Abstract

With the draft sequence of human chromosome 19 recently completed, we have focused on construction of parallel sets of mouse BAC clone contigs for comparative sequencing. Our goal was to create a series of contigs spanning all mouse DNA with syntenic homology to the 46 Mb gene-rich euchromatic portion of human chromosome 19 (H19) (Figure 1). We sought as a first priority to identify mouse clones that span regions of H19 related to identified genes; we did not target "gene deserts" including the centomere or large pericentric regions of H19p and H19q. To generate BAC contigs in regions containing orthologous mouse genes, we used conserved, expressed sequences that were identified as BLAST matches from sequenced, ordered H19 clones to design mouse overgo probes. We designed mouse probes based on the position of the related human sequences, to generate markers spaced at an average distance of 50-75 kb in most regions (Figure 2). These markers were hybridized in pools to the RPCI-23 (C57NBL/6) BAC library at complete (~12X) genome coverage, in order to blanket syntenically homologous mouse segments with overlapping sets of BACs. To cover regions occupied by the numerous, tandemly clustered gene families that are concentrated in H19, we designed family-specific probes and used BAC end sequences to assemble the positively hybridizing clones into contigs. Human gene order and spacing was used as a model to facilitate selection of mouse markers with appropriate spacing, and as a guide to permit rapid assembly of the overlapping mouse clones (Figure 3). Map assembly was verified by restriction mapping of contig members, generating information on clone integrity, length, and overlap that facilitated selection of an optimal tiling path (Figure 4). The deep BAC coverage and detailed restriction maps permitted adjacent contigs to be joined, and allowed relationships between mouse and human regions to be established with a significant degree of precision.

Using conserved gene sequences, and later, BAC end sequences, as our only sources of probes, we generated ~ 40 Mb of restriction-mapped mouse BAC contigs. These BAC contigs represent >90% of H19-related gene-containing regions in the mouse genome and span all 15 identified chromosome 19-related mouse homology segments. The 15 segments range from ~ 600 kb to more than 13 Mb in length and correspond to specific regions of mouse chromosomes 7, 8, 9, 10, and 17, respectively (Table 1; Figure 1). Table 2 displays the human chromosome 19 sequencing tiling path, listed with mouse EST sequence matches, overgo probe sequences designed from these ESTs, and mouse clones from the mouse sequencing tiling path linked through those conserved probes to the human clones. Table 3 displays probes that were used to isolate BAC clones spanning the conserved, clustered H19 gene families. The mouse sequencing tiling path selected to span each homology segment, with hotlinks to the underlying restriction maps, is displayed in Table 4. The mouse BAC clones displayed in Table 4 are presently being sequenced at the D.O.E.’s Joint Genome Insititute. The resulting comparative maps (P arm map, Q arm map) illustrate the alignment of mouse contigs with the human chromosome map and sequence in graphical form, and provides a overview of the completed comparative physical map. Alignment of these mouse BAC contigs with the sequenced human chromosome 19 clone map refined the positions and lengths of the known homology segments and revealed several micro-rearrangements, such as inversions, differential expansions, and other evolutionary changes throughout otherwise well conserved portions of the mouse and human maps.