The bovine lactoferrin gene : defining the minimal promotor region : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry
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Lactoferrin is an 80 kDa glycoprotein with two lobes, each of which bind a single iron atom. Originally isolated from milk, lactoferrin has since been identified in a variety of exocrine secretions and in the secretory granules of neutrophils. A number of functions have been proposed for lactoferrin, some of which are related to the capacity of the protein to bind iron tightly but reversibly. The proposed functions include iron transport in the gut, antimicrobial activity and modulation of the activity of the immune system. The synthesis of lactoferrin in the mammary gland is developmentally regulated with changes in protein concentration in milk being correlated with changes in lactoferrin mRNA in mammary tissue. Most species studies have identified high levels of lactoferrin during involution and late pregnancy into early lactation. The amounts of lactoferrin during the lactational phase are much lower, especially in bovine milk. The regulation of bovine lactoferrin expression was studied in the belief that knowledge of the factors influencing expression will provide insight into the function of lactoferrin in the mammary gland. A 2.5 kb fragment of bovine genomic DNA, including the region immediately upstream of the transcription start point, has been subcloned into luciferase reporter gene vectors. The 2.5 kb fragment has been sequenced and a number of putative response elements identified. Promoter activity was tested by transient expression in the human endometrial carcinoma cell line RL 95-2. 5'- and 3'- deletion analysis of the promoter was used to establish regions which confer transcriptional regulation and the minimal promoter region. A recent report on the sequence of the cDNA for caprine (goat) lactoferrin suggests that the transcription start point for the mRNA for this protein may be further upstream than that reported for the mRNA of bovine lactoferrin. In view of the high level of sequence identity between the two cDNA's in this region an attempt was made to reinvestigate the transcriptional start point for bovine lactoferrin using DNA footprinting.