Biomaterial Database

Sequential transcription-translation of simian virus 40 by using mammalian cell extracts. 1981

C L Cepko, and U Hansen, and H Handa, and P A Sharp

Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980) were tested for their efficiency and fidelity in directing protein synthesis in reticulocyte lysates. Simian virus 40 deoxyribonucleic acid (DNA), cleaved by various restriction endonucleases, was used as the template. Successful translation of the small tumor antigen t, as well as the capsid proteins VP1, VP2, and VP3, was detected by immunoprecipitation analysis. Although no synthesis of large T antigen was detected, use of this technology allows detection of large T synthesis resulting from the correct splicing of as little as 0.2% of the in vitro RNA transcripts, making it ideal for use as an in vitro splicing assay. Transcripts synthesized in vitro were used as messages at least as efficiently as were viral messenger RNA's (mRNA's) synthesized in vivo; and in the case of small t, there was more efficient translation of small t mRNA synthesized in vitro than of small t mRNA synthesized in vivo. The transcripts that served as mRNA's for the various polypeptides were identified by using the following two criteria. (i) The sensitivity of synthesis of a given protein to digestion of the template DNA with restriction enzymes allowed the localization of the promoter and coding regions. (ii) Translation of size-fractionated RNA allowed confirmation of the transcript-mRNA assignments. With these techniques we found that VP2, VP3 and, in some cases, VP1 synthesis resulted from the initiation of translation at internal AUG codons. In fact, families of polypeptides were produced by initiation of translation at AUG codons within sequences coding for VP1 and T, presumably as a result of transcription initiation events that generated 5' ends immediately upstream from these AUGs. Application of this technology for the identification of coding regions within cloned DNA fragments is discussed.

UI	MeSH Term	Description	Entries
D009876	Operon	In bacteria, a group of metabolically related genes, with a common promoter, whose transcription into a single polycistronic MESSENGER RNA is under the control of an OPERATOR REGION.	Operons
D002478	Cells, Cultured	Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others.	Cultured Cells,Cell, Cultured,Cultured Cell
D002522	Chlorocebus aethiops	A species of CERCOPITHECUS containing three subspecies: C. tantalus, C. pygerythrus, and C. sabeus. They are found in the forests and savannah of Africa. The African green monkey is the natural host of SIMIAN IMMUNODEFICIENCY VIRUS and is used in AIDS research.	African Green Monkey,Cercopithecus aethiops,Cercopithecus griseoviridis,Cercopithecus griseus,Cercopithecus pygerythrus,Cercopithecus sabeus,Cercopithecus tantalus,Chlorocebus cynosuros,Chlorocebus cynosurus,Chlorocebus pygerythrus,Green Monkey,Grivet Monkey,Lasiopyga weidholzi,Malbrouck,Malbrouck Monkey,Monkey, African Green,Monkey, Green,Monkey, Grivet,Monkey, Vervet,Savanah Monkey,Vervet Monkey,Savannah Monkey,African Green Monkey,Chlorocebus cynosuro,Green Monkey, African,Green Monkeys,Grivet Monkeys,Malbrouck Monkeys,Malbroucks,Monkey, Malbrouck,Monkey, Savanah,Monkey, Savannah,Savannah Monkeys,Vervet Monkeys
D004262	DNA Restriction Enzymes	Enzymes that are part of the restriction-modification systems. They catalyze the endonucleolytic cleavage of DNA sequences which lack the species-specific methylation pattern in the host cell's DNA. Cleavage yields random or specific double-stranded fragments with terminal 5'-phosphates. The function of restriction enzymes is to destroy any foreign DNA that invades the host cell. Most have been studied in bacterial systems, but a few have been found in eukaryotic organisms. They are also used as tools for the systematic dissection and mapping of chromosomes, in the determination of base sequences of DNAs, and have made it possible to splice and recombine genes from one organism into the genome of another. EC 3.21.1.	Restriction Endonucleases,DNA Restriction Enzyme,Restriction Endonuclease,Endonuclease, Restriction,Endonucleases, Restriction,Enzymes, DNA Restriction,Restriction Enzyme, DNA,Restriction Enzymes, DNA
D004279	DNA, Viral	Deoxyribonucleic acid that makes up the genetic material of viruses.	Viral DNA
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D000956	Antigens, Viral	Substances elaborated by viruses that have antigenic activity.	Viral Antigen,Viral Antigens,Antigen, Viral
D000957	Antigens, Viral, Tumor	Those proteins recognized by antibodies from serum of animals bearing tumors induced by viruses; these proteins are presumably coded for by the nucleic acids of the same viruses that caused the neoplastic transformation.	Antigens, Neoplasm, Viral,Neoplasm Antigens, Viral,T Antigens,Tumor Antigens, Viral,Viral Tumor Antigens,Virus Transforming Antigens,Large T Antigen,Large T-Antigen,Small T Antigen,Small T-Antigen,T Antigen,T-Antigen,Viral T Antigens,Antigen, Large T,Antigen, Small T,Antigen, T,Antigens, T,Antigens, Viral Neoplasm,Antigens, Viral T,Antigens, Viral Tumor,Antigens, Virus Transforming,T Antigen, Large,T Antigen, Small,T Antigens, Viral,T-Antigen, Large,T-Antigen, Small,Transforming Antigens, Virus,Viral Neoplasm Antigens
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012326	RNA Splicing	The ultimate exclusion of nonsense sequences or intervening sequences (introns) before the final RNA transcript is sent to the cytoplasm.	RNA, Messenger, Splicing,Splicing, RNA,RNA Splicings,Splicings, RNA