Gangfeng Xu

A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutations.

Abstract Overlapping cDNA clones from the translocation break-point region (TBR) gene, recently discovered at the neurofibromatosis type 1 locus and found to be interrupted by deletions and a t(17;22) translocation, have been sequenced. A 4 kb sequence of the transcript of the TBR gene has been compared with sequences of genomic DNA, identifying a number of small exons. Identification of splice junctions and a large open reading frame indicates that the gene is oriented with its 5′ end toward the centromere, in opposition to the three known active genes in the region. PCR amplification of a subset of the exons, followed by electrophoresis of denatured product on native gels, identified six variant conformers specific to NF1 patients, indicating base pair changes in the gene. Sequencing revealed that one mutant allele contains a T→C transition changing a leucine to a proline; another NF1 allele harbors a C→T transition changing an arginine to a stop codon. These results establish the TBR gene as the NF1 gene and provide a description of a major segment of the gene.

Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus

Three new neurofibromatosis type 1 (NF1) mutations have been detected and characterized. Pulsed-field gel and Southern blot analyses reveal the mutations to be deletions of 190, 40, and 11 kb of DNA. The 11 kb deletion does not contain any of the previously characterized genes that lie between two NF1 translocation breakpoints, but it does include a portion of a rodent/human conserved DNA sequence previously shown to span one of the translocation breakpoints. By screening cDNA libraries with the conserved sequence, we identified a number of cDNA clones from the translocation breakpoint region (TBR), one of which hybridizes to an approximately 11 kb mRNA. The TBR gene crosses at least one of the chromosome 17 translocation breakpoints found in NF1 patients. Furthermore, the newly characterized NF1 deletions remove internal exons of the TBR gene. Although these mutations might act by compromising regulatory elements affecting some other gene, these findings strongly suggest that the TBR gene is the NF1 gene.

The neurofibromatosis type 1 gene encodes a protein related to GAP

cDNA walking and sequencing have extended the open reading frame for the neurofibromatosis type 1 gene (NF1). The new sequence now predicts 2485 amino acids of the NF1 peptide. A 360 residue region of the new peptide shows significant similarity to the known catalytic domains of both human and bovine GAP (GTPase activating protein). A much broader region, centered around this same 360 amino acid sequence, is strikingly similar to the yeast IRA1 product, which has a similar amino acid sequence and functional homology to mammalian GAP. This evidence suggests that NF1 encodes a cytoplasmic GAP-like protein that may be involved in the control of cell growth by interacting with proteins such as the RAS gene product. Mapping of the cDNA clones has confirmed that NF1 spans a t(1;17) translocation mutation and that three active genes lie within an intron of NF1, but in opposite orientation.

The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae

Sequencing of the neurofibromatosis gene (NF1) revealed a striking similarity among NF1, yeast IRA proteins, and mammalian GAP (GTPase-activating protein). Using both genetic and biochemical assays, we demonstrate that this homology domain of the NF1 protein interacts with ras proteins. First, expression of this NF1 domain suppressed the heat shock-sensitive phenotype of yeast ira1 and ira2 mutants. Second, this NF1 domain, after purification as a glutathione S-transferase (GST) fusion protein, strongly stimulated the GTPase activity of yeast RAS2 and human H-ras proteins. The GST-NF1 protein, however, did not stimulate the GTPase activity of oncogenic mutant ras proteins, H-rasVal-12 and yeast RAS2Val-19 mutants, or a yeast RAS2 effector mutant. These results establish that this NF1 domain has ras GAP activity similar to that found with IRA2 protein and mammalian GAP, and therefore may also regulate ras function in vivo.

The gene encoding the oligodendrocyte-myelin glycoprotein is embedded within the neurofibromatosis type 1 gene.

In the course of efforts to identify the neurofibromatosis type 1 gene (NF1), three genes were found embedded within an intron of NF1. The cDNA sequence of one of these genes (OMGP) encodes oligodendrocyte-myelin glycoprotein. OMGP spans at least 2.7 kb of genomic DNA, and it maps within 4 kb of the breakpoint of a balanced chromosomal translocation carried by an individual with NF1. OMGP is similar in genomic structure to two other expressed genes, EVI2A and EVI2B, which lie approximately 20 and 5 kb telomeric of the OMGP locus, respectively. All three genes have the same transcriptional orientation and are contained within one intron of NF1, which is transcribed off the opposite strand. Whether altered expression of OMGP might play a role in the clinical heterogeneity of NF1 is as yet unclear.

Characterization of human adenylate kinase 3 (AK3) cDNA and mapping of the AK3 pseudogene to an intron of the NF1 gene.

We have isolated cDNA clones for human adenylate kinase isozyme 3 (AK3) with a genomic probe from the neurofibromatosis type 1 (NF1) region. Three overlapping clones isolated from a human frontal-cortex cDNA library gave rise to a consensus sequence of 1.7 kb. The open reading frame identified in this sequence predicted a peptide of 223 residues. A database search revealed striking homology, about 58% amino acid sequence identity, between this predicted protein and bovine AK3. Human AK3 protein also showed significant homology to other members of the adenylate kinase family isolated from various species. Genomic Southern analysis suggested that multiple AK3 loci exist in the human genome, including one located in an intron of NF1 on chromosome 17. The chromosome-17 locus appears to be a processed pseudogene, since it is intronless and contains a polyadenylate tract; it nevertheless retains coding potential because the open reading frame is not impaired by any observed base substitutions.

The neurofibromatosis type 1 (NF1) gene: identification and partial characterization of a putative tumor suppressor gene

The NF1 gene has been isolated and partially characterized. The discovery that NF1 functions as a ras GTPase activator protein has led to new opportunities for understanding the pathology of this disease. The approximately 11 kilobase (kb) NF1 consensus cDNA sequence contains an open reading frame encoding a peptide of 2818 amino acids. DNA blot and polymerase chain reaction analysis indicate that the NF1 gene consists of over 50 exons spanning 300 kb of chromosome 17.

Erratum: A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutations (Cell 62, 193-201, 1990; July 13 issue)

[This corrects the article on p. 492 in vol. 41.].