Molecular cloning of the cDNA for ligatin.
We describe the first isolation and sequence of a partial cDNA clone encoding ligatin, a trafficking receptor for phosphoglycoproteins. The clone was isolated from a human U937 promonocyte lambda gt11 cDNA library using rabbit antiserum to rat ileal ligatin. RNA blot hybridization revealed that the intact receptor transcript in human cells is 2.4 kilobases (kb). DNA sequencing together with expression of protein fusion products in Escherichia coli demonstrated that the cloned segment begins with a 1.2 kb open reading frame potentially encoding a 7.5 x 10(3) Mr section of the 10 x 10(3) Mr receptor followed by a 3' tail of 948 bases. The 225 bases of coding sequence correspond to the carboxyl region of ligatin and contain a potential acceptor site for asparagine-linked glycosylation. Neither a poly(A) sequence nor polyadenylation signal was found at the 3' end of the clone. In the 3' untranslated region there is a paired consensus sequence (TGAGnnnTTTTTCA) that is analogous to a conserved 12 base-pair sequence present in clusters in several growth-controlled mRNAs, including those for c-fos and beta-actin. The identity of this clone as ligatin was confirmed immunologically using antisera to an encoded fusion protein and three independent regions of its derived sequence. In addition, one of these antibodies produced a punctate immunofluorescence pattern within the cytosol of U937 cells in a similar fashion to anti-ligatin serum.
Jakoi
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Department of Anatomy, Duke University Medical Center, Durham, North Carolina 27710.
Brown
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Ho
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Snyderman
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D015151:Immunoblotting; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics*; D008969:Molecular Sequence Data; D012313:RNA / Q000032:analysis / Q000235:genetics; D051381:Rats
D008565:Membrane Proteins; C013090:ligatin; D012313:RNA; D004247:DNA