A functional assay for heterozygous mutations in the GTPase activating protein related domain of the neurofibromatosis type 1 gene

Chikashi Ishioka, R. Ballester, M. Engelstein, M. Vidal, J. Kassel, I. The, A. Bernards, J. F. Gusella, S. H. Friend

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)841-847
Number of pages7
JournalOncogene
Volume10
Issue number5
Publication statusPublished - 1995 Jan 1
Externally publishedYes

Keywords

  • Functional assay in yeast FASAY
  • NF1

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cancer Research

Fingerprint Dive into the research topics of 'A functional assay for heterozygous mutations in the GTPase activating protein related domain of the neurofibromatosis type 1 gene'. Together they form a unique fingerprint.

Cite this