RESUMO
We describe a simple, rapid technique for simultaneously isolating large numbers of cDNAs encoding secreted proteins. The technique makes use of a facile genetic selection performed in a strain of Saccharomyces cerevisiae deleted for its endogenous invertase gene. A cDNA cloning vector which carries a modified invertase gene lacking its leader sequence is used in conjunction with this strain. Heterologous secreted genes fused appropriately upstream of this defective invertase provide the necessary signals to restore secretion, allowing the yeast to grow on sugars such as sucrose or raffinose. This microbial growth selection facilitates scanning cDNA libraries containing millions of clones, enabling the wholesale identification of novel secreted proteins without the need for specific bioassays. The technique is similar to one previously described (Klein et al. (1996) Proc. Natl. Acad. Sci. USA 93, 7108-7113). We describe results using a cDNA library derived from activated human peripheral blood mononuclear cells (PBMC). Genes identified from this library encoded signal sequences of proteins of diverse structure, function, and cellular location such as cytokines, type 1 and type 2 transmembrane proteins, and proteins found in intracellular organelles. In addition, a number of novel secreted proteins were identified, including a chemokine and a novel G-protein-coupled receptor. Since signal sequences possess features conserved throughout evolution, the procedure can be used to isolate genes encoding secreted proteins from both eukaryotes and prokaryotes.
Assuntos
DNA Complementar/isolamento & purificação , Vetores Genéticos , Sinais Direcionadores de Proteínas , Proteínas/metabolismo , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Quimiocinas/genética , Glicosídeo Hidrolases/genética , Humanos , Interferon gama/genética , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Seleção Genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , beta-FrutofuranosidaseAssuntos
Clonagem Molecular/métodos , DNA Complementar/isolamento & purificação , Glicosídeo Hidrolases/genética , Sinais Direcionadores de Proteínas/genética , Sequência de Bases , Primers do DNA , DNA Complementar/genética , Eletroporação , Escherichia coli/genética , Biblioteca Gênica , Vetores Genéticos , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , Biossíntese de Proteínas , Sinais Direcionadores de Proteínas/biossíntese , Saccharomyces cerevisiae/genética , beta-FrutofuranosidaseRESUMO
System responsiveness (SR) is defined as the elapsed time until a system responds to user control. SR fluctuates over time, so it must be described statistically with mean (MSR) and standard deviation (SDSR). In this paper, we examine SR in virtual environments (VEs), outlining its components and methods of experimental measurement and manipulation. Three studies of MSR and SDSR effects on performance of grasp and placement tasks are then presented. The studies used within-subjects designs with 11, 12, and 10 participants, respectively. Results showed that SDSR affected performance only if it was above 82 ms. Placement required more frequent visual feedback and was more sensitive to SR. We infer that VE designers need not tightly control SDSR and may wish to vary SR control based on required visual feedback frequency. These results may be used to improve the human-computer interface in a wide range of interactive graphical applications, including scientific visualization, training, mental health, and entertainment.
Assuntos
Análise e Desempenho de Tarefas , Interface Usuário-Computador , Apresentação de Dados , HumanosRESUMO
By the genetic selection of mouse cDNAs encoding secreted proteins, a B7-like cDNA clone termed mouse GL50 (mGL50) was isolated encoding a 322-aa polypeptide identical with B7h. Isolation of the human ortholog of this cDNA (hGL50) revealed a coding sequence of 309 aa residues with 42% sequence identity with mGL50. Northern analysis indicated GL50 to be present in many tissues including lymphoid, embryonic yolk sac, and fetal liver samples. Of the CD28, CTLA4, and ICOS fusion constructs tested, flow cytometric analysis demonstrated only mouse ICOS-IgG binding to mGL50 cell transfectants. Subsequent phenotyping demonstrated high levels of ICOS ligand staining on splenic CD19+ B cells and low levels on CD3+ T cells. These results indicate that GL50 is a specific ligand for the ICOS receptor and suggest that the GL50-ICOS interaction functions in lymphocyte costimulation.