Abstract
N-ethyl-
N-nitrosourea (ENU) mutagenesis has led to the elucidation of several regulator genes for melanocyte and skin development. Here we characterized a mutant from ENU mutagenesis with similar phenotype as that of
Splotch mutant, including exencephaly, spina bifida and abnormal limbs in homozygotes as well as white belly spotting and occasionally loop-tail in heterozygotes. This novel mutant was named as
Sp
xG
. Through genome-wide linkage analysis in backcross progenies with microsatellite markers, the
Sp
xG
was confined to a region between D1MIT415 and D1MIT7 on chromosome 1, where notable
Pax3 gene was located. Direct sequencing revealed that
Sp
xG
carried a nucleotide A894G missense transition in exon 6 of
Pax3 gene that resulted in Asn to Asp substitution at amino acid 269 within the highly-conserved homeodomain (HD) DNA recognition module, which was the first point mutation found in this domain in mice. This N269D mutation impaired the transactivation capacity of Pax3 protein, but exerted no effect on Pax3 protein translation. The characterization of the new mutation expanded our understanding the transactivation and DNA-binding structure of Pax3 protein.