Leishmania major Friedlin chromosome 1 has an unusual distribution of protein-coding genes.

Leishmania are evolutionarily ancient protozoans (Kinetoplastidae) and important human pathogens that cause a spectrum of diseases ranging from the asymptomatic to the lethal. The Leishmania genome is relatively small [ approximately 34 megabases (Mb)], lacks substantial repetitive DNA, and is distributed among 36 chromosomes pairs ranging in size from 0.3 Mb to 2.5 Mb, making it a useful candidate for complete genome sequence determination. We report here the nucleotide sequence of the smallest chromosome, chr1. The sequence of chr1 has a 257-kilobase region that is densely packed with 79 protein-coding genes. This region is flanked by telomeric and subtelomeric repetitive elements that vary in number and content among the chr1 homologs, resulting in an approximately 27.5-kilobase size difference. Strikingly, the first 29 genes are all encoded on one DNA strand, whereas the remaining 50 genes are encoded on the opposite strand. Based on the gene density of chr1, we predict a total of approximately 9,800 genes in Leishmania, of which 40% may encode unknown proteins.

Optimization of restriction fragment DNA mapping.

Consider a mapping project in which overlap of clonal segments is inferred from complete multiple restriction digests. The fragment sizes of the clones are measured with some error, potentially leading to a map with erroneous links. The number of errors in the map depends on the number and types of enzymes used to characterize the clones. The most critical parameter is the decision rule k, or the criterion for declaring clone overlap. Small changes in k may cause an order of magnitude change in the amount of work it takes to build a map of given completion. We observe that the cost of an optimal mapping strategy is approximately proportional to the target size. While this finding is encouraging, considerable effort is nonetheless required: for large-scale sequencing projects with up-front mapping, mapping will be a non-negligible fraction of the total sequencing cost.

A physical map of human chromosome 7: an integrated YAC contig map with average STS spacing of 79 kb.

The construction of highly integrated and annotated physical maps of human chromosomes represents a critical goal of the ongoing Human Genome Project. Our laboratory has focused on developing a physical map of human chromosome 7, a approximately 170-Mb segment of DNA that corresponds to an estimated 5% of the human genome. Using a yeast artificial chromosome (YAC)-based sequence-tagged site (STS)-content mapping strategy, 2150 chromosome 7-specific STSs have been established and mapped to a collection of YACs highly enriched for chromosome 7 DNA. The STSs correspond to sequences generated from a variety of DNA sources, with particular emphasis placed on YAC insert ends, genetic markers, and genes. The YACs include a set of relatively nonchimeric clones from a human-hamster hybrid cell line as well as clones isolated from total genomic libraries. For map integration, we have localized 260 STSs corresponding to Genethon genetic markers and 259 STSs corresponding to markers orders by radiation hybrid (RH) mapping on our YAC contigs. Analysis of the data with the program SEGMAP results in the assembly of 22 contigs that are "anchored" on the Genethon genetic map, the RH map, and/or the cytogenetic map. These 22 contigs are ordered relative to one another, are (in all but 3 cases) oriented relative to the centromere and telomeres, and contain > 98% of the mapped STSs. The largest anchored YAC contig, accounting for most of 7p, contains 634 STSs and 1260 YACs. An additional 14 contigs, accounting for approximately 1.5% of the mapped STSs, are assembled but remain unanchored on either the genetic or RH map. Therefore, these 14 "orphan" contigs are not ordered relative to other contigs. In our contig maps, adjacent STSs are connected by two or more YACs in > 95% of cases. With 2150 mapped STSs, our map provides an average STS spacing of approximately 79 kb. The physical map we report here exceeds the goal of 100-kb average STS spacing and should provide an excellent framework for systematic sequencing of the chromosome.

Integration of physical, genetic and cytogenetic maps of human chromosome 7: isolation and analysis of yeast artificial chromosome clones for 117 mapped genetic markers.

An important goal for the human genome project is to assemble fully integrated physical, genetic and cytogenetic maps for each human chromosome. Towards that end, we have isolated yeast artificial chromosome (YAC) clones containing 117 of the 119 genetic markers that constitute a recently constructed, detailed genetic map of human chromosome 7. Analysis of these clones reveals numerous examples where adjacent genetic markers have been physically connected, either in individual YACs or in multi-YAC contigs. At present, the 117 genetic markers are contained in fewer than 80 YAC contigs, with most of these contigs uniquely ordered relative to one another based on the genetic map positions of the corresponding markers. These YACs and YAC contigs are estimated to contain approximately 60-85% of the DNA from human chromosome 7. YACs representing 36 genetic markers were mapped by fluorescence in situ hybridization (FISH) to metaphase chromosomes, allowing assignment of these genetic markers to cytogenetic bands along chromosome 7 and placement of the centromere within the genetic map. Together, these studies provide genetically and cytogenetically anchored YAC clones covering the majority of chromosome 7 that will be useful both for the positional cloning of genes and as a framework for assembling a complete YAC-based physical map of the chromosome.