Isolation of chromosome clusters from metaphase-arrested HeLa cells

We have developed a simplified approach for the isolation of metaphase chromosomes from HeLa cells. In this method, all the chromosomes from a cell remain together in a bundle which we call a “metaphase chromosome cluster”. Cells are arrested to 90–95% in metaphase, collected by centrifugation, extracted with non-ionic detergent in a low ionic strength buffer at neutral pH, and homogenised to strip away the cytoskeleton. The chromosome clusters which are released can then be isolated in a crude state by pelleting or they can be purified away from nearly all the interphase nuclei and cytoplasmic debris by banding in a Percoll TM density gradient. — This procedure has the advantages that it is quick and easy, metaphase chromatin is recovered in high yield, and Ca ++ is not needed to stabilise the chromosomes. Although the method does not yield individual chromosomes, it is nevertheless very useful for both structural and biochemical studies of mitotic chromatin. The chromosome clusters also make possible biochemical and structural studies of what holds the different chromosomes together. Such information could be useful in improving chromosome isolation procedures and for understanding suprachromosomal organisation of the nucleus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47359/1/412_2004_Article_BF00327351.pd

Linkage analysis in von Recklinghausen neurofibromatosis (NF1) with DNA markers for chromosome 17

The mutant gene causing von Recklinghausen neurofibromatosis (NF1) was recently shown to map to chromosome 17. We have used additional markers for chromosome 17 to narrow further the location of the gene defect. A preliminary multipoint linkage analysis suggests that the NF1 gene is located on the long arm of chromosome 17, flanked by D17Z1 and NGFR. Linkage analysis with the human oncogene homolog erbA1, which maps to this region, suggests that this cancer-related gene is not the primary cause of NF1

Genetic linkage of von Recklinghausen neurofibromatosis to the nerve growth factor receptor gene

von Recklinghausen neurofibromatosis (VRNF) is one of the most common inherited disorders affecting the human nervous system. VRNF is transmitted as an autosomal dominant defect with high penetrance but variable expressivity. The disorder is characterized clinically by hyperpigmented patches of skin (café au lait macules, axillary freckles) and by multiple tumors of peripheral nerve, spinal nerve roots, and brain (neurofibromas, optic gliomas). These tumors can cause disfigurement, paralysis, blindness, and death. We have determined the chromosomal location of the VRNF gene by genetic linkage analysis using DNA markers. The VRNF gene is genetically linked to the locus encoding nerve growth factor receptor, located on the long arm of chromosome 17 in the region 17q12→17q22. However, crossovers with the VRNF locus suggest that a mutation in the nerve growth factor receptor gene itself is unlikely to be the fundamental defect responsible for the VRNF phenotype