The simian virus 40 (SV40) mutant ts A1499 contain an 81-base-pair deletion in the region of A gene encoding the C-terminal portion of the large T antigen. This mutant is particularly interesting, since it is a temperature-sensitive mutant that is apparently able to separate the lytic growth and transforming functions of the SV40 large T antigen at 38.5°C. We report the isolation of a tsA1499 revertant (tsA 1499-Rev) which is no longer temperature sensitive for lytic growth but still contains the 81-base-pair deletion of tsA 1499. Marker rescue experiments with tsA 1499-Rev or wild-type strain 830 (wt830) DNAs revealed that the original tsA 1499 mutant contained a second mutation within the HindIII-Fnu4HI restriction fragment between 0.425 and 0.484 map units. Sequencing of this DNA fragment from the tsA 1499, tsA 1499-Rev, and wt830 viruses revealed that tsA 1499 contained a single-base transversion (C to G) at 0.455 map units (nucleotide 4261). This transversion resulted in the creation of a new RsaI cleavage site in the tsA 1499 DNA and predicts an arginine-to-threonine substitution at amino acid position 186 in the mutant large T antigen. The DNA sequence of the tsA 1499-Rev HindIII-Fnu4HI fragment was identical to that of wt830. To determine whether tsA 1499 was temperature sensitive for lytic growth solely as a result of the newly discovered point mutation or because of a combination of the point and deletion mutations, a series of viruses were constructed which contained the point mutation, the deletion mutation, both mutations, or neither. This was done by ligating the PstI and B DNA fragments from either tsA 1499 or wt830 and transfecting the ligated DNA into BSC-1H monkey kidney cells. This experiment revealed that all viruses containing the point mutation (the tsA 1499 PstI A DNA fragment) were temperature sensitive for lytic growth, regardless of the presence of the 81-base-pair deletion (the tsA 1499 PstI B DNA fragment). This newly discovered point mutation, at nucleotide 4261, is therefore unique, since to our knowledge it is the first tsA mutation to be described in the 0.455-map-unit region of the SV40 genome. We then tested the effect of this unique mutation on the ability of the SV40 virus to transform cultured rat cells to anchorage independence. We found that a mutant virus that contained only the point mutation, tsA 3900, was able to transform F111 rat cells to anchorage independence equally well at the permissive and nonpermissive temperatures for lytic growth with an efficiency of transformation similar to that of a wild-type strain of SV40 (wt830). Since the tsA lytic phenotype of tsA 3900 is tight and the anchorage independence assay for transformation is stringent, the apparent separation of the lytic growth and transforming functions of tsA 3900 is striking.
All Science Journal Classification (ASJC) codes
- Insect Science