PMC

The processes involved in the construction of the genome map. A flow chart detailing the inter-related activities involved in the construction of the physical map of L. major Friedlin. Rounded boxes indicate computer-based analyses.

Minimum tile set for chromosome 4. L. major Friedlin cosmid clones were fingerprinted, as detailed in Methods, prior to contig assembly by CONTASP and FPC v. 2.8.2 software. Subcontigs shown to contain chromosome 4 marker loci were joined by (1) the application of additional software packages: MAPSUB (identified loose fingerprint matches), EXTEND (identified potential end–end joins), and (2) hybridization data (assigned and joined subcontigs), to yield an integrated map. The contig display from FPC formed the basis of this diagram. All clones that constitute the contig, including those that have been buried, are shown as horizontal lines. DNA hybridization probes are shown along the top (see Methods for probe descriptions). A minimum tile set (•) was chosen for DNA sequencing purposes. The scale bar represents 26 consensus bands (∼35 kb).

PFGE separation of L. major Friedlin chromosomes. (a) Separation of the larger chromosomes: Agarose-embedded L. major Friedlin DNA was prepared as described in Methods and subjected to PFGE [0.8% agarose (GIBCO), Bio-Rad DRII apparatus, 100 V, 1000- to 100-sec ramp, 250 hr, 4°C; electrophoresis buffer was changed after 90 hr]. The chromosomes of S. cerevisiae YNN295 (Bio-Rad) and H. wingiae (Promega) were used as molecular mass markers. Sizes are indicated. The chromosomes were visualized under UV after staining with ethidium bromide. (b) Ideogram of the L. major Friedlin karyotype. Numbering of the chromosomes was derived from this study and . Lines indicate the relative mobilities of selected chromosomes in panels a and c. (c) Separation of the smaller chromosomes: Agarose-embedded L. major Friedlin DNA was prepared as above but separated under different PFGE conditions [1.0% agarose (GIBCO), Bio-Rad DRII apparatus, 150 V, 140- to 90-sec ramp, 110 hr, 4°C].