Results

To construct a BAC contig of the bovine polled gene region we started to screen a bovine BAC library by hybridization of 12 different heterologous human IMAGE cDNA clones (Table 1).

Table 1 Human cDNA hybridization probes within the bovine BAC contig.

human gene

symbol Genname IMAGE-ID RZPD clone ID TIAM1 T-cell lymphoma invasion and

metastasis 1

3197030 IMAGp 998 O157814 SOD1 superoxid dismutase 1 436140 IMAGp 998 B131026 HUNK Hormonally upregulated

Neu-associated kinase 768063 IMAGp 998 H161890 C21orf108 Chromosome 21 open reading

frame 108

25729 IMAGp 998 G19138 C21orf59 Chromosome 21 open reading

frame 59

124398 IMAGp 998 E07121 SYNJ1 Synaptojonin-1 2038462 IMAGp 998 M235017 OLIG2 Oligodendrocyte lineage

transcription factor 2170611 IMAGp 998 P045361 IL10RB Interleukin 10 receptor, beta 842859 IMAGp 998 E042085 GART Glycinamide ribonucleotide

formyltransferase 2901218 IMAGp 998 J037162 SON SON DNA-binding protein 1696332 IMAGp 998 N134307 KCNE2 potassium voltage-gated

channel, Isk-related family, member 2

2308895 IMAGp 998 A245722

DSCR1 Down syndrome critical region gene 1

324006 IMAGp 998 B07734

The physical localizations of six representative gene associated BAC clones were established by RH mapping and FISH on BTA1q12 (DRÖGEMÜLLER et al., 2002) prior to the beginning of a chromosome walking strategy. Further sequence tagged site (STS) probes that allowed the gradual joining of the individual emerging contigs into one large contig were generated from the obtained BAC end sequences of appropriate clones. Overlaps between clones were determined by STS content analysis. In total, 109 new STS markers were generated (Appendix 1, Table 2). The complete BAC contig consisted of 92 clones (Figure 1).

The physical mapping information derived from the contig assembly was refined by taking into account estimated BAC insert sizes from fingerprint analyses and pulsed field gels. The average insert size of the 92 BAC clones was 162 kb (range from 30 to 200 kb). The entire contig spans approximately 4 Mb and can be covered with a minimal tiling path of 32 clones (Figure 1).

The clone-based physical map was anchored to the linkage and RH map of BTA1 by STS content mapping of five previously described bovine microsatellites (ARO9, ARO24, TGLA49, SOD1MICRO2, BM6438) and two EST markers (EST0601, EST1413) (Figure 1).

During construction of the bovine contig primers were designed for 25 HSA21q22 genes from corresponding bovine EST sequences (Appendix 1, Table 3). PCR analysis of all 92 BAC clones with the gene-specific EST primer pairs revealed positive clones and the localization of these genes on the contig (Figure 1).

In total 165 BAC end sequences with an average read length of 726 bp totalling approximately 120 kb of genomic survey sequences were generated. Thus, the BAC end sequences cover approximately 3% of the studied genomic region. The sequence information of 165 BAC ends has been deposited in the EMBL nucleotide database under accession nos.

AJ698510 - AJ698674. Sequence alignments revealed 8 pairs of identical BAC ends. The end sequences contain an average GC content of 44.3% marginally exceeding the value of 41%

that is generally accepted as the average GC content in mammalian genomes. The GC content analysis further suggests that BTA1q12 is indeed closely related to HSA21q22, which has a GC content of 43.2% in the corresponding 4 Mb region. An analysis of repetitive sequences revealed that 39.1% of the BAC end sequences consisted of bovine repetitive DNA, mainly LINE (18.9%) and SINE (14.9%) elements, only 3.4% were of retroviral origin (LTRs), and 1.3% represented DNA transposons. In 56 cases, all or the majority of the BAC end

sequences represented repetitive sequences and were therefore discarded for STS design. The repeat masked BAC end sequences were subjected to BLAST comparisons against the sequence of the human genome (build 34.3). The obtained matches confirmed the homology between the cloned chromosomal region in cattle with HSA21q22. Significant and unique matches (e-value < 10^-5) against human genomic sequences were observed for 38 (23%) bovine BAC end sequences. All but one of the 38 matches mapped to the expected location on HSA21q22 (Appendix 1, Table 4). All these BLAST matches corresponded well with the overall clone order in the bovine BAC contig and confirmed the correct assembly. In some cases the BLAST searches revealed the presence of genes within BAC end sequences and confirmed the previously obtained mapping results (Appendix 1, Table 4). The C21orf62 and SFRS15 genes could be localized in silico by this approach on the contig for the fist time (Figure 1). Only one single sequence (380C19-SP6) matched to a different human chromosome during the BLAST search. This unexpected BLAST result probably indicates a chimeric clone, as this BAC has been anchored in the contig by 4 STS markers and a gene specific bovine EST primer pair (Figure 1).

In total, the construction of this contig confirms the mapping of 15 previously mapped BTA1 genes and provides 16 new chromosomal assignments of bovine orthologs to the human genes SFRS15, C21orf45, C21orf108, C21orf63, C21orf59, C21orf66, C21orf62, IFNGR2, C21orf4, SON, MRPS6, C21orf82, C21orf51, KCNE1, DSCR1, and CLIC6. The gene order of the 31 assigned genes in the bovine BAC clone contig (Figure 1) corresponds exactly to the gene order of the NCBI HSA21q22 map (http://www.ncbi.nlm.nih.gov/mapview/; build 34.3), which lists 50 gene loci in the interval between KRTAP8P1 and CLIC6. Of these 50 loci 7 represent computer predicted hypothetical genes and 5 pseudogenes while 38 genes have at least some experimental evidence. The physical size of the investigated region and the distances between the mapped genes seems to be conserved between human and cattle. A high degree of gene order conservation can also be observed with respect to annotated murine genes. Some of the mapped bovine genes are assigned to the linkage map of mouse chromosome (MMU) 16. The current NCBI sequence map of MMU 16 (http://www.ncbi.nlm.nih.gov/mapview/; build 32.1) lists 19 of the 31 analyzed genes in a similar order as in cattle or human.

383 K23 63 O12 496 H4 394 A5 386 F4 352 O20 44 B5 292 J15 234 N12 506 K17 23 E5 506 K15 311 D23 320 O18 26 I16 180 G7

569 F23 161 B10

420 E6 76 J4 351 B8 219 G21 21 K598 P9

* 100 kb 100 kb Figure 1 Physical map of the isolated bovine BAC contig on BTA1q12. All mapped loci are indicated vertically on the top. Previously published BTA1 mapping results are marked by one (genes), two (ESTs), or three (microsatellites) asterisks. Underlined gene markers were initially assigned by human cDNA hybridization probes. Two framed genes were localized on the contigin silico. RPCI-42 BAC clones are given horizontally below the markers by continuous lines with their corresponding abbreviated clone names. A single chimeric BAC is shown by a dashed horizontal line. A minimal tiling path of 32 clones is indicated by thick lines. Bovine microsatellite, EST, and STS markers are represented by vertical solid lines. Bovine markers that are associated to corresponding human genes are plotted by dotted vertical lines and linked to 31 genes on the 4 Mb sequence segment of HSA21q22 (NCBI build 34.3) at the bottom. Comparative mapping of 31 gene- associated markers revealed a complete conservation of the gene order across the entire 4 Mb interval between Bos taurus and Homo sapiens.