Abstract. 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 (ti10-' 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

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.

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Constitutive heterochromatin is that part of the chromosome which does not decondense or unravel during interphase (for a review, see reference 2). It is commonly associated with centromeres and contains large amounts of satellite DNA, a class of highly repetitive DNA composed of tandem arrays of simple sequence repeats (for a review, see references 3, 26, and 47). Constitutive heterochromatin is generally associated with transcriptional inactivity. One indication of this is the low density of major gene loci in constitutive heterochromatin (25). Furthermore, genes translocated to the vicinity of heterochromatin are frequently repressed or variegated. In Drosophila melanogaster, this repression has been correlated with the spread of the condensed state from the heterochromatic region to the translocated euchromatin (29), and the probability of repression has been observed to increase with increasing proximity to the heterochromatin. This type of cis effect is classically referred to as position effect or position effect variegation. A similar phenomenon occurs in mice in which autosomal loci translocated to a heterochromatic X chromosome also are observed to be inactivated (6).The molecular mechanism underlying classical position effects remains obscure. A detailed molecular analysis of genes modulated within heterochromatin may provide insights into this intriguing phenomenon. In the long term, such studies may allow the identification of cis-acting elements which play a role in exerting position effects. In this study, we describe a cell line, LC2-3, which can serve as a valuable model system for such studies. LC2-3 is a cell line derived from thymidine kinase-deficient (tk-) mouse L cells (LMTK-cells) satellite DNA was found to immediately flank these tk inserts and, using the tk DNA as a marker, the adjacent satellite DNA was shown to undergo a high frequency of DNA rearrangements (5), a process which is reminiscent of the known fluidity of satellite DNA. In the current study, we characterized three pairs of tk-revertant and tk+ rerevertant derivatives of LC2-3. Our results indicate that one of the tk genes in LC2-3 is probably not expressed but can be reactivated in association with DNA rearrangements and changes in DNA methylation. Through these and other studies in the future, we hope to obtain insights into the molecular mechanism mediating tk repression and, furthermore, to determine whether and how the flanking centromeric heterochromatin may participate in this proc- 5-AzaC treatments. Treatments with 5-azacytidine (5-azaC) were performed by plating 106 cells into a 100-mm dish containing medium supplemented with 5-azaC at a concentration of 0, 1, 3, or 10 ,uM. Cells were exposed to the nucleotide analog for 2 days and subsequently grown for 2 days in medium without 5-azaC. Cells were then harvested from each dish and replated at a density of 5 x 106 cells per 100-mm dish in HAT medium and 200 cells per 100-mm dish in BrdU medium. After 7 to 10 days of growth, colonies were fixed in methanol-acetic ...

Following fertilization in Xenopus eggs, the dorsal-ventral asymmetry of the egg is established and a rapid cell cycle is begun. In the mid to late blastula period this initial asymmetry is translated into differentiation of the dorsal mesoderm initiated by the vegetal dorsalizing center; as suggested by experiments of Nieuwkoop. About this time the cell cycle undergoes a modification coincident with several events including the onset of cell motility. We report here on experiments describing how the dorsal-ventral asymmetry is produced in the early period. In particular we discuss the role of the sperm, its associated aster, the cortex, and redistribution of cytoplasmic contents. This analysis furthers our understanding of UV effects on dorsalization and the mechanism of twinning. We report also on evidence for a cytoplasmic clock regulating the cycle of DNA synthesis and cytokinesis in early cleavage, as well as on further experiments on the mechanism of cell cycle changes in the midblastula period.

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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.