Isolation and characterisation of the 5' region of the bovine lactoferrin gene : a dissertation presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry
Lactoferrin is an iron-binding protein found in a variety of mammalian secretions and polymorphonuclear leucocytes. Many biological roles have been proposed for lactoferrin however despite extensive research, the precise function(s) of this protein remain unclear. Lactoferrin appears to be regulated in a highly specific manner, dependent upon the developmental stage, the tissue and the species being investigated. RNA analyses have suggested that transcriptional regulation may be a significant factor controlling the expression of bovine lactoferrin within the mammary gland. A detailed knowledge of the molecular mechanisms and factors involved in the transcriptional regulation of the bovine lactoferrin gene will provide insights into the biological role of this protein. It was with this primary aim in mind that an unamplified bovine genomic library was screened for the presence of bovine lactoferrin 5' regulatory sequences. This resulted in the isolation of a clone which was characterised by detailed restriction mapping and found to contain sequences complementarity to bovine lactoferrin exon I and exon II sequences. Analysis of this clone indicated that is contained ~ 10 kb of DNA 5' to the transcriptional start site of bovine lactoferrin. A ~2.85 kb fragment which hybridised to an exon I probe was isolated and subjected to dideoxy sequencing. ~2.5 kb of DNA 5' to the lactoferrin coding sequence was identified within this fragment. A computer sequence homology search indicated that several putative consensus binding sites for transcription factors may be located within this DNA sequence. Fragments prepared from this subclone by PCR were subcloned into reporter gene expression constructs. The transcriptional activity of one of these constructs was investigated using COS cells and transient reporter gene expression studies. This construct, containing 565 bp of 5' bovine lactoferrin sequence, exhibited promoter activity. The successful isolation of this promoter will enable further investigations directed towards elucidating the transcriptional regulation of the bovine lactoferrin gene to be performed.