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Title: Structure and expression of ruminant α-lactalbumin genes
Author: Vilotte, J. L.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1991
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α-lactalbumin is one of the major milk protein in ruminants. It induces synthesis of lactose in the mammary gland and is related to lysozyme. The present study describes the structure and analysis of expression of ruminant α-lactalbumin genes. The structure of the goat α-lactalbumin gene was determined and found to be similar to that of the known homologous genes with four exons. Analysis of bovine, goat and sheep genomic DNA revealed the occurrence of α-lactalbumin-related sequences. Southern-blotting analysis and determination of the structure of such related sequences suggested that most correspond to pseudogenes as judged by their lack of homology to regions 5' to intron 2 of the α-lactalbumin-encoding gene. Such a complex genomic organization, so far limited to ruminants, has also been reported for lysozyme. According to recent data, these genes are linked and so may have co-evolved. Expression of the bovine gene with 738 and 300 bp of 5' and 3' flanking regions, respectively, was investigated in vitro in BHK cells using the SV40 enhancer/promoter sequences. The transcription unit of this gene was found to be functional as judged by the secretion of the bovine protein in the medium of transformed cells. Transient expression of the CAT gene placed under the transcriptional control of the α-lactalbumin promoter was also investigated. Unexpectedly, this led to the discovery of a highly significant influence of temperature of the calcium-phosphate-DNA co-precipitate on the DNA uptake by the cells. The same incidence was observed with other plasmids. It suggests that this external factor should be controlled to ensure optimal conditions. Finally, expression of the bovine gene was tested in transgenic mice. Expression of the bovine gene was found to be tissue-specific and developmentally regulated. Its level in some transgenic lines was close to that of the endogenous gene, but unrelated to the number of copies integrated. These results suggest that some important cis-regulatory elements involved in its regulation are located within the injected fragment. Analysis of the expression of three constructs derived from the same gene with shortened 5'-flanking regions (477 bp, 220 bp or 53 bp in length, respectively) suggests that important cis-regulatory elements involved in the control of the transcriptional level are located within a 257 bp fragment (position -477 to -220 with regards to the CAP site). The potential usefulness of the bovine gene for driving the expression of a linked gene was investigated. Most of the transcription unit of the bovine gene was replaced by an ovine trophoblastin cDNA, leading to an intronless hybrid gene. Expression of this transgene was observed in one out of the four lines tested at levels which compare to that observed with similar constructs based on other milk-protein genes. These results suggest that the bovine gene could be used to manipulate the milk composition and to identify some of the cis-regulatory elements involved in its hormonal regulation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available