Cloning and characterization of a thermolabile v-src gene for use in reversible transformation of mammalian cells.
The use of temperature-sensitive (ts) src mutants for studies of cell transformation and differentiation has been limited by the availability of cloned ts-src genes that are inactivated at temperatures compatible with growth of mammalian cells. In this report, we describe the cloning and characterization of the tsLA90src gene, which displays tight thermal sensitivity at 39.5 degrees C. Nucleotide sequence comparison of tsLA90 and wild-type src genes from the Schmidt-Ruppin subgroup A and D strains of Rous sarcoma virus (RSV) revealed four amino acid differences in tsLA90src. Substitution of one of these residues (Lys-280) from tsLA90src with its wild-type homolog (Glu-280) caused a reversion to a wild-type src phenotype. The cloned tsLA90 gene, designated tsUP1, was introduced into avian and mammalian retroviral vectors. Chicken embryo fibroblasts and immortalized mouse 3T3 cells infected with these viral vectors displayed a temperature-dependent transformed phenotype as assessed by cell morphology, secretion of plasminogen activator, transcriptional activation of the primary response genes, Egr-1 and TIS 10, and stimulation of tyrosine phosphorylation. In addition, chicken myoblasts (infected with RSVtsUP1) showed a temperature-dependent differentiation into myotubes. Thus, this cloned src gene should be ideally suited for inducing reversible transformation and differentiation of mammalian cells in culture.
Maroney
A C
AC
Howard Hughes Medical Institute, Department of Microbiology, University of Pennsylvania, School of Medicine, Philadelphia 19104.
Qureshi
S A
SA
Foster
D A
DA
Brugge
J S
JS
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics*; D001483:Base Sequence; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic; D002642:Chick Embryo; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics; D051766:Early Growth Response Protein 1; D005347:Fibroblasts; D015967:Gene Expression Regulation, Viral; D016391:Genes, src; D017874:Immediate-Early Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology / Q000502:physiology; D009838:Oligodeoxyribonucleotides; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D016133:Polymerase Chain Reaction / Q000379:methods; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis; D014764:Viral Proteins; D016335:Zinc Fingers / Q000235:genetics
D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; D014764:Viral Proteins; C074810:SRC protein, Rous sarcoma virus; D019000:Phosphotyrosine; D014443:Tyrosine; D015688:Oncogene Protein pp60(v-src)