BRIEF REPORTS rate images of DAPI-banded chromosomes and FITC-tar- P450RAI (CYP26A1) Mapsto Human geted chromosomes were acquired and merged using im- age analysis software, pseudo-colored blue (DAPI) and yel- Chromosome 10q23–q24 and Mouse low (FITC) as previously described (1), and overlaid Chromosome 19C2-3 electronically. Two individual PAC clones were mapped, and the sig- Jay A. White,* , † Barbara Beckett,* nals were shown to be localized to 10q23–q24 for each Stephen W. Scherer,‡ Jo-Anne Herbrick,‡ clone (Fig. 1A). The regional assigment of the genomic and Martin Petkovich* , † , § ,1 probe was determined by the analysis of 20 well-spread metaphases. Positive hybridization signals at 10q23–q24 * Cancer Research Laboratories, †Department of Pathology, were observed in greater than 90% of the cells. Addition- and §Department of Biochemistry, Queen’s University, ally, greater than 90% of the positive spreads showed sig- Kingston, Ontario K71 3N6, Canada; and ‡Department nals on both homologues. The 10q23–q24 band assigment of Genetics, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada was determined by measuring the fractional chromosome length and by analyzing the banding pattern generated by Received October 6, 1997; accepted December 1, 1997 the DAPI-counterstained image. We have also mapped the mouse P450RAI gene and found it to localize to chromosome 19C2-3 (Fig. 1B), a re- gion known to be sytenic to a portion of human chromo- Cytochrome P450 enzymes constitute a large, diverse some 10. This assignment was determined by the analysis superfamily of proteins involved in the generation and ca- of 20 metaphase spreads from mouse ES cells. Positive tabolism of biologically active molecules (see Ref. 2 for re- signals were observed in greater than 20% of the spreads. view). We have recently cloned and characterized a novel The mouse chromosomal mapping was performed using cytochrome, P450RAI (CYP26A1), 2 from zebrafish (10), two independent mouse genomic clones, 15 and 17 kb, iso- mouse (B. Beckett and M. Petkovich, unpublished data), lated by screening a mouse genomic library using the full- and human (9). This enzyme appears to be involved in the length mouse P450RAI cDNA (Beckett et al., unpublished extrahepatic metabolism of retinoic acid (a derivative of results). Sequence analysis has shown that these two vitamin A). In vitro, metabolic studies have shown that clones encompass the entire open reading frame, introns, P450RAI can markedly increase the rate of conversion of and both 5 and 3  untranslated regions of the mP450RAI all-trans-retinoic acid to more polar metabolites, including gene. The mapping of the mouse gene was performed es- 4-oxo-retinoic acid and 4-OH-retinoic acid. In addition, in sentially as described for the human gene with the follow- situ hybridization and Northern blot analysis, showing de- ing modifications. Normal mouse embryonic stem cell (ES) velopmental expression, suggest that P450RAI plays a role cell chromosomes were used. In addition, dual-color FISH in normal developmental processes (10). To investigate the using both biotin-labeled probe for mouse chromosome 19 possible relationship between the P450RAI gene locus and and digoxigenin-labeled mP450RAI was used to verify previously characterized disease loci, fluorescence in situ chromosomal identity (Fig. 1B). The band assignment was hybridization (FISH) was utilized to determine the chro- determined by measuring the fractional chromosome mosomal localization of the P450RAI gene. length and analyzing the DAPI-counterstained image. The A portion of the human P450RAI cDNA was used as a assignment of mP450RAI to chromosome 19C2-3 and probe to identify P-1 artificial chromosome (PAC) clones hP450RAI to chromosome 10q23–q24 supports our hy- that corresponded to P450RAI. Five PAC clones were iso- pothesis that the mouse and human P450RAI cDNAs we lated and purified. FISH mapping was performed, utilizing have isolated are homologues. This is in conjunction with two of the five PAC clones as previously described (7). both structural and functional data on the zebrafish, Briefly, plasmid DNA was purified from the PAC clones mouse, and human cDNAs (9, 10, and Beckett et al., un- and used to generate biotinylated probe using a nick-trans- published results), which clearly show these cDNAs to be lation reaction. In situ hybridization was performed on homologues. metaphase chromosomes isolated from normal human To map more closely the human gene for P450RAI, a lymphocytes. Chromosomes were counterstained with pro- YAC panel was screened using polymerase chain reaction pidium iodide and 4,6-diamidin-2-phenylindol-dihydro- (PCR). Briefly, oligonucleotides designed from one of the chloride (DAPI) (5). After hybridization, biotinylated probe introns of the human P450RAI gene were used to amplify was detected using avidin – fluorescein isothiocyanate. Im- a 229-bp fragment from positive YAC clones. Using this ages from the metaphase chromosome FISH preparations methodology we initially found positive signals for were captured digitally using a thermoelectrically cooled hP450RAI on YACs 967c12 and 905f4. Further analysis charge-coupled camera (Photometrics, Tuscon, AZ). Sepa- has more closely defined the region of localization to a small portion on the end of YAC 804f2 (Fig. 1C). This local- 1 To whom correspondence should be addressed at Cancer Re- ization corresponds closely to markers D10S185 and search Laboratories, Room 355, Botterell Hall, Queen’s University, D10S1755. Kingston, Ontario K7L 3N6, Canada. Telephone: (613) 545-6791. Currently, efforts to map chromosome 10 have generated Fax: (613) 545-6830. E-mail: petkovic@post.queensu.ca. genetic linkage maps, physical maps, as well as localiza- 2 The HUGO/GDB-approved symbol for the gene described in this paper is CYP26A1. tion of disease gene loci. Interestingly, several disease loci 270 GENOMICS 48, 270–272 (1998) ARTICLE NO. GE975157 0888-7543/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.