Assignment of solute carrier family 26 (sulfate transporter), member 2 (SLC26a2) to river buffalo (Bubalus bubalis, 2n = 50) chromosome 9q26 (BBU9q26) by fluorescence in situ hybridization and R-banding

G. Kierstein, L. Iannuzzi, A. Silva, M.P. Schneider, B.G. Baumgartne, B. Brenig
2003 Cytogenetic and Genome Research  
Rationale and significance To investigate the possible cause of a bone dysplasic phenotype in a Brazilian water buffalo population we used a candidate gene approach. The SLC26a2 gene has been associated with diastrophic dysplasia (DTD) in human and was shown to code for a novel sulfate transporter, thus symbolized as DTDST (Hästbacka, 1994) . Mutations in the SLC26a2 gene, beside other genes like those encoding cartilage oligomeric matrix protein (COMP), type IX collagen (COL9A1, COL9A2, and
more » ... 9A1, COL9A2, and COL9A3) and matrilin (MATN3) have been shown to cause pseudoachondroplasia and multiple epiphyseal dysplasia. Both syndromes constitute a bone dysplasia family, which is genetically and phenotypically heterogeneous (Briggs and Chapman, 2002) . Materials and methods A bubaline ÏFIX ® II library (Silva et al., 1999) was screened by PCR using oligonucleotides based on the bovine sequence of the SLC26a2 gene (Brenig et al., 2003) . Phage DNA was isolated with a Qiagen Plasmid Purifi-cation Kit according to the manufacturer's instructions and was subcloned into pGEM ® -4Z (Promega GmbH, Mannheim) vector. Subsequent sequencing was carried out on a LI-COR DNA Sequencer Model 4000L (MWG) as described previously (Middendorf et al., 1992) . Concavalin A stimulated peripheral blood lymphocytes were cultured for three days in RPMI medium at 37.8°C (CO 2 incubator) and treated with 5-BrdU (15 Ìg/ml) and Hoechst 33258 (30 Ìg/ml) 6 h before harvesting (late incorporation) to obtain R-banding pattern preparations. Colcemid treatment (0.05 Ìg/ml) lasted for 1 h. After hypotonic treatment (20 min with 0.5 % KCl at 37.5°C) and three fixations (the third overnight) in methanol/ acetic acid 3:1, cell suspension was dropped on cleaned, wet and cool slides. A day later, slides were stained with Hoechst 33258 (20 Ìg/ml) for 10 min, then washed with distilled water, mounted in 2× SSC (pH 7.0) with a glass coverslip, exposed to UV light for 30 min and washed with distilled water. Slides were then treated for FISH-technique with a BAC-clone containing SLC26a2 for about three days (weekend) in presence of bovine COT-1 DNA (with 1 h of annealing step at 37.5°C before the in situ hybridization) and allocated in a moist chamber. Biotin-14-dATP was incorporated into 1 Ìg of probe DNA by nick-translation (Gibco-BRL, BioNick). After the detection step with FITC-avidin and anti-avidin antibody (Oncor), slides were mounted with Antifade/Hoechst 33258 dye (3 Ìg/ml). Both R-banded metaphases (RBH-banding) and fluorescent FITC-signals were separately captured by a CCD-camera (Sensys, Photometrics) and processed by superimposing FITC-signals on RBH-banded preparations. Chromosome identification and banding followed the river buffalo standard karyotype (Iannuzzi, 1994) . Probe name(s): Ï 6-1 Probe type: bubaline genomic ÏFIX ® II phage Insert size: app. 20 kb Vector: pGEM ® -4Z (Promega GmbH, Mannheim) Proof of authenticity: sequence accession no. AY350740 Gene reference: SLC26a2
doi:10.1159/000076318 pmid:15008140 fatcat:ou3wp7mrxragpeepct3igyl5im