Comparative analysis of the gene-dense ACHE/TFR2 region on human chromosome 7q22 with the orthologous region on mouse chromosome 5

Abstract

Chromosome 7q22 has been the focus of many cytogenetic and molecular studies aimed at delineating regions commonly deleted in myeloid leukemias and myelodysplastic syndromes. We have compared a gene-dense, GC-rich sub-region of 7q22 with the orthologous region on mouse chromosome 5. A physical map of 640 kb of genomic DNA from mouse chromosome 5 was derived from a series of overlapping bacterial artificial chromosomes. A 296 kb segment from the physical map, spanning ACHE: to Tfr2, was compared with 267 kb of human sequence. We identified a conserved linkage of 12 genes including an open reading frame flanked by ACHE: and Asr2, a novel cation-chloride cotransporter interacting protein Cip1, Ephb4, Zan and Perq1. While some of these genes have been previously described, in each case we present new data derived from our comparative sequence analysis. Adjacent unfinished sequence data from the mouse contains an orthologous block of 10 additional genes including three novel cDNA sequences that we subsequently mapped to human 7q22. Methods for displaying comparative genomic information, including unfinished sequence data, are becoming increasingly important. We supplement our printed comparative analysis with a new, Web-based program called Laj (local alignments with java). Laj provides interactive access to archived pairwise sequence alignments via the WWW. It displays synchronized views of a dot-plot, a percent identity plot, a nucleotide-level local alignment and a variety of relevant annotations. Our mouse-human comparison can be viewed at http://web.uvic.ca/~bioweb/laj.html. Laj is available at http://bio.cse.psu.edu/, along with online documentation and additional examples of annotated genomic regions.

Figure 1

Overview of the refined chromosome 7q22 physical map for the region spanning the genes PAI1 and CYP3A4, with a direct comparison with 640 kb of orthologous genomic sequence from mouse chromosome 5. Black rectangles represent completely sequenced regions, gray rectangles represent partially sequenced or PCR-mapped clones and gray hatches represent parts of clones whose overlapping distances are not known. (A) Representative genes and clones provide further refinement to the human gene map previously described (11). A minimal tiling path determined by hybridization, PCR and in silico methods clearly establishes MUC3 on the telomeric side of ACHE and links the HRBL end of AF053356 to the CYP3A4 locus (see http://www.genet.sickkids.on.ca/chromosome7/ for a more detailed physical map including additional genes, further clones and experimental details). (B) Comparative human–mouse genomic map showing conserved synteny of at least 22 genes. The composite gene map shows human genes (upper case letters), all of which have a mouse ortholog, as well as the mouse gene Cyp3a13, whose human ortholog is yet to be determined. The orientation of the mouse BAC clones in relation to chromosome 5 was inferred from the location of the mouse Cyp3a gene locus on a refined chromosome 5 map (http://genome.nhgri.nih.gov/chr7/comparative; 8). PCR mapping determined that a TSC-22-like gene, THG-1, is next to HRBL in both human and mouse followed by ZE03F02 and FLJ20257 (Ze03f02 and D5Wsu46e) whose order has not yet been determined. None of the genes shown between FLJ20257 and CYP3A4 has been identified in our partial mouse sequence data for BACs 139n8 and 493b1 (D5Wsu46e to Cyp3a13).

Figure 2

(A) Dot-plot display of mouse (x-axis) and human (y-axis). (B) PIP. Nucleotide positions are shown for the mouse sequence on the x-axis, and percent sequence identity with the corresponding human sequence (50–100%) is shown on the y-axis. The purple and orange shading on the upper and lower halves of the PIP represent the locations of exons on the + and – strands, respectively, while the pink and light orange areas represent UTRs. Red stripes indicate regulatory regions that have been functionally characterized. Genes are labeled based on cDNA, EST and comparative genomic information. RepeatMasker2 (A.F.A.Smit and P.Green, unpublished results) was used to locate repeat elements.

Figure 2

(A) Dot-plot display of mouse (x-axis) and human (y-axis). (B) PIP. Nucleotide positions are shown for the mouse sequence on the x-axis, and percent sequence identity with the corresponding human sequence (50–100%) is shown on the y-axis. The purple and orange shading on the upper and lower halves of the PIP represent the locations of exons on the + and – strands, respectively, while the pink and light orange areas represent UTRs. Red stripes indicate regulatory regions that have been functionally characterized. Genes are labeled based on cDNA, EST and comparative genomic information. RepeatMasker2 (A.F.A.Smit and P.Green, unpublished results) was used to locate repeat elements.

Figure 2

(A) Dot-plot display of mouse (x-axis) and human (y-axis). (B) PIP. Nucleotide positions are shown for the mouse sequence on the x-axis, and percent sequence identity with the corresponding human sequence (50–100%) is shown on the y-axis. The purple and orange shading on the upper and lower halves of the PIP represent the locations of exons on the + and – strands, respectively, while the pink and light orange areas represent UTRs. Red stripes indicate regulatory regions that have been functionally characterized. Genes are labeled based on cDNA, EST and comparative genomic information. RepeatMasker2 (A.F.A.Smit and P.Green, unpublished results) was used to locate repeat elements.

Figure 3

Using Laj to view complete and partial comparative genomic sequences from human chromosome 7q22 (y-axis) and the orthologous region in the mouse (x-axis). (A) View of the entire ACHE/TFR2 genomic region. The overall scale bar, dot-plot, relevant annotations, PIP (labeled as in Fig. 2B) and local alignment text are all displayed in the same window. The first message box below the menu bar displays information about the object the mouse is pointing at, in this case a hyperlink to the UniGene cluster Mm.21836. A local alignment in the ACHE/ASR2 region has been selected by clicking on it, which has caused the local alignment to be highlighted in red and the clicked position to be marked with a red circle. Information about these is provided in the second message box. The scrollable panel at the bottom of the window displays the selected local alignment in a nucleotide-level text format, with the white cursor corresponding to the marked position. (B) ‘Zooming in’ on the region selected in (A). The marked position lies in a gene flanked by ACHE and ASR2 that contains UniGene cluster Mm.21836, and the mouse pointer is currently resting on a B1 element found in the 3′ UTR of this gene. The purple and orange shading on the upper and lower halves of the PIP represent the locations of exons on the + and – strands, respectively, while the pink and light orange areas represent UTRs. Similar shading of the exons and UTRs appears in the top row (mouse sequence) of the text panel. Hyperlinked annotations include UniGene cluster Mm.21836 (blue bar above mouse pointer) flanked by Ache and Asr2 UniGene clusters, cDNAs, alternatively spliced ESTs (all blue bars), LocusLinks (pink bars) and a GeneCard for ACHE (orange bar). (C) Display of unordered partial mouse data (y-axis) aligned to complete human sequence (x-axis). Eight mouse contigs from BAC 139n8 orthologous to the PCOLCE/HRBL region on human clone AF053356 (11) are displayed, separated by horizontal gray lines. An alignment (highlighted in red) that contains the 5′ end of the IRS3L gene has been selected, and the mouse pointer is currently resting on a hyperlink (purple bar) to the PubMed entry for a paper about the mouse Irs3 gene (54). The orange-shaded text highlights the beginning of the IRS3L ORF and UTR. (See http://web.uvic.ca/~bioweb/laj.html for a display of the ordered contig data.)