New technologies and their application to the analysis of
chromosome 1
Over the past few years a number of technologies have become available for the rapid analysis of DNA and mRNA. The resources and methodologies for these techniques are applicable to the whole genome however several studies have concentrated on chromosome 1. This report is intended to direct the reader to relevant references and highlight chromosome 1-specific applications using two new technologies, microarrays of DNA and two-dimensional gel electrophoresis.
DNA Microarrays
DNA arrayed on a solid support such as nylon membrane has been used for a number of years in hybridisation experiments and is routinely used for the identification of genomic clones for chromosome 1 (for example see Williams et al., this report). In a different strategy, photolithography and combinatorial chemistry have been used to synthesise many thousands of different DNA sequences on a solid surface (Fodor et al., 1993). Flourescently labeled DNA is hybridized to the array and specifically bound molecules are detected with a fluorescent scanner. These arrays have been used to re-sequence known genes (Hacia et al., 1996), for expression profiling experiments (Lockhart et al., 1996) and for genotyping (Wang et al., 1998). However none of these arrays are specifically aimed at chromosome 1. An alternative system is to deposit presynthesised DNA (e.g. oligonucleotides, cloned DNA, PCR products) on the array surface using a high density gridding robot. The hybridisation is essentially the same as above using fluorescently labeled DNA, a suitable light source to activate the fluorochromes (usually a laser) and a detection system to quantitate the fluorescence from the specifically bound molecules. This has been successfully used to monitor the level of multiple mRNAs in yeast and cancer cell lines (DeRisi et al., 1996 and DeRisi et al., 1997). In theory it would be possible to produce an array of chromosome 1 genes to specifically monitor the expression of genes from this chromosome.
A second application of gridded arrays is comparative genomic hybridisation (CGH). Cytogenetic CGH has been used to identify a number of lesions in DNA from tumours, for example the amplification of chromosome 1 in sarcomas (Forus et al., this report and references therein). A microarray of genomic clones representing the whole genome would offer an alternative to cytogenetic CGH, with possibly higher resolution, however suitable clones are not yet available for the entire genome. In the interim it will be possible to use low density arrays from the whole genome (for instance with clones every 5 Mb) or localized high density arrays from specific regions. One such high density array with a cluster of clones every 500 Kb is being produced for chromosome 1 (see Aubin et al., this report). The array will be used to analyze cancer cell lines and tumours to identify deletions and amplifications on chromosome 1. An array containing genomic clones covering the whole of chromosome 1 will be possible when the mapping is complete.
Gel based comparison approaches
Wimmer and colleagues (1996) had previously reported utilizing two-dimensional electrophoresis to separate and quantitate chromosome 1 NotI-EcoRV-derived genomic fragments. Two of the 346 fragments were cloned and shown to map back to 1p35-p36.1 and to 1p13.3-p21 by FISH thus, opening a route to identify fragments of chromosome 1 that were either deleted or amplified in tumours. Indeed, Almeida and co-workers (1998) used this approach to identify GAC1, a gene that was amplified and over expressed in two of eight malignant gliomas and in one of eight unselected glioblastomas multiforme. GAC1 maps to 1q32.1 and is a candidate for the targeted in 1q32.1 amplifiction in malignant gliomas. In this report Zhu and colleagues describe two out of ten chromosome 1 derived fragments that were completely or partially lost in neuroblastoma cell lines and tumours. One of the fragments mapped to 1p13-21 and contains a CpG island that exhibits similarity to the hamster homeobox gene Alx3, while another maps to 1p36 and contains the promoter sequence for p73. These results suggest a role for both of these genes in neuroblastomas and await confirmation by other investigators.