Identification and order of sequential mutations in beta-actin genes isolated from increasingly tumorigenic human fibroblast strains.

We have sequenced the mutant beta-actin gene of a tumorigenic human fibroblast cell line (HuT-14T) and found that it carries three mutations that alter the amino acids at positions 36, 83, and 244 as well as a 22-base-pair "insertion" sequence, in the 5' intron, not present in a wild-type gene. The less tumorigenic cell line HuT-14, a progenitor of HuT-14T, has the same codon-244 mutation and the insertion sequence but not the other two mutations. A nontumorigenic cell line that is related to HuT-14 but that has no beta-actin mutations does carry the intron-length polymorphism. We conclude that the mutation at codon 244 occurred first in a beta-actin allele already bearing the 22-base-pair intron insert and that mutations at codons 36 and 83 arose subsequently during the selection for the HuT-14T phenotype. Rat-2 cells synthesize the appropriate charge-variant species of mutant actin protein when transfected with either the singly or the triply mutated beta-actin gene.