5 Zijlstra C. et al. (1996) Mamm Genome 7, 280±4.

6 Chevalet C. et al. (1997) Comp Appl Biosci 13, 69±73.

7 Chevalet C. & Corpet F. (1986) Cytogenet Cell Genet 43,

132±9.

8 Toldra F. & Etherington D. (1988) Meat Sci 23, 1±7.

9 Wang X. et al. (1987) Cytogenet Cell Genet 46, 710±1.

10 Rettenberger G. et al. (1995) Genomics 26, 372±8.

11 FroÈnicke L. et al. (1996) Mamm Genome 7, 285±90.

12 Goureau A. et al. (2000) Mamm Genome 11, 796±9.

13 Chauhan S.S. et al. (1993) J Biol Chem 268, 1039±45.

14 Itoh R. et al. (1999) DNA Res 6, 137±40.

15 Santamaria I. et al. (1998) Cancer Res 58, 1624±30.

16 BroÈmme D. et al. (1999) Biochemistry 38, 2377±85.

17 Adachi W. et al. (1998) Invest Ophthalmol Vis Sci 39,

1789±96.

18 Goureau A. et al. (1996) Genomics 36, 252±62.

19 Jùrgensen C.B. et al. (1997) Mamm Genome 8, 423±7.

20 Winterù A.K. et al. (1998) Mamm Genome 9, 366±72.

21 UniGene (2001) National Center for Biotechnology Infor-

mation, Bethesda, USA, World Wide Web (URL: http://

www.ncbi.nlm.nih.gov/UniGene/).

Correspondence: L. Fontanesi (e-mail: luca.fontanesi@stpa.unibo.it)

FISH, RH and linkage assignment of the porcineABCD3 (PXMP1) gene to the distal end ofchromosome 4q

A. Stratil*, S. KubõÂcÏkova ², L. J. Peelman³,G. Reiner§, P. Musilova ², M. Van Poucke³, J. Rubesϲ

and H. Geldermann§

*Institute of Animal Physiology and Genetics, Academy of Sciences

of the Czech Republic, LibeÏ chov, Czech Republic. ²Veterinary

Research Institute, Hudcova 70, Brno, Czech Republic. ³Ghent

University, Faculty of Veterinary Medicine, Department of Animal

Nutrition, Genetics, Breeding and Ethology, Merelbeke, Belgium.§Institute of Animal Husbandry and Breeding,

Department of Animal Breeding and Biotechnology, University

of Hohenheim, Garbenstrasse 17, Stuttgart, Germany

Accepted 13 May 2001

Source/description: The 70 kDa peroxisomal membrane protein

(PXMP1; PMP70) is a member of a family of half-ATP-binding

cassette (ABC) transporter proteins located in the human per-

oxisomal membrane1. In human, the gene (symbol ABCD3 ±

for ATP-binding cassette, subfamily D, member 3; or PXMP1) is

localized on chromosome 1p22-p212. To approach the porcine

ABCD3 gene we used PCR with primers (Pair 1) designed from

the human sequence of exons 13 and 141 (EMBL accession

numbers X83479 and X83480). The ampli®ed porcine frag-

ment was sequenced and from the sequence new primers were

designed (Pair 2).

Primer sequences: Pair 1: Forward: 5¢-CTG GGC GTG AAA

TGA CTA GAT-3¢Reverse: 5¢-AGA CCA TTG TGC GCT CAT ATT-3¢

Pair 2: Forward: 5¢-GTA AGC TGG GAA ACG TTG AAT C-3¢Reverse: 5¢-GTC AGA CAG CAG GCC TCT AAC-3¢

PCR conditions/cloning/sequencing: The PCR, using Pair 1

primers, was performed in 25 ll reactions containing 100 ng

genomic DNA, reaction buffer, 2á0 mM MgCl2, 2% DMSO,

200 lM each dNTP, 10 pmol each primer and 1 U LA poly-

merase (Top Bio, Prague, Czech Republic). Ampli®cation con-

ditions were 2 min at 95 °C, followed by 30 cycles of 55 °C

(45 s), 68 °C (2 min) and 94 °C (45 s), with ®nal extension at

68 °C (7 min). A single fragment (�2 kb) was observed on

agarose gel electrophoresis. The fragment was cloned and

subcloned (plasmid pUC18; Escherichia coli DH5a) and

sequenced (ALFexpress Sequencing System, Pharmacia Bio-

tech, Uppsala, Sweden). The sequence was homologous to parts

of exons 13 and 14 (EMBL X83479 and X83480) (94% iden-

tity) and the intervening intron of the human ABCD3 gene. The

porcine sequence, without the primer sequences, has been

deposited in the EMBL database under accession number

AJ309827. It is of interest to note that SINE (PRE-1) is present

within the intron ¯anked by sequences of direct repeats

(GAGGAGTCTCCTTTTCTT).

For the PCR with Pair 2 primers, Taq polymerase was used

and concentration of MgCl2 was 1á5 mM. After initial

denaturation at 95 °C followed 30 cycles (55 °C ± 45 s, 72 °C ±

1 min, 94 °C ± 45 s), with ®nal extension at 72 °C (7 min).

A single�950 bp fragment was ampli®ed, the sequence of which

agreed with the corresponding sequence of the larger fragment.

Polymorphism/Mendelian inheritance/allele frequencies: In the

�2 kb PCR fragment (Pair 1 primers) polymorphism was

revealed after restriction with HpaII and RsaI. Using HpaII, two

polymorphic loci were detected: HpaII-1 and HpaII-2; with RsaI

one locus was found (Fig. 1). The electrophoretic pattern, after

restriction with HpaII, for homozygotes AA in both HpaII loci

Figure 1 Agarose gel (2%) showing genotypes in the porcine ABCD3

gene after restriction of the �2 kb PCR fragment with HpaII (locus

HpaII-1: diagnostic fragments 881 and �820 bp; locus HpaII-2:

diagnostic fragments 473 and 386 bp) and RsaI. The genotypes (AA,

AB, BB) are given at the top of each lane. PCR, undigested PCR

fragment; M, 1000-100 bp marker.

323Brief notes

Ó 2001 International Society for Animal Genetics, Animal Genetics, 32, 316±331

(i.e. HpaII-1 and HpaII-2), had the following fragments (sizes

determined according to the sequence plus the sequences of the

primers): 881, 473, 257, 198, 74, 67 and 25 bp. In allele

HpaII-1 B the 881 bp fragment was cut to �800 + 80 bp

fragments (base transition was not identi®ed). In allele HpaII-2

B the 473 bp fragment was cut to 386 + 87 bp fragments

(transition A G in position 1048 of the EMBL AJ309827

sequence).

After restriction with RsaI two alleles were observed: A ±

fragments 799, 656, 290 and 230 bp (sizes according to the

sequence plus primers); in allele B the 799 bp fragment was cut

to 573 + 226 bp (transition A G in position 1090 of the EMBL

AJ309827 sequence). All polymorphic sites studied are within

an intron.

Polymorphism at the HpaII-2 locus could easily be tested also

with Pair 2 primers (allele A ± fragments 616, 271 and 74 bp;

in allele B the 271 bp fragment was cut to 184 + 87 bp). The

RsaI polymorphism was also present in the shorter PCR frag-

ment, but as there was some overlapping of zones in homozy-

gote BB and heterozygote AB, the genotypes could not be

differentiated reliably.

Codominant inheritance in all three studied loci was con-

®rmed in the Hohenheim Meishan ´ Pietrain pedigree3. Allele

frequencies in different breeds of pigs are presented in Table 1.

As alleles of HpaII-2 and RsaI loci co-segregated without

exception (A with A, and B with B) only frequencies for HpaII-1

and HpaII-2 are given.

By linkage analysis, using the CRI-MAP software package4,

in the Hohenheim pedigree Meishan ´ Pietrain, the ABCD3

gene was localized to a chromosome 4 linkage group5. The

results are as follows (distances are in Kosambi cM; sex aver-

age):

MYC ± 31.3 ± V-ATPase ± 6.3 ± ATP1B1±5.4 ± ATP1A2±2.9

± SDHC ± 6.4 ± PKLR ± 7.9 ± EAL ± 4.4 ± AMPD1±0.0 ±

NGFB ± 4.0 ± TSHB ± 38.9 ± ABCD3.

Radiation hybrid mapping: This was performed with the use of

the INRA-University of Minnesota porcine Radiation Hybrid

panel (IMpRH)6,7. PCR with Pair 2 primers was used to screen a

panel of 118 hybrid clones. Further details are given in an

earlier paper8. In Fig. 2 a clone from chromosome 4 is shown to

which ABCD3 was mapped. The most signi®cantly linked

marker (2pt analysis) is S0161 (14 cR; LOD � 18á67).

Physical mapping: Recombinant plasmid with the �2 kb ABCD3

insert was labelled with DIG-11-dUTP (Roche Diagnostics,

Mannheim, Germany) using nick-translation and used for

standard Fluorescence in situ hybridization (FISH)9. Immun-

odetection was performed with the use of ¯uorescent antibody

enhancer set for DIG detection (Roche Diagnostics, Mannheim,

Germany). The porcine chromosomes were counterstained with

propidium iodide and DAPI (Sigma, St Louis, MO, USA). The

G-like pattern generated by DAPI staining was used for chro-

mosome identi®cation and for regional assignment. Most

precise assignment of ABCD3 was on chromosome 4q24-q25

(Fig. 3). Background signals, i.e. sites with >2 signals were not

observed. There is good agreement between physical assign-

ment, radiation hybrid mapping and linkage analysis of the

porcine ABCD3 gene.

Comments: Human ABCD3 is localized on chromosome 1p22-

p212 and another gene, UOX, on 1p2210. Porcine UOX is

localized on chromosome 6q26-q3211. Assignment of porcine

ABCD3 to chromosome 4q24-q25 extends a synteny group

conserved on porcine chromosome 4 and human chromosome

1. These results indicate that the evolutionary chromosomal

breakpoint is located between ABCD3 and UOX.

Table 1 Allele frequencies at loci HpaII-1 and HpaII-2 of the ABCD3

gene in different breeds of pigs.

HpaII-1 HpaII-2

Breed n A B A B

Large White 14 0á89 0á11 0á11 0á89

Landrace 12 0á63 0á37 0á42 0á58

Czech Meat Pig 15 0á87 0á13 0á27 0á73

Pietrain 20 0á97 0á03 0á57 0á43

Black Pied Prestice 7 1á00 0á00 0á21 0á79

Hampshire 6 1á00 0á00 0á17 0á83

Duroc 15 1á00 0á00 0á03 0á97

Meishan 9 1á00 0á00 0á67 0á33

Figure 2 Radiation hybrid mapping of the porcine ABCD3 to a

chromosome 4 clone. Distances are in cR.

Figure 3 Fluorescence in situ hybridization (FISH) with the porcine

ABCD3 plasmid clone to a porcine metaphase. One double and one

single signals are shown (indicated by arrows) on chromosome

4q24-q25.

324 Brief notes

Ó 2001 International Society for Animal Genetics, Animal Genetics, 32, 316±331

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,

MO.

5 BlazÏkova P. et al. (2000) Anim Genet 31, 416±8.

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: stratil@iapg.cas.cz)

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