Multiple transcripts coding for the menkes gene: evidence for alternative splicing of Menkes mRNA.
We isolated cDNA fragments from four human cell lines that had sequences for the Menkes Cu-transporting ATPase (ATP7A). Primers designed to generate a 4.8 kb cDNA with the complete open reading frame generated a 1.9 kb cDNA in addition to the expected 4.8 kb product. Sequence analysis revealed that the 1.9 kb cDNA encoded one of the six Cu-binding sites and two of the eight transmembrane domains of ATP7A. Stop and start codons were also present. More striking, however, was an unusual union between exons 2 and 16 that retained an in-frame reference to exon 23. The 1.9 kb cDNA thus appeared to be a truncated Menkes mRNA that coded for an ATP7A variant that lacked exons 3-15. A 530 bp probe specific for exon 23 that avoided sequences in the exon 3-15 region hybridized to a 5.5 kb band on Northern blot analysis. Western blotting provided immunochemical evidence for the presence of both a 170 kDa and a 57 kDa protein with ATP7A sequences in detergent extracts of Caco-2 and induced BeWo cells. Extracts from non-induced BeWo cells, which lack the capacity to express the Menkes gene (MNK), showed neither protein. In a cell-free reticulocyte lysate, a plasmid containing the 1.9 kb cDNA insert directed the synthesis of a 59 kDa protein with antigenic properties of ATP7A. These studies provide evidence that non-Menkes cells have the capacity to synthesize more than one MNK mRNA. The one characterized in this report codes for a 57-59 kDa protein that lacks the core structure of the ATP7A protein. The smaller variant could be an alternative spliced form of MNK mRNA.
Reddy
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MC
Department of Biochemistry and Biophysics and Faculty of Nutrition, Texas A&M University, College Station, TX 77843-2128, USA.
Harris
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ED
D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D027682:Cation Transport Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D000073840:Copper-Transporting ATPases; D018076:DNA, Complementary; D005091:Exons; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007706:Menkes Kinky Hair Syndrome / Q000201:enzymology / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases