Isolation and characterization of a MAP-2 complementary-DNA clone encoding epitopes of Alzheimer neurofibrillary tangles
Dammerman, Marilyn Malinda
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For the purpose of characterizing protein domains sharing epitopes with Alzheimer neurofibrillary tangles (NFT), a human brain complementary DNA (cDNA) library in lambda gt11 was screened with 10 monoclonal antibodies to NFT (Yen et al., 1985). The largest of the clones isolated is 1.7 kilobases and is recognized by four anti-NFT antibodies. This clone encodes 569 amino acids, or approximately 30%, of human microtubule-associated protein 2 (MAP-2), a 200-kilodalton protein which forms a 40 to 90 nanometer extension perpendicular to the microtubule wall.;In adult rat brain messenger RNA (mRNA), the MAP-2 cDNA hybridizes to a 9.5 kilobase transcript. In mRNA from fetal rat brain and from human neuroblastoma cells, the MAP-2 probe hybridizes to two prominent transcripts of 9.5 and 6 kilobases at high stringency. Several additional transcripts are detected in neuroblastoma mRNA upon prolonged exposure. The MAP-2 probe does not detect a transcript in non-neuronal RNAs.;Hybridization of the probe, at high stringency, to human genomic DNA digested with several restriction enzymes is consistent with detection of a single gene containing at least one intron. At reduced stringency, one additional high molecular weight fragment is seen per digest, suggesting detection of a related gene. Hybridization to genomic DNAs from a variety of species indicates that the MAP-2 gene has not been highly conserved through evolution. DNA sequence analysis demonstrates that the MAP-2 cDNA consists entirely of translated sequence, with no termination signal present. The predicted amino acid sequence has a net negative charge and is rich in glutamic acid, proline and lysine residues. Comparison of the partial human MAP-2 sequence with that of a complete mouse MAP-2 cDNA (Lewis et al., 1988) reveals that the human clone encodes sequences near to, but not encompassing, the amino terminus of the MAP-2 molecule, which is distal to the microtubule. A search of the GenBank database revealed no other highly related sequences.;Studies carried out in collaboration with Dr. Jack Erlichman demonstrate that the first 76 amino acids of the MAP-2 region encoded by the clone contain a binding site for type II cyclic AMP-dependent protein kinase. The antigenic determinants detected by the four anti-NFT antibodies that recognize the clone have been assigned to a 51-amino-acid region which begins 13 amino acids downstream from the kinase binding region.
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