Isolation, sequencing and relative quantitation by fluorescent-ratio PCR of feline beta-lactoglobulin I, II, and III cDNAs.
Beta-lactoglobulin (betaLG) is a whey protein found in the milk of most mammals, except those of humans and rodents. In ruminants, only one type of betaLG is expressed in milk, although the presence of pseudogenes has been reported. The milk of other mammals (dog, horse, dolphins) contains two types of betaLG: type I is related to ruminant betaLG, while type II has a strong similarity to the ruminant pseudogenes. The presence of three types of betaLG has been described only in cat milk. For the first time, we have cloned, sequenced, and characterized the three types of feline betaLG cDNAs from a mammary gland sample of a lactating cat. Since no specific probe could be easily used to differentiate them by Northern 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 betaLG cDNA was quantified in two cats by capillary electrophoresis of the restricted RT-PCR product, labeled with fluorescent primers. We observed the same percentage of expression of the three betaLG genes in two cats. The differences of expression could be due to changes in the promoter and 3' non-coding region affecting the level of transcription and the mRNA stability. The FR-PCR technique was shown to be reproducible and accurate even for small percentage differences, being very useful when a small amount of sample is available.
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Unitat de Genètica i Millora, Departament de Patologia i Producció Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
Sánchez
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Coll
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Folch
J M
JM
D000818:Animals; D001483:Base Sequence; D002415:Cats; D018076:DNA, Complementary / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods; D005260:Female; D005453:Fluorescence; D005786:Gene Expression Regulation; D007774:Lactation; D007782:Lactoglobulins / Q000235:genetics* / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000502:physiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D020033:Protein Isoforms / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D018076:DNA, Complementary; D007782:Lactoglobulins; D020033:Protein Isoforms