We synthesized the 60-amino acid polypeptide corresponding to the sequence of the Drosophia antennapedia gene homeobox. This peptide (pAntp) recognized the consensus motif for binding to the promoter region ofHox-1.3. pAntp mechanically introduced into mammalian nerve cells provoked a dramatic morphological differentiation of the neuronal cultures. Moreover, pAntp directly added to already differentiated neuronal cultures penetrated the cells and further augmented their morphological differentiation. Examination of live and fixed neurons in classical and confocal fluorescence microscopy demonstrated that pAntp was captured at all regions of the nerve cells and accumulated in the nuclei. In addition, the effect of pAntp on neurite extension was blocked in the presence of the protein synthesis inhibitor cycloheximide. Thus, our results demonstrate that neurons possess an efficient uptake system for the antennapedia homeobox peptide and suggest that binding of pAntp to consensus motifs present in nerve cell nuclei influences neuronal morphogenetic programs.Studies on developmental mutants in Drosophila have demonstrated that several DNA binding proteins encoded by homeotic genes are endowed with morphogenetic functions (1, 2). In Drosophila at least, the expression of specific homeotic genes is responsible for the formation of cell assemblies exhibiting precise and defined morphologies. The DNA binding properties ofthese proteins is due to a sequence of about 60 amino acids called the homeobox.The homeobox sequences have been highly conserved during evolution and genes containing homeobox sequences are present in all vertebrates, including mammals (3, 4). In vertebrates, as in Drosophila, homeobox gene expression is not limited to the period during which the general features of body organization are established. In particular, a number of these genes are expressed in the nervous system rather late during development and, in some cases, through adulthood (5-7).In vitro studies have shown that the regulatory effect of homeotic proteins on gene expression depends on the specific binding of the homeobox domain to consensus DNA sequences found in the promotors or enhancers of several reporter genes and homeobox-containing genes (8-10). It has also been clearly demonstrated that the 60-amino acid homeobox polypeptide alone, isolated from the flanking regions, which are necessary for the activation or repression of transcription, has per se a high affinity for such consensus motifs (11).Thus, one possible way to study directly, within the live cell, the role of a homeotic protein family, as defined by the primary structure of the homeobox (e.g., antennapedia-or engrailed-like) and by the recognition of identical binding sites, on neural development would be to synthesize and inject the 60-amino acid homeotic polypeptides into the cells. In fact, such peptides could act as competitive inhibitors of endogeneous homeotic proteins with similar binding specificities.In the experiments presented herein we have used this...

Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it is in tight association with genomic RNA and the tRNA primer. NC protein is necessary for the dimerization and encapsidation of genomic RNA, the annealing of the tRNA primer to the primer binding site (PBS) and the initial strand transfer event. Due to the general nature of NC protein-promoted annealing, its use to improve nucleic acid interactions in various reactions can be envisioned. Parameters affecting NC-promoted nucleic acid annealing of NCp7 from HIV-1 have been analyzed. The promotion of RNA:RNA and RNA:DNA annealing by NCp7 is more sensitive to the concentration of MgCl2 than the promotion of DNA:DNA hybridization. Stimulation of complex formation for all three complexes was efficient at 0-90 mM NaCl, between 23 and 55 degrees C and at pH values between 6.5 and 9.5, inclusive. Parameters affecting NCp7-promoted hybridization of tRNA(Lys,3) to the PBS, which appears to be specific for NC protein, will be discussed. Results implicate the basic regions of NCp7, but not the zinc fingers, in promoting the annealing of complementary nucleic acid sequences. Finally, NCp7 strand transfer activity aids the formation of the most stable nucleic acid complex.

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An increasing number of eukaryotic transcription factors interacting specifically with DNA comprise a dimerization motif called the "leucine zipper". These leucine zipper proteins form homodimers and/or heterodimers with another protein containing a leucine zipper motif. The leucine zipper of the oncoprotein Jun is particular in that Jun may form homodimers as well as heterodimers with the oncoprotein Fos, which are however more stable than the Jun-Jun homodimers. Leucine zipper dimerization is thought to occur through a coiled-coil arrangement of parallel alpha-helices, but the rules governing the specificity of homo- and/or heterodimerization are still largely unknown. To address this question in the case of the Jun leucine zipper, we constructed a fusion protein containing the amino-terminal DNA binding domain of the LexA repressor from Escherichia coli fused to the Jun leucine zipper. This hybrid protein (LexA-JunZip) is stable in E. coli and confers much tighter repression in vivo than the DNA binding domain of LexA alone. DNA binding competition experiments with synthetic Jun and Fos leucine zipper peptides in vitro showed that the leucine zipper mediated dimerization of LexA-JunZip is essential for DNA binding of the fusion protein. The purified LexA-JunZip protein dimerizes in vitro with a dimerization constant of 2 x 10(7) M-1 at 5 degrees C. Dimerization is very sensitive to temperature, since the dimerization constant drops at 20 degrees C to 2 x 10(6) M-1 and at 30 degrees C to only 3 x 10(5) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)

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