Physical, Transcriptional and Comparative Mapping on the Human X Chromosome

Abstract

Progress in the study of the human genome has resulted in the production of a complete clone map and associated 'working draft' sequence. This will underpin the completion of the sequence itself, the annotation of genes and other features, and application of this new found knowledge. This thesis focuses on the evolving methods to determine the map, and to use the emerging sequence for the study of genes, incorporating new studies of other genomes to enhance progress in understanding and interpretation to biology and medicine. The success of the endeavours is necessarily accompanied by the development and evolution of new technologies and by critical assessment of the progress in acquiring knowledge of the genomic information. Evolution of mapping technologies included the development of the larger insert bacterial cloning systems (PACs) and (BACs), and an increase in available landmarks both from Y AC maps and RH maps. The work described in chapter 3, followed this evolution and was applied to construct a 6 Mb sequence-ready bacterial clone contig map in Xq22. A minimum set of clones was chosen for genomic sequencing. The resulting sequence map was compared to previously published maps and analysed both for common repeats, and previously unidentified low copy repeats. The availability of the emerging sequence of the human genome provided a resource for identification of the features encoded within. In chapter 4, the sequence of a 7 Mb region in Xq23-24 was analysed for the presence of genes using a combination of sequence similarity searches against both protein and DNA databases, and ab initio gene prediction. Predicted genes were confirmed where possible, by generating novel cDNA sequence. The region contained 33 confirmed genes (of which 14 were confirmed during this study), 11 predicted genes and 20 pseudogenes. Comparative genome sequence analysis is a powerful method both for aiding human gene identification and identifying other features encoded within the human genome such as regulatory elements. Comparing the genomes of two or more species also provides insights into the evolution of the species since the divergence from a common ancestor. Sequence from a 1 Mb region in human Xq24 was compared in two other species, mouse and zebrafish. In chapter 5, bacterial clone contigs for sequencing were constructed in the mouse by designing mouse-specific STSs orthologous to human sequence for clone isolation. In chapter 6, bacterial clone contigs for sequencing were constructed in zebrafish using STS from exons of human genes to identify zebrafish BAC clones by reduced stringency hybridisation. Comparative analysis of the region showed that humans and mice are more highly conserved than humans and zebrafish, in terms of gene content and organisation. A combination of comparative sequence analysis tools identified 14 novel potential conserved sequences between human and mouse, one of which was also conserved in zebrafish.