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PubMed1985

Heparin-binding fragments of fibronectin are potent inhibitors of endothelial cell growth.

Two heparin-binding proteolytic fragments of fibronectin--an amino-terminal 29-kd segment and a carboxyl-terminal 40-kd segment-are apparently specific, potent inhibitors of the growth of cultured bovine aortic endothelial cells and inhibit growth in a reversible, dose-dependent manner. In contrast, native fibronectin at higher dosages neither inhibits nor interferes with the effects of the 29-kd fragment. The data, therefore, suggest that fibronectin fragments may participate in the regulation of vascular growth.

Animals↗
PubMed1997

The cyclic AMP receptor promoter DNA complex: a comparison of crystal and solution structure by quantitative molecular electrooptics.

The complexes formed between the cyclic AMP receptor and three different promoter DNA fragments, including a synthetic 30 bp fragment with the sequence used for determination of the crystal structure, have been analysed in solution by measurements of the electric dichroism (ED) at an ionic strength of 105 mM, using a special instrument based on cable discharge. The ED of the protein is negligible and, thus, the ED of the complexes is determined by the DNA and its orientation relative to the protein. The complex formed between the cyclic AMP receptor with the 30 bp fragment is characterized by a positive ED, indicating that the electric dipole is perpendicular relative to the direction of the helix; moreover, the dipole changes its nature from an induced one for the free DNA to a permanent one of 3.0 x 10(-27) Cm for the complex; both the limiting value of the ED +0.3 and the dichroism decay time constant of 62 ns found for the complex (free DNA: 52 ns; 20 degrees C) demonstrate bending of the DNA double helix. All these parameters are calculated quantitatively from the crystal structure: bead model simulations are used to derive the coefficients of rotational diffusion and to define the center of diffusion, which is the reference for calculation of the dipole vector; the dipole vector is then the basis for calculation of the limit value of the dichroism; the time constants are derived from the diffusion coefficients of the bead model The calculated parameters are in very satisfactory agreement with the experimental ones, demonstrating agreement of the structures in the crystal and in solution with respect to their essential features. These results also demonstrate the utility of electrooptical procedures for a quantitative comparison of crystal or model structures with structures in solution. While the crystal structure has been determined only for the complex with the 30 bp promoter fragment, it has been relatively simple to extend measurements of the ED to complexes formed with a 40 bp and a 203 bp promoter fragment: the data obtained for a 40 bp fragment with the consensus binding sequence are quite similar to those obtained for the 30 bp promoter, whereas the data obtained for the 203 bp promoter clearly show a much higher degree of protein induced bending with a bending angle of approximately 180 degrees.

Binding Sites↗
PubMed2004

Correlation of percutaneous liver biopsy fragmentation with the degree of fibrosis.

BACKGROUND: Although fragmentation of a liver biopsy specimen has been considered to be suggestive of cirrhosis, the evidence for this is difficult to find in the published literature. AIM: To determine whether fragmentation of percutaneous liver biopsy specimens correlates with the degree of fibrosis. METHODS: One hundred and eighty-six patients underwent percutaneous liver biopsy prospectively. The specimens were measured for the length and number of fragments. The extent of fibrosis was scored by a pathologist blind to the clinical data. Length and fragmentation data were compared between the different stages. RESULTS: The overall median fragment length was 1.85 cm and the median fragment number was four. Specimens with advanced fibrosis (stages III-IV) had more fragments than those with no or mild fibrosis (stages 0-II) (P < 0.0001). The aggregate fragment length decreased with increasing stage of fibrosis (P < 0.0001). Specimens with greater than 12 fragments were seen only with advanced fibrosis. CONCLUSIONS: Fragmentation of percutaneous liver biopsy specimens is common and increases with progression from early to advanced fibrosis. Fibrotic specimens fragment more often and more extensively.

Adult↗
PubMed1996

Assembly of high-resolution restriction maps based on multiple complete digests of a redundant set of overlapping clones.

An approach to restriction-site mapping and contig building that uses fragment-size data from multiple complete digests of a set of clones that oversample a genomic region is presented. Maps containing both fragment-length data and clone-end data are maintained for each restriction enzyme. Synchronization between the maps for the different enzymes is achieved by requiring the clone-end maps for all enzymes to be compatible. Basic concepts that underlie multiple-complete-digest mapping--including the match/merge approach to map incorporation, extension vs assimilation, ambiguity, and clone-end compatibility--are presented. An initial application of multiple-complete-digest mapping to real data on a set of cosmid clones suggests that this mapping method has exceptional power to produce accurate maps that are well suited to the needs of large-scale DNA-sequencing projects.

Algorithms↗
PubMed2000

Crystallization of the F41 fragment of flagellin and data collection from extremely thin crystals.

Flagellin, which constructs supercoiled filaments of the bacterial flagellum, is very difficult to crystallize because of its strong tendency to polymerize. We therefore crystallized the F41 fragment of flagellin, which does not polymerize because terminal regions that play important roles in polymerization are cleaved off. F41 was crystallized by the hanging drop vapor diffusion method in a mixture of polyethylene glycol, glycerol, and isopropanol, with a reservoir solution covered with silicon oil. The two key factors for success in growing sufficiently large crystals were isopropanol and silicon oil, which worked well to reduce the otherwise very high nucleation rate that resulted in hundreds of tiny crystals. The crystals were grown to very thin plates with thickness less than 10 microm, which made the collection of diffraction data very difficult. Freezing and annealing of the crystals and irradiation at synchrotron beamlines had to be carried out by specific methods and under specific conditions for its structure analysis at 2.0-A resolution.

Antigens, Bacterial↗
PubMed2002

The NH2-terminal domain of Golgin-160 contains both Golgi and nuclear targeting information.

Golgin-160 is a member of the golgin family of Golgi-localized membrane proteins. The COOH-terminal two-thirds of golgin-160 is predicted to form a coiled-coil, with an NH(2)-terminal "head" domain. To identify the Golgi targeting information in golgin-160, full-length and deletion constructs tagged with green fluorescent protein were generated. The head domain alone was targeted to the Golgi complex in the absence of assembly with endogenous golgin-160. Further truncations from both ends of the head domain narrowed the Golgi targeting information to 85 amino acids between residues 172 and 257. Surprisingly, certain truncations of the head domain also specifically accumulated in the nucleus. Both a nuclear localization signal (masked in the full-length protein) and information for nuclear retention contributed to the nuclear localization of these truncations. Because the golgin-160 head is cleaved by caspases during apoptosis, we examined the localization of epitope-tagged proteins corresponding to all potential caspase cleavage fragments. Our data suggest that three of six fragments could be targeted to the nucleus, provided that they are released from Golgi membranes after cleavage. The finding that both Golgi and nuclear targeting information is present in the same region of golgin-160 suggests that this protein may have more than one function.

Active Transport, Cell Nucleus↗
PubMed1997

EcoRI restriction fragment-length polymorphism of the human immunoglobulin variable lambda 8 (IGLV8) subgroup reveals a gene family.

The human immunoglobulin lambda locus (IGL) maps on chromosome 22q11.1-q11.2 and directs the synthesis of lambda-type Ig light chains. This locus is formed by three gene clusters (VA, VB and VC) that encompass the variable coding genes and the J-C cluster plus the joining segments and the constant genes. Recently the variable lambda gene clusters were mapped by the contig methodology which located all the known functional v-lambda genes and pseudogenes. The 30 functional v-lambda genes described so far were subgrouped into ten families (V lambda I to V lambda X), but RFLP studies have estimated that the germline repertoire contains about 70 genes. Based on sequence comparisons, we defined specific oligonucleotide primers for the unique IGLV8S1 gene described. The cloned 244 bp product obtained from genomic DNA with these primers was sequenced and used as probe in Southern hybridization EcoRI RFLP analysis of Brazilian people. We detected the IGLV8S1 gene in a 3.7 kb EcoRI restriction fragment present in all the individuals analyzed, in agreement with the physical map of the IGL locus. Moreover, we detected an 8.0 kb EcoRI monomorphic fragment and a 6.0 kb EcoRI polymorphic fragment. These data suggest that the IGLV8 subgroup is a gene family.

Amino Acid Sequence↗
PubMed1996

The use of differential display-PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages.

The differential display (DD)-PCR technique has been modified to identify prokaryotic cDNA fragments that are differentially induced by facultative intracellular bacteria in response to the intracellular environment of eukaryotic cells. Several DD-PCR fragments identified from the intracellular bacterium Legionella pneumophila were induced at 4 h post-infection of the U937 macrophage-like cells. From these, a 700 bp fragment was cloned and sequenced. Neither the DNA sequence nor the predicted protein sequence from the open reading frame has similarity to other sequences in genetic databases. Transcription of the chromosomal locus containing the 700 bp fragment (eml, for early stage macrophage-induced locus) was induced by intracellular bacteria during the first few hours post-infection of macrophages but the expression was downregulated by 12 h post-infection. Transcription of eml was not growth phase-related in vitro, and was not affected by in vitro stress stimuli. A 3.7 kb EcoRI genomic fragment containing the 700 bp DD-PCR product was cloned. Six mini-Tn 10 insertions in the 3.7 kb EcoRI fragment were recombined into the L. pneumophila chromosome. Compared to the wild-type strain, five of the eml isogenic mutants had a similar phenotype of reduced cytopathicity to the U937 cells, showed a 100-fold increase in killing by macrophages during the first 5 h of the intracellular infection, and showed a 100-fold increase in killing during the first 24h of infection of the amoeba Hartmanella vermiformis. The 6th mutant had a phenotype indistinguishable from the wild-type strain. The cytopathicity defect of the mutants to the U937 cells was restored to wild-type levels by complementation of the mutants with a plasmid containing the 3.7 kb EcoRI fragment. These data showed that the 3.7 kb fragment containing eml is a novel L. pneumophila locus whose expression is uniquely induced by non-stress stimuli during early stages of the intracellular infection of phagocytic cells. Expression of this locus is required for survival of L. pneumophila within macrophages and within amoebae during early stages of the infection.

Animals↗
PubMed1985

Neuropharmacological characterization of fragment B from tetanus toxin.

Tetanus toxin and Fragment B from tetanus toxin were assayed for activity on the mouse phrenic nerve-hemidiaphragm preparation. Both molecules produced blockade of neuromuscular transmission, but the parent molecule was at least two orders of magnitude more potent than the fragment. Experiments were done to determine whether the toxicity attributed to Fragment B was authentic or due to contamination with the parent molecule. Analysis of the fragment by high-performance liquid chromatography and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed trace contamination. Removal of the major contaminant (1-2%) did not abolish toxicity of the material. However, in pharmacological experiments with native toxin and its fragment, the latter behaved indistinguishably from the former. At equiactive concentrations, both were antagonized by Fragment C and both were antagonized by lysosomotropic agents (ammonium chloride and methylamine hydrochloride). In addition, monoclonal antibodies directed against epitopes in Fragment C neutralized both native toxin and the material presumed to be Fragment B. The studies with antagonists and antibodies suggest that the toxicity apparently associated with Fragment B was in fact due to trace contamination with the parent molecule. In experiments on planar lipid bilayers, Fragment B formed pH-dependent channels. This activity was not abolished by monoclonal antibodies directed against epitopes in Fragment C. The data indicate that Fragment B retains the ability to form channels in membranes, but in the absence of Fragment C it retains little ability to paralyze neuromuscular transmission.

Animals↗
PubMed2004

The multiassembly problem: reconstructing multiple transcript isoforms from EST fragment mixtures.

Recent evidence of abundant transcript variation (e.g., alternative splicing, alternative initiation, alternative polyadenylation) in complex genomes indicates that cataloging the complete set of transcripts from an organism is an important project. One challenge is the fact that most high-throughput experimental methods for characterizing transcripts (such as EST sequencing) give highly detailed information about short fragments of transcripts or protein products, instead of a complete characterization of a full-length form. We analyze this "multiassembly problem"-reconstructing the most likely set of full-length isoform sequences from a mixture of EST fragment data-and present a graph-based algorithm for solving it. In a variety of tests, we demonstrate that this algorithm deals appropriately with coupling of distinct alternative splicing events, increasing fragmentation of the input data and different types of transcript variation (such as alternative splicing, initiation, polyadenylation, and intron retention). To test the method's performance on pure fragment (EST) data, we removed all mRNA sequences, and found it produced no errors in 40 cases tested. Using this algorithm, we have constructed an Alternatively Spliced Proteins database (ASP) from analysis of human expressed and genomic sequences, consisting of 13,384 protein isoforms of 4422 genes, yielding an average of 3.0 protein isoforms per gene.

Algorithms↗
PubMed2001

Fragment assembly with double-barreled data.

For the last twenty years fragment assembly was dominated by the "overlap - layout - consensus" algorithms that are used in all currently available assembly tools. However, the limits of these algorithms are being tested in the era of genomic sequencing and it is not clear whether they are the best choice for large-scale assemblies. Although the "overlap - layout - consensus" approach proved to be useful in assembling clones, it faces difficulties in genomic assemblies: the existing algorithms make assembly errors even in bacterial genomes. We abandoned the "overlap - layout - consensus" approach in favour of a new Eulerian Superpath approach that outperforms the existing algorithms for genomic fragment assembly (Pevzner et al. 2001 InProceedings of the Fifth Annual International Conference on Computational Molecular Biology (RECOMB-01), 256-26). In this paper we describe our new EULER-DB algorithm that, similarly to the Celera assembler takes advantage of clone-end sequencing by using the double-barreled data. However, in contrast to the Celera assembler, EULER-DB does not mask repeats but uses them instead as a powerful tool for contig ordering. We also describe a new approach for the Copy Number Problem: "How many times a given repeat is present in the genome?". For long nearly-perfect repeats this question is notoriously difficult and some copies of such repeats may be "lost" in genomic assemblies. We describe our EULER-CN algorithm for the Copy Number Problem that proved to be successful in difficult sequencing projects.

Algorithms↗
PubMed2003

Molecular genetic diversity of the French-American grapevine hybrids cultivated in North America.

French-American hybrid grapevines are most popular in eastern and mid-western North America: they are hardy cultivars derived from crosses between the European Vitis vinifera and American wild vines. The aim of this study was to characterize their genetic background using 6 microsatellite (SSR) markers and a set of 33 diagnostic RAPD markers. The latter were reproducible with different PCR thermal cyclers. Two SSR loci were found to be synonymous, VrZAG47 and VVMD27. The DNA profile frequencies estimated for each cultivar were much lower with multi-locus SSR data than that obtained from multi-fragment RAPD data. There was no significant correlation between the multi-locus DNA profile frequencies derived from SSRs and those from RAPDs. Estimates of genetic diversity derived from SSRs were generally higher and the average similarity between cultivars was generally lower than values reported for subgroups of V. vinifera, in accordance with expectations for hybrid cultivars. The phenetic relationships depicted by UPGMA (unweighted pair-group method with arithmetic averaging) and neighbor-joining analyses of microsatellite data were congruent and, to a large extent, in agreement with the known pedigree or history of each cultivar. A major dichotomy was observed between one group where the known genetic background was dominated by the North American Vitis riparia and Vitis labrusca, and another one where the genetic background was dominated by the European V. vinifera. Two Kulhmann varieties thought to be synonymous were found to be different, though closely related.

Algorithms↗
PubMed1994

Improved estimation of DNA fragment length from gel electrophoresis data using a graphical method.

A computer program has been developed for computing DNA fragment size from its electrophoretic mobility using a graphical method. The program uses DNA marker data and selects the semilogarithmic linear range (sl-range), i.e., the linear portion of the semilogarithmic curve (mobility vs. log of DNA fragment length). Over this range a linear interpolation is derived for calculating the size of a DNA fragment whose mobility falls in the sl-range. The program also derives a hyperbolic interpolation formula that covers the entire range for determining the size of a DNA fragment whose mobility is beyond the semilogarithmic linear range. The method described in this paper is sensitive, accurate and reliable. This program can also be used to compute protein or polypeptide size from sodium dodecyl sulfate polyacrylamide gel electrophoresis data. The DOS version of the DNASIZE program is freely available from Netserver at EMBL or from BioTechNet by EMail.

DNA↗
PubMed1991

Crystallization and preliminary X-ray diffraction data of the Fab fragment of a monoclonal antibody against apamin, a bee venom neurotoxin.

Fab fragments of anti-apamin monoclonal antibodies have been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group P21 with cell dimensions a = 99.0 +/- 0.3 A, b = 137.1 +/- 0.4 A, c = 76.0 +/- 0.2 A and beta = 92.9 +/- 0.9 degrees. They most likely contain four molecules in the asymmetric unit (Vm = 2.39 A3/Da). The possibility of the existence of non-crystallographic symmetry is discussed.

Antibodies, Monoclonal↗
PubMed1985

Crystallization and preliminary crystallographic data of the Fab fragment of an anti-phenylalanine hydroxylase monoclonal antibody.

Two crystal habits, one rod shaped and the other square prismatic, of the Fab fragment of a monoclonal anti-phenylalanine hydroxylase antibody have been grown using the method of vapour phase diffusion against polyethylene glycol 6000. The square prisms diffract to better than 2.8 A, belong to the space group P1 and have unit cell parameters a = 41.8 A, b = 50.3 A, c = 114.7 A, alpha = 97.6 degrees, beta = 91.7 degrees, gamma = 91.0 degrees, while the rod-shaped crystals belong to the space group P212121, have unit cell parameters a = 105.6 A, b = 119.8 A, c = 82.2 A and diffract to 3.5 A resolution.

Antibodies, Monoclonal↗
PubMedDate not supplied

Size calculation of restriction enzyme HaeIII-generated fragments detected by probe YNH24 by comparison of data from two laboratories: the generation of fragment-size frequencies.

Restriction fragment-length polymorphism of locus D2S44 detected by the highly polymorphic probe YNH24 and restriction endonuclease HaeIII can be used to improve parentage testing when representative fragment-size frequencies can be obtained. By joining the results of different laboratories, it is possible to set up a meaningful databank. Therefore, the same randomly chosen samples were tested for the HaeIII RFLP detected by probe YNH24 in Düsseldorf (DUS) and Amsterdam (AMS). The results of the different fragment-size calculations obtained by using internal markers and a computerized system (DUS-cad and AMS-cad), and by using external markers and manual calculations (DUS-man), were analyzed. Comparing these results, no statistically significant differences were seen. The results obtained with probe YNH24 and enzyme HaeIII in Düsseldorf and Amsterdam can be used to attain a sufficient number of samples to generate relevant fragment-size frequencies.

DNA↗
PubMed1997

Agrin binds to the nerve-muscle basal lamina via laminin.

Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel. The first 130 amino acids from the NH2 terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH2-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.

Agrin↗