Happy mapping

Abstract: Cryptosporidium parvum is a protozoan of the phylum Apicomplexa, class Coccidia. It is an obligate intracellular parasite infecting the gut epithelia of a wide range of mammals, and is transmitted from host to host via thick-walled oocysts in the faeces. The first case of human cryptosporidiosis was reported as recently as 1976, and it is now recognized as a major cause of gastro-intestinal illness worldwide. In most cases, the body mounts a strong immune response and the parasite is eradicated from the gut after ∼seven days of diarrhea. Infections in the immunosuppressed (e.g., AIDS patients), however, can be persistent and eventually fatal (for review, see Fayer et al. 1997). Massive outbreaks have occurred when the drinking water for entire communities has been contaminated with oocysts (e.g., MacKenzie et al. 1994). The Cryptosporidium genome is small for a coccidian (Tilley and Upton 1997), consisting of eight chromosomes with a total size estimate of between 9.6 (Caccio et al. 1998) and 10.4 Mb (Blunt et al. 1997). The low copy number and genome-wide distribution of both 5S and cytoplasmic rDNA genes (four to five and five copies, respectively; Taghi-Kilani et al. 1994; Le Blancq et al. 1997) is similar to that of several other apicomplexans, such as Plasmodium. This differs markedly from the usual eukaryotic pattern of hundreds or thousands of tandem copies. The ∼60%–70% AT content of the genome (Jenkins and Petersen 1997; Piper et al. 1998) is much lower than that of most Plasmodium spp. (Weber 1988). C. parvum appears to contain neither mitochondria (although they have been observed in other Cryptosporidium spp.) nor the plastid commonly found in apicomplexan parasites (Fayer et al. 1997; Kohler et al. 1997). Around 15–20 complete genes have been sequenced (e.g., Gooze et al. 1991; Nelson et al. 1991; Kim et al. 1992; Lally et al. 1992; Jenkins et al. 1993; Ranucci et al. 1993; Taghi-Kilani et al. 1994; Khramstov et al. 1995, 1996; Steele et al. 1995), and many others have been identified during the UCSF Cryptosporidium EST project (http://www.embl-ebi.ac.uk/parasites/news.html). Like most apicomplexans, Cryptosporidium appears to have few introns—currently only one has been reported (Caccio et al. 1997). The combination of its medical importance and small, unusual genome make C. parvum an ideal candidate for genomic studies, but these have been hampered by the absence of a detailed map and large insert clone library. The second of these problems was addressed recently with the release of the C. parvum PAC library (Piper et al. 1998). It has only recently become possible, however, to distinguish all eight Cryptosporidium chromosomes electrophoretically, and mapping to date has been limited to the placing of 20 markers on specific chromosomes (Caccio et al. 1998). This paper describes the construction of a physical genome map of C. parvum using HAPPY mapping (Dear and Cook 1993; Dear 1997; Dear et al. 1998), an in vitro technique that involves breaking intact genomic DNA at random, segregating the fragments into aliquots by limiting dilution and measuring the frequency of cosegregation of markers among the aliquots (Fig. ​(Fig.1).1). Closely linked markers are rarely separated by an intervening break and therefore tend to cosegregate. In this way, it is analogous both to traditional linkage mapping, which measures the frequency of recombination between markers during meiosis, and to radiation hybrid mapping (Cox et al. 1990). HAPPY mapping, however, does not suffer from the inaccuracies inherent in biological methods (recombination hotspots or hybrid instability), and the AT content of the genome should be largely immaterial. Constructing and screening a HAPPY mapping panel is relatively simple, and only small quantities of DNA are needed. All of the above suggest that HAPPY mapping is ideal for the study of otherwise intractable parasite genomes. Genomic studies of Cryptosporidium should be greatly aided by the use of this map in conjunction with the PAC library. Accession numbers and primer sequences for all markers are available at http://www.mrc-lmb.cam.ac.uk/happy/CRYPTO/crypto-genome.html. In addition, a limited number of aliquots of the mapping panel can be made available to researchers wishing to map their own markers. Figure 1 How HAPPY mapping works. Intact genomic DNA is broken by irradiation (I) to give a pool of random fragments, which are size selected by PFGE (II). A mapping panel of 96 aliquots is taken (III) from this pool. Each aliquot contains ∼1 haploid genome’s ...

Abstract: A theoretical approach for linkage mapping the genome of any higher eukaryote is described. It uses the polymerase chain reaction, oligonucleotides of random sequence and single haploid cells. Markers are defined and then the DNA of a single sperm is broken at random (eg by gamma-rays) and physically split into 3 aliquots. Each aliquot is screened for the presence of each marker. Closely-linked markers are more likely to be found in the same aliquot than unlinked markers. The entire process is repeated with further sperm and the frequency that any two markers co-segregate determined. Closely-linked markers co-segregate from most cells; unlinked markers do so rarely. A map can then be constructed from these co-segregation frequencies. A specific application for determining the order and distance between sets of closely-linked and previously-defined markers is also described.

Abstract: 1: Human linkage mapping. 2: Linkage mapping of plant and animal genomes. 3: Linkage mapping of quantitative trait loci in plants and animals. 4: Radiation hybrid mapping. 5: HAPPY mapping. 6: Construction and use of somatic cell hybrids. 7: The use of flow-sorted chromosomes in genome mapping. 8: The use of microdissected chromosomes in genome mapping. 9: Fluorescence in situ hybridization. 10: Contig assembly by fingerprinting. 11: Chromosome wallking. 12: Long-range restriction mapping of genomic DNA. Appendix 1 Core list of suppliers. Appendix 2 Non-commercial source for genome mapping. Appendix 3 Bioinformatics for genome mapping