Tau protein binds to microtubules through a flexible array of distributed weak sites.

Tau protein plays a role in the extension and maintenance of neuronal processes through a direct association with microtubules. To characterize the nature of this association, we have synthesized a collection of tau protein fragments and studied their binding properties. The relatively weak affinity of tau protein for microtubules (approximately 10(-7) M) is concentrated in a large region containing three or four 18 amino acid repeated binding elements. These are separated by apparently flexible but less conserved linker sequences of 13-14 amino acids that do not bind. Within the repeats, the binding energy for microtubules is delocalized and derives from a series of weak interactions contributed by small groups of amino acids. These unusual characteristics suggest tau protein can assume multiple conformations and can pivot and perhaps migrate on the surface of the microtubule. The flexible structure of the tau protein binding interaction may allow it to be easily displaced from the microtubule lattice and may have important consequences for its function.

Modulation of tk expression in mouse pericentromeric heterochromatin.

We have obtained a mouse transformant cell line containing two herpes viral thymidine kinase (tk) genes integrated in pericentromeric heterochromatin. Restriction analysis of tk- revertant and tk+ rerevertant derivatives suggest that one of the two tk genes is repressed in tk- cells, but is reactivated in tk+ rerevertants. The results of Northern analysis indicated that repression-activation is probably controlled at the transcriptional level. To examine the molecular basis for this repression, we cloned the tk gene from a tk- revertant cell line. Then, using the cloned tk gene as donor DNA to select for tk+ transformants, we found that it has a transfection efficiency indistinguishable from the viral tk gene. This indicates that repression is probably not mediated via any DNA sequence changes within the tk gene. The results of further studies by restriction analysis, azacytidine treatments, and secondary DNA transfection assays demonstrated that tk repression is associated with changes in DNA methylation. Surprisingly, derepression of the tk gene was accompanied by rearrangements in the flanking DNA. The latter result suggests that the flanking DNA may exert cis effects on tk gene expression. Additional studies with this system may provide insights into the molecular basis underlying position effects in heterochromatin.

High frequency DNA rearrangements associated with mouse centromeric satellite DNA.

A DNA transformed mouse cell line, generated by the microinjection of a pBR322 plasmid containing the herpes thymidine kinase (tk) gene, was observed to exhibit a high frequency of DNA rearrangement at the site of exogenous DNA integration. The instability in this cell line does not appear to be mediated by the tk inserts or the immediately adjacent mouse DNA, but instead may be a consequence of the larger host environment at the chromosomal site of tk insertion. Results obtained from restriction analysis, in situ chromosome hybridizations, and cesium chloride density-gradient fractionations indicate that the tk inserts are organized as a single cluster of direct and inverted repeats embedded within pericentromeric satellite DNA. To determine the molecular identity of the flanking host sequences, one of the mouse-tk junction fragments was cloned, and subsequent restriction and sequence analyses revealed that this DNA fragment consists almost entirely of classical mouse satellite DNA. On the basis of these observations, we suggest that the instability in this cell line may reflect the endogenous instability or fluidity of satellite DNA.

Germ cell-specific proteins interact with the 3' untranslated regions of Prm-1 and Prm-2 mRNA.

The testis-specific mouse protamine genes (Prm-1 and Prm-2) are transcribed in haploid round spermatids, their mRNAs stored as cytoplasmic ribonucleoprotein particles and translated about 1 week later in elongating spermatids. We have compared the in vitro translational efficiencies of deproteinized Prm-1 mRNA isolated from purified populations of germ cells and found that Prm-1 mRNA from round spermatids translates as efficiently as Prm-1 mRNA from elongating spermatids, suggesting that translation of Prm-1 mRNA is normally repressed in round spermatids. Previous studies in transgenic mice have shown that the 3' UTR of Prm-1 mRNA is necessary and sufficient for its translational control (Braun et al., 1989). In this manuscript, we have used an RNA band shift assay to identify an activity, present in cytoplasmic fractions of meiotic spermatocytes and postmeiotic round spermatids, that binds the 3'UTRs of both Prm-1 and Prm-2 mRNA. We have used 3' UTR deletion variants to map the binding site to a 22-nt region within the Prm-1 3' UTR and to a 20-nt region within the Prm-2 3' UTR. uv cross-linking of the RNA band shift activities detected with the Prm-1 and Prm-2 3' UTRs generated the same two RNA/protein complexes of 53 and 55 kDa. The presence of the binding activity in the cell type and subcellular compartment associated with Prm-1 and Prm-2 mRNA storage suggest that the activity may be actively engaged in translational repression of these mRNAs.