Goals Genomic DNA Sequencing Clone Libraries Sequence Annotation

Genome Project

Clone Libraries

P. carinii libraries were constructed in pLorist6Xh and pWEB, to provide a scaffold for assembly and in plasmid shotgun libraries for sequencing. Construction of a fosmid and a 10kb library is underway.

pLorist6Xh (Gibson, T.J. A. Rosenthal, and R.H. Waterston (1987) Gene 53: 283-286) using Form 1 rat Pc. This vector contains hygromycin and neomycin resistance markers, and a multiple cloning site with an XhoI restriction site. Sau 3A partially digested Form 1 Pc DNA was ligated in XhoI- digested pLorist6Xh vector after a 2 nucleotide fill-in of both vector and Pc DNA to inhibit self-ligation and produce compatible ends between target and vector. The cosmids were packaged with GigaPack XL and transfected into XL1 Blue MR. Approximately 4000 colonies were picked and transferred to (40) 96-well microtiter plates, grown and replicated onto nylon membranes using a 96 pin replicator and a frame to permit stamping of clones in 16 microtiter plates as 4 x 4 plate arrays of 1536 clones. The entire 3,840 clones were stamped onto 2.5 nylon membranes containing 1536 clones each arrayed in a 4 plate tetrad arrangement permitting an alphanumeric identity for each clone. Bacterial colonies were grown overnight by overlay of the membranes on top of LB-kanamycin agar in large glass dishes. The colonies were subsequently lysed with a sodium dodecyl-alkaline solution, neutralized, and washed. The Pc DNA was fixed to the membranes by UV-crosslinking.

Library characterization. All purified Pc populations contain varying degrees of host cell contamination. Hybridization of 42% of the library (1536 of 3840 clones) with rat cell DNA revealed 12.1% positive clones (186/1536), an acceptable contamination level. Although the library had good representation of known Pc genes and was estimated at 10 genome equivalents, one unusual set of clones was identified; 12 of the original 1536 hybridized to both MSG and rat probes (0.8%), suggesting chimerization. That there may be some chimeras in the library was also supported by sequence data from one clone which yielded what appeared to be sequences off of two T7 primers, suggesting the presence of 2 vectors in the cosmid. Chimeras can lead to poor assembly and misassembly of portions of the genome and this library was not used for assembly in the Project.

pWEB. Form 1 P. carinii was again selected for cloning into the cosmid vector, pWEB (Epicentre Technologies, Madison, WI). The organisms were obtained from a Lewis rat maintained at the Cincinnati VAMC VMU and seeded with rats infected with Form 1. The lung homogenate containing the organisms was treated with ammonium chloride (0.85% aqueous) to lyse erythrocytes and some host cells, extensively filtered through a series of 10 um filters (~ 12 filtrations) to further reduce host cell contamination, enumerated by microscopic analysis, treated with 10 ug/ml DNAse at 37C for 30 minutes, washed with 250 mM EDTA then 125 mM EDTA, centrifuged at 1000xg, resuspended in 0.5ml 125 mM EDTA and 1 ml 1.2% low melt agarose and digested overnight in a solution of SDS and proteinase K at 55C (Cushion MT, Zhang J, Kaselis M, et al. 1993. J. Clin. Microbiol. 31: 1217-1223).

DNA was purified from the agarose block using Phaselock PLG 1 Light tubes (5-3 Prime, Boulder, CO), size selected at >40 kb by agarose electrophoresis through low melt agarose, treated with gelase, ethanol precipitated and ligated and packaged according to vendor protocol (EpiCentre). 1152 colonies on LB-carbenicillin plates were picked into 12-96 well plates, permitted to grow overnight, and gridded onto nylon membranes for characterization.

Library characterization. One of 18 clones evaluated for insert size and empty vectors, contained no insert and the remaining inserts were 35-40 kb. Hybridization of 1152 clones with rat DNA showed < 1% rat contamination; with msgB, 4% representation; and with the glucan synthase probe, 1.4% hybridization. No evidence for chimerization has been observed to date.

Sequencing libraries. (Clones not available) The original strategy for sequencing was to create a physical map from an 8-10x cosmid library and to use this minimum tile as the source of DNA for shotgun libraries. Shotgun libraries were to be constructed with nebulized cosmid DNA, which would be cloned into a blunt-end vector and sequenced. Problems resulting from this approach included the sequencing of unacceptable amounts of the pWEB cosmid vector DNA; significant contamination of the sequencing libraries with bacterial host DNA; and a cloning bias of some regions of the P. carinii genome. A new strategy for sequencing the P. carinii genome utilizing a modified vector and sticky-end cloning with subsequent southern hybridization-based screening of inserts was found to provide a higher percentage of assembled sequence than the previous blunt-end strategy and is described [Sesterhenn et al. J. Eukaryot. Microbiol. 2003;50 Suppl:663-5]. This method has been superseded by shotgun sequencing of entire chromosomes cut from pulsed field gels using a pSMART low copy vector and a DNA amplification strategy (www.Lucigen.com). The results from these libraries make up the bulk of the data available on this website.

Goals Genomic DNA Sequencing Clone Libraries Sequence Annotation