Reticulocyte Translation System Research Articles

De novo synthesis of neuron-specific enolase in a rabbit reticulocyte translation system programmed by poly(A)-RNA from rat brain.

Poly(A)-RNA prepared from the brains of 30-day-old male rats has been shown to direct the synthesis of neuron-specific enolase (NSE) in a cell-free system derived from rabbit reticulocytes. The newly synthesized polypeptides were immunoprecipitated and analyzed on SDS-polyacrylamide slab gels. Autoradiography indicated the synthesis of a product that comigrated with purified NSE and was recognized only by the anti-NSE antisera. Similar immunoprecipitation of reticulocyte lysates programmed with total RNA derived from embryonic chick heart failed to indicate the synthesis of NSE. These results show that the mRNA coding for NSE contains a poly(A) sequence and that brain-specific factors are not required for its translation.

The coupling of transcription from influenza virions to translation in vitro.

The optimum conditions for the coupling of fowl plague virus (FPV) transcription to an in vitro reticulocyte translation system have been established and shown to be close to those required for maximum RNA synthesis by purified FPV virions. Products have been characterized by the peptides they yield on limited proteolysis in SDS and it has been shown that virus nucleoprotein (NP) and matrix (M) protein are made. The smallest virus coded polypeptide, the non-structural protein (NS), is made in only small amounts in the coupled system although it is a major virus coded product of infected cells early in infection.

De novo biosynthesis of an enzymatically active precursor form of bovine pancreatic RNase.

The de novo biosynthesis of RNase (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) was studied in a cell-free rabbit reticulocyte translation system using a polyadenylylated fraction of mRNA isolated from bovine pancreas. Analysis of the [35S]methionine-labeled translation products of pancreas mRNA by polyacrylamide gel electrophoresis indicated the presence of several proteins, one of which corresponded to 16,500 daltons, or approximately 2800 daltons greater than native RNase A. This protein was specifically precipitated from the mixture of products by anti-RNase antibodies. Partial sequence determination of the NH2-terminal region of the anti-RNase antibody-precipitable species indicated that it is a precursor form of RNase A with 25 additional amino acids on its NH2 terminus. The precursor nature of the protein was confirmed by demonstration that a mixture of RNase A and a glycosylated form of the enzyme, RNase B, is formed when translation of the mRNA is conducted in the presence of dog pancreas membranes. Assay of the putative precursor form of RNase for catalytic activity with polycytidylic acid as substrate indicated that the protein has a specific enzymatic activity identical to that of native RNase A.

Recombinant avian oncoviruses I. Alterations in the precursor to the internal structural proteins

The synthesis of the gag precursor protein (Pr76) was studied in a number of recombinant avian oncoviruses, which were selected for recombination between the env and pol genes or the env and src genes. Such studies show that the electrophoretic mobility of the gag precursor protein of recombinant viruses (ΔPr76) was greater than that of the parental gene product (Pr76) in 16 of 24 cases. Viruses derived from recombination between endogenous (RAV-0) and exogenous viruses (RSV), as well as between two exogenous viruses, showed the ΔPr76 phenotype. In an mRNA-dependent rabbit reticulocyte translation system, 35 S RNA isolated from PR-RSV-C directed the synthesis of Pr76, while RNA isolated from a recombinant between PR-RSV-C and RAV-0 directed the synthesis of ΔPr76. These observations show that the synthesis of ΔPr76 is due to an alteration in the genome related to recombination. An analysis of the RNase T 1-resistant oligonucleotides demonstrated a crossover near the 5′ end of the genome (which may be within the gag gene) in two recombinant virus clones which synthesize ΔPr76 in infected cells; but no crossover was detected near the 5′ end of the genome in a third recombinant virus clone which synthesizes Pr76 in infected cells. Our data suggest that the synthesis of ΔPr76 is a consequence of recombination near the 5′ end of the genome.