TY - JOUR
T1 - Influence of neighboring base sequence on mutagenesis induced by in vitro misincorporation in the lacI gene of Escherichia coli
AU - Maldonado-Rodriguez, Rogelio
AU - Espinosa-Lara, Mercedes
AU - Beattie, Kenneth L.
N1 - Funding Information:
This researchw ass upportebdy N.I.H. Grants GM25530 and GM30590 and by Grant Q-1006 from the RobertA . WelchF oundationR.. M.-R. receivedfe llowships upportf rom COFAA-IPN, M6xico and ColgateP almolive,M 6xico. K.L.B. was recipiento f ResearchC areerD evelopment Award CA00891f romthe NationaCl ancerIn sti-tute.
Funding Information:
* Present address: Depto. de Bioquimica, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional, Prol. de Carpio y Plan de Ayala, Mexico, D.F. 113404 (Mexico), Tel. (905) 396-3716. ** Recipient of Research Career Development Award CA00891 from the National Cancer Institute.
PY - 1991/12
Y1 - 1991/12
N2 - Genetic and electrophoretic assays of misincorporation were used to assess the effect of DNA sequence on mutagenesis arising from in vitro DNA synthesis within the lacI gene of Escherichia coli. The viral strand of a derivative of phage M13 containing the entire lacI gene was annealed with a series of synthetic oligonucleotides complementary to the N-terminal region of the lacI gene. Each primer-template was incubated with E. coli DNA polymerase I (Klenow fragment) under conditions favoring misincorporation, wherein one of the 4 dNTPs was lacking ('minus' reaction) or present at very low concentration ('micro' reaction). The extent of elongation of each primer was assessed by gel electrophoresis, and lacI mutants arising during the misincorporation reactions were detected by a transfection assay in which i- base substitutions within the in vitro synthesized strand were selectively recovered by the use of uracil-containing templates. Direct dideoxy sequencing of the '-A' reaction products and sequence analysis of i- mutant progeny revealed a vast predominance of single and non-tandem multiple base transitions. The addition of small quantities of dATP to a '-A' reaction increased the mutation yield and broadened the distribution of base substitutions along the template. We detected a general bias towards increased base substitution at template positions flanked by G · C base pairs or 5′-pyrimidine, 3′-purine nearest neighbors, although considerable site-to-site variation in the occurence of base substitutions was seen, even within identical nearest neighbor contexts.
AB - Genetic and electrophoretic assays of misincorporation were used to assess the effect of DNA sequence on mutagenesis arising from in vitro DNA synthesis within the lacI gene of Escherichia coli. The viral strand of a derivative of phage M13 containing the entire lacI gene was annealed with a series of synthetic oligonucleotides complementary to the N-terminal region of the lacI gene. Each primer-template was incubated with E. coli DNA polymerase I (Klenow fragment) under conditions favoring misincorporation, wherein one of the 4 dNTPs was lacking ('minus' reaction) or present at very low concentration ('micro' reaction). The extent of elongation of each primer was assessed by gel electrophoresis, and lacI mutants arising during the misincorporation reactions were detected by a transfection assay in which i- base substitutions within the in vitro synthesized strand were selectively recovered by the use of uracil-containing templates. Direct dideoxy sequencing of the '-A' reaction products and sequence analysis of i- mutant progeny revealed a vast predominance of single and non-tandem multiple base transitions. The addition of small quantities of dATP to a '-A' reaction increased the mutation yield and broadened the distribution of base substitutions along the template. We detected a general bias towards increased base substitution at template positions flanked by G · C base pairs or 5′-pyrimidine, 3′-purine nearest neighbors, although considerable site-to-site variation in the occurence of base substitutions was seen, even within identical nearest neighbor contexts.
UR - http://www.scopus.com/inward/record.url?scp=0025993035&partnerID=8YFLogxK
U2 - 10.1016/0027-5107(91)90076-Z
DO - 10.1016/0027-5107(91)90076-Z
M3 - Artículo
SN - 0027-5107
VL - 251
SP - 217
EP - 226
JO - Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
JF - Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
IS - 2
ER -