mapping of the porcine agouti-related protein (agrp) gene to chromosome 6
TRANSCRIPT
Acknowledgements: We gratefully acknowledge provision of the
radiation hybrid panel by Drs Martine Yerle and Denis Milan
(INRA, Castanet-Tolosan, France). We would like to thank
Marie Datlova and Ing. Gabriela PursÏova for excellent technical
assistance. This work was supported by Grant Agency of the
Czech Republic (Grant no. 523/00/0669) and Grant Agency of
the Ministry of Agriculture of the Czech Republic (MZE-M03-
99-1).
References1 GaÈrtner J. et al. (1998) Genomics 48, 203±8.
2 GaÈrtner J. et al. (1993) Genomics 15, 412±4.
3 Geldermann H. et al. (1996) J Anim Breed Genet 113,
381±7.
4 Green P. et al. (1990) Documentation for CRI-MAP, Version
2.4. Washington University, School of Medicine, St Louis,
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6 Yerle M. et al. (1998) Cytogenet Cell Genet 82, 182±8.
7 Hawken R.J. et al. (1999) Mamm Genome 10, 824±30.
8 Stratil A. et al. (2001) Anim Genet 32, 110±2.
9 Trask B.J. (1991) Method Cell Biol 35, 3±35.
10 Yeldandi A.V. et al. (1992) Cytogenet Cell Genet 61, 121±2.
11 Rettenberger G. et al. (1996) Chromosome Res 4, 147±50.
Correspondence: A. Stratil (e-mail: [email protected])
Mapping of the porcine agouti-relatedprotein (AGRP ) gene to chromosome 6
K. S. Kim and M. F. Rothschild
Department of Animal Science, Iowa State University, 2255 Kildee
Hall, Ames, IA 50011, USA
Accepted 13 May 2001
Source/description: Agouti-related protein (AGRP) is a neuro-
peptide that mediates the orexigenic and metabolic effects of
leptin signalling via binding to and inhibiting of central mela-
nocortin receptors1. Primers were designed in the regions of
sequence conserved among the human, mouse and bovine
AGRP genes (GenBank accession nos. U89485, U89486 and
AJ002025, respectively). The sequence of the porcine poly-
merase chain reaction (PCR) products was identi®ed as the
porcine AGRP gene spanning exons 1 and 3 and showed 91 and
87% exonic identity to the corresponding human and bovine
AGRP sequences, respectively. Using this sequence (GenBank
accession no. AF220543), pig speci®c primers were designed.
Sequence analysis of the PCR products from several individual
pigs of different breeds detected an intronic nucleotide substi-
tution situated in a DrdI restriction enzyme recognition site.
Primer sequences: Primers derived from other species sequences
(800 bp)
Forward primer: 5¢-GAA GGG CAT C(A/G)G AAG GCC TG-3¢.
Reverse primer: 5¢-TAC CCA GCT TGC GGC AGT AG-3¢.The porcine-speci®c primers (600 bp)
Forward primer: 5¢-GTG GTT CTG CCC TCA CAT CAT C-3¢.Reverse primer: 5¢-CAT GGT ACC TGG TGT CCC AGA C-3¢.
PCR conditions: Both PCR reaction were performed using
12.5 ng of porcine genomic DNA, 1x PCR buffer, 1.5 mM MgCl2,
0.125 mM dNTP, 0.3 lM of each primer, and 0.35 U Taq DNA
polymerase (Promega, Madison, WI, USA) in a 10-ll ®nal
volume. The PCR pro®le included 2 min at 94 °C; 35 cycles of
30 s at 94 °C, 1 min at 56 °C, 1 min 30 s at 72 °C; and a ®nal
15 min extension at 72 °C in a Robocycler (Stratagene, La Jolla,
CA, USA).
Polymorphisms: The DrdI digestion of the 600 bp PCR product
produced allelic fragments of 600 bp (allele 1), or 420 and
180 bp (allele 2) and this restriction fragment length poly-
morphism (RFLP) analysis was used to genotype animals from
PiGMaP reference families and the Iowa State University herd.
Mendelian inheritance/allele frequencies: Mendelian segregation
of DrdI PCR-RFLP was observed in 4 three-generation PiGMaP
families2. Genotyping of 54 unrelated animals from several
breeds in the Iowa State University herd determined allele
frequencies of the polymorphism. Allele 1 was observed with
a frequency of 1 in Hampshire (n � 9), Duroc (n � 9) and
Chester White (n � 9), 0.83 and 0.88 in Large White (n � 9)
and Landrace (n � 8), respectively, but was not observed in
Meishan (n � 10) (Fig. 1).
Chromosomal location/linkage: The AGRP was assigned to
chromosome 6 (P � 1.00) and the (1/2)p12-(1/2)p14 region
(P � 0.81) by PCR analysis of a pig-rodent somatic cell hybrid
panel3. Two-point and multipoint linkage analyses were per-
formed using CRIMAP 2.4 against other genotypes in the
PiGMaP Linkage database (http://www.resSpecies.org). Most
signi®cant linkages between AGRP and PiGMaP markers were
obtained from microsatellite S0087 (recombination frac-
tion � 0.00 and LOD � 3.01) and S0297 (recombination
fraction � 0.00 and LOD � 3.31) on chromosome 6.
Figure 1 DrdI PCR-restriction fragment length polymorphism (RFLP) of
the porcine AGRP gene. Lane 1 is molecular marker, lane 2, the
heterozygote; lane 3 and 5, allele 1 homozygotes; lane 4 and 6, allele 2
homozygotes. The arrows indicate each allele.
325Brief notes
Ó 2001 International Society for Animal Genetics, Animal Genetics, 32, 316±331
Comments: As the human AGRP maps to HSA16, which is
known to share homology with pig chromosome 6p, the
assignment of the AGRP to porcine chromosome 6 is in
accordance with the previous results obtained by chromosome
painting between human and pig4.
Acknowledgements: This work is supported by PIC International
Group and the Iowa Agriculture and Home Economics Experi-
mental Station, Ames, paper no. J-19015, project no. 3600,
as well as by Hatch Act and State of Iowa funds. Support by the
EC for the PiGMaP DNA and bioinformatics support by
A. Archibald and associates of the Roslin Institute is greatly
appreciated.
References1 Shutter J.R. et al. (1997) Genes Develop 11, 593±602.
2 Archibald A. et al. (1995) Mamm Genome 6, 157±75.
3 Yerle M. et al. (1996) Cytogenet Cell Genet 73, 194±202.
4 Goureau A. et al. (1996) Genomics 36, 252±62.
Correspondence: M.F. Rothschild (e-mail: [email protected])
FISH assignment of two equine BAC clonescontaining SRY and ZFY
K. Hirota*, F. Piumi², F. Sato³, N. Ishida³,G. Gue rin§, N. Miura* and T. Hasegawa³
*Department of Molecular Genetics, Laboratory of Racing
Chemistry, 1731-2 Tsuruta-Cho, Utsunomiya 320-0851, Japan.²INRA, Centre de Recherche de Jouy, Laboratoire mixte INRA-CEA
de Radiobiologie et d'Etude du GeÂnome, 78352 Jouy-en-Josas
Cedex, France. ³Equine Research Institute, Japan Racing Associ-
ation, 321-4 Tokami-Cho, Utsunomiya 320-0856, Japan. §INRA,
Centre de Recherche de Jouy, Laboratoire de GeÂne tique biochi-
mique et de Cytoge ne tique, 78352 Jouy-en-Josas Cedex, France
Accepted 19 May 2001
Source/description: The INRA equine BAC library1 (G. GueÂrin
et al., unpublished data) was screened with two pairs of primers
derived from previously reported Y-chromosome linked genes
for sex-determining protein (SRY; GenBank AB004572)2 and
zinc ®nger protein (ZFY; GenBank AF133198)3. These two
clones, 616B11(SRY) and 397C2(ZFY) were mapped by
f luorescence in situ hybridization (FISH).
PCR conditions for BAC screening at INRA: Thirty seconds at
94 °C, 30 s at 60 °C and 30 s at 72 °C, 35 cycles.
PCR Mix ®nal concentrations: MgCl2: 1.5 mM, dNTPs: 200 lM
each, primers: 1 lM, Taq: 0.035 U.
Primer sequence: SRY forward primer, ESRYF002: 5¢-CTTAA-
GCTTCTGCTATGTCCAGAGTATCC-3¢.
SRY reverse primer, ESRYR480:
5¢-GCGGTTTGTCACTTTTCTGTGGCATCTT-3¢.Ampli®ed fragment size: 429 bp2.
ZFY forward primer, P1-5EZ:
5¢-ATAATCACATGGAGAGCCACAAGCT-3¢.ZFY reverse primer, P2-3EZ: 5¢-GCACTTCTTTGGTATCTGA-
GAAAGT-3¢.Ampli®ed fragment size: 450 bp3.
FISH assignment: Each BAC clone was labelled with biotin by
nick-translation. The BrdU incorporated metaphases were
prepared by a whole blood culture as previously reported4,
and were R-banded by UV-irradiation followed by Hoechst
33258 staining as described by Takahashi et al.5 The FISH
procedure was performed by a common method for suppres-
sion in situ hybridization and the detection using FITC-avidin.
The images of the observation were photographed using the
positive ®lms. The equine SRY gene was assigned to the distal
region of Yq13, adjacent to Yq14 and the equine ZFY gene
was assigned to the terminal band, Yq15. No obvious signals
could be observed on X and other chromosomes in both FISH
(Fig. 1).
Comments: This is the ®rst report indicating a physical assign-
ment to the equine Y-chromosome. Lack of SRY is thought to
be a primary genetic basis of mares with XY-gonadal dysgen-
esis, called XY-mares2. In the diagnostic procedure, failure in
PCR ampli®cation of SRY is a strong evidence of certain alter-
ation of this gene with the successful ampli®cation of other sex-
linked genes, such as ZFX/ZFY. Although, Shiue et al. reported
the gene order among SRY, ZFY and STS-Y by analysis using
somatic cell hybrids6, the distances between any two genes
were still unknown. In cattle, ZFY was assigned to the short
arm (Yp13) of Y-chromosome7 in contrast to SRY on the long
arm (Yq11)8. It is interesting that the equine SRY and ZFY
localize to the different bands on the same arm of Y-chromo-
some. This observation is consistent with humans, where both
genes map to the same band (Yp11.3), but certain differences
with the ruminants existed. These two assigned loci should be
important landmarks for linkage and other analysis on the
equine Y-chromosome.
References1 Godard S. et al. (1998) Mamm Genome 9, 633±7.
2 Hasegawa T. et al. (2000) J Vet Med Sci 62, 1109±10.
3 Senese C. et al. (1999) Anim Genet 30, 390±1.
4 Hirota K. et al. (2001) Anim Genet, in press.
5 Takahashi E. et al. (1991) Human Genet 88, 119±21.
6 Shiue Y.L. et al. (1999) Chromosome Res 8, 45±55.
7 Xiao C. et al. (1998) Mamm Genome 9, 125±30.
8 Cui X. et al. (1995) Anim Sci Technol 66, 441±4.
Correspondence: K. Hirota (e-mail: [email protected])
326 Brief notes
Ó 2001 International Society for Animal Genetics, Animal Genetics, 32, 316±331