Developmentally regulated expression of specific tau sequences.

Tau protein undergoes a shift in its molecular mass and its electrophoretic complexity during early postnatal development. We have sequenced a tau cDNA from an adult rat brain expression library and have found two inserted sequences. One of these inserts predicts a fourth repeated sequence homologous to the other three in the carboxyl end of tau that have the property of microtubule binding. Oligonucleotide probes directed against the insert hybridized only to tau mRNA at postnatal time points, even though tau is first expressed as early as embryonic day 13. A probe directed against the junction revealed expression of non-insert-containing tau mRNA from embryonic day 14 until postnatal day 8, after which time there was an abrupt decline in the expression of this immature form. Comparison of the developmentally expressed tau sequences with those sequences obtained directly from Alzheimer paired helical filaments revealed the presence of both the mature and the immature tau mRNA sequences.

Epitopes that span the tau molecule are shared with paired helical filaments.

Tau protein has been shown to be an integral component of Alzheimer paired helical filaments (PHF). However, the extent to which tau is incorporated into PHF has not been clear because the antibodies used to label PHF generally do not have precisely defined epitopes. Here we define the antigenic sites for five monoclonal antibodies that react with tau and cross-react with SDS-extracted neurofibrillary tangles. The reactive sites were determined by screening a lambda gt11 sublibrary expressing small fragments of the tau sequence. The mapped epitopes were found to span almost the entire length of tau, suggesting that PHF contains tau in its entirety or nearly in its entirety. One antibody was found to cross-react with microtubule-associated protein 2, implying some degree of homology between the two proteins.

Human GAP-43: its deduced amino acid sequence and chromosomal localization in mouse and human.

The growth-associated protein (GAP-43) is considered a crucial component of an effective regenerative response in the nervous system. Its phosphorylation by protein kinase C correlates with long-term potentiation. Sequence analysis of human cDNAs coding for this protein shows that the human GAP-43 gene is highly homologous to the rat gene; this homology extends into the 3'-untranslated region. However, the human protein contains a 10 amino acid insert. Somatic cell hybrids demonstrate localization of the GAP-43 gene to human chromosome 3 and to mouse chromosome 16.

Partial sequence of MAP2 in the region of a shared epitope with Alzheimer neurofibrillary tangles.

A 3.3-kilobase DNA complementary to human microtubule-associated protein 2 (MAP2) was sequenced by the dideoxy method. The 3' end terminates at an internal EcoRI site before the polyA tail. Due to the arrangement of the cDNA insert in the lambda gt11 vector, the MAP2 fragment is not fused to beta-galactosidase when expressed. The Chou Fasman algorithm for the initial 58 amino acids from the first in-frame methionine predicts an alpha helix. Beyond this point, a series of turns is predicted until amino acid 160. The frequent presence of basic residues in proximity to serines or threonines is consistent with multiple phosphorylation sites. The minimum specificity determinant for Ca2+/calmodulin-dependent kinase is repeated 13 times. The sequence of a region containing a MAP2 epitope that is shared with the Alzheimer neurofibrillary tangle was determined by DNase treatment of the cDNA and antibody selecting the small resultant clones in a lambda gt11 sublibrary. Likewise, a MAP2 epitope that is not shared with the neurofibrillary tangle also has been located. Both epitopes are in the projection portion of the molecule. A bovine MAP2 cyanogen bromide fragment, which contains the epitope shared with the neurofibrillary tangle, is partially insoluble under aqueous conditions, probably due to the aggregation of oppositely charged residues. Thus, rapid cleavage of MAP2 to small peptides is probably necessary in vivo to prevent the aggregation of larger cleavage fragments.

The primary structure and analysis of the squid kinesin heavy chain.

We report the cDNA sequence of the squid kinesin heavy chain and compared the predicted amino acid sequence with that of the Drosophila heavy chain as reported by Yang, J.T., Laymon, R.A., and Goldstein, L.S. B. (1989) Cell 56, 879-889). We compared the two kinesin sequences with regard to the predicted physicochemical parameters of hydrophobicity, charge, and propensities of the secondary conformations. A comparison of the sequences from the two species reveals the head, stalk, and tail domains because a reduced degree of conservation demarcates the stalk. The charge profile indicates that the head region is nearly neutral, the stalk region acidic, and the tail is basic. The Fourier transform analysis of the hydrophobic profile of the stalk shows predominant peaks at 1/3.5 and 1/2.3, which are indexed as the second and third orders of the period 7 residue. As in the Drosophila sequence, the rod domain is divided into an amino and a carboxyl subdomain by a predicted hinge region. We show that the disposition of hydrophobic residues is distinct in these two subdomains. In particular, the heptad repeat is more regular in the amino-terminal rod domain than in the carboxyl-terminal rod domain. The tail region is positively charged, a feature that is consistent with the known electrostatic interaction between the heavy chain and negatively charged surfaces such as glass coverslips and latex beads. Three monoclonal antibodies to the kinesin heavy chain have been mapped to a region within the carboxyl terminus of the stalk.