Large-scale sequencing of two regions in human chromosome 7q22: analysis of 650 kb of genomic sequence around the EPO and CUTL1 loci reveals 17 genes

Abstract

We have sequenced and annotated two genomic regions located in the Giemsa negative band q22 of human chromosome 7. The first region defined by the erythropoietin (EPO) locus is 228 kb in length and contains 13 genes. Whereas 3 genes (GNB2, EPO, PCOLCE) were known previously on the mRNA level, we have been able to identify 10 novel genes using a newly developed automatic annotation tool RUMMAGE-DP, which comprises >26 different programs mainly for exon prediction, homology searches, and compositional and repeat analysis. For precise annotation we have also resequenced ESTs identified to the region and assembled them to build large cDNAs. In addition, we have investigated the differential splicing of genes. Using these tools we annotated 4 of the 10 genes as a zonadhesin, a transferrin homolog, a nucleoporin-like gene, and an actin gene. Two genes showed weak similarity to an insulin-like receptor and a neuronal protein with a leucine-rich amino-terminal domain. Four predicted genes (CDS1-CDS4) CDS that have been confirmed on the mRNA level showed no similarity to known proteins and a potential function could not be assigned. The second region in 7q22 defined by the CUTL1 (CCAAT displacement protein and its splice variant) locus is 416 kb in length and contains three known genes, including PMSL12, APS, CUTL1, and a novel gene (CDS5). The CUTL1 locus, consisting of two splice variants (CDP and CASP), occupies >300 kb. Based on the G, C profile an isochore switch can be defined between the CUTL1 gene and the APS and PMSL12 genes.

Figure 1

Location of the EPO and CUTL1 contigs in the 7q22 region. The position of representative genetic markers [(•) genes (in red) and STSs (unmarked)] with respect to genomic clones in the region are shown. The clones preceded by C represent CEPH–Généthon mega-YACs. Clones preceded by an E are from the HSC7E chromosome 7-specific YAC library. Three PAC clones are also shown (preceded by H_DJ). Information on additional markers represented on these genomic clones is available on the World Wide Web at http://www.genet.sickkids.on.ca/chromosome7/. The shaded area represents an interval within the contig where the orientation of the markers is still not confirmed.

Figure 2

Map of the sequenced contigs. Sequenced clones are drawn as open rectangles containing the clone names. Arrows indicate the genes and their transcriptional directions. The genes are (1) ZAN, (2) EPO, (3) CDS1, (4) CDS2, (5) GNB2, (6) ACTL6, (7) TFR2, (8) CDS3, (9) PCOLCE, (10) CDS4, (11) LRN, (12) IRS3L, (13) HRBL, (14) CDS5, (15) PMSL12, (16) APS, and (17) CUTL1. The GC content is drawn below the gene arrows with a step of 1000 and a sliding window of 100. (A) EPO contig. The single sequencing gap is indicated by a star. (B) CUTL1 contig. The cloning gap is not drawn to scale.

Figure 3

Graphic output of RUMMAGE results of the EPO contig. The vertical line represents the sequencing gap in the EPO contig. Rectangles define the regions in which matches were found. Corresponding matches are connected by green lines. Repeat structures are derived from Repeatmasker, Censor; exon predictions from GENESCAN, GRAIL2, FEXHB, MZEF, XPOUND; promotor prediction from ProScan; motif matches from DPS; poly(A) signals from Pol II; protein motifs from PROSITE; BLAST matches from BLASTS of various databases, Exon sampler. For references, see Methods. The graphic and tabular output of the automated first-pass annotation of both the EPO and the CUTL1 contig with RUMMAGE-DP is viewable via our Home page at http://genome.imb-jena.de/.

Figure 4

Phylogenetic tree of act genes. Sequences were aligned using CLUSTALW. The phylogenetic tree was constructed using PHYLIPP. The random seed value was 75 with 15× to jumble.

Figure 5

The splice variants of CDS3. Rectangles connected by lines indicate the different gene structures of the splice variants. The minimal EST coverage of the splice variants is drawn below the gene structures. The gene location on the EPO contig, together with the transcription direction, is given above the line representing the sequence.

Figure 6

EST coverage of the total EPO contig. All matches of ESTs to the EPO contig in either strand are shown as lines. Matches of the same EST are represented by lines covering the whole region within the matches. Regions of the genes found are drawn above the EST matches.