Abstract
alpha 1-Microglobulin (A1M) and bikunin are plasma proteins which are present both as free molecules and as complexes with either IgA heavy chains for A1M or the H1, H2, and H3 heavy chains of the inter-alpha-inhibitor family for bikunin. Mature A1M and bikunin originate from the cleavage of an A1M/bikunin precursor (ABP) synthesized from a single gene with liver-specific expression. Five kilobases of the 5'-flanking region of the human ABP gene were sequenced. Deletion mutants of this region subcloned upstream of a CAT reporter gene were transfected into HepG2 hepatoma cells. A segment covering the -2.7- to -2.8-kb area is required for full activity of the ABP gene. This segment contains a cluster of six elements (boxes 1-6, 5' to 3') which are potential binding sites for the liver-enriched trans-acting factors HNF-1, HNF-4, HNF-3, HNF-1, HNF-3, and HNF-4, respectively. This cluster enhances the activity of heterologous minimal promoters in a position- and distance-independent fashion in HepG2 cells. This enhancer activity is restricted to liver cells as the cluster is unable to activate promoters in Chinese hamster ovary (CHO) or HeLa cells. By band-shift experiments we have shown that the liver-enriched transcription factors HNF-1, or HNF-3, do bind to boxes 1 and 4, or 3, respectively. The combination of a weak promoter and a strong distant and liver-specific enhancer distinguishes the ABP gene from most other plasma protein genes expressed in hepatocytes.
Highlights
From the SZnstitut National de la Sante etde la Recherche Medicale Unit-78 and the European Institutefor Peptide Research, Boisguillaume 76233, France, the llUniti des Virus Oncogenes, Centre National de la Recherche Scientifique UA 1149, Znstitut Pasteur, Paris 75724, France, and the **ZnstitutNational de la Sante etde la Recherche Midicak, Unit-136, Marseille 13288, France a1-Microglobulin (AlM) andbikunin are plasma a free state (Mr31,000) as well as covalently bound to some proteins which are present both as free molecules and as complexeswith eitherIgA heavy chains for A I M or the H1, HZ, and H3heavy chains of the inter-a-inhibitor family for bikunin
By mRNA (1.25 kb) [7] is transcribed by a single copy gene (8, band-shift experiments we have shown that the liver- 9) with hepatocyte-restricted expression [10]
Several reports have dealt with the sequence and organization of thehuman ABP gene andits 5"flanking region [8,9],no functional analysis of the ABP gene promoter has yet been reported
Summary
Nucleotide Sequence of the 5“Flanking Region in the Human ABP Gene-A XEMBL-3 clone containing part of the human ABP gene was isolated by screening a genomic library withahuman 179-bp cDNA probe corresponding to ABP gene exon I. To verify the transcription start site of the ABP gene from an ABPICAT construct,poly(A)+RNAs isolated from HepG2 cells transfected with p-2929/57CAT were hybridized with a CAT gene-specificprimer (Fig. 3, leftpanel) and treatedwith reverse transcriptase. A Strong Enhancer Containing Several Liver-specific Elements Is Located in a Distal 5”Flanking Region of the ABP Gene-We further analyzed the activity of the positive regulatory element described above. Potential liver-specific elements found in the 5"flanking sequence of the human AlMlbikuningene and comparison with published consensus
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