RESUMEN
The whey acidic protein (WAP) is the major whey protein of rodent, rabbit and camel. Recently, it was identified in the milk of swine (Simpson et al., 1998. J. Mol. Endocrinol. 20, 27-35). In this paper, the cloning of the pig WAP cDNA and of bacterial artificial chromosome (BAC) construct containing the entire porcine WAP gene is reported. The comparison of the coding sequence of the pig WAP gene to rodent or lagomorph WAP sequence already published demonstrated that only exon sequences are partially conserved. The porcine WAP gene was localized on the subtelomeric region of the chromosome 18. The estimation of the expression of the swine WAP gene in the mammary gland from lactating animals revealed a high level of expression. In order to compare the expression level of the porcine WAP gene from the large genomic fragment which contained 70 kb downstream and 50 kb upstream the pig WAP gene or the smaller one (1 kb downstream and 2.4 kb upstream), these two genomic fragments were transfected in HC11 cell line. The BAC construct was expressed 15 times higher than the plasmid when reported to the integrated copy number. This report suggests that the HC11 cell line is a useful tool to identify the regulatory sequences of milk protein genes.
Asunto(s)
Genes/genética , Proteínas de la Leche/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Bandeo Cromosómico , Mapeo Cromosómico , Clonación Molecular , ADN/química , ADN/genética , ADN/aislamiento & purificación , Expresión Génica , Hibridación Fluorescente in Situ , Lactancia , Glándulas Mamarias Animales/metabolismo , Ratones , Datos de Secuencia Molecular , ARN/genética , ARN/metabolismo , Conejos , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Porcinos , Transcripción GenéticaRESUMEN
The transferrin gene is expressed in all mammals, mainly in the liver. A rabbit genomic library was screened using cDNA probes, and 8kb of 5' flanking sequence of the rabbit transferrin gene was cloned upstream of the cat reporter gene. The first 200 nucleotides of this promoter were sequenced. The rabbit transferrin promoter is highly homologous to the human and murine ones. Its functional activity was tested in the human hepatic cell line HepG2. Using transitory transfections in these cells, a proximal positive region, a negative region and a distal positive region located between -3.6 and -4.0kb were identified. This distal positive region sequence is highly conserved with the the human gene enhancer sequence, and contains an HNF3alpha binding site, the mutation of which totally abolished its effect in HepG2 and HuH7 cell lines. The rabbit transferrin 5' flanking sequence thus shows a promoter organization very similar to that of the human gene, and the HNF3alpha binding site in the distal positive region presents the same functional importance in both genes.