Butyrophilin is expressed in mammary epithelial cells from a single-sized messenger RNA as a type I membrane glycoprotein.

We investigated the expression of butyrophilin in eukaryotic cells with a view to determining the number of mRNA species, the incorporation of the peptide chain into microsomes, and the topology of the processed protein in biological membranes. Butyrophilin is synthesized from a single sized mRNA in both bovine and murine lactating mammary tissue and associates with microsomal membranes with a type I topology (Nexo.Ccyto) via a single hydrophobic anchor in the middle of the sequence. Several isoelectric variants of the protein were detected in cellular membranes from lactating bovine mammary tissue and in the milk-fat-globule membrane. We found no evidence for soluble forms of butyrophilin in postmicrosomal supernatants. The 66-kDa protein appears to be subjected to limited proteolysis, giving rise to a 62-kDa fragment lacking the C terminus and to other more minor fragments of lower Mr in the milk-fat-globule membrane. Antipeptide antibodies to epitopes within the N- and C-terminal domains were used to show that butyrophilin retains a type I topology in plasma membranes when expressed in insect cells from a baculovirus vector, and in secreted milk-fat globules. These data do not agree with previous suggestions that butyrophilin may exist in cytoplasmic soluble forms, or be reorganized in the plane of the lipid bilayer during secretion in lipid droplets from mammary cells. The results are discussed with reference to the role butyrophilin may play as the principal scaffold for the assembly of a complex with xanthine oxidase and other proteins that functions in the budding and release of milk-fat globules from the apical surface during lactation.

Structure and sequence of the bovine butyrophilin gene.

The complete sequence of the bovine butyrophilin gene (BTN) is described and compared with the mouse gene (Btn). Both genes contain seven exons separated by six introns, and the organisation of exons is closely associated with structural domains of the protein. Individual exons of BTN and Btn are 68-87% similar in sequence. There are no canonical TATA or CCAAT boxes associated with the transcription initiation sites in the genes of either species. However, a number of potential binding sites for transcription factors were identified in the 5'-flanking DNA, some of which may function in regulating expression of the gene in mammary tissue. Conservation of a 110-bp region in the promoters of BTN and Btn may have some functional significance. Cloning and sequencing of BTN provides an additional mammary-specific gene promoter that may be used for driving the expression of transgenes in the lactating mammary gland, and for determining the basis for tissue-specific gene expression. In addition, the sequence of BTN may be used to map intragenic polymorphisms and identify quantitative trait loci in commercial livestock.

Structural organization and mammary-specific expression of the butyrophilin gene.

Butyrophilin is a glycoprotein of the immunoglobulin superfamily that is secreted in association with the milk-fat-globule membrane from mammary epithelial cells. As a first step towards determining the possible function(s) of this protein in lactation, the mouse butyrophilin gene (Btn) has been cloned from a 129-ES cell genomic library. Over 14 kb of DNA was sequenced, including the entire transcriptional unit of the gene, and 4.6 kb and 1.1 kb of the 5' and 3' flanking region, respectively. In addition, the overall structure of the bovine gene (BTN) was determined by amplification of genomic DNA by the polymerase chain reaction. Both Btn and BTN comprise seven exons and six introns. The signal sequence and two immunoglobulin-like folds of the exoplasmic domain and the membrane anchor are encoded by separate exons, and the cytoplasmic domain is encoded by two short exons and a large terminal exon that also includes 3' untranslated sequence. The butyrophilin gene appears to have evolved from a subset of genes in the immunoglobulin superfamily and genes encoding the B30.2 domain, which is conserved in a family of zinc-finger proteins. Murine butyrophilin mRNA was detected specifically in the mammary gland by RNase protection analysis. Expression increased during the last half of pregnancy and was maximal during lactation. The 5' flanking region of Btn was analyzed for putative regulatory elements and is different from the promoters of other mammary-specific genes. Btn should be useful for determining the mechanisms underlying mammary-specific gene expression and potentially for the production of heterologous proteins in the milk of transgenic animals.

The bovine butyrophilin gene maps to chromosome 23.

Chromosomal assignment of the bovine butyrophilin gene (BTN) was performed by analysis of DNA from somatic hybrid cell lines using the polymerase chain reaction. The gene was assigned to bovine chromosome 23 using two sets of primers specific for bovine BTN.

A continuous culture of pluripotent fetal hepatocytes derived from the 8-day epiblast of the pig.

Continuous cultures of pluripotent parenchymal hepatocytes were derived from the epiblasts of 8-day-old pig blastocysts. The cells were polygonal and had phase-contrast dark, granular cytoplasm with prominent nuclei and nucleoli. These feeder-dependent cell cultures differentiated into large, multicellular, secretory, duct-like structures or formed small canaliculi between individual cells. Alternatively, the cells accumulated droplets that stained intensely with Oil Red O, a lipid-specific stain. Alpha-fetoprotein (AFP), albumin, and beta-fibrinogen mRNAs were expressed as the cells differentiated in culture. Serum-free medium that was conditioned by the cells contained transferrin, AFP, and albumin. The growth and viability of the cells were inhibited by transforming growth factor beta 1 (TGF beta 1) at concentrations > or = 1 ng/ml. The cell cultures grew slowly with doubling times of 2 to 3 d. One of the cultures, pig inner cell mass-19 (PICM-19), was passaged continuously for over 2 yr [> 100 population doublings (PD)] and appears to be an infinitely self-renewing cell population. The stem cell characteristics of the epiblast-derived fetal hepatocytes indicate that the cells may be unique for investigations of liver differentiation and organogenesis.