SHORT COMMUNICATION Chromosomal mapping of adenosine receptor genes in chicken suggests clustering of two members of the gene family Alexei Sazanov 1 , Matthew R. Atkinson 2 , Johannes Buitkamp 1 & Ruedi Fries 1 1 Lehrstuhl fu Èr Tierzucht der Technischen Universita Èt Mu Ènchen, Alte Akademie 12, 85350 Freising-Weihenstephan, Germany; Tel: (49) 8161 713228; Fax: (49) 8161 713107; E-mail: Ruedi.Fries@tierzucht.tum.de; 2 Neurobiology Division, The Garvan Institute of Medical Research, St Vincent'sHospital, Darlinghurst, Sydney, NSW, 2010, Australia  Correspondence Received 30 October 1999; received in revised form and accepted for publication by M. Schmid 14 December 1999 Key words: adenosine receptor genes, chicken, ¯uorescence in-situ hybridization, Gallus gallus, microchromosome, paralogous chromosome regions Adenosine and adenosine receptor ligands in¯uence numerous processes in the chicken including angio- genesis in the developing embryo- and chorioallan- toic membrane (Dusseau et al. 1986). Adenosine also plays an important role in the development and growth regulation of chicken sympathetic neurons (Wakade et al. 1995). Human genes have been identi®ed for four adenosine receptor subtypes, namely ADORA1 (adenosine A 1 receptor), ADOR- A2A (adenosine A 2a receptor), ADORA2B (adeno- sine A 2b receptor) and ADORA3 (adenosine A 3 receptor) (Townsend-Nicholson et al. 1995a, 1995b, Atkinson et al. 1997). Sequence homology between the adenosine receptor subtype genes suggests divergence from one ancestral adenosine receptor gene. Here, we report the cloning of cosmids representing adenosine receptor genes and their chromosomal mapping in the chicken genome. Three fragments representing ADORA1 (450 bp), ADORA2B (350 bp) and ADORA3 (350 bp) were am- pli®ed from chicken genomic DNA with degenerate oligonucleotides and sequenced. The primers were designed based on the alignment of known adenosine receptor amino acid sequences. The ADORA1 frag- ment was produced by oligos #708 and #711, the ADORA2B fragment by oligos #709 and #711 and the ADORA3 fragment by oligos #710 and #711. The oligonucleotide sequences were as follows: Oligo #708: 59-GTCGAATTCACICCI(A/T/C)- TITT(T/C)GGITGGAA-39; Oligo #709: 59-CCCGAATTCTA(T/C)ATGGTI- TA(T/C)T(A/T)(T/C)AA(T/C)TT-39; Oligo #710: 59-GTGGAATTC(A/T)TIGA(T/C)TA- (T/C)ATGGTIT(A/T)(T/C)TT-39; Oligo #711: 59-CTGGAAAAGCTTIGC(G/A)TAI- AC(G/A/T)ATIGG(G/A)TTCAT-39. The sequences obtained from the chicken adenosine receptor fragments were aligned with known mam- malian adenosine receptor sequences for subtype veri®cation. Screening of a gridded chicken cosmid library (Buitkamp et al. 1998) with the PCR products yielded ®ve cosmids for ADORA1, two for ADORA2B and eight for ADORA3. Cosmid D17- 237 that had been isolated with the ADORA3 probe was investigated by direct sequencing on both strands using primers that were designed based on the ADORA3 cDNA sequence that became available in the meantime (EMBL accession number AF115332). The resulting sequence was 100% identical to the deposited cDNA sequence (data not shown). Two cosmids were investigated by PCR for the presence of ADORA1 and ADORA2B sequences, respectively, using primers, which were designed based on chicken sequences with EMBL accession numbers V12601 and U28380, respectively. Cosmid Chromosome Research 8: 173±176, 2000. # 2000 Kluwer Academic Publishers. Printed in the Netherlands 173