Experimental investigations and finite element simulations on the measuring error of Prescale pressure measurement films of the mono-sheet type ‘S’ were done with perfect Hertzian contact bodies in ...contact situations similar to the wheel-rail contact. The sources of the systematic error were identified and quantified.
•Measurement of Hertzian contact with Prescale films MS, HS, HHS.•Finite element simulation of contact situation with film.•Prescale overestimate the contact area.•Systematic error in measurement due to continuum mechanics and geometric effects.
The complete genomic DNA sequence of the highly attenuated vaccinia strain modified vaccinia Ankara (MVA) was determined. The genome of MVA is 178 kb in length, significantly smaller than that of the ...vaccinia Copenhagen genome, which is 192 kb. The 193 open reading frames (ORFs) mapped in the MVA genome probably correspond to 177 genes, 25 of which are split and/or have suffered mutations resulting in truncated proteins. The left terminal genomic region of MVA contains four large deletions and one large insertion relative to the Copenhagen strain. In addition, many ORFs in this region are fragmented, leaving only eight genes structurally intact and therefore presumably functional. The inserted DNA codes for a cluster of genes that is also found in the vaccinia WR strain and in cowpox virus and includes a highly fragmented gene homologous to the cowpox virus host range gene, providing further evidence that a cowpox-like virus was the ancestor of vaccinia. Surprisingly, the central conserved region of the genome also contains some fragmented genes, including ORF F5L, encoding a major membrane protein, and ORFs F11L and O1L, encoding proteins of 39.7 and 77.6 kDa, respectively. The right terminal genomic region carries three large deletions: all classical poxviral immune evasion genes and all ankyrin-like genes located in this region are fragmented except for those encoding the interleukin-1β receptor and the 68-kDa ankyrin-like protein B18R. Thus, the attenuated phenotype of MVA is the result of numerous mutations, particularly affecting the host interactive proteins, including the ankyrin-like genes, but also involving some structural proteins.
The crystal structure of restriction endonuclease Bam HI complexed to DNA has been determined at 2.2 angstrom resolution. The DNA binds in the cleft and retains a B-DNA type of conformation. The ...enzyme, however, undergoes a series of conformational changes, including rotation of subunits and folding of disordered regions. The most striking conformational change is the unraveling of carboxyl-terminal α helices to form partially disordered "arms." The arm from one subunit fits into the minor groove while the arm from the symmetry related subunit follows the DNA sugar-phosphate backbone. Recognition of DNA base pairs occurs primarily in the major groove, with a few interactions occurring in the minor groove. Tightly bound water molecules play an equally important role as side chain and main chain atoms in the recognition of base pairs. The complex also provides new insights into the mechanism by which the enzyme catalyzes the hydrolysis of DNA phosphodiester groups.
FokI is a member of an unusual class of bipartite restriction enzymes that recognize a specific DNA sequence and cleave DNA nonspecifically a short distance away from that sequence. Because of its ...unusual bipartite nature, FokI has been used to create artificial enzymes with new specificities. We have determined the crystal structure at 2.8A resolution of the complete FokI enzyme bound to DNA. As anticipated, the enzyme contains amino- and carboxy-terminal domains corresponding to the DNA-recognition and cleavage functions, respectively. The recognition domain is made of three smaller subdomains (D1, D2 and D3) which are evolutionarily related to the helix-turn-helix-containing DNA-binding domain of the catabolite gene activator protein CAP. The CAP core has been extensively embellished in the first two subdomains, whereas in the third subdomain it has been co-opted for protein-protein interactions. Surprisingly, the cleavage domain contains only a single catalytic centre, raising the question of how monomeric FokI manages to cleave both DNA strands. Unexpectedly, the cleavage domain is sequestered in a 'piggyback' fashion by the recognition domain. The structure suggests a new mechanism for nuclease activation and provides a framework for the design of chimaeric enzymes with altered specificities.
Influenza vaccine production is dependent on the availability of embryonated hen eggs for virus growth. This is an extremely cumbersome system with many disadvantages with respect to selection of ...virus variants and presence of adventitious viruses. We have developed an alternative cell culture system which allows rapid production of large volumes of vaccine. The World Health Organisation (WHO) approved Vero cell line was used in serum-free culture to grow a multitude of influenza strains to high titre. This system could be scaled-up to allow vaccine production with a 1200 litre fermenter volume. A purification scheme was developed which resulted in a high purity whole virus vaccine. This was demonstrated to be at least as immunogenic as a conventional egg-derived preparation in a mouse model.
The crystal structure of the HincII restriction endonuclease-DNA complex shows that degenerate specificity for blunt-ended cleavage at GTPyPuAC sequences arises from indirect readout of ...conformational preferences at the center pyrimidine-purine step. Protein-induced distortion of the DNA is accomplished by intercalation of glutamine side chains into the major groove on either side of the recognition site, generating bending by either tilt or roll at three distinct loci. The intercalated side chains propagate a concerted shift of all six target-site base pairs toward the minor groove, producing an unusual cross-strand purine stacking at the center pyrimidine-purine step. Comparison of the HincII and EcoRV cocrystal structures suggests that sequence-dependent differences in base-stacking free energies are a crucial underlying factor mediating protein recognition by indirect readout.
The interaction of BamHI endonuclease with DNA has been studied crystallographically, but has not been characterized rigorously in solution. The enzyme binds in solution as a homodimer to its ...recognition site GGATCC. Only six base-pairs are directly recognized, but binding affinity (in the absence of the catalytic cofactor Mg2+) increases 5400-fold as oligonucleotide length increases from 10 to 14 bp. Binding is modulated by sequence context outside the recognition site, varying about 30-fold from the bes t (GTG or TAT) to the worst (CGG) flanking triplets.BamHI,EcoRI and EcoRV endonucleases all have different context preferences, suggesting that context affects binding by influencing the free energy levels of the complexes rather than that of the free DNA. Ethylation interference footprinting in the absence of divalent metal shows a localized and symmetrical pattern of phosphate contacts, with strong contacts at NpNpNpGGApTCC. In the presence of Mg2+, first-order cleavage rate constants are identical in the two GGA half-sites, are the same for the two nicked intermediates and are unaffected by substrate length in the range 10-24 bp. DNA binding is strongly enhanced by mutations D94N, E111A or E113K, by binding of Ca2+ at the active site, or by deletion of the scissile phosphate GpGATCC, indicating that a cluster of negative charges at the catalytic site contributes at least 3-4 kcal/mol of unfavorable binding free energy. This electrostatic repulsion destabilizes the enzyme-DNA complex and favors metal ion binding and progression to the transition state for cleavage.
Type II restriction endonucleases are characterized by the remarkable specificity with which they cleave specific DNA sequences. Surprisingly, their protein sequences are in most cases unrelated, and ...no recurring structural motif has yet been identified. We have determined the structure of restriction endonuclease BamHI at 1.95 A resolution. BamHI shows striking resemblance to the structure of endonuclease EcoRI (refs 3, 4), despite the lack of sequence similarity between them. We also observe some curious differences between the two structures, and propose an evolutionary scheme that may explain them. The active site of BamHI is structurally similar to the active sites of EcoRI and EcoRV (ref. 5), but the mechanism by which BamHI activates a water molecule for nucleophilic attack may be different.
The ospC gene was amplified by the polymerase chain reaction from each of 76 Lyme disease Borrelia strains. Restriction fragment length polymorphism (RFLP) analysis demonstrated 33 distinct RFLP ...types; two additional RFLP types were identified from published ospC sequences. For each RFLP type, at least one ospC gene was sequenced and the degree of sequence relatedness examined by construction of an ospC gene tree. The genes were extremely diverse, with sequence identity ranging from 74.4% to 99.0%; the majority of changes are localized within the central portion of the molecule. A comparison of ospC sequences suggests that recombination occurs frequently between ospC alleles; this genetic exchange is proposed to be mediated by lateral transfer of ospC sequences. Evidence indicates that recombination occurs between ospC genes from the same Borrelia species (i.e. B. afzelii and B. garinii) as well as between different Borrelia species (i.e. B. afzelii and B. garinii, B. burgdorferi and genogroup DN127).
Etherified melamine resins are well known from the lacquer and paint industry. Up to now, important characterization in terms of composition and structure has been a time-consuming procedure. Now, ...fast analysis can also be achieved by use of
13
C nuclear magnetic resonance spectroscopy. A special polarization transfer technique was applied to overcome the inherent low signal/noise ratio of
13
C NMR spectra. The melamine content of the resin was determined by combustion analysis. This combination of methods allows for fast resin analysis with accuracy as yet unmatched and shows good correlation with established techniques.
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