DNA clone of avian Fujinami sarcoma virus with temperature-sensitive transforming function in mammalian cells.

摘要

We have molecularly cloned an integrated proviral DNA of Fujinami sarcoma virus (FSV) into a lambda phage vector and further subcloned it into plasmid pBR322. The source of provirus was a quail nonproducer cell clone transformed by FSV. The FSV strain used is temperature sensitive in the maintenance of transformation of avian cells. The recombinant plasmid was shown to contain an entire FSV genome by fingerprinting the hybrids formed with 32P-labeled FSV RNA. This analysis also revealed a previously undetected env-related sequence in FSV which represents the 3' end of the gp85 env gene. A physical map of cloned FSV DNA identifying sites of several restriction enzymes is described. Upon transfection, FSV DNA cloned in pBR322 transformed mouse NIH-3T3 cells, which proved to be temperature sensitive in maintaining transformation. Phosphorylation but not synthesis of p140, the only known gene product of FSV, was also temperature sensitive in these cells. The correlation between transformation and phosphorylation of p140 suggests that phosphorylation of p140 is necessary for transformation of mouse cells, as was shown previously for avian cells. These results provide direct genetic evidence that the mechanisms for maintaining transformation of mammalian and avian cells involve the same FSV gene product, p140. Homology was detected by hybridization between transformation-specific sequences of FSV DNA and certain restriction endonuclease-resistant fragments of cellular DNA of two avian species, chicken and quail. Under the same conditions homology was also detected with DNA of non-avian species, although apparently to a lower degree than with avian cells.