A functional assay for heterozygous mutations in the GTPase activating protein related domain of the neurofibromatosis type 1 gene.
The GTPase-activating protein related domain of the human neurofibromatosis type 1 protein (NF1GRD) can down-regulate RAS in Saccharomyces cerevisiae. Using a technique termed the FASAY method, for Functional Analysis of Separated Alleles in Yeast, we designed a rapid method for detection of heterozygous NF1GRD loss-of-function mutations. In our method, PCR amplified NF1GRD cDNA is directly cloned into a centromeric vector by homologous recombination in a cdc25 temperature-sensitive mutant strain expressing human Ha-ras. This strain is dependent on the Ha-ras for growth, allowing a simple growth assay for NF1GRD loss-of-function mutations. In a test of our method, two alternatively spliced NF1GRD cDNAs (type I and II) inhibited yeast growth whereas four mutants with amino acid substitutions at highly conserved residues did not. This simple method thus permits the rapid screening for heterozygous germline or somatic NF1GRD mutations. In an initial application of this method, no mutations disrupting NF1GRD function were detected in lymphoblasts from 11 previously untested neurofibromatosis type 1 patients.
Ishioka
C
C
MGH Cancer Center, Massachusetts General Hospital, Charlestown 02129.
Ballester
R
R
Engelstein
M
M
Vidal
M
M
Kassel
J
J
The
I
I
Bernards
A
A
Gusella
J F
JF
Friend
S H
SH
D001483:Base Sequence; D002471:Cell Transformation, Neoplastic; D020690:GTPase-Activating Proteins; D016514:Genes, Neurofibromatosis 1; D011905:Genes, ras; D006579:Heterozygote; D008969:Molecular Sequence Data; D009154:Mutation; D011506:Proteins / Q000235:genetics* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins