Chromosome 1 Map, Sequence and Variation

The construction of well characterised sequence-ready physical maps has been central to the generation of high quality genomic sequence by the Human Genome Project. The technological advances that made possible a clone based sequencing approach to large genomes have included the use of large insert bacterial clones and the development of high throughput fingerprinting techniques. The first part of this thesis is devoted to development and application of these improvements in technology. The adaptation of fluorescent technologies and their application to existing fingerprinting methods described in this work has resulted in a fingerprinting technique which improves upon levels of data accuracy, increases throughput and incorporates of increased levels of safety and automation. The initial application of this and other restriction digest fingerprinting methods to the assembly of large insert P1-artificial chromosome clones (PACs) was also evaluated. PACs were used to construct a 1.4 Mb contig across a region of chromosome 13q12 that includes the breast cancer susceptibility gene BRCA2. These experimental and technical developments were then utilised within a hierarchical mapping strategy to construct a 13 Mb contig of human chromosome 1pcen-1p13. The finished sequence generated by the clone based sequencing strategy provides the basis for the elucidation of genic features and the motifs that influence their regulation within the human genome sequence. Detailed analysis of the finished genomic sequence from 1pcen-1p13 is described. These analyses include the characterisation of base composition and determination of repeat content within the region, as well as identification of known and novel genes by manual annotation. The majority of differences between individuals can be attributed to allelic sequence variation. The characterisation of sequence differences and comprehension of how they may affect the expression and function of genes will be crucial for the study of molecular alterations in human disease. A subset of highly similar genes within 1pcen-1p13, in addition to seven other genes of interest, were investigated by developing and assessing assays to determine sequence variation. The particular challenges of investigating gene families where sequences are nearly identical were explored, and enable better resolution of new and previously available data. The consequences that these sequence changes may have upon gene function is also discussed, and this provides an example of the ways in which knowledge of genomic sequence can be analysed to support new areas of structural and functional research