Mammalian Genome 8, Brief Data Reports 227 The leptin receptor (LEPR) gene maps to bovine Chromosome 3q33 M. Pfister-Genskow, 1 H. Hayes, 2 A. Eggen, t'2 M.D. Bishop 1 ~DNA Research & Testing Laboratory, ABS Global, Inc., DeForest, Wisconsin 53532, USA 2Laboratoire de Grnrtique biochimique et de Cytogrnrtique, INRA-CRJ, 78350, Jouy-en-Josas, France Received: I September 1996 / Accepted: 20 October 1996 Species: Bovine Locus name: Leptin receptor Locus symbol: LEPR Map position: Chromosome (Chr) 3q33 Method of mapping: Fluorescence in situ hybridization (FISH), R banding. Database deposit information: GenBank accession number U62385 Molecular reagents: Oligonucleotide primers were designed by Fig. 1. R-banded whole metaphase spread after in situ hybridization with aligning sequences encoding the long forms of the leptin receptor a YAC clone containing the bovine LEPR gene; arrows indicate the spe- intracellular domain of the mouse and human (GenBank accession cific hybridization signal. number U46135 and U43168, respectively). Primer sequences hormone, leptin is expressed in the mouse choroid plexus and were: forward primer 5'-CATGGAAAAATAAAGATGAGATG- resembles a class I cytokine receptor . Several theories have 3'; and reverse primer 5'-CAAAAGCACACCACTCTCTC-3'. been proposed in which the binding of leptin to its receptor may PCR amplification was conducted in a reaction volume of 25 txl act to control appetite, fuel storage, and energy expenditure by containing 30 ng of template DNA, 0.5 p~M each primer, 50 p~M influencing hypothalamic function [1,9,10]. Several alternatively each dNTP, 1.5 mM MgC12, 1 x PCR buffer (10 mM Tris-HC1, pH spliced transcripts of LEPR were identified in mouse, resulting in 8.3~ 50 mM KC1) and 0.5 units AmpliTaq DNA Polymerase (Perkin the severely obese phenotype found in diabetic (db/db) mice . A Elmer). Samples were overlaid with 50 Ixl of mineral oil. Cycling missense mutation within the rat LEPR gene causes the fatty (fa/fa) conditions were 3 min at 94~ (initial denaturation), followed by phenotype [ 11 ]. As the first step toward the molecular understand- 30 cycles of 1 min at 94~ 1 min at 56~ and 1 rain at 72~ and ing of the regulatory genes involved with feed intake and feed a final extension of 4 min at 72~ An expected 496-bp PCR efficiency in cattle, we report the mapping of the bovine LEPR product was observed electrophoretically with human, mouse, and gene to Chr 3q33. The physical mapping of the bovine LEPR gene bovine genomic DNA. The bovine 496-bp PCR fragment was to Chr 3q33 is consistent with ZOO-FISH data . Recent dis- cloned and sequenced (Promega; GenBank accession number coveries indicate that mutations or defects in the leptin receptor U62385). Comparison of the bovine PCR product sequence with may result in obesity. Understanding the role of the circulating human and mouse LEPR DNA sequences [1,2] indicated 81% and hormone leptin and its receptor in energy regulation in ruminants 75% sequence identity, respectively. A bovine YAC library pre- could prove useful to animal breeding programs. viously described , containing 21,000 YAC clones, was screened in a two-step PCR approach with the previously men- References tioned primers. In the first step, DNA from 28 superpools (pool of 1. Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, 768 YAC clones corresponding to 8 microtiter plates) was sub- Richards G J, Campfield LA, Clark Fr, Deeds J, Muir C, Sanker S, jected to amplification in order to identify the positive superpool. Moriarty A, Moore K J, Smutko JS, Mays GG, Woolf EA, Monroe CA, After identifying the positive superpool(s), the location of the posi- Tepper RI (1995) Cell 83, 1263-1271 2. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis S J, Lakey tive YAC clone within the superpool was determined by screening ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, the pools of rows (8), columns (12), and plates (8) for each positive Morgenstern JP (1996) Cell 84, 491-495 superpool by PCR. Five clones were positive for the bovine LEPR 3. Libert F, Lefort A, Okimoto J, Womack J, Georges M (1993) Genom- gene. For FISH mapping, approximately 2 txg of total yeast DNA ics 18, 270-276 of each clone was labeled (BioNick, Boehrmger Mannheim) and 4. Hayes H, Petit E, Lemieux N, Dutrillaux B (1992) Cytogenet Cell applied to a R-banded chromosome spread after denaturation at Genet 61,286-288 100~ for 10 min and prehybridized at 37~ for 30-40 min. In situ 5. Lemieux N, Dutrillaux B, Viegas-Pequignot E (1992) Cytogenet Cell hybridization and detection of hybridization signals were per- Genet 59, 311-312 formed as previously described . Chromosomes were identif- 6. Di Berardino D, Hayes H, Fries R, Long S (eds). (1990) ISCNDA (1989): International System for Cytogenetic Nomenclature of Domes- ied by R-banding according to Lemieux and associates  and tic Animals. Cytogenet Cell Genet 53, 65-79 numbered according to the standard RBG-banded karyotypes of 7. Chung WK, Power-Kehoe L, Chua M, Leibel RL (1996). Genome Res ISCNDA . Specific signals were observed on 30 metaphases for 6, 431-438 LEPR gene, and a each of the five YAC clones possessing the 8. Chua S, Chung WK, Wu-Peng XS, Zhang Y, Liu S, Tartaglia L, Leibel common signal was observed on bovine Chr 3q33 (Fig. 1). RL (1996) Science 271,994-996 Previously identified homologs: The LEPR gene is located on 9. Coleman DL (1982) Diabetes 31, 1~5 mouse Chr 4 , human Chr lp , and rat Chr 5 . 10. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman J Discussion: The receptor for the adipose tissue-derived circulating (1994) Nature 372, 425432 l 1. Phillips MS, Liu Q, Hammond HA, Dugan V, Hey PJ, Caskey CT, Hess JF (1996) Nature Genet 13, 18-19 Correspondence to: M. Pfister-Genskow 12. Hayes H (1995) Cytogenet Cell Genet 71, 168-174
Mammalian Genome – Springer Journals
Published: Mar 23, 2009
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