DNase I cleavage of adenoviral nucleoprotein.

Cleavage products resulting from DNase I treatment of adenoviral nucleoprotein were examined by gel electrophoresis, Southern blotting and hybridization to cloned restriction fragments derived from various regions of the viral genome. DNase I produced specific double-stranded cleavages in DNA of purified adenoviral cores and in DNA of intranuclear viral chromatin at early and late times of infection. At least some of these sites were also cleaved by DNase I in purified viral DNA, showing that sequence specificity of DNase I cleavage may contribute to the observation of specific double-stranded DNase I cleavage sites in adenoviral nucleoprotein. In addition, sites were observed which were specific either for cores or for intranuclear chromatin. In contrast to many cellular genes which have been characterized, there was no obvious relationship between DNase I cleavage sites and other features of the viral genome such as promoters or polyadenylation sites.     

In vitro selection of nucleoprotein enzymes.

Natural nucleic acids frequently rely on proteins for stabilization or catalytic activity. In contrast, nucleic acids selected in vitro can catalyze a wide range of reactions even in the absence of proteins. To augment selected nucleic acids with protein functionalities, we have developed a technique for the selection of protein-dependent ribozyme ligases. After randomizing a previously selected ribozyme ligase, L1, we selected variants that required one of two protein cofactors, a tyrosyl transfer RNA (tRNA) synthetase (Cyt18) or hen egg white lysozyme. The resulting nucleoprotein enzymes were activated several thousand fold by their cognate protein effectors, and could specifically recognize the structures of the native proteins. Protein-dependent ribozymes can potentially be adapted to novel assays for detecting target proteins, and the selection method's generality may allow the high-throughput identification of ribozymes capable of recognizing a sizable fraction of a proteome.     

Specific nucleoprotein complexes within adenovirus capsids.

Adenoviral DNA was examined within capsids by dimethyl sulfate footprinting. Protein-DNA interactions were visualized through ligation-mediated PCR (LM-PCR). Signals for protein binding were found adjacent to both inverted terminal repeats (ITR). There were no indications of close protein binding at several other loci of the viral genome. Therefore, adenovirus type 5 seems to contain sequence- or locus-specific DNA binding proteins within the virion.     

Nuclear import and export of influenza virus nucleoprotein.

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.     

Isolation and characterization of adenovirus core nucleoprotein subunits.

Digestion of adenovirus type 2 (Ad2) or Ad5 cores with micrococcal nuclease generated four nucleoprotein species that could be resolved by electrophoresis in low-ionic-strength polyacrylamide gels: these nucleoproteins displayed mobilities equivalent to those of DNA fragments of 900 to 1,025, 775 to 850, 650 to 725, and 525 to 600 base pairs (bp) and thus were readily distinguishable from HeLa cell mononucleosomes. The DNA fragments associated with the core nucleoprotein species were more than 250 to 90 bp long. Nucleoproteins containing 150, 120, or 90 bp of DNA were the most stable. Polypeptide VII was associated with each of the nucleoprotein species liberated from Ad2 cores. These data suggest that polypeptide VII and viral DNA of 90 to 150 bp comprise the unit particle of the Ad2 or Ad5 core nucleoproteins.     

The structure of nucleoprotein cores released from adenovirions.

The morphology, protein composition and DNA organization of nucleoprotein core complexes isolated from type 5 adenovirions have been examined by electron microscopy and biochemical techniques. The morphology of such core structures is in some ways strikingly similar to that exhibited by cellular chromatin. 'Native' core preparations contain compact and less highly-folded forms: the latter appear as thick fibres, 150-300A in diameter. Upon exposure to 0.4M NaCl, adenovirus cores undergo a transition to a beaded string form, reminiscent of nucleosomes. Of the three arginine-rich proteins, polypeptides V, VII and mu present in 'native' cores, only polypeptide VII remains associated with viral DNA in the presence of 0.4M NaCl. We therefore conclude that the nucleosome-like beads are constructed solely of polypeptide VII. The results of micrococcal nuclease digestion experiments suggest that polypeptide VII is sufficient to protect some 100-300bp of adenoviral DNA.     

Ionic and nonionic interactions in adenoviral nucleoprotein complexes.

Ionic and nonionic interactions between the adenoviral histone-like proteins and DNA were examined by determining effects of ionic strength and urea concentration on disruption of viral nucleoprotein. The viral proteins were as susceptible to dissociation by salt in the presence of urea as histones. Nonionic interactions between viral proteins appeared more extensive than those between histones.     

Partitioning of zinc and copper within subnuclear nucleoprotein particles.

Nuclei from frozen calf thymus suspended in buffer were analyzed for metal content prior to and after repeated washing. After three such extractions about 0.1 micrograms Zn/mg DNA and 0.025 micrograms Cu/mg DNA remained tightly associated with chromatin. This level of metal was essentially unchanged with subsequent washings. Digestion of extracted nuclei with micrococcal nuclease yielded soluble nucleoprotein containing zinc and copper. Metal enriched regions of chromatin appeared to be preferentially solubilized by digestion, and the solubilized metal was only partially dializable either with or without EDTA. Metal profiles generated from gel (A-5m) chromatography analysis of chelated and non-chelated solubilized chromatin were distinctive in that copper was undetectable (by flame AA) while zinc was associated only with low molecular weight products when EDTA was used. In contrast, both metals were detected with higher molecular weight oligonucleosomes in the absence of chelating agents. Additionally, the two metals localized within nucleoprotein peaks and these metal-containing regions were only resolved by gel chromatography when EDTA was omitted throughout the procedure. A discrete Cu-rich species in a region of the profile suggests a subset of Cu-rich nucleoprotein complexes.     

Physical properties of nucleoprotein cores from adenovirus type 5.

Analytical ultracentrifugation, thermal denaturation, and electron microscopy have been used to study nucleoprotein core particles, obtained from disrupted type 5 adenovirus and partially purified on glycerol density gradients. Electron microscopy at low salt concentrations has shown that the cores are homogeneous particles with characteristic structures, which vary with conditions of observation from a fairly loose network of fibers to a highly condensed, compact particle. Sedimentation measurements in the analytical ultracentrifuge, both by boundary and by band techniques, show that the cores are relatively homogeneous in solution and have sedimentation coefficients near 185 S at low salt concentrations, about 243 S in 1 or 2 M NaCl, and 376 S in 1 mM MgCl2. Correlation of sedimentation data with electron microscopic observations suggests that the 185 S particle has a loose, fibrous structure, while the faster species are more highly condensed particles. The melting temperature of the cores in 5 mM Tris/HCl is 79 degrees C, which is 10 degrees C higher than the Tm for purified, viral DNA. This indicates that the protein enhances the stability of DNA in the nucleoprotein complex.     

Oxytricha telomeric nucleoprotein complexes reconstituted with synthetic DNA.

The telomere binding protein from macronuclei of Oxytricha nova binds macronuclear DNA in vitro, protecting the 3'-terminal single-stranded (T4G4)2 tail from chemical and enzymatic probes. We have used synthetic oligodeoxynucleotides to study the binding properties of the telomere protein. It binds at the 3' end of single-stranded oligonucleotides that have the sequence (T4G4)n, where n greater than or equal to 2, reconstituting the methylation protection seen with macronuclear DNA. Three oligonucleotide.protein complexes are resolved in nondenaturing gels, all specific for this sequence. Single-stranded oligonucleotides that have one or more repeats of the sequence C4A4 are also recognized, forming a single complex. The dissociation constant for (T4G4)4 is about 19 nM, and for macronuclear DNA is at least 20-fold lower. The basis for this difference is not fully understood, but it is not simply due to the absence of a (C4A4)2.5.(G4T4)2.5 region on the oligonucleotide. Transversions of T's to A's or of G's to C's in the 3' tail portion prevent binding. Changing T's to dU's does not prevent binding, indicating that the hydrophobic 5-methyl group is not required for binding as had been suggested from the salt-stability of the complex. The properties of the DNA-protein complex suggest a revised model for telomere synthesis in Oxytricha.     

Myc and Max function as a nucleoprotein complex.

The Myc family of oncoproteins are thought to regulate proliferation and differentiation in a wide variety of cell types. Recent studies show that Myc proteins form sequence-specific DNA-binding complexes with Max, a new member of the helix-loop-helix leucine zipper protein class. The properties of the Myc-Max complex suggest a mechanism for Myc's function in both normal and neoplastic cell behavior.     

Feulgen nucleal reaction. I. Quantitative extraction of fuchsin from Feulgen-stained nucleoprotein

A new extraction method for the quantitative determination of the fuchsin contained in a Feulgen-stained nucleoprotein sample has been introduced. The method is based on the following facts: (1) Treatment of a Feulgen-stained nucleoprotein sample with hot acid or alkali brings about a splitting of the linkage between fuchsin-SO(2) and the hydrolyzed nucleic acid moiety of nucleoprotein through aldehyde groups. (2) It also effectuates the formation of fuchsin from the liberated fuchsin-SO(2). (3) The fuchsin is made colorless by the treatment, but is restored to its original pink colored state when the pH of the acidic or alkaline medium is adjusted to 4.6. (4) The fuchsin, either pink colored or decolorized by alkali, can be extracted from an aqueous phase by amyl alcohol. A linear relationship was found to exist between the amount of fuchsin extracted by the FEM from a Feulgen-stained nucleoprotein sample and its DNA content. This relationship holds over a wide range of DNA concentration. From experiments utilizing this method, knowledge may be gained about the mechanism of the Feulgen reaction in situ which can lead to an improvement of the reaction in the field of cytochemistry.     

Histone Sequence Database: a compilation of highly-conserved nucleoprotein sequences.

By searching the current protein sequence databases using sequences from human and chicken histones H1/H5, H2A, H2B, H3 and H4, a database of aligned histone protein sequences with statistically significant sequence similarity to the search sequence was constructed. In addition, a nucleotide sequence database of the corresponding coding regions for these proteins has been assembled. The region of each of the core histones containing the histone fold motif is identified in the protein alignments. The database contains >1300 protein and nucleotide sequences. All sequences and alignments in this database are available through the World Wide Web at http://www.ncbi.nlm.nih.gov/Baxevani/HISTO NES.