RESUMEN
Symmetry-breaking bifurcations have been studied for convection in a nonrotating spherical shell whose outer radius is twice the inner radius, under the influence of an externally applied central force field with a radial dependence proportional to 1/r(5). This work is motivated by the GeoFlow experiment, which is performed under microgravity condition at the International Space Station where this particular central force can be generated. In order to predict the observable patterns, simulations together with path-following techniques and stability computations have been applied. Branches of axisymmetric, octahedral, and seven-cell solutions have been traced. The bifurcations producing them have been identified and their stability ranges determined. At higher Rayleigh numbers, time-periodic states with a complex spatiotemporal symmetry are found, which we call breathing patterns.
RESUMEN
As an alternative to existing methods for the detection of new insertions during a transposon mutagenesis, we adapted the method of vectorette ligation to genomic restriction fragments followed by PCR to obtain genomic sequences flanking the transposon. By combining flies containing a defined genomic transposon with an excess of flies containing unrelated insertion sites, we demonstrate the specificity and sensitivity of the procedure in the detection of integration events. This method was applied in a transposon-tagging screen for BJ1, the Drosophila homolog of the vertebrate gene Regulator of Chromosome Condensation (RCCI). Genetic mobilization of a single genomic P element was used to generate preferentially new local insertions from which integrations into a genomic region surrounding the BJ1 gene were screened. Flies harboring new insertions were phenotypically selected on the basis of the zeste1-dependent transvection of white. We detected a single transposition to a 13-kb region close to the BJ1 gene among 6650 progeny that were analyzed. Southern analysis of the homozygous line confirmed the integration 3 kb downstream of BJ1.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster/genética , Animales , Secuencia de Bases , Cruzamientos Genéticos , Cartilla de ADN , Femenino , Genoma , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Mapeo RestrictivoRESUMEN
A humanized IgG antibody (D1.3) which retains murine complementarity determining regions specific for the antigen lysozyme has been expressed in CHO-DUKX cells. Heavy and light chain containing plasmids were co-transfected into CHO-DUKX cells and stable clones were grown in DMEM/F12 medium supplemented with 5% foetal calf serum. D1.3 antibody was purified from culture supernatants by Protein G chromatography. With the recombinant D1.3 antibody as a model, this cell culture system was shown to glycosylate the IgG Fc region in a similar manner to IgG isolated from serum. The neutral, core fucosylated biantennary oligosaccharides found are present in serum IgG and no novel carbohydrate sequences were detected. The degree of terminal agalactosylation was also similar to normal serum, in contrast to the increased levels found in rheumatoid serum. Furthermore, those oligosaccharides which lack only one terminal Gal are exclusively galactosylated on the GlcNAc(beta1,2) Man(alpha1,6) Man(beta1,4) antenna. Unambiguous identification of the exact glycosylation pattern of the antibody was carried out by a combination of specific exoglycosidase digestions, gel permeation chromatography of 2-aminobenzamide derivatives, high pH anion exchange chromatography and methylation analysis followed by gas-liquid chromatography-mass spectrometry.
Asunto(s)
Células CHO/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Animales , Anticuerpos Monoclonales , Sitios de Unión , Células CHO/metabolismo , Secuencia de Carbohidratos , Cromatografía de Gases/métodos , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Cricetinae , Glicoproteínas/análisis , Glicoproteínas/química , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Glicosilación , Humanos , Inmunoglobulina G/genética , Espectrometría de Masas/métodos , Metilación , Ratones , Datos de Secuencia Molecular , Muramidasa/genética , Muramidasa/inmunología , Oligosacáridos/análisis , Oligosacáridos/química , Polisacáridos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , ortoaminobenzoatos/químicaRESUMEN
The competing incorporation of [14C]Phe (cognate) and [3H]Leu (miscognate) into polypeptide material was measured. Kinetic analysis revealed that at the very beginning of protein synthesis the error fraction was 3 to 10 times larger than the "system-inherent" error, which is the error fraction found on the synthesized proteins. However, if a cognate tRNA, namely either deacylated tRNAPhe or AcPhe-tRNAPhe were prebound to the ribosomal P site, no enhanced error fraction was observed at the beginning. Furthermore, the initial rate of protein synthesis was increased by a factor of 10 to 50, regardless as to whether the ribosomes were precharged with tRNAPhe or AcPhe-tRNAPhe. In contrast, preincubation of ribosomes with non-cognate tRNA such as deacylated tRNALys had no effect. We conclude that codon-anticodon interaction at the P site facilitates the precise exposure of the adjacent codon at the ribosomal A site, thus contributing to the velocity and precision of the decoding process.
Asunto(s)
Anticodón/metabolismo , Codón/metabolismo , Escherichia coli/metabolismo , Leucina/metabolismo , Fenilalanina/metabolismo , ARN Mensajero/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , CinéticaRESUMEN
In a classical translocation experiment, deacylated RNA is bound to the ribosomal peptidyl-tRNA site (P site) and N-acetyl-phenylalanyl-tRNA (AcPhe-tRNA) to the aminoacyl-tRNA site (A site); upon addition of elongation factor (EF-G) and GTP, AcPhe-tRNA is translocated from the A to the P site. Here, we demonstrate a model reaction for a spontaneous, EF-G independent translocation. If AcPhe-tRNA is bound to the P site and Phe-tRNA to the A site at 15 mM Mg2+, then at 37 degrees C up to 60% of the AcPhe2-tRNA formed is found at the P site without the addition of EF-G. We demonstrate the following: 1) the spontaneous translocation is not merely illusory as a result of Phe-tRNA binding directly to the P site; 2) it is not mimicked by release of AcPhe2-tRNA from an A site and rebinding to a P site of another ribosome; 3) it is not caused by an EF-G contaminant present in the 70 S preparation, since without EF-G the spontaneous translocation works equally well in the presence of guanyl-5'-yl imidodiphosphate or fusidic acid; 4) AcPhe2-tRNA evidently has a higher affinity for the P site than AcPhe-tRNA, thus promoting the spontaneous translocation; and 5) peptide-bond formation favors the subsequent translocation. Addition of EF-G increases the initial rate by a factor of 13. Furthermore, at 15 mM Mg2+, 37 degrees C and in the presence of EF-G and GTP, Phe-tRNA cannot be translocated from the A to the P site, if the P site is occupied by deacylated tRNA. With the spontaneous translocation system, all reactions of the elongation cycle are cooperatively interconnected; i.e. upon binding of aminoacyl-tRNA to the A site, a significant portion of the ribosomes performs a complete round of the elongation cycle without the addition of elongation factor EF-G.