Preparation of a "functional library" of African green monkey DNA fragments which substitute for the processing/polyadenylation signal in the herpes simplex virus type 1 thymidine kinase gene.
Open Access
- 1 April 1983
- journal article
- research article
- Published by Taylor & Francis in Molecular and Cellular Biology
- Vol. 3 (4), 643-653
- https://doi.org/10.1128/mcb.3.4.643
Abstract
Fragments of African green monkey (Cercopithecus aethiops) DNA (3.5 to 18.0 kilobases) were inserted downstream from the thymidine kinase (TK, tk) coding region in pTK206/SV010, a gene construct which lacks both copies of the hexanucleotide 5'-AATAAA-3' and contains a simian virus 40 origin of replication, allowing it to replicate in Cos-1 cells. No polyadenylated tk mRNA was detected in Cos-1 cells transfected by pTK206/SV010. The ability of simian DNA fragments to restore tk gene expression was examined by measuring the incorporation of [125I]iododeoxycytidine into DNA in Cos-1 cells transfected by pTK206/SV010 insertion derivatives. tk gene expression was restored by the insertion in 56 of the 67 plasmids analyzed, and the level of expression equaled or exceeded that obtained with the wild-type tk gene in 30 of these. In all plasmids examined that showed restoration of tk gene expression, polyadenylated tk mRNA of discrete size was detected. The sizes of these tk mRNAs were consistent with the existence of processing and polyadenylation signals within the inserted DNA fragments. The frequency with which inserted fragments restored tk gene expression suggests that the minimal signal for processing and polyadenylation is a hexanucleotide (AAUAAA or a similar sequence). LTK- cells were biochemically transformed to TK+ with representative insertion constructs. pTK206/SV010 transformed LTK- cells at a very low frequency; the frequency of transformation with insertion derivatives was 40 to 12,000 times higher.Keywords
This publication has 49 references indexed in Scilit:
- Transcripts from the Cellular Homologs of Retroviral Oncogenes: Distribution Among Chicken TissuesMolecular and Cellular Biology, 1982
- Phenotypic Switching in Cells Transformed with the Herpes Simplex Virus Thymidine Kinase GeneMolecular and Cellular Biology, 1982
- Reiteration Frequency of the Histone Genes in the Genome of the Amphibian, Xenopus laevisEuropean Journal of Biochemistry, 1976
- 3′ Non-coding region sequences in eukaryotic messenger RNANature, 1976
- Deoxythymidine kinase induced in HeLa TK- cells by herpes simplex virus type I and type II. II. Purification and characterization.Journal of Biological Chemistry, 1976
- Detection of specific sequences among DNA fragments separated by gel electrophoresisJournal of Molecular Biology, 1975
- Biogenesis and characterization of histone messenger RNA in HeLa cellsJournal of Molecular Biology, 1972
- Purification of Biologically Active Globin Messenger RNA by Chromatography on Oligothymidylic acid-CelluloseProceedings of the National Academy of Sciences, 1972
- Calcium-dependent bacteriophage DNA infectionJournal of Molecular Biology, 1970
- Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation eventBiochemistry, 1970