Cytogenet Cell Genet 84:121-124 (1999) Cytogenetics and Cell Genetics

Synteny mapping of five human 7 on bovine 4 and 21

E. Antoniou,^ J.E. Womack,'^ and M.D. Grosz^ *Fort Keogh Livestock and Range Research Laboratory, Miles City MT; ''Department of Veterinary Pathobiology, Texas A&M University, College Station TX (USA)

Abstract. Five genes oti human (HSA 7) protein 1 (TFC0UP2). Four genes mapped to BTA 4 (GNAI1, were assigned to bovine chromosome 21 (BTA 21) and 4 (BTA TACI. RELN, PRKAR2B) while one mapped to BTA 21 4) using a bovine-rodent somatic hybrid cell panel. These five {TFC0UP2). This study eonfirms the synteny conservation genes were aipha-I subunit of adenylate cyclase-inhibiting between HSA 7 and BTA 4, finely maps the breakpoints of eon- G-protein (GNAIl), alpha/beta preprotachykinin (TACl), ree- served synteny on HSA 7 and defines a new synteny conserva- lin {RELN).c-AMP dependant protein kinase type II beta regu- tion between HSA 7 and BTA 21. latory chain (PRKAR2B) and apolipoprotein Al regulatory

A complete and accurate comparative map of high resolu- aries of human chromosome 7 q arm eonserved segments. Two tion is necessary for the implementation of comparative posi- segments of HSA7 have been shown to be homologous to BTA tional candidate mapping. However, human chromosomes can 4 (http://bos.cvm.tamu.edu; Chowdhary et al., 1996; Hayes. be homologous to more than one bovine chromosome (Chowd- 1995; Solinas-Toldo et al., 1995) according to the Texas hary et al., 1996; Hayes, 1995; Solinas-Toldo et al.. 1995). nomenclature (Popescu et al.. 1996). We report the assignment Recent studies have shown even more complex situations of five genes that lie on HSA 7; alpha-i subunit of adenylate where several interrupted regions ofa human chromosome are eyclase-inhibitingG-protein (GNAII). alpha/beta preprotachy- conserved on one bovine chromosome (Aleyasin et al, 1997; kinin {TACl), reelin (RELN), c-AMP dependent protein kinase Beever et al.. 1997; Sonstegard et al., 1997). This observed type I! beta regulatory chain (PRICAR2B) and apolipoprotein complexity underscores the difficulty in using human gene Al regulatory protein 1 {TFC0UP2) to BTA 4 and 21 and maps in a comparative positional gene approach. refine the boundaries ofconserved synteny between BTA 4. 21, The accurate definition of boundaries of conserved synteny and 25 with HSA 7. is therefore vital to the use of eomparative gene maps in eattle. The objective of our study is to accurately define the bound- Materials and methods

DNA cxiraciions Bovine genomic DNA and cell line DNA were prepared as described previously (Li et al., 1992). This research was conducted under a ctxiperative agreement between USDA-ARS and the Monfana Agricultural Experiment Station. The U.S. Department of Agri- Primer sequences culture. Agricultural Research Service, Northern Plains Area, is an Equal Oppor- Primers Tor GNAIl. TACl and PRKAR2B were designed from pub- tunity/Affirmative Action employer and all Agency services are available without lished bovine cDNA sequences (Luo ct al.. 1990; Nawa et al., 1983; Nukada discrimination. et al.. 1986) and arc located in the 3' iintranslaled region (3'UTR) ot" the Received 19 October 1998; revision accepted 11 January 1999. respective cDNA. Primer sequences are as follows: GNAIl-F (5'-GCAAAG- Request reprints from Dr. E Antoniou. For! Keogh Livestock and Range TGAACAGCATTCCA-3) and GNAIl-R (5-AAGGAAAAATCCCCA- Research Laboratory. Route I. Box 2021. Miles Cily, MT 59301 (USA); AATG-3 ), TACl-F (5'-AATTrCTCCCCAAAGCACAG-3') and TACl-R telephone: 406-232-8234; fax; 4U6-232-8209; e-mail; erida/larrl.ars.usda.gov. (5'-TGAAACATGCTGCAGGGATA-3 ). PRKAR2B-F (5-TATTGGAGC-

FaK + 4l 61 306 1234 e 1999 S. Kaiser .'\G, Basel Accessible online al: E-mail karger^torger.ch 0301-0171/99/0842-4)121517.50/0 http://BioMedNct.com/karger www, karger. com AAGACCTGTAGTGA-3'). and PRKAR2B-R (5'-AAACCTGAAATC- Correlation analysis {Chevalet and Corpet, 1986) assigned ACAGAA.'\ATGC-3'). The PCR product sizes are 217 bp for GNAI 1.220 bp RELN, GNAI1. TAC 1, and PRKAR2B to eattle chromosome 4 for TACl and 10(1 bp for PRKAR2B. Primers for RELN and TFC01IP2 were designed from the consensus coding sequences computed (Hi^iiis et while TFC0UP2 is assigned to cattle chromosome 21 (Ta- al., 1992) using RELN human (DeSilva et al., 1997) and mouse (D'Arcangelo ble 1). The amplification profiles in the somatic cell hybrid ctal.. 1995) or TFCOUP2 human (Ladias etal.. 1991). mouse {Jonk et al.. panel of the three referenee markers for chromosome 4 1994). rat {unpublished, GenBank accession number .•\F003944). and chick- (MAF50. BM6458, RM188) used to compute the correlation en (Lutz et al.. 1994) cDNA sequences. Primer sequences are as follows: scores are different. This may be explained by chromosome RELN-F (5'-TCTnTTCCTGTGCACCCAC-3 ) and RELN-R (5-CTG- GTTACCAAACTGGGTGTC-3), TFC0UP2-F (ACCTCAGATGCC- breaks that resulted in the retention of one reference marker in TGTGGTC) and TFC0UP2-R (CCT.\CCAAACGGACGAAAAA). After a given cell line while another marker was not retained. This sequencing of the PCR fragments, new primers specific to the bovine variation resulted in different correlation scores between the sequences of RELN and TFC0UP2 were designed and used subsequently to genes reported in this manuscript and MAF50, BM6458. and assign these genes. Their sequences arc as follow; RELNBOV-F (5'-GAG- ATGGCATCCCAGACATT-3'), RELNBOV-R (5'-AAGACGAGAGCC- RM188. Based on the higher correlation between PRKAR2B TGGAACTG-3'), TFC0UP2B0V-F (5'-CCTCAGATGCCTGTGGTC- and MAF50 (0.916) or BM6458 (0.906) compared to the corre- TC-3-), and TFCOUP2BOV.R (5'-CAAATCGTGTTGGrrGGTTG-3). lation between PRKAR2B and RM 188 (0.590), it is reasonable The PCR product sizes are 117 bp for TFC0UP2 and 67 bp for RELN. to predict that PRKAR2B is positioned closer to MAF50 (47.4 cM) and BM6458 (68.3 cM) than to RM188 (24.7 cM). The PCR amplifications correlation value between RELN and MAF50 (1.00) is higher The polymerase chain reactions were performed using 50 ng of bovine. rodent or hybrid cell genomic DNA in a bufTer containing 10 mM Tris-HCI than between RELN and BM6558 (0.829) or RM 188 (0.645). pH 8.3. 50 mM KCl. 0.001% gelatin (iv/v). 1.5 mM MgCl;, 0.5 unit oiTaq Therefore, it is plausible to expect RELN to be closer to MAF piiiymerase {Promega. Madisitn. Wis.). 300 f)M dNTPs. and 2 fiM of forward 50. TFC0UP2 has only a correlation value of 0.809 with the and reverse primers. A touchdown PCR protocol was used: 2 min at 94''C. BTA21 marker ETH131 (32.3 cM) when compared with a cor- then 20 cycles for 30 s at 94°C, 30 s at 65''C and decreasing 0.5°C every relation of 1.00 with CSSM 18(81.5 cM) and consequently can eycie, 30 sat 72''C, followed by 20 cycles for 30 sat 94''C, 30 sat 55''C, 30 s at 72''C. A final extension step at 72''C for 10 min was performed. The be expected to be closer to CSSM 18. reaction products were electrophorcsed on 3 % agarose geis. Databases were searched for the location on HSA 7 of genes previously mapped to bovine chromosomes. Our results, com- Cloning and sequencing PCR fragments were cloned in the plasmid pCR 2.! using the TA cloning bined with the already pubiished data, allow us to refine the kit {Invitrogen) and sequeneed using the Sequeiiase DNA sequencing kit (Amersham/USB).

Assignmenl of genes to bovine chromosome PCR amplifications were conducted on 31 cell lines from a bovine- Table 1. Correlation analysis of HSA 7 genes rodent somatic cell hybrid panel previously described (Womack and Moll, with bovine chronuwomes 1986). The rodent DNA that is the somatic cell background did not yield a Chromosome TAC 1' RELN TFC0tF2 fragment with any of the primer pairs. Only one fragmeni wa.s amplified from [he bovine DNA for all primer pairs used. For testing synteny. ihe cor- 1 0.408 0.4O8 0.1S4 relation coefficients proposed by Chevalet and Corpet (1986) were calcu- 2 0.446 0.487 0.247 lated. When the number of cell lines is 30. the correlation coefficient must be 3 0.022 0.O43 0.008 higher than 0.87 to declare synteny, for an error level of 0.003. The power of 4 0.9t6 1.000 0.508 this test is 0.79 (Chevalet and Corpet, 1986). 5 0.067 0.000 0.075 6 0.595 0.615 0.464 7 0.000 0.000 0.000 8 0.079 0.302 0.242 9 0.323 0.395 0.389 10 0.000 0.057 0.100 Results and discussion 11 0.046 0.087 0.223 12 0.244 0.333 0.057 13 0.078 0.200 0.098 PCR fragments were sequenced to verify the specificity of 14 0.323 0.395 0.508 the reactions. The RELN PCR fragment translated sequence is 15 0.279 0.373 0.000 100% identical to the human protein. TACl. PRKAR2B and 16 0.398 0.480 0.309 17 0.204 0.288 0.344 GNAII PCR fragment sequences are 100% identical to the 18 0.077 0.034 0.058 bovine cDNA 3' UTR sequences. The bovine TFC0UP2 PCR 19 0.239 0.129 0.221 fragment translated sequence is 97% identical to the human 20 0.321 0.271 0.383 21 0.832 0.7t3 1.000 apolipoprotein Al regulatory protein 1 (TFCOUP2). Since 22 0.406 0.333 0.318 human TFC0UP2 protein is 85% identical to the human coup 23 0.204 0.257 0.438 transeription factor I (Genbank accession number PI0589), 24 0.071 0.167 0.380 25 0.000 0.000 0.181 there was a possibility that the bovine PCR fragment repre- 26 0.000 0.099 0.048 sented a homolog of human coup transcription faetor I. How- 27 0.000 0.057 0.423 ever, the bovine PCR fragment translated sequence is only 89 % 28 O.t64 0.223 0.167 29 0.000 0.000 0.042 identical with the human coup transcription factor I protein X 0.046 0.137 0.000 and therefore the PCR fragment corresponds most likely to the bovine apolipoprotein Al regulator)' protein I gene ' GNAIl and l'RKAR2B display the same (TFC0UP2). corretalicHi values as TACl.

12: Cyiogenet Cell Genet 84:121 -124 (1999) boundaries of the conserved segments of HSA 7 {Fig. 1, Ta- BTA 4 HSA 7 q arm BTA 25 ble 2). The most centromeric fragment contains the malate dehydrogenase (MDH2) and elastin (ELN) genes and ends between ELN and GNAIl (4-14 cM interval, transcript map; http://www.ncbi.nlm.nih.gov/SCIENCE96/). The next con- served segment starts between TACl and the erythropoietin gene (EPO) (2-4 cM interval. YAC map; http://www.nhgri.nih. gov/DIR/GTB/CHR7/) and ends between the plasminogen ac- tivator inhibitor 1 (PAIl) gene and TFCOUP2(1 cM interval, transcript map). Interestingly. EPO and PAI1 map on HSA 7 to the same 1 cM interval (Table 2) while they are separated by at least 7 cM on BTA 25 (http://spinal.tag.csiro.au). Consequent- ly, there eould be genes on BTA 25 located between EPO and PAI I that do not map between these two genes on HSA 7. BTA 22 Alternatively, the larger genetic distance in eattle could also be due to a recombination hot spot between the two genes. The following segment is conserved on BTA 21 and to our knowl- edge, it is the first time a conservation between HSA 7 and BTA 21 is described. This region is small (1-2 cM) and therefore would not be detected by ZOO-FISH experiments (Hayes, 1995). The telomeric boundary of this fragment is between the TFC0UP2 and RELN (1-7 cM interval, transcript map). The most telomeric homology fragment to BTA 4 also contains Fig. 1. Conserved synteny between human chromosome 7 (HSA 7) and PRKAR2B and extends at least up to the cystic fibrosis trans- bovine chromosomes 4 (BTA 4). 21 (BTA 2!), and 25 (BTA 25). The genes membrane conductance regulator gene (CFTR; Wallis et al.. mapped in this study are in bold type. 1994).

Table 2. Comparafive localization of genes in human and bovine ordered by relative position on HS.A 7, Breakpoints in conserved synteny are identified by a line

Bovine Gene symbol Gene name Human location

chromosome FISH NLH transcript NHGRI YAC Closest polymorpbie map' Contig'' markers in NHGRI YAC contig"

25* MDH2 Malate debydrogenase 7qll-Hi22' 84-90 cM NA NA 25' ELN EJastin 7qll.2' 84-90 cM NA NA

4' GNAIl Alpha 1 subunii of adenylate NA 94-98 cM NA NA cyclase-inhibiting G-prolein 4' TACl Alph-beta preprotachykinin 7q21-M|22'' 109-111 cM J 125-126 cM

25" EPO Erythrqjoieiin 7q22' 111-112 cM K 128-129 cM 25" PAll Plasminogen aaivator inhibitor 1 7q22' lll-I12cM I. 129 cM

H' TFCOUP2 Apolip(^rotein Al regulatory NA 112-113cM NA NA protein I

4' RELN Reelin NA 114-119 cM N 131-132cM 4' PRKAR2B c-AMP dependant protein kinase NA 119-t20eM N 138-139 cM type 11 beta regulatory cbain 4' CFTR CysiJc fibrosis transmembrane 7q31' 125-126 cM 0 144 cM conductance regulat(»-

Http: //bos. c vm. tam u.edu/htmls/Bov25.html Http: //www .ncbi.nlm.nih.g ov/Entrez/Gen ome/org Ji tm I Mttp://www.ncbi nlm.nih.gov/SCIENCE96/ Http7/www.nb gri.nih.gov/DrR/GTB/CHR7/ Into'val is defined by the closest AFM markers centromene and telocnerjc of tbe gene. This study.

Cytogenet Cell Genet 84:121-124 (1999) 123 The identification of the above listed boundaries through til a more complete comparative map is established, use of the the mapping of HSA 7 genes does not preclude the existence of comparative candidate gene approach to quantitative traits loci additional regions ofconserved synteny with BTA 4, 21. 25 or (QTL) identification will be severely limited. other bovine chromosomes. Just as this work identifies a novel conserved fragment between HSA 7 and BTA 21, the addition of bovine genes to physical and genetic maps will undoubtedly Acknowledgments define more, previously unknown regions of eonser\'ation. Un- We thank Jan Johnson for assistance in preparing the cell line DNAs.

References

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