Expression Of Beta-lactoglobulin Gene Research Articles

Hormonal regulation of platypus Beta-lactoglobulin and monotreme lactation protein genes

Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, Beta-lactoglobulin (BLG), a whey protein and monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians.

Polymorphisms of beta-lactoglobulin promoter region in three Sicilian goat breeds

Several beta-lactoglobulin (BLG) polymorphisms have been described within the proximal promoter region and coding region of the caprine gene, although no genetic variants affecting the protein amino acid composition and/or expression level have been characterized so far. Binding sites for several transcription factors (TFs) are present in the BLG promoter region. The aims of this work were to sequence the full-length promoter region of three Sicilian goat breeds in order to identify polymorphisms, analyze the identified haplotypes, search for differences between breeds for the presence of polymorphisms in this gene region, search for putative TFs binding sites, and check if polymorphisms lay within the identified TFs binding sites. The promoter region of BLG gene in Sicilian goat breeds showed high level of polymorphism due to the presence of 36 single nucleotide polymorphisms (SNPs). Association between polymorphic sites was computed within the whole sample analyzed and 18 haplotypes were inferred. Binding sites for three milk protein binding factors (MPBFs) and four nuclear factor-I (NF-I) were found within BLG promoter region based on the ovine sequence. The identification of some SNPs within TFs binding sites allowed hypothesizing the loss of TFs. Further studies are in progress to evaluate the effect of these mutations on binding affinity of TFs, the functional interaction of the TFs with the goat BLG promoter, and the relationship of the polymorphisms with BLG gene expression and milk production and composition.

Induction of lactogenesis in transgenic virgin pigs: evidence for gene and integration site-specific hormonal regulation.

Five month-old transgenic female pigs from three lines carrying the mouse whey acidic protein (WAP) gene and nontransgenic female littermates were implanted with slow-release estrogen and progesterone pellets. Histological analysis of biopsies taken at the time of implantation and 4 weeks later revealed that mammary alveolar development had occurred upon hormonal stimulation in vivo. beta-Casein and beta-lactoglobulin mRNA was found in all induced animals, and WAP mRNA was detected in two of the three transgenic pigs. Differential hormonal regulation between the transgenes in the three lines and also between endogenous milk protein genes was observed in induced mammary tissue cultured in vitro. In the presence of insulin, hydrocortisone, and PRL, beta-casein and WAP mRNA levels increased in all transgenic pigs. In contrast, beta-lactoglobulin mRNA had reached or exceeded lactational levels in response to the in vivo induction, and no further increase was observed in vitro. This suggests that the regulation of the beta-lactoglobulin gene is distinct from that of beta-casein and WAP. Differences were also observed during pregnancy; whereas beta-lactoglobulin gene expression was induced in early pregnancy, a time when PRL levels are low, WAP mRNA levels increased sharply around parturition. Finally, the observation that hormonal regulation of WAP transgenes greatly differed between the three lines suggests that chromatin surrounding the integration site can modify the response of transcription elements.

Ovine β-lactoglobulin messenger RNA: Nucleotide sequence and mRNA levels during functional differentiation of the mammary gland

The nucleotide sequence of ovine beta-lactoglobulin mRNA has been determined by chemical sequencing of two cDNA recombinant plasmids and primer extension products. Ovine beta-lactoglobulin mRNA consists of a 540 nucleotide coding region, flanked by 39 nucleotide 5' and 206 nucleotide 3' non-coding regions including a 20 nucleotide poly A tail. The deduced 180 amino acid sequence of pre-beta-lactoglobulin is in agreement with the previously published amino acid sequence of signal peptide and mature protein. Northern blot analysis of poly A+ RNAs from the lactating mammary glands of porcine, rabbit and rat species, allowed us to identify a homologous RNA to beta-lactoglobulin mRNA solely in the porcine species. We also detected a mRNA transcript of a size similar to that of beta-lactoglobulin mRNA in hepatic poly A+ RNA from female rat liver treated by estrogens. Furthermore, we have examined the levels of beta-lactoglobulin mRNA during the functional differentiation of the mammary gland and after hormonal stimulation. During the last third of pregnancy, the expression of beta-lactoglobulin gene is significantly more elevated than that of alpha s1- or beta-casein whose mRNA levels were found to change very slightly during this period. Both beta-lactoglobulin and casein mRNAs showed a rapid response and a wide range of change in response to cortisol treatment. However, there was a significant difference in the rate at which these processes occurred, suggesting that beta-lactoglobulin gene expression is regulated independently of the casein genes.