Jeffery L. Cole

Construction of a transcription map surrounding the BRCA1 locus of human chromosome 17

We have used a combination of methods (exon amplification, direct selection, direct screening, evolutionary conservation, island rescue-PCR, and direct sequence analysis) to survey approximately 600 kb of genomic DNA surrounding the BRCA1 gene for transcribed sequences. We have cloned a set of fragments representing at least 26 genes. The DNA sequence of these clones reveals that 5 are previously cloned genes; the precise chromosomal location of 2 was previously unknown, and 3 have been cloned and mapped by others to this interval. Three other genes, including BRCA1 itself, have recently been mapped independently to this region. Sequences from 11 genes are similar but not identical matches to known genes; 5 of these appear to be the human homologues of genes cloned from other species. Another 7 genes have no similarity with known genes. In addition, 39 putative exons and 14 expressed sequence tags have been identified and mapped to individual cosmids. This transcript map provides a detailed description of gene organization for this region of the genome.

Physical mapping of the cystic fibrosis region by pulsed-field gel electrophoresis

The gene for cystic fibrosis (CF) is known to be flanked by the closely linked DNA markers met and J3.11 on chromosome 7. Using the technique of pulsed-field gel electrophoresis, we have constructed a complete overlapping restriction map of approximately 3000 kb of DNA in this region. The met and J3.11 probes are found to be between 1300 and 1800 kb apart, which compares well with their genetic distance of 1-2 cM. The CF gene must be located within this interval, and the availability of this physical map should be of considerable utility in mapping additional clones as the search for the gene proceeds.

Expression of the neurofibromatosis type 1 (NF1) gene during mouse embryonic development.

The von Recklinghausen neurofibromatosis type 1 (NF1) gene was identified by positional cloning and found to be a tumor suppressor gene expressed most abundantly in brain. One isoform of NF1 (type 2 NF1) contains an additional 21 amino acids inserted into a region of the protein involved in the regulation of p21-ras. To study the role of the NF1 gene in mammalian development, the expression of the NF1 gene and protein product, neurofibromin, during mouse embryonic development was determined. NF1 mRNA and neurofibromin expression was detectable by Northern and Western analysis, respectively, after day 10 of murine embryogenesis and remained elevated throughout development. Type 2 NF1 mRNA expression predominated before day 10, after which time, type 1 (lacking the insertion) NF1 mRNA was the predominant isoform detected. The protein expression of the type 2 isoform was similar to overall neurofibromin expression by Western blot analysis with greatest expression in adult brain. Despite a similar tissue distribution pattern, type 2 neurofibromin was not found to be associated with brain cytoplasmic microtubules in the same fashion as the uninserted type 1 isoform. Collectively, these experiments suggest that the switch from type 2 to type 1 neurofibromin isoform predominance during embryogenesis may have significant functional consequences.

Identification of the cystic fibrosis gene: Chromosome walking and jumping

An understanding of the basic defect in the inherited disorder cystic fibrosis requires cloning of the cystic fibrosis gene and definition of its protein product. In the absence of direct functional information, chromosomal map position is a guide for locating the gene. Chromosome walking and jumping and complementary DNA hybridization were used to isolate DNA sequences, encompassing more than 500,000 base pairs, from the cystic fibrosis region on the long arm of human chromosome 7. Several transcribed sequences and conserved segments were identified in this cloned region. One of these corresponds to the cystic fibrosis gene and spans approximately 250,000 base pairs of genomic DNA.