8s J . EUK. MICROBIOL., VOL. 44, NO. 6, NOVEMBER-DECEMBER 1997

Constructing a Physical Map of the Pneurnocysfk Genome

JONATHAN ARNOLD'' and MELANIE T. CUSHION' ' Universip of Georgia, Athens, GA 30602 and 2Universiry of Cincinnati College of Medicine, Cincinnati,OH 45267-0560

The relatively small genome size of Pneumocystis carinii (Pc) (-7.7 Mbp) permits two basic approaches for a genome project; direct genomic sequencing or creation of a physical map with an ordered cosmid clone collection followed by subsequent sequencing of the cosmid library. The strategy favored by the Pc community at the 5th IWOP is physical mapping followed by subsequent sequencing. Physical maps of PC genomes will assist in: (i) isolating pathenogenicity islands in the PC genome; (ii) creating reagents for other experiments in the Pc community; (iii) isolating genes in metabolic cycles useful for targeted drug development; and (iv) isolating new families of repeated DNA sequences for biotyping Pc populations El].

Physical Maps. A physical map is defined as a partial ordeiing of distinguishable DNA fragments by their position along a chromosome. Examples include the cytological maps of Drosophila rnelanogaster and Homo supiens, an ordered clone collection of Aspergillus nidulans, or the entire DNA sequence of a chromosome of Sacchurornyces cerevisiae. The purpose of a physical map is to cany out reverse genetics systematically, to provide rapid access to any DNA fragment (or gene) of biological interest, and to assist in the determination of a genome's entire sequence. Good physical maps lend themselves to automated reconstruction with robots and computers. Good physical maps are also independent of the biological material used to construct them, like a DNA sequence, and should be easily linked to other resources, like genetic maps and sequence databases. Finally, a good physical map should allow the recovery of any segment of DNA, by such methods as the Polymerase Chain Reaction (PCR).

Physical Mapping Strategy. One efficient physical mapping strategy that lends itself to automation involves: (i) embedding Pc populations from a host in agarose plugs followed by protease and detergent treatment to release intact Pc chromosomal DNA [3]; (ii) separating Pc chromosomes by pulsed field electrophoresis [3]; (iii) in vitro packaging of partially digested chromosomal DNA into two cosmid vectors [7]; (iv) arraying cosmid clones on high-density nylon membranes with a robotics system, ISAAC (Intelligent System for Automated Assembly of Chromosomes) [6]; (v) hybridization of intact chromosomes with cosmid clones arrayed on membranes to verify chromosomal assignment of cosmid clones; to identify DNA repeats in clones; and to make the libraries chromosome-specific [2]; (vi) probing the membranes with a panel of cosmid clones having unique assignments to individual chromosomes, thereby assigning a digital "call number" to each clone [6 ] ; and (vii) ordering clones by their digital call numbers with computer algorithms so that clones with similar call numbers fall next to each other on the physical map [ 5 ] .

Community Reagents from Physical Mapping Strategy. This physical mapping strategy initially will provide 2 cosmid vectors with the capability of fungal transformation, from which an 8-genome equivalent chromosome-specific library with 1500 cIones stored in 15 microtitre plates will be generated. The robotics system, ISAAC, will then be used to generate chromosome walks across each chromosome with markers every 29 kb, and the clones constituting the walks will be rearrayed in 3 microtitre plates with ISAAC to reconstruct the entire Pc genome in vitro. A portrait of repeats will be generated by the strategy above along each chromosome walk. An existing cDNA library can then be overlayed on the chromosome walks by sequencing of ends of cDNAs to map

expressed sequences onto the chromosome walks and then cross-linked to physical maps of other Pc genomes, like P. carinii f. sp. hominis. These chromosome walks have all the properties of a good physical map. The maps are generated in a highly automated fashion, can be rendered independent of the library by sequencing the ends of cosmids, and tied to other resources, like the cDNA library. If markers are spaced every 29 kb or at a slightly higher density, then any intervening DNA fragment can be reached by long distance PCR. The final physical map can then provide an armature for further genomic sequencing by the Pc community.

Informatics and Databases. Methods exist [ 5 ] for assembling the physical maps and assessing their statisticat reliability [5,6]. These tools are largely incorporated into the Fungal Genome Database, in which a curator can keep track of the physical mapping experiment. The resulting maps can then be presented to the Pc community over the World Wide Web at http://fungus.genetics.uga.edu:5080 using the Fungal Genome Database [4], an independent Web Page, or both.

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