Estimation of mutation rates based on the analysis of polypeptide constituents of cultured human lymphoblastoid cells.
A subclone of a human diploid lymphoblastoid cell line, TK-6, with consistently high cloning efficiency has been used to estimate the rates of somatic mutations on the basis of protein variation detected by two-dimensional polyacrylamide gel electrophoresis. A panel of 267 polypeptide spots per gel was screened, representing the products of approximately 263 unselected loci. The rate of human somatic mutation in vitro was estimated by measuring the proportion of protein variants among cell clones isolated at various times during continuous exponential growth of a TK-6 cell population. Three mutants of spontaneous origin were observed, giving an estimated spontaneous rate of 6 x 10(-8) electrophoretic mutations per allele per cell generation (i.e., 1.2 x 10(-7) per locus per cell generation). Following treatment of cells with N-ethyl-N-nitrosourea, a total of 74 confirmed variants at 54 loci were identified among 1143 clones analyzed (approximately 601,000 allele tests). The induced variants include 65 electromorphs which exhibit altered isoelectric charge and/or apparent molecular weight and nine nullimorphs for each of which a gene product was not detected at its usual location on the gel. The induced frequency for these 65 structural gene mutants is 1.1 x 10(-4) per allele. An excess of structural gene mutations at ten known polymorphic loci and repeat mutations at these and other loci suggest nonrandomness of mutation in human somatic cells. Nullimorphs occurring at three heterozygous loci in TK-6 cells may be caused by genetic processes other than structural gene mutation.
Chu
E H
EH
Department of Human Genetics, University of Michigan, Ann Arbor 48109.
Boehnke
M
M
Hanash
S M
SM
Kuick
R D
RD
Lamb
B J
BJ
Neel
J V
JV
Niezgoda
W
W
Pivirotto
S
S
Sundling
G
G
D002460:Cell Line; D002461:Cell Line, Transformed; D004591:Electrophoresis, Polyacrylamide Gel; D005038:Ethylnitrosourea / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008957:Models, Genetic; D009154:Mutation; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification
D011506:Proteins; D005038:Ethylnitrosourea