Mutation in the Sp1 motif of the bovine leptin gene affects
Department of Genetics and Animal Breeding, Agricultural University of Poznan, Poznan, Poland
Department of Molecular Biology, Institute of Genetics and Animal Breeding, Jastrze˛biec, 05-552 Wo
lka Kosowska, Poland
Received: 12 May 2005 / Accepted: 26 August 2005
Leptin is expressed mainly by adipocytes and plays a
crucial role in the regulation of energy expenditure,
food intake, and adiposity. Using PCR-heteroduplex
analysis and sequencing, we investigated a C/G
substitution in the promoter region of the bovine
leptin gene. Application of the electrophoretic
mobility shift assay showed that the C ﬁ G trans-
version decreased the leptin gene promoter binding
capacity for nuclear proteins. With real-time PCR
and Western blotting, we showed that the leptin
expression level was higher in cattle with the CC
than with the GG genotype.
Leptin is involved in the regulation of energy
expenditure, food intake, and adiposity (Flier 1997).
Expression of this gene is regulated by glucocor-
ticoids (Slieker et al. 1996), insulin, b-adrenergic
agonists (de Vos et al. 1995), and fasting (Considine
et al. 1996). It has been shown that the Sp1 tran-
scriptional factor binds to a consensus sequence in
the promoter of leptin gene ()101 to )83), and
mutation in this region abolishes the binding and
reduces promoter activity in the rat (Mason et al.
1998). Fukuda and Iritani (1999) postulated that the
region from )101 to )83 of the rat leptin gene is
responsible for stimulation of transcription by
glucose and insulin. The most important regula-
tion sites such as Sp1, C/EBP, and TATA are lo-
cated in the first 200 bp upstream from the
transcription start site of the bovine leptin gene.
This region is conserved evolutionarily (Liefers et
al. 2005). In cattle the Sp1 transcription factor has
a putative binding site to nucleotides from )114 to
)102 (Taniguchi et al. 2002). Several studies have
shown numerous polymorphisms in the promoter
region of the bovine leptin gene (Konfortov et al.
1999; Pomp et al. 1997; Haegeman et al. 2000;
Lagonigro et al. 2003). Recently, a new polymor-
phism at position )105 was identified (Leifers
2004). The aim of this study was to investigate the
effect of the C/G mutation at position )105 on
leptin gene expression in cattle.
Materials and methods
Animals and DNA isolation. We analyzed 172
young Fresian bulls from 11 paternal half-sib fami-
lies. All the animals were maintained under stan-
dard conditions in the experimental farm Jastrze˛biec
and fed ad libitum a total mixed ration (TMR)
composed of 75% corn silage, 20% concentrates, and
5% hay. All experimental procedures involving ani-
mals were approved by the Local Ethics Commission
(permission No. 67/2001). Approximately 10 ml of
blood was withdrawn from each animal by an
authorized veterinarian into test tubes containing
EDTA. DNA was isolated from blood by the
method of Kanai et al. (1994).
Genotyping. Polymerase chain reaction-hetero-
duplex (PCR-HD) analysis was applied for genotyp-
ing (n = 172) with the use of a Hoefer SE 600
electrophoresis apparatus (Pfizer, New York, NY).
The following PCR primers were used to amplify a
300-bp fragment (nt )272 to +28) of the bovine leptin
(LEP) gene: forward, 5¢-AGGTGTGATTTTCCAG
Correspondence to: Krzysztof Flisikowski; E-mail: krzysztof.
DOI: 10.1007/s00335-005-0068-1 Volume 17, 77À82 (2006) Ó Springer Science+Business Media, Inc. 2006