Mapping of dentin-specific acidic phosphoprotein and integrin-binding sialoprotein in sheep defines an inversion breakpoint with respect to human chromosome 4Q

Mapping of dentin-specific acidic phosphoprotein and integrin-binding sialoprotein in sheep defines an inversion breakpoint with respect to human chromosome 4Q

Joanne M. Lumsden¹, Eric A. Lord¹, Karen L. Hirst², Michael J. Dixon² and Grant W. Montgomery¹

¹AgResearch Molecular Biology Unit, Department of Biochemistry and the Centre for Gene Research, University of Otago, PO Box 56, Dunedin, New Zealand. Send correspondence to G.W.M. Fax [phone], E-mail: [e-mail]

²School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom

ABSTRACT

Genes from sheep chromosome 6 map to human chromosome 4 in the region extending from 4p16 to 4q26. However, there is an inversion of gene order in the central portion of the chromosome with one breakpoint close to secreted phosphoprotein 1 (SPP1). Genes for SPP1, integrin-binding sialoprotein (IBSP) and dentin-specific acidic phosphoprotein (DMP1) are located close together in a YAC contig in the human. RFLP markers were developed for DMP1 and IBSP in sheep and located on the sheep linkage map to further define the breakpoint region. There were no recombinants between SPP1 and IBSP indicating that these loci are close together in sheep, as in humans. DMP1 was located approximately 80 cM from SPP1 in sheep, 7 cM from the microsatellite BMC4203. In the human YAC contig, the order of these genes is SPP1-IBSP-DMP1 with 340 kb separating SPP1 and IBSP and 150 kb between IBSP and DMP1. Therefore, one breakpoint for the inversion in gene order between the sheep and the human has been narrowed to a region of 150 kb on the human map.

INTRODUCTION

Chromosomal rearrangements provide one mechanism for evolution of species. Identification of common breakpoints between species may help identify some of the mechanisms responsible. In the mouse, genes from human chromosome 4 map to mouse chromosomes 3 and 5 with a breakpoint close to SPP1. Similarly, genes from PDGFRA to SPP1 are located on pig chromosome 8, and there is a breakpoint close to SPP1 (Archibald et al., 1995). Genes from a large portion of human chromosome 4 extending from 4p16 to 4q26 map to sheep chromosome 6 (Lord et al., 1996). However, gene order varies and the region on sheep chromosome 6 from PDGFRA to BMP3 appears to be inverted with a breakpoint close to SPP1.

The area around SPP1 is the focus of researchers mapping the human autosomal dominant disorder of dentin formation, dentinogenesis imperfecta type II (DGI1) (Aplin et al., 1995). The mutated gene involved has been mapped to a 3.2-cM region surrounding SPP1. Recently a novel dentin-specific acidic phosphoprotein (DMP1) has been found to be tightly linked to DGI1, but has been excluded as a candidate for the DGI1 locus (Hirst et al., 1997b). DMP1 and integrin-binding sialoprotein (IBSP) have been mapped to within 470 kb of SPP1 on a human YAC contig (Aplin et al., 1995). The aim of this study was to locate genes close to SPP1 from human chromosome 4 on the sheep linkage map and to further define the breakpoint region.

MATERIAL AND METHODS

An RFLP for DMP1 was identified after hybridizing a bovine cDNA clone B012 (Hirst et al., 1997a) with Southern filters of sheep DNA samples digested with HindIII. A human cDNA clone for IBSP (B6-5g; Fisher et al., 1990) was used to identify an EcoRV RFLP for this gene. Conditions for preparing Southern filters of restricted DNA and hybridization of filters have been described (Montgomery et al., 1992). The final washing stringency was 0.5 x SSC at 63°C for 10 min. The RFLPs were typed in the AgResearch International Mapping Flock, which comprises nine three-generation families (six to 17 offspring per family) (Crawford et al., 1995). The resulting segregation data were analyzed using CRI-MAP (Lander and Green, 1987) together with other markers typed in this flock (Table I).

Table I
- Pairwise linkage data for dentin-specific acidic phosphoprotein (DMP1) and integrin-binding sialoprotein (IBSP) with markers on sheep chromosome 6.

Locus 1	Locus 2	Recombination fraction	Zmax1
DMP1	BMC4203	0.07	10.39
DMP1	BMP3	0.18	4.46
DMP1	SPP1	0.50	0.00
DMP1	OarAE101	0.30	0.46
IBSP	BMC4203	0.30	0.35
IBSP	BMP3	0.50	0.00
IBSP	SPP1	0.00	7.53
IBSP	OarAE101	0.00	10.84

¹Maximum lod score.

RESULTS AND DISCUSSION

There were no recombinants between SPP1 and IBSP (Z_max = 7.53, q = 0.00), indicating that these loci are close together in sheep, as in humans. DMP1 is located approximately 80 cM from SPP1 in sheep, 7 cM from the microsatellite BMC4203 (Z_max = 10.39, q = 0.07). Figure 1 shows the current sheep chromosome 6 linkage map, including IBSP and DMP1.

Figure 1
- The sheep chromosome (Chr) 6 linkage map compared with the orthologous region from human Chr 4. The sheep Chr 6 linkage map was calculated from the data presented in Lord et al. (1996), adding the loci for dentin-specific acidic phosphoprotein (DMP1) and integrin-binding sialoprotein (IBSP). The human gene order is that predicted by the Genetic Location Database (Ldb) (Collins et al., 1996).

Comparison of the sheep chromosome 6 map and the equivalent region on human chromosome 4, with respect to gene order, is difficult because many genes have only been physically assigned in the human, rather than included in linkage maps. The Genetic Location Database (Ldb) is an analytical database for generating an integrated map (or summary map) from partial maps of physical, genetic, regional, mouse homology and cytogenetic data (Collins et al., 1996). Figure 1 shows the putative arrangement of genes along human chromosome 4 as determined by this algorithm with an inversion between SPP1 and DMP1. Mapping data for two genes on human chromosome 4 suggest alternative locations to those shown in Ldb. Firstly, IBSP is placed distal to CCNA while YAC content mapping (Aplin et al., 1995) and linkage mapping (Crosby et al., 1996) place it close to DMP1 and SPP1. Secondly, CSN2 is predicted to lie in the interval between PDE6B (4p16.3) and PDGFRA/KIT (4q11-q12). More recent data using fluorescence in situ hybridization (McConkey et al., 1996) suggest that this gene maps to the interval 4q13-q21 and would therefore lie in the interval SPP1-PDGFRA/KIT.

The linkage data for IBSP in sheep support a location of IBSP close to both SPP1 and the microsatellite marker OarAE101 in agreement with the physical map from analysis of human YACs (Aplin et al., 1995). In sheep, the casein gene complex lies between PDGFRA/KIT and ALB (Lord et al., 1996). Another difference in gene order between the human and sheep maps is the position of GNRHR which lies distal to SPP1 in humans, but between PDGFRA/KIT and ALB in sheep. More accurate linkage maps will be required to determine if the large rearrangement between sheep and human is a simple inversion, or if there are smaller rearrangements in gene order within the inversion.

PDE6B has been mapped to the telomere of both human chromosome 4 and sheep chromosome 6, indicating that the rearrangement between the sheep and human chromosomes consists of two breakpoints and one inversion. One breakpoint lies between IBSP and DMP1. The human YAC contig containing SPP1, IBSP and DMP1 indicates that the order of these genes is SPP1-IBSP-DMP1 with 340 kb separating SPP1 and IBSP and 150 kb between IBSP and DMP1. Therefore, one inversion breakpoint has been narrowed to a region of 150 kb. The second breakpoint must lie between PDGFRA/KIT and PDE6B, but no genes have been mapped to this interval in sheep.

In mice, DMP1, IBSP and SPP1 all map to chromosome 5 indicating that the breakpoint creating mouse chromosomes 5 and 3 is different from the breakpoint causing the inversion on sheep chromosome 6. Mouse chromosome 5 appears to have undergone further rearrangement because Pdeb, which is equivalent to PDE6B, lies between SPP1 and ALB on this chromosome. In pigs, the order of loci on chromosome 8 is SPP1-CSN1S1-ALB-PDGFRA showing that there have been further rearrangements of gene order when compared to sheep. Ellegren and associates (1993) suggest that these rearrangements occurred after the Ruminantia/Nonruminantia split within the Order Artiodactyla. To date, IBSP and DMP1 have not been mapped in pigs.

There have been a number of breakpoints and chromosomal rearrangements in the region of genes from human chromosome 4 during evolution of ungulate and rodent species. The results from this study narrow down one breakpoint to a region of 150 kb on human chromosome 4. It will be of interest to see whether there is some common structural feature in the region of these breakpoints as more breakpoints are identified and DNA sequence becomes available for human chromosome 4.

ACKNOWLEDGMENTS

This work has been funded by AgResearch through a grant from the New Zealand Foundation for Research Science and Technology.

RESUMO

Gens do cromossomo 6 de carneiro ocupam posição similar no cromossomo 4 humano na região que vai de 4p16 a 4q26. Contudo, há uma inversão na ordem dos gens na porção central do cromossomo, com um ponto de quebra próximo à fosfoproteína 1 secretada (SPP1). Os gens para SPP1, sialoproteína ligada a integrina (IBSP) e fosfoproteína ácida dentina-específica (DMP1) estão localizados muito próximos em um contig YAC em humanos. Marcadores para RFLP foram desenvolvidos para DMP1 e IBSP em carneiros e localizados no mapa de ligações para posterior definição da região do ponto de quebra. Não houve recombinantes entre SPP1 e IBSP, indicando que esses loci estão intimamente relacionados no carneiro, como em humanos. DMP1 se localizou a aproximadamente 80 cM do SPP1 no carneiro e a 7 cM do microsatélite BMC4203. No contig YAC humano, a ordem desses gens é SPP1 - IBSP - DMP1, com 340 kb separando SPP1 de IBSP e 150 kb entre IBSP e DMP1. Portanto, um ponto de quebra para a inversão na ordem do gene do carneiro em relação ao homem foi reduzido para uma região de 150 kb no mapa humano.

(Received July 8, 1998)

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Publication Dates

• Publication in this collection
  02 June 1999
• Date of issue
  Mar 1999

History

• Received
  08 July 1998