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A cDNA was cloned coding for human placental 5’-nucleotidase. The 3547-cDNA contains an open reading frame that encodes a 574-residue polypeptide with a calculated size of 63375 Da. The NH2-terminal 26 residues comprises a signal peptide, which is followed by the NH2-terminal sequence of the purified protein. Four potential N-linked glycosylation sites are found in the molecule, accounting for a larger mass of the mature form (71 kDa). The predicted structure contains a hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post-translational modification by glycophosholipid. To confirm this possibility, we tried to isolate and characterize the membrane-anchoring domain of 5’-nucleotidase. BrCN-cleaved fragments of the protein were extracted with hexane and subjected to HPLC, resulting in purification of a single component of 2.3 kDa. Chemical analyses revealed that the purified fragment contains the tetradecapeptide Lys-Ile-Tyr-Pro-Ala-Val-Glu-Gly-Arg-Ile-Lys-Phe-Ser, ethanolamine, glucosamine, mannose, inositol, palmitic acid, and stearic acid. The peptide sequence determined is identified at positions 540-523 in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 548, containing the hydrophobic amino acid sequence. Thus, it is concluded that the nmature 5’-nucleotidase lacks the predicted COOH-terminal peptide extension (524-548), which has been replaced by the glycophospholipid functioning as the membrane anchor of 5’-nucleotidase. [ABSTRACT FROM AUTHOR]

Structural analysis of 55 nearly full-length cDNA clones revealed heterogeneity in the 5'-untranslated regions of rat liver alkaline phosphatase mRNAs. The 5' extremities diverged into two totally unrelated sequence stretches at the position 88 nucleotides upstream of the initiation codon ATG. These two sequences, referred to as E1 and E2, were assigned on the genome about 36 000 base pairs (36 kbp) and 10 kbp upstream, respectively, of the exon coding for the 5'-most part of the common region. The gene consisted of 13 exons, including E1 and E2, and spanned about 56 kbp. The 11 exons (E3 to E13) following E1 and E2 were shared in common by the El-type and the E2-type mRNAs. Analyses by SI nuclease mapping and primer extension revealed the presence of two independent transcription-initiation sites specific to each of the El and E2 sequences. These results are interpreted as indicating a possible alternative usage of two leader exons, hence the presence of two independent promoters. Structural features of these putative promoters are described in the context of transcriptional fundamental and regulatory cis-elements. [ABSTRACT FROM AUTHOR]