Construction and characterisation of a genomic PAC library of the intestinal parasite Cryptosporidium parvum

Abstract

Cryptosporidium par6um is an obligate-intracellular parasite of the gut mucosa. It infects many mammals, and can be considered an emerging pathogen in man, the first case of human cryptosporidiosis being reported as recently as 1976 [1]. Little is known about the molecular genetics of Cryptosporidium, partly due to our inability to continuously culture it in vitro. Oocysts for study are usually obtained by extraction from the faeces of infected calves or lambs [1]. Karyotype analysis suggests that the Cryptosporidium genome consists of eight chromosomes of :1–2 Mb [2,3], giving a genome size of 10.4 Mb [2]. Several partial and complete gene sequences have been reported [4–15], and an expressed sequence tag (EST) sequencing project is currently in progress (http:// www.embl-ebi.ac.uk/parasites/news.html). Several small insert Cryptosporidium libraries, both genomic [6,9] and cDNA [4,5,12–15], have been reported. However, no large-insert libraries have hitherto been available, impeding mapping and sequencing efforts. The vector chosen for constructing this library was the P1 artificial chromosome (PAC) vector pCYPAC2 [16], which differs by only a single NotI restriction site from pCYPAC1 [17]. PAC vectors are based on the P1 vector system [18,19], but are introduced into the host cell by electroporation rather than viral transformation. A PAC library can be manipulated exactly as one would a P1 library and has the same desirable features, e.g. clonal stability over many generations and Abbre6iations: EST, expressed sequence tag; PAC, P1 artificial chromosome; PFGE, pulsed field gel electrophoresis; STS, sequence tagged site. * Corresponding author. Tel.: +44 1223 402190; fax: +44 1223 412178; e-mail: phd@mrc-lmb.cam.ac.uk 1 Note: Nucleotide sequence data reported in this paper are available in the EMBL, DDJB and GenBankTM databases under the accession numbers G35127–G35275 and G35338– G35349.