Source/description: The human collagen, type XVI, alpha 1 (COL16A1) gene has been physically assigned to human chromosome 1p341,2 starting at 31 786 941 bp and ending at 31 838 742 bp. The human COL16A1 gene consists of 71 exons spanning about 51.8 kb encoding the alpha chain of type XVI collagen. The collagens form 2 major classes: the fibril-forming collagens and the non-fibril-forming collagens.
COL16A1 (collagen type XVI) is part of the non-fibril-forming collagens with features resembling those of members of the fibril-associated collagens with interrupted triple helices subgroup,1 which may serve as molecular bridges that are important for the organization and stability of extracellular matrices.3 The expression of COL16A1 has been demonstrated in human fibroblasts, keratinocytes, chondrocytes and corneas1,4,5.
The equine CHORI-241 BAC library was screened to isolate a BAC clone containing the COL16A1 gene. High density BAC colony filters were probed according to the CHORI protocols (http://www.chori.org/bacpac/) with a heterologous
32P-labelled insert of a human COL16A1 cDNA clone (IMAGp998C217560) provided by the Resource Center/Primary Database of the German Human Genome Project (http://www.rzpd.de/). A positive equine genomic BAC clone designated CH241-15G6 with an insert of approximately 210 kb containing the COL16A1 gene was identified.
BAC DNA was prepared from 100-ml overnight cultures using the Qiagen Midi plasmid kit according to the modified protocol for BACs (Qiagen, Hilden, Germany).
The BAC ends were sequenced using the ThermoSequenase kit (Amersham Biosciences, Freiburg, Germany) and a LI-COR 4300 automated sequencer (LI-COR, Inc., Lincoln, NE, USA). The BAC end sequences were deposited in the EMBL nucleotide database (Accession nos. AJ871799, AJ871800). BLASTN analysis of the SP6 BAC end against build 35.1 of the human genome revealed a significant match on HSA 1p35, located approximately 48 kb downstream of human COL16A1 gene (identity = 83%, BLAST E-value 2e-44) starting at 31 886 205 bp.
Primer sequences: The two CH241_15G6 BAC end sequences were used to design two pairs of equine PCR primers (GenBank accession nos. AJ871799 and AJ871800) using the PRIMER3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) to give products of 209 bp and 202 bp respectively.
CH241_15G6_SP6: F: 5’-TCT TTC ATG CAT TTG GAA GG-3’
R: 5’-TAA CAC TGG ATC ACG GACT G-3’
CH241_15G6_T7: F: 5’-TCT ATT TCT TGG CCA GGT TG-3’
R: 5’-CTG CCA GCT CTA GTC CTC TG-3’
Using an exonic primer pair designed in high conserved regions from an alignment of human sequence of COL16A1 exons 6 and 8 and canine genome sequence of COL16A1 exons 6 and 8 (Accession nos. Homo sapiens, gi: 51511461; Canis familiaris: NW_139841), we amplified a PCR product of approximately 940 bp from BAC clone CH241-15G6.
COL16A1_Ex6-8: F: 5'-TTC TGG GCT TGG ATG CTG AG-3' COL16A1_Ex6-8: R: 5'-GAC CTT GCC TTC AGT CTG TG-3'
Subsequent sequence analyses of this PCR revealed a single 642 bp read (Accession no. AJ879504) which gave a significant match (BLASTN analysis against build 35.1 of the human genome) on HSA 1p35, located in intron 6 and exon 7 of human COL16A1 gene (identity = 77%, BLAST E-value 2e-35) starting at 31 832 467 bp.
Chromosome location: Equine metaphase spreads for fluorescence in situ hybridization (FISH) on GTG-banded chromosomes were prepared using pokeweed mitogen stimulated blood lymphozytes. DNA from the equine BAC clone CH241-15G6 was digoxigenin labelled by nick translation using a dig-nick translation mix (Roche Diagnostics, Mannheim, Germany). FISH on the GTG-banded horse chromosomes was performed using 750 ng of labelled BAC DNA. As competitors in this experiment, 20 µg sheared total equine DNA and 10 µg salmon sperm DNA were
Assignment of COL16A1 to ECA 2p15.1-p15.3
75
used. After hybridization overnight, signal detection was performed using a Digoxigenin-FITC Detection Kit (Qbiogene, Heidelberg, Germany). The chromosomes were counterstained with DAPI (4',6’-diaminidino-2-phenylindole) and embedded in propidium iodide/antifade. Metaphase chromosomes that had been previously photographed using a CCD camera were re-examined after hybridization using a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) equipped for fluorescence. Identification of chromosomes followed the international system for chromosome nomenclature of the domestic horse (ISCNH 1997).6 The equine genomic BAC clone containing the COL16A1 gene was most precisely located to ECA2p15.1-p15.3 by examination of metaphase chromosomes of 30 cells (Fig. 1).
Radiation hybrid mapping/PCR conditions: To confirm the chromosome location of the BAC clone, the Texas A&M University equine 5,000 radiation hybrid panel7 was used to map the COL16A1. Two independent PCR reactions using the previously described primers for both BAC ends were performed in a total of 20 µl using 25 ng of RH cell line DNA, 15 pmol of each primer and 0.75 U Taq polymerase (Qbiogene, Heidelberg, Germany). Samples were denatured at 94°C for 4 min, followed by 35 cycles under the following conditions: denaturation for 30 s at 94°C, annealing for 60 s at 58°C (SP6) and at 61°C (T7), respectively, and extension for 40 s at 72°C. The PCR was completed with a final cooling at 4°C for 10 min. PCR products were separated on a 1.5% agarose gel. After scoring positive signals, a two-point analysis was performed using RHMAPPER-1.228 (http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) against 861 equine markers typed previously on the equine RH5,000panel.9 The retention frequency of the sequence tagged site (STS) marker of the SP6 BAC end sequence was 8.7% and the two-point analysis revealed complete linkage of CH241-15G6_SP6 at a distance of 0.0 cR to HDAC1 and close linkage to the microsatellite TKY024 at a distance of 7.26 cR. The retention frequency of the STS marker of the T7 BAC end sequence was 9.8% and the two-point analysis revealed complete linkage of CH241-15G6_T7 at a distance of 0.00 cR to HDAC1. The corresponding LOD scores were > 12.0.
TKY024 has been previously located on ECA 2p14-p16 by FISH and by RH mapping at 65.2 cR from the beginning of RBBP4 on ECARH02b.9,10 HDAC1 was annotated on HSA1p34 approximately 590 kb downstream of COL16A1 (sequence human genome build 35.1).
Comment: The RH and FISH mapping results of the equine COL16A1 gene on ECA2p15.1-p15.3 agree with comparative mapping of the current equine-human comparative RH and cytogenetic map9 of ECA 2p, which shows conserved synteny to HSA 1p.
Figure 1 Chromosomal assignment of the equine BAC CH241-15G6 containing
the COL16A1 gene by FISH analysis. (a) GTG-banded horse metaphase (b) double signals visible on both ECA2 chromosomes are indicated by arrows.
Acknowledgements: This study was supported by grants of the German Research Council, DFG, Bonn (DI 333/12-1).
Assignment of COL16A1 to ECA 2p15.1-p15.3
77 References
1 Pan T.-C. et al. (1992) Proc Natl Acad Sci USA 89, 6565-9.
2 Yamaguchi N. et al. (1992) J Biochem 112, 856-63.
3 Shaw L.M. et al. (1991) Trends Biochem Sci 16, 191-4.
4 Kassner A. et al. (2004) J Mol Biol 339, 835-53.
5 Jun A.S. et al. (2001) Arch Ophthal 119, 1629-34.
6 Bowling A.T. et al. (1997) Chromosome Res 5, 433-43.
7 Chowdhary B.P. et al. (2002) Mamm Genome 13, 89-94.
8 Slonim D. et al. (1997) J Comp Biol 4, 487-504.
9 Chowdhary B.P. et al. (2003) Genome Res 13, 742-51.
10 Hirota K. et al. (2001) Anim Genet 32, 160-2.
Correspondence: Ottmar Distl (ottmar.distl@tiho-hannover.de)
Chapter 5
doi:10.1111/j.1365-2052.2005.01330.x