding protein 1 (DDB1), was mapped to the centromeric region of the tomato chromosome 2. This region was previously shown to harbor the HP-1 gene, encoding the high pigment-1 (hp-1) and the high pigment-1^w (hp-1^w) mutant phenotypes. Recent results also show that the A. thaliana DDB1 protein interacts both genetically and biochemically with the protein encoded by DEETIOLATED1, a gene carrying three tomato mutations that are in many respects isophenotypic to hp-1: high pigment-2 (hp-2), high pigment-2^j (hp-2^j) and dark green (dg). The entire coding region of the DDB1 gene was sequenced in an hp-1 mutant and its near-isogenic normal plant in the cv. Ailsa Craig background, and also in an hp-1^w mutant and its isogenic normal plant in the GT breeding line background. Sequence analysis revealed a single A⁹³¹-to-T⁹³¹ base transversion in the coding sequence of the DDB1 gene in the hp-1 mutant plants. This transversion results in the substitution of the conserved asparagine at position 311 to a tyrosine residue. In the hp-1^w mutant, on the other hand, a single G²³⁹²-to-A²³⁹² transition was observed, resulting in the substitution of the conserved glutamic acid at position 798 to a lysine residue. The single nucleotide polymorphism that differentiates hp-1 mutant and normal plants in the cv. Ailsa Craig background was used to design a pyrosequencing genotyping system. Analysis of a resource F₂ population segregating for the hp-1 mutation revealed a very strong linkage association between the DDB1 locus and the photomorphogenic response of the seedlings, measured as hypocotyl length (25<LOD score<26, R²=62.8%). These results strongly support the hypothesis that DDB1 is the gene encoding the hp-1 and hp-1^w mutant phenotypes.Communicated by R. Hagemann     

The A locus that controls anthocyanin accumulation in pepper encodes a MYB transcription factor homologous to Anthocyanin2 of Petunia

Pepper plants containing the dominant A gene accumulate anthocyanin pigments in the foliage, flower and immature fruit. We previously mapped A to pepper chromosome 10 in the F₂ progeny of a cross between 5226 (purple-fruited) and PI 159234 (green-fruited) to a region that corresponds, in tomato, to the location of Petunia anthocyanin 2 (An2), a regulator of anthocyanin biosynthesis. This suggested that A encodes a homologue of Petunia An2. Using the sequences of An2 and a corresponding tomato expressed sequence tag, we isolated a pepper cDNA orthologous to An2 that cosegregated with A. We subsequently determined the expression of A by Northern analysis, using RNA extracted from fruits, flowers and leaves of 5226 and PI 159234. In 5226, expression was detected in all stages of fruit development and in both flower and leaf. In contrast, A was not expressed in the sampled tissues in PI 159234. Genomic sequence comparison of A between green- and purple-fruited genotypes revealed no differences in the coding region, indicating that the lack of expression of A in the green genotypes can be attributed to variation in the promoter region. By analyzing the expression of the structural genes in the anthocyanin biosynthetic pathway in 5226 and PI 159234, it was determined that, similar to Petunia, the early genes in the pathway are regulated independently of A, while expression of the late genes is A-dependent.Communicated by R. Hagemann     

Restoration of gene function by homologous recombination: from PCR to gene expression in one step

We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete lambdaPR or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage lambda Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding beta-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.     

Aminoimidazo[1,2-a]pyridines as a new structural class of cyclin-dependent kinase inhibitors. Part 1: Design, synthesis, and biological evaluation

We have identified a novel structural class of protein serine/threonine kinase inhibitors comprised of an aminoimidazo[1,2-a]pyridine nucleus. Compounds from this family are shown to potently inhibit cyclin-dependent kinases by competing with ATP for binding to a catalytic subunit of the protein. Structure-based design approach was used to direct this chemical scaffold toward generating potent and selective CDK2 inhibitors. The discovery of this new class of ATP-site directed protein kinase inhibitors, aminoimidazo[1,2-a]pyridines, provides the basis of new medicinal chemistry tool in search for an effective treatment of cancer and other diseases that involve protein kinase signaling pathways.     

Global proteomic approach unmasks involvement of keratins 8 and 18 in the delivery of cystic fibrosis transmembrane conductance regulator (CFTR)/deltaF508-CFTR to the plasma membrane

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF gene (cftr). Physiologically, CF is characterized by an abnormal chloride secretion in epithelia due to a dysfunction of a mutated cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a cAMP-dependent chloride channel whose most frequent mutation, deltaF508, leads to an aberrantly folded protein which causes a dysfunction of the channel. However, a growing number of reports suggest that modifier genes and environmental factors are involved in the physiology of CF. To identify proteins whose expression depends on wild-type WT-CFTR or deltaF508-CFTR, we chose a global proteomic approach based on the use of two-dimensional gel electrophoresis (2-DE) and mass spectrometry. The experiments were carried out with HeLa cells stably transfected with WT-CFTR (pTCFWT) or deltaF508-CFTR (pTCFdeltaF508). These experiments unmasked keratin 8 (K8) and 18 (K18) which were differentially expressed in pTCFWT vs. pTCFdeltaF508. An immunoblot of K18 confirmed the 2-DE results. Intracellular localization experiments of WT-CFTR, deltaF508-CFTR, K8, and K18 suggest that the expression of these proteins are linked, and that the concentrations of K8 and K18 and/or their distribution may be involved in the traffic of WT-CFTR/deltaF508-CFTR. A functional assay for CFTR revealed that specifically lowering K18 expression or changing its distribution leads to the delivery of functional deltaF508-CFTR to the plasma membrane. This work suggests a novel function of K18 in CF.     

Mass spectrometric analysis of Plasmodium falciparum erythrocyte membrane protein-1 variants expressed by placental malaria parasites

Surface proteins from Plasmodium falciparum are important malaria vaccine targets. However, the surface proteins previously identified are highly variant and difficult to study. We used tandem mass spectrometry to characterize the variant antigens (Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)) expressed on the surface of malaria-infected erythrocytes that bind to chondroitin sulfate A (CSA) in the placenta. Whereas PfEMP1 variants previously implicated as CSA ligands were detected, in unselected parasites four novel variants were detected in CSA-binding or placental parasites but not in unselected parasites. These novel PfEMP1 variants require further study to confirm whether they play a role in placental malaria.     

Activation and inhibition of cyclin-dependent kinase-2 by phosphorylation; a molecular dynamics study reveals the functional importance of the glycine-rich loop

Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin-dependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160-CDK2/Cyclin A/ATP, inhibited pT14-; pY15-; and pT14,pY15,pT160-CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine-rich loop (G-loop) positioned opposite the activation T-loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G-loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho-group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X-ray data.     

PANDORA: keyword-based analysis of protein sets by integration of annotation sources

Recent advances in high-throughput methods and the application of computational tools for automatic classification of proteins have made it possible to carry out large-scale proteomic analyses. Biological analysis and interpretation of sets of proteins is a time-consuming undertaking carried out manually by experts. We have developed PANDORA (Protein ANnotation Diagram ORiented Analysis), a web-based tool that provides an automatic representation of the biological knowledge associated with any set of proteins. PANDORA uses a unique approach of keyword-based graphical analysis that focuses on detecting subsets of proteins that share unique biological properties and the intersections of such sets. PANDORA currently supports SwissProt keywords, NCBI Taxonomy, InterPro entries and the hierarchical classification terms from ENZYME, SCOP and GO databases. The integrated study of several annotation sources simultaneously allows a representation of biological relations of structure, function, cellular location, taxonomy, domains and motifs. PANDORA is also integrated into the ProtoNet system, thus allowing testing thousands of automatically generated clusters. We illustrate how PANDORA enhances the biological understanding of large, non-uniform sets of proteins originating from experimental and computational sources, without the need for prior biological knowledge on individual proteins.