8 resultados para Non-line-of-sight
em National Center for Biotechnology Information - NCBI
Resumo:
A transposon based on the transposable element Minos from Drosophila hydei was introduced into the genome of Drosophila melanogaster using transformation mediated by the Minos transposase. The transposon carries a wild-type version of the white gene (w) of Drosophila inserted into the second exon of Minos. Transformation was obtained by injecting the transposon into preblastoderm embryos that were expressing transposase either from a Hsp70-Minos fusion inserted into the genome via P-element-mediated transformation or from a coinjected plasmid carrying the Hsp70-Minos fusion. Between 1% and 6% of the fertile injected individuals gave transformed progeny. Four of the insertions were cloned and the DNA sequences flanking the transposon ends were determined. The "empty" sites corresponding to three of the insertions were amplified from the recipient strain by PCR, cloned, and sequenced. In all cases, the transposon has inserted into a TA dinucleotide and has created the characteristic TA target site duplication. In the absence of transposase, the insertions were stable in the soma and the germ line. However, in the presence of the Hsp70-Minos gene the Minos-w transposon excises, resulting in mosaic eyes and germ-line reversion to the white phenotype. Minos could be utilized as an alternative to existing systems for transposon tagging and enhancer trapping in Drosophila; it might also be of use as a germ-line transformation vector for non-Drosophila insects.
Resumo:
The production of subtle or conditional mutations in mice through the combined use of site-specific and homologous recombination has become an increasingly widespread experimental paradigm in mammalian genetics. Embryonic stem cells containing recombinase transgenes that were expressed in the male germ line, but not in other tissues or in the embryonic stem cells themselves, would substantially simplify the production of such alleles. Here we show that transgenes comprised of the mouse protamine 1 promoter and the Cre recombinase coding sequence mediate the efficient recombination of a Cre target transgene in the male germ line, but not in other tissues. Embryonic stem cell lines generated from one of these transgenic strains were transfected with targeting vectors that included loxP-flanked selectable markers, and homologously recombined alleles containing the marker and functional loxP sites were isolated. These results establish the potential of the system for substantially reducing the time, effort, and resources required to produce homologously recombined alleles in mice that have been secondarily rearranged by a site-specific recombinase.
Resumo:
We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.
Resumo:
Very-long-baseline interferometry images of the nuclear region of the radio galaxy Cygnus A reveal a pronounced "core" and a knotty jet and counterjet. The knots are moving away from the core at apparent speeds which are subluminal for h = 1 [h = H0/100 km.s-1.Mpc-1;1 parsec (pc) = 3.09 x 10(16)m] and about c for h = 0.5. The jet is aligned with the outer, kiloparsec-scale jet to within 2 degrees. The counterjet has a total flux density at 5 GHz of about one-fifth of that of the jet. In the context of the twin relativistic jet model for active galactic nuclei, the jet in Cygnus A is oriented at an angle to our line of sight of 35-80 degrees and 55-85 degrees, and the intrinsic velocity of the jet fluid is 0.4-0.6c and 0.6-1c for h = 1 and h = 0.5, respectively.
Resumo:
The compact steep-spectrum sources (CSSs) are an interesting class of objects which are of subgalactic dimensions; they occur more frequently in high-frequency surveys because their spectra often turn over at lower frequencies. We have estimated the symmetry parameters of a well-defined sample of CSSs and compared these with the larger 3CR sources of similar luminosity to understand the evolution and the consistency of CSSs with the unified scheme. We suggest that the majority of CSSs are likely to be young sources advancing outward through an asymmetric, inhomogeneous environment to form the larger ones. The radio properties of the CSSs are consistent with the unified scheme, where the axes of the quasars are seen closer to the line of sight while the radio galaxies lie closer to the plane of the sky. We discuss how radio polarization observations may be used to probe whether the physical conditions in the central regions of the CSSs are different from the larger ones. We present a simple scenario where the depolarization and high rotation measures seen in many CSSs can be consistent with the low rotation measures of cores in the more extended quasars and suggest further observations to test this scenario.
Resumo:
To study the molecular basis for the clinical phenotype of incomplete penetrance of familial retinoblastoma, we have examined the functional properties of three RB mutations identified in the germ line of five different families with low penetrance. RB mutants isolated from common adult cancers and from classic familial retinoblastoma (designated as classic RB mutations) are unstable and generally do not localize to the nucleus, do not undergo cyclin-dependent kinase (cdk)-mediated hyperphosphorylation, show absent protein “pocket” binding activity, and do not suppress colony growth of RB(−) cells. In contrast, two low-penetrant alleles (661W and “deletion of codon 480”) retained the ability to localize to the nucleus, showed normal cdk-mediated hyperphosphorylation in vivo, exhibited a binding pattern to simian virus 40 large T antigen using a quantitative yeast two-hybrid assay that was intermediate between classic mutants (null) and wild-type RB, and had absent E2F1 binding in vitro. A third, low-penetrant allele, “deletion of RB exon 4,” showed minimal hyperphosphorylation in vivo but demonstrated detectable E2F1 binding in vitro. In addition, each low-penetrant RB mutant retained the ability to suppress colony growth of RB(−) tumor cells. These findings suggest two categories of mutant, low-penetrant RB alleles. Class 1 alleles correspond to promoter mutations, which are believed to result in reduced or deregulated levels of wild-type RB protein, whereas class 2 alleles result in mutant proteins that retain partial activity. Characterization of the different subtypes of class 2 low-penetrant genes may help to define more precisely functional domains within the RB product required for tumor suppression.
Resumo:
Although the zebrafish possesses many characteristics that make it a valuable model for genetic studies of vertebrate development, one deficiency of this model system is the absence of methods for cell-mediated gene transfer and targeted gene inactivation. In mice, embryonic stem cell cultures are routinely used for gene transfer and provide the advantage of in vitro selection for rare events such as homologous recombination and targeted mutation. Transgenic animals possessing a mutated copy of the targeted gene are generated when the selected cells contribute to the germ line of a chimeric embryo. Although zebrafish embryo cell cultures that exhibit characteristics of embryonic stem cells have been described, successful contribution of the cells to the germ-cell lineage of a host embryo has not been reported. In this study, we demonstrate that short-term zebrafish embryo cell cultures maintained in the presence of cells from a rainbow trout spleen cell line (RTS34st) are able to produce germ-line chimeras when introduced into a host embryo. Messenger RNA encoding the primordial germ-cell marker, vasa, was present for more than 30 days in embryo cells cocultured with RTS34st cells or their conditioned medium and disappeared by 5 days in the absence of the spleen cells. The RTS34st cells also inhibited melanocyte and neuronal cell differentiation in the embryo cell cultures. These results suggest that the RTS34st splenic–stromal cell line will be a valuable tool in the development of a cell-based gene transfer approach to targeted gene inactivation in zebrafish.