RESUMO
1. A new cloning procedure is described for cDNA synthesis from mRNA released by in vitro translation of polysomes in a cell-free amino acid incorporating system. The usefulness of the method lies in the feasibility of employing nanogram amounts of mRNA. 2. Complementary DNA is synthesized derectly in the translation mixture simply by adjusting the concentration of some components and removing ribosomes by boiling and centrifugation. 3. As an example, we report here the construction and characterization of cDNA clone corresponding to chick alfa (1) procollagen starting from a collagen-synthesizing polysome fraction obtained from chick embryos
Assuntos
Embrião de Galinha , Animais , Clonagem Molecular , DNA Recombinante/biossíntese , Técnicas In Vitro , Polirribossomos/metabolismo , RNA Mensageiro/metabolismo , Colágeno/metabolismoRESUMO
When a rabbit reticulocyte lysate is incubated in the presence of vaccina cores, protein synthesis is umpaired at the level of the initiation step and the polyribosomes are depolymerized. However, when the same system is coupled with virus transcription: a) protein synthesis is restored, b) the initiation step is not inhibited, and c) the polyribosomes are not disaggregated. A viral factor activated in the absence of virus transcription and not activated when RNA synthesis occurs may be involved in the early mechanism of protein synthesis inhibition by vaccinia virus