Structural and Functional Analysis of the Rabbit Erythroid-Specific Lipoxygenase Gene Promoter

[摘要] The rabbit erythroid-specific lipoxygenase (RBC 15-LOX) is a member of a family of enzymes which catalyse the addition of molecular oxygen to certain polyunsaturated fatty acids. It functions in the inactivation and degradation of mitochondria during the final stages of erythroid maturation, following extrusion of the nucleus. The protein has been purified to homogeneity and extensively characterised (Rapoport et al, 1979), and both cDNA and genomic clones have recently been obtained (Thiele et al, 1987). RBC 15-LOX expression shows several interesting features, including an erythroid-specific accumulation of its mRNA (Thiele et al, 1987), and translational inactivation of the messenger (Thiele et al, 1982) . Data presented in this thesis includes the sequencing of the recently-cloned RBC 15-LOX gene in the region surrounding the ATG translation initiation codon, and mapping of the transcription initiation site to a position 28nt upstream of the ATG codon by a combination of primer extension and S1 nuclease protection analyses. No transcripts arising from the available 2.7kb of sequences upstream of this point have been detected by Northern blot, primer extension or SI nuclease protection analyses. A unique mRNA sequence, matching the genomic sequence immediately downstream of the putative transcription initiation site, has been obtained by primer extension mRNA sequencing. Functional analysis of 5' flanking sequences reveals two regions which affect transient expression of a cis-linked bacterial chloramphenicol acetyItransferase (CAT) reporter gene in established murine erythroleukaemia (MEL) and murine fibroblast cell lines. A proximal region within 150nt of the transcription initiation site functions as a promoter in both erythroid and non-erythroid cell lines, elevating CAT enzyme activity 7- to 8-fold above background, and can respond to a heterologous Friend Murine Leukaemia Virus enhancer. A distal region between 1.0 and 2.7kb upstream of the transcription initiation site confers a 6- to 8-fold cell type-specific effect upon CAT expression from the RBC 15-LOX promoter in the same assay system. The proximal region contains several sequence motifs which are also seen in the 5' flanking sequences of a variety of other genes, including: a TATA-like sequence; two CACCC motifs; a GGGCGG element; and a sequence resembling the cognate binding site for the CTF/NF-1 family of transcription factors. In vitro DNasel footprinting of this proximal region using nuclear protein extracts from erythroid and non-erythroid murine cells shows protection of both the proximal CACCC sequence and the CTF/NF-l-like element. Future experimental strategies for investigating a possible role for the regulation of transcription in the erythroid-specific accumulation of RBC 15-LOX mRNA are discussed. Also considered are aspects of the data presented here which, in conjunction with the work of others, may have implications for the translational inactivation of RBC 15-LOX transcripts, and for the evolution and structure-function relationships of the lipoxygenase enzyme family.

[发布机构] University:University of Glasgow

[关键词] Molecular biology, Genetics