Mapping the heparin-binding site on the 13-14F3 fragment of fibronectin

Fibronectin, a multifunctional glycoprotein of the extracellular matrix, plays a major role in cell adhesion. Various studies have revealed that the human 13th and 14th fibronectin type III domains (labeled (13)F3 and (14)F3 here) contain a heparin-binding site. Mapping of the heparin-binding sites of (13-14)F3, (13)F3, and (14)F3 by NMR chemical shift perturbation, isothermal titration calorimetry, and molecular modeling show that (13)F3 provides the dominant heparin-binding site and that the residues involved are within the first 29 amino acids of (13)F3. Predictions from earlier biochemical and modeling studies as well as the x-ray structure of (12-14)F3 were tested. It was shown that the positively charged residues that project into the solvent from the ABE face of the triple-stranded beta sheet on (13)F3 are involved in binding, but (14)F3 does not appear to contribute significantly to heparin binding.     

Functional characterization of Drosophila melanogaster peptide O-xylosyltransferase, the key enzyme for proteoglycan chain initiation and member of the core 2/I N-acetylglucosaminyltransferase family

Chondroitin and heparan sulfates are essential players in animal development and are synthesized by a series of glycosyltransferases, the first of which is UDP-alpha-D-xylose:proteoglycan core protein beta-D-xylosyltransferase (EC ). In the present study, a Drosophila melanogaster gene (CG17771), previously designated as a homologue of core 2 and I beta1,6-N-acetylglucosaminyltransferases, was shown to encode an active peptide O-xylosyltransferase. A novel coupled assay using matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated transfer of xylose to the peptide DDDSIEGSGGR. Analysis of sequences of various peptide O-xylosyltransferase and beta1,6-N-acetylglucosaminyltransferase sequences indicates that they are members of a large multifunctional protein family with a range of roles in beta-glycosylation of either peptide or glycan substrates. Because in contrast to mammals, there is only one fly peptide O-xylosyltransferase gene, it is anticipated that, given the key roles of proteoglycans, the hereby designated oxt gene is essential for viability.     

FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter

We have applied laser UV photo-footprinting to characterise kinetically complexes involving the activator protein FIS, RNA polymerase and the tyrT promoter of Escherichia coli. FIS photo-footprints strongly to three binding sites upstream of the core promoter. The polymerase photo-footprints in the near-consensus -35 hexamer on the non-template strand of DNA in a fashion similar to that of stable complexes involving the lacUV5 promoter. The kinetics of the interactions of polymerase alone with the tyrT promoter differ from those observed previously at the lacUV5 promoter. In the absence of FIS, we observe an upstream polymerase-induced signal at -122 within FIS site III that occurs subsequent to changes in the core promoter region and is strongly dependent on negative supercoiling. These observations support the proposal that the upstream region of the promoter is wrapped around the polymerase. We propose that the wrapped DNA allows the polymerase to overcome, at least in part, the barrier to DNA untwisting imparted by the G+C-rich discriminator. We further suggest that FIS plays a similar role and may facilitate polymerase escape.     

Borna disease virus accelerates inflammation and disease associated with transgenic expression of interleukin-12 in the central nervous system

Targeted expression of biologically active interleukin-12 (IL-12) in astrocytes of the central nervous system (CNS) results in spontaneous neuroimmunological disease of aged mice. Borna disease virus (BDV) can readily multiply in the mouse CNS but does not trigger disease in most strains. Here we show that a large percentage of IL-12 transgenic mice developed severe ataxia within 5 to 10 weeks after infection with BDV. By contrast, no disease developed in mock-infected IL-12 transgenic and wild-type mice until 4 months of age. Neurological symptoms were rare in infected wild-type animals, and if they occurred, these were milder and appeared later. Histological analyses showed that the cerebellum of infected IL-12 transgenic mice, which is the brain region with strongest transgene expression, contained large numbers of CD4(+) and CD8(+) T cells as well as lower numbers of B cells, whereas other parts of the CNS showed only mild infiltration by lymphocytes. The cerebellum of diseased mice further showed severe astrogliosis, calcifications and signs of neurodegeneration. BDV antigen and nucleic acids were present in lower amounts in the inflamed cerebellum of infected transgenic mice than in the noninflamed cerebellum of infected wild-type littermates, suggesting that IL-12 or IL-12-induced cytokines exhibited antiviral activity. We propose that BDV infection accelerates the frequency by which immune cells such as lymphocytes and NK cells enter the CNS and then respond to IL-12 present in the local milieu causing disease. Our results illustrate that infection of the CNS with a virus that is benign in certain hosts can be harmful in such normally disease-resistant hosts if the tissue is unfavorably preconditioned by proinflammatory cytokines.     

Physical and genetic mapping in the grasses Lolium perenne and Festuca pratensis

A single chromosome of the grass species Festuca pratensis has been introgressed into Lolium perenne to produce a diploid monosomic substitution line 2n = 2x = 14. In this line recombination occurs throughout the length of the F. pratensis/L. perenne bivalent. The F. pratensis chromosome and recombinants between it and its L. perenne homeologue can be visualized using genomic in situ hybridization (GISH). GISH junctions represent the physical locations of sites of recombination, enabling a range of recombinant chromosomes to be used for physical mapping of the introgressed F. pratensis chromosome. The physical map, in conjunction with a genetic map composed of 104 F. pratensis-specific amplified fragment length polymorphisms (AFLPs), demonstrated: (1) the first large-scale analysis of the physical distribution of AFLPs; (2) variation in the relationship between genetic and physical distance from one part of the F. pratensis chromosome to another (e.g., variation was observed between and within chromosome arms); (3) that nucleolar organizer regions (NORs) and centromeres greatly reduce recombination; (4) that coding sequences are present close to the centromere and NORs in areas of low recombination in plant species with large genomes; and (5) apparent complete synteny between the F. pratensis chromosome and rice chromosome 1.     

A Drosophila SNAP-25 null mutant reveals context-dependent redundancy with SNAP-24 in neurotransmission

The synaptic protein SNAP-25 is an important component of the neurotransmitter release machinery, although its precise function is still unknown. Genetic analysis of other synaptic proteins has yielded valuable information on their role in synaptic transmission. In this study, we performed a mutagenesis screen to identify new SNAP-25 alleles that fail to complement our previously isolated recessive temperature-sensitive allele of SNAP-25, SNAP-25(ts). In a screen of 100,000 flies, 26 F(1) progeny failed to complement SNAP-25(ts) and 21 of these were found to be null alleles of SNAP-25. These null alleles die at the pharate adult stage and electroretinogram recordings of these animals reveal that synaptic transmission is blocked. At the third instar larval stage, SNAP-25 nulls exhibit nearly normal neurotransmitter release at the neuromuscular junction. This is surprising since SNAP-25(ts) larvae exhibit a much stronger synaptic phenotype. Our evidence indicates that a related protein, SNAP-24, can substitute for SNAP-25 at the larval stage in SNAP-25 nulls. However, if a wild-type or mutant form of SNAP-25 is present, then SNAP-24 does not appear to take part in neurotransmitter release at the larval NMJ. These results suggest that the apparent redundancy between SNAP-25 and SNAP-24 is due to inappropriate genetic substitution.     

Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex

The heterodimeric nuclear cap-binding complex (CBC) binds to the 5' cap structure of RNAs in the nucleus and plays a central role in their diverse maturation steps. We describe the crystal structure at 2.1 A resolution of human CBC bound to an m(7)GpppG cap analogue. Comparison with the structure of uncomplexed CBC shows that cap binding induces co-operative folding around the dinucleotide of some 50 residues from the N- and C-terminal extensions to the central RNP domain of the small subunit CBP20. The cap-bound conformation of CBP20 is stabilized by an intricate network of interactions both to the ligand and within the subunit, as well as new interactions of the CBP20 N-terminal tail with the large subunit CBP80. Although the structure is very different from that of other known cap-binding proteins, such as the cytoplasmic cap-binding protein eIF4E, specificity for the methylated guanosine again is achieved by sandwiching the base between two aromatic residues, in this case two conserved tyrosines. Implications for the transfer of capped mRNAs to eIF4E, required for translation initiation, are discussed.