Characterization of the NF1 gene and its gene product, neurofibromin.

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Publication Type dissertation
School or College School of Medicine
Department Human Genetics
Author Xu, Gangfeng Cloning
Title Characterization of the NF1 gene and its gene product, neurofibromin.
Date 1994-08
Description The neurofibromatosis 1 gene (NFl) spans approximately 350 kb of genomic DNA. To develop a comprehensive screen for NFl mutations, I constructed a genomic DNA contig that included the entire NFl cDNA sequence. Using these genomic clones, I identified all intron-exon boundaries of the NFl coding region and established that the gene comprises 59 exons. The flanking intronic sequences obtained enabled me and others to design intron-based oligonucleotide primers to amplify NFl exons. I also discovered a locus homologous to NFl on chromosome 15. In spite of its high degree of sequence homology with NF1, frame-shifting deletions in some exons of the chromosome 15 locus destroy the intact coding sequence. NFl encodes a p21ras GTPase-activating protein (GAP). Since p21ras-GTP is a major regulator of cellular proliferation, mutant neurofibromins with diminished GAP activity might deregulate the ras-GTP level and contribute to the development of tumors. With this hypothesis in mind, I found an amino acid substitution (altering Lys-1423, in the NFl GAP-related-domain [NF1-GRD]) in three tumor samples: a colorectal adenocarcinoma, bone marrow from a myelodysplastic syndrome patient, and an anaplastic astrocytoma. I further proved that this substitution causes a drastic decrease in GAP activity. Since I also found somatic mutations in NFl in some breast cancer samples, I constructed several NF1 -deficient breast epithelial cell lines using an antisense-cDNA method. However, I have found no evidence that these cells are tumorigenic. To further explore the tumor-suppressive role of NF1, I overexpressed full-length NF 1 cDNA, as well as wild type and mutant (K1423E) NF1-GRD, in a colon carcinoma cell line, and demonstrated that overexpression of all three constructs suppressed the tumor phenotype. I also demonstrated that Raf-1 kinase (a downstream effector of p21ras) activity was reduced in cells with overexpressed neurofibromin and that reintroducing an activated Raf-1 kinase domain can reverse tumor suppression. I therefore concluded that tumor suppression by neurofibromin is not due to GAP activity, but rather to tight binding to p21ras-GTP.
Type Text
Publisher University of Utah
Subject cDNA; Genes
Subject MESH Neurofibromatoses; Genetics
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "Characterization of the NF1 gene and its gene product, neurofibromin." Spencer S. Eccles Health Sciences Library. Print version of "Characterization of the NF1 gene and its gene product, neurofibromin." available at J. Willard Marriott Library Special Collection. RC39.5 1994 .X8.
Rights Management © Gangfeng Xu.
Format Medium application/pdf
Identifier us-etd2,6382
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available).
ARK ark:/87278/s6417bj2
Setname ir_etd
Date Created 2012-04-23
Date Modified 2012-04-23
ID 192409
Reference URL https://collections.lib.utah.edu/ark:/87278/s6417bj2