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UBC Theses and Dissertations

Characterization of the human ceruloplasmin cDNA and gene Koschinsky, Marlys Laverne


A cDNA for human ceruloplasmin was identified in a human liver cDNA library by screening with mixtures of synthetic oligonucleotides complementary to two regions of the ceruloplasmin mRNA. The resulting clone (phCP-1) contained DNA coding for amino acid residues 202 - 1046 of the protein, followed by a 3' untranslated region of 123 bp and a poly(A) tail. To isolate additional clones extending in a 5' direction, two randomly-primed human liver cDNA libraries were constructed in the bacteriophage vectors λgt10 and λgtll. From the former library, a clone was isolated (λhCP-1) that contained DNA coding for a putative signal peptide consisting of 19 amino acid residues, followed by DNA encoding residues 1 - 380 of plasma ceruloplasmin. From the λgtll library, six ceruloplasmin cDNA clones were purified, two of which were shown to contain 10 and 38 bp of non-coding sequence extending 5' to λhCP-1. Blot hybridization analysis using cDNA probes showed that ceruloplasmin mRNA from the human hepatoma cell line HepG2 is 3700 nucleotides in size, while human liver RNA contained an additional hybridizing species of 4500 nucleotides in size. Ceruloplasmin genomic DNA clones (spanning a region of approximately 45 Kbp) were obtained by the screening of several human genomic phage libraries using cDNA probes. These clones were initially characterized by restriction endonuclease mapping. Using DNA sequence analysis, the positions of intron/exon boundaries were determined. To date, 14 exons (average size of 183 bp) have been identified in the ceruloplasmin gene, corresponding to nucleotide residues 1 - 2565 of the coding sequence. The majority of the 14 introns localized within this region were located in analogous positions in the factor VIII gene, thereby suggesting that these two proteins have evolved from a common ancestral gene. At least 4 exons have been localized within the 5' untranslated region of the human ceruloplasmin gene, although typical eukaryotic promoter elements have not yet been identified. The significance of this novel organization remains unclear at present. In addition to the wild-type gene, a processed pseudogene for human ceruloplasmin was identified and contained DNA corresponding to the functional gene sequence encoding the carboxy-terminal 563 amino acid residues and the 3' untranslated region. The pseudogene appears to have arisen from a processed RNA species, since intervening sequences coincident with those of the functional gene have been removed with the exception of a short segment of intronic sequence which denotes the 5' boundary of the pseudogene. Based on genomic Southern blot anlysis performed under high stringency conditions, the pseudogene seems to comprise the only sequence in the human genome that is closely related to the wild-type gene. Using somatic cell hybridization, the pseudogene was localized to human chromosome 8; this differs from the location of the wild-type ceruloplasmin gene, which has been mapped to chromosome 3.

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