Surveying Saccharomyces genomes to identify functional elements by comparative DNA sequence analysis.

Comparative sequence analysis has facilitated the discovery of protein coding genes and important functional sequences within proteins, but has been less useful for identifying functional sequence elements in nonprotein-coding DNA because the relatively rapid rate of change of nonprotein-coding sequences and the relative simplicity of non-coding regulatory sequence elements necessitates the comparison of sequences of relatively closely related species. We tested the use of comparative DNA sequence analysis to aid identification of promoter regulatory elements, nonprotein-coding RNA genes, and small protein-coding genes by surveying random DNA sequences of several Saccharomyces yeast species, with the goal of learning which species are best suited for comparisons with S. cerevisiae. We also determined the DNA sequence of a few specific promoters and RNA genes of several Saccharomyces species to determine the degree of conservation of known functional elements within the genome. Our results lead us to conclude that comparative DNA sequence analysis will enable identification of functionally conserved elements within the yeast genome, and suggest a path for obtaining this information.

Theories and applications for sequencing randomly selected clones.

Theory is developed for the process of sequencing randomly selected large-insert clones. Genome size, library depth, clone size, and clone distribution are considered relevant properties and perfect overlap detection for contig assembly is assumed. Genome-specific and nonrandom effects are neglected. Order of magnitude analysis indicates library depth is of secondary importance compared to the other variables, especially as clone size diminishes. In such cases, the well-known Poisson coverage law is a good approximation. Parameters derived from these models are used to examine performance for the specific case of sequencing random human BAC clones. We compare coverage and redundancy rates for libraries possessing uniform and nonuniform clone distributions. Results are measured against data from map-based human-chromosome-2 sequencing. We conclude that the map-based approach outperforms random clone sequencing, except early in a project. However, simultaneous use of both strategies can be beneficial if a performance-based estimate for halting random clone sequencing is made. Results further show that the random approach yields maximum effectiveness using nonbiased rather than biased libraries.

Comparative genomic sequence analysis of the human and mouse cystic fibrosis transmembrane conductance regulator genes.

The identification of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 1989 represents a landmark accomplishment in human genetics. Since that time, there have been numerous advances in elucidating the function of the encoded protein and the physiological basis of cystic fibrosis. However, numerous areas of cystic fibrosis biology require additional investigation, some of which would be facilitated by information about the long-range sequence context of the CFTR gene. For example, the latter might provide clues about the sequence elements responsible for the temporal and spatial regulation of CFTR expression. We thus sought to establish the sequence of the chromosomal segments encompassing the human CFTR and mouse Cftr genes, with the hope of identifying conserved regions of biologic interest by sequence comparison. Bacterial clone-based physical maps of the relevant human and mouse genomic regions were constructed, and minimally overlapping sets of clones were selected and sequenced, eventually yielding approximately 1.6 Mb and approximately 358 kb of contiguous human and mouse sequence, respectively. These efforts have produced the complete sequence of the approximately 189-kb and approximately 152-kb segments containing the human CFTR and mouse Cftr genes, respectively, as well as significant amounts of flanking DNA. Analyses of the resulting data provide insights about the organization of the CFTR/Cftr genes and potential sequence elements regulating their expression. Furthermore, the generated sequence reveals the precise architecture of genes residing near CFTR/Cftr, including one known gene (WNT2/Wnt2) and two previously unknown genes that immediately flank CFTR/Cftr.

High-throughput plasmid DNA purification for 3 cents per sample.

To accommodate the increasingly rapid rates of DNA sequencing we have developed and implemented an inexpensive, expeditious method for the purification of double-stranded plasmid DNA clones. The robust nature, high throughput, low degree of technical difficulty and extremely low cost have made it the plasmid DNA preparation method of choice in both our expressed sequence tag (EST) and genome sequencing projects. Here we report the details of the method and describe its application in the generation of more than 700 000 ESTs at a rate exceeding 16 000 per week.

High throughput fingerprint analysis of large-insert clones.

As part of the Human Genome Project, the Washington University Genome Sequencing Center has commenced systematic sequencing of human chromsome 7. To organize and supply the effort, we have undertaken the construction of sequence-ready physical maps for defined chromosomal intervals. Map construction is a serial process composed of three main activities. First, candidate STS-positive large-insert PAC and BAC clones are identified. Next, these candidate clones are subjected to fingerprint analysis. Finally, the fingerprint data are used to assemble sequence-ready maps. The fingerprinting method we have devised is key to the success of the overall approach. We present here the details of the method and show that the fingerprints are of sufficient quality to permit the construction of megabase-size contigs in defined regions of the human genome. We anticipate that the high throughput and precision characteristic of our fingerprinting method will make it of general utility.