Isolation, sequencing and relative quantitation by fluorescent-ratio
PCR of feline ␤-lactoglobulin I, II, and III cDNAs
Ramona N. Pena, Armand Sa´nchez, Agustina Coll, Josep M. Folch
Unitat de Gene`tica i Millora, Departament de Patologia i Produccio´ Animals, Facultat de Veterina`ria, Universitat Auto`noma de Barcelona,
08193 Bellaterra, Spain
Received: 10 December 1998 / Accepted: 31 January 1999
Abstract. Beta-lactoglobulin (␤LG) is a whey protein found in
the milk of most mammals, except those of humans and rodents. In
ruminants, only one type of ␤LG is expressed in milk, although the
presence of pseudogenes has been reported. The milk of other
mammals (dog, horse, dolphins) contains two types of ␤LG: type
I is related to ruminant ␤LG, while type II has a strong similarity
to the ruminant pseudogenes. The presence of three types of ␤LG
has been described only in cat milk. For the first time, we have
cloned, sequenced, and characterized the three types of feline ␤LG
cDNAs from a mammary gland sample of a lactating cat. Since no
specific probe could be easily used to differentiate them by North-
ern Blotting, we developed a new technique named Fluorescent-
Ratio PCR (FR-PCR) in order to assess their level of expression
during lactation. The relative amounts of each feline ␤LG cDNA
was quantified in two cats by capillary electrophoresis of the re-
stricted RT-PCR product, labeled with fluorescent primers. We
observed the same percentage of expression of the three ␤LG
genes in two cats. The differences of expression could be due to
changes in the promoter and 3Ј non-coding region affecting the
level of transcription and the mRNA stability. The FR-PCR tech-
nique was shown to be reproducible and accurate even for small
percentage differences, being very useful when a small amount of
sample is available.
Beta-lactoglobulin (␤LG) is the major whey protein in the milk of
ruminants. It is also present in the milk of other mammals, but not
in that of humans or rodents (Hambling et al. 1992). However, in
humans there is a placental protein, PP14, that shares a high level
of homology with ␤LG at the protein structure level and in the
intron-exon distribution. Both proteins are members of the super-
family of hydrophobic molecule transporters or Lipocalins (Jul-
kunen et al. 1988), which also includes the mouse Plasma Retinol-
Binding Protein (RBP) and the rat Major Urinary Protein (MUP).
The Lipocalins family is predominantly composed of low-
molecular-mass proteins that have a strong affinity for hydropho-
bic molecules. A conserved three-dimensional structure, rather
than strong sequence similarity, and similar gene organization are
the defining characters of this family. The physiological function
of ␤LG is unclear, though it is suspected to be involved in retinol
and fatty acids transport in the milk (Flower 1996; Pe´rez and Calvo
In livestock ruminants, ␤LG is encoded by a single gene, al-
though the presence of a ␤LG pseudogene has been described in
the cow (Passey and MacKinlay 1995) and goat (Folch et al.
1996). In the milk of these species, ␤LG exists predominantly as
an 18-kDa dimer. In other species including dogs, dolphins, don-
keys, and horses, two structurally different monomeric forms exist,
called type I and type II ␤LG. These have been sequenced at the
protein level in dogs (Pervaiz 1986; Halliday et al. 1991), dolphins
(Pervaiz and Brew 1986), horses (Godovac-Zimmermann et al.
1985), and donkeys (Godovac-Zimmermann et al. 1990). Type I
and type II ␤LG proteins are highly related, displaying, in each
species, about 80% sequence identity. Moreover, type I ␤LGs have
a high degree of homology with the ruminant ␤LG proteins, while
type IIs are highly homologous with the product that would be
encoded by the ruminant pseudogenes. Many type I ␤LGs have
been sequenced at the DNA level, but only the type II DNA
sequence is known in horse (Masel et al. unpublished).
Furthermore, the presence of three forms of ␤LG protein in
milk (named type I, II, and III) has been described only in cats.
Feline ␤LG proteins exhibit a highly polymorphic pattern, with up
to five different variants identified in each form by protein elec-
trophoresis analysis (Halliday et al. 1993). The amino acid se-
quences of feline ␤LG I and III are related to type I ␤LG proteins,
while feline ␤LG II belongs to type II ␤LG protein group.
This information suggests that, in species with more than one
type of ␤LG protein, there may be more than one active gene, each
one producing a different type of ␤LG. In order to investigate this
further, the cDNAs for the feline ␤LG forms I, II, and III were
synthesized from a lactating cat mammary gland sample. Sequenc-
ing and characterization of the three cDNAs is reported.
In donkey and horse, ␤LG type II protein is a minor component
of the total ␤LG protein observed (Godovac-Zimmerman et al.
1990). However, so far, more precise quantitative studies of both
types of ␤LG have not been reported in any species. The relative
ratio of expression of each ␤LG in cat during lactation was deter-
mined using a new quantification technique named Fluorescent-
Ratio PCR (FR-PCR). This technique uses fluorescent primers and
capillary electrophoresis to quantitate the various cDNA products
amplified by RT-PCR. The results are reproducible and accurate,
demonstrating the reliability of this method even with minor rela-
tive mRNA differences.
Materials and methods
Isolation and cloning of cDNAs.
Total mRNA was isolated from a
sample of mammary gland from a cat at mid-lactation using oligo(dT)/
cellulose chromatography in a micro-spin column (QuickPrep Micro
mRNA Purification Kit, Pharmacia and Upjohn Diagnosys, Kalamazoo,
MI). The first strand of the cDNA was synthesized with the M-MuLV
retrotranscriptase (Ready-to-go You Prime cDNA Kit, Pharmacia and Up-
Johns Diagnosys, Kalamazoo, MI) with a modified oligo(dT) primer that
contained 30 thymidines followed by an adapter sequence of 20 bp with
target sites for several restriction enzymes (5Ј-GGCCACGCGT-
The cDNA was amplified by PCR with a degenerate primer CATE1F1
The nucleotide sequence data reported in this paper have been submitted to
GenBank and have been assigned the accession numbers AF073781,
AF073782, AF073783, AF073784, and AF073785.
Correspondence to: R.N. Pena
Mammalian Genome 10, 560–564 (1999).
© Springer-Verlag New York Inc. 1999