Mcm. Reddy et Ed. Harris, MULTIPLE TRANSCRIPTS CODING FOR THE MENKES GENE - EVIDENCE FOR ALTERNATIVE SPLICING OF MENKES MESSENGER-RNA, Biochemical journal, 334, 1998, pp. 71-77
We isolated cDNA fragments from four human cell lines that had sequenc
es 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 ana
lysis 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 st
art codons were also present. More striking, however, was an unusual u
nion between exons 2 and 16 that retained an in-frame reference to exo
n 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 spe
cific for exon 23 that avoided sequences in the exon 3-15 region hybri
dized to a 5.5 kb band on Northern blot analysis. Western blotting pro
vided immunochemical evidence for the presence of both a 170 kDa and a
57 kDa protein with ATP7A sequences in detergent extracts of Caco-2 a
nd induced BeWo cells. Extracts from non-induced BeWo cells, which lac
k the capacity to express the Menkes gene (MNK), showed neither protei
n. 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 c
ells have the capacity to synthesize more than one MNK mRNA. The one c
haracterized in this report codes for a 57-59 kDa protein that lacks t
he core structure of the ATP7A protein. The smaller variant could be a
n alternative spliced form of MNK mRNA.