Historic Prussian blue (PB) pigment is easily obtained as an insoluble precipitate in quantitative yield from an aqueous mixture of Fe 3+ and [Fe II (CN) 6 ] 4− (Fe 2+ and [Fe III (CN) 6 ] 3−). It has been found that the PB pigment is inherently an agglomerate of 10-20 nm nanoparticles, based on powder x-ray diffraction (XRD) line broadenings and transmission electron microscopy (TEM) images. The PB pigment has been revived as both organic-solvent-soluble and water-soluble nanoparticle inks. Through crystal surface modification with aliphatic amines, the nanoparticles are stably dispersed from the insoluble agglomerate into usual organic solvents to afford a transparent blue solution. Identical modification with [Fe(CN) 6 ] 4− yields water-soluble PB nanoparticles. A similar ink preparation is applicable to Ni-PBA and Co-PBA (nickel and cobalt hexacyanoferrates). The PB (blue), Ni-PBA (yellow), and Co-PBA (red) nanoparticles function as three primary colour inks.

By random transposon TnS insertions, we previously identified six virulence-associated SaIl fragments, B, D, F, G, H, and P, in the 230-kilobase plasmid pMYSH6000 of Shigellaflexneri 2a. In this study, we analyzed the sites of 134 independent TnS insertions on four contiguous Sall fragments, B, P, H, and D, of pMYSH6000 and identified five virulence-associated regions; four were associated with inducing a positive Sereny test (Ser), invasion into epithelial cells (Inv), binding to Congo red (Pcr), and inhibition of bacterial growth (Igr), and one was associated with the Ser and Inv but not with the Pcr or Igr phenotypes. Hybridization studies revealed that these virulence-associated DNA regions were highly conserved among 15 other virulence plasmids of four species of Shigella and enteroinvasive Escherichuz coli. These data indicate that at least seven separate genetic determinants on the virulence plasmid are required for fuDl expression of the virulence phenotype of shigeliae.Shigellae are enteroinvasive bacteria that cause bacillary dysentery in humans and monkeys. These organisms invade colonic epithelial cells, multiply intracellularly, and spread to adjacent cells (9). The genetic determinants required for these abilities are located on at least three separate sites of the chromosome (3-5, 18) and on a 100-to 140-megadalton (MDa) plasmid. Commonly, plasmids of shigellae and enteroinvasive Escherichia coli (EIEC) contain genetic regions that are required in the early steps of the invasion process (18, 19); loss of the plasmid or deletion of an essential region consistently leads to loss of virulence (13,21).Expression of virulence in shigellae is dependent on temperature (12). Shigellae grown at 37°C are fully virulent, whereas bacteria grown at 30°C neither invade epithelial cells (12) nor provoke keratoconjunctivitis in guinea pigs (24). Making use of this property, Hale et al. (6) identified at least seven plasmid-coded, virulence-associated peptides produced by Shigella flexneri 2a and 5 and EIEC strain 0143. By Western blot (immunoblot) analysis of extracts of whole cells, four peptides of 78, 62, 43, and 38 kDa were recognized by convalescent-phase monkey antisera. These workers proposed that these proteins function as components of the invasion phenotype and are expressed on the bacterial surface. Oaks et al. (15) identified an additional plasmid-encoded surface peptide of 140 kDa which was also specifically recognized by convalescent-phase human or monkey sera. To identify the genetic regions associated with invasion, Maurelli et al. (14) shotgun-cloned Sau3A digests of the plasmid DNA into a cosmid vector, which was subsequently introduced into plasmid-free S. flexneri 5. A clone containing a 37-kilobase (kb) minimum sequence necessary for invasion was isolated. This recombinant clone also produced the four virulence-associated peptides described by Hale et al. (6). A DNA fragment coding for three antigenic proteins of 57, 43, and 39 kDa was cloned into a A expression vector by Buysse et al. (1). T...

In vitro replication of DNA containing the polyoma (Py) virus orign of replication has been carried out with cell-free extracts prepared from mouse FM3A cells. The in vitro system required the Py virus-encoded large tumor (T) antigen, DNA containing the Py virus origin of replication, ATP, and an ATP-regenerating system. The replication reaction was inhibited by aphidicolin, suggesting the involvement of DNA polymerase a in this system. Simian virus 40 (SV40) T antigen could not substitute for the Py T antigen. Cell extracts prepared from HeLa cells, a source that replicates SV40 DNA in the presence of SV40 T antigen, replicated Py DNA poorly. Cell-free extracts of monkey and human cells that replicate SV40 DNA have been described (4-7). The in vitro replication of SV40 DNA requires, in addition to suitably prepared cell extracts, circular DNA containing the viral replication origin (ori) and purified SV40 large tumor (T) antigen. Since SV40 T antigen and DNA containing the SV40 replication origin are the only viral components required, this system should be useful for identifying and characterizing the eukaryotic proteins involved in DNA replication. In previous reports (7-9), it was shown that DNA polymerase a (pol a) and DNA primase are essential for the replication of SV40 DNA in vitro and that the source of these enzymes is important in determining whether SV40 DNA replication will occur. In contrast to extracts of human cells, mouse cell extracts supplemented with SV40 T antigen did not support replication of DNA containing the SV40 origin unless supplemented with the pol a-primase complex from HeLa cells.In this report we describe the establishment of a system that replicates Py DNA in vitro; it requires mouse cell extract, the Py replication origin, and Py T antigen. In addition, we have confirmed and further defined the important role played by pol a-primase in determining the species specificity of papovavirus DNA replication. MATERIALS AND METHODSPurification of Py T Antigen. Py T antigen was purified from CV-1 cells infected with the helper-dependent recombinant adenovirus vector Ad-SVR587 (10) as follows. Cells were infected with wild-type adenovirus [2-5 plaque-forming units (pfu) per cell] and Ad-SVR587 recombinant virus (a gift from S. Mansour, T. Grodzicker, and R. Tjian) (5-10 pfu per cell). After incubation for 36 hr at 370C, cells were scraped from thirty 150-mm plates into -3 ml of cold Dulbecco's phosphate-buffered saline (PBS: Ca2+-and Mg2+-free), washed twice with cold PBS, suspended in 10 volumes of pH 9.0 buffer [20 mM Tris HCl, pH 9.0/0.2 M NaCl/1 mM EDTA/1 mM dithiothreitol/10% (vol/vol) glycerol/1% (vol/vol) Nonidet P-40 (NP-40)/1 mM phenylmethylsulfonyl fluoride (PMSF)], and lysed on ice for 10: min. The lysate was centrifuged for 10 min at 2000 rpm in a Sorvall H-6000A rotor, and the supernatant was then centrifuged for 10 min at 20,000 rpm in a Sorvall SS34 rotor. The supernatant was mixed with 0.5 volume of pH 6.8 buffer (0.1 M Tris'HCl, pH 6.8/1 mM EDTA/1 mM dithiothre...

A protein, which facilitates assembly of a nucleosome-like structure in vitro, was previously partially purified from mouse FM3A cells [Ishimi, Y. et al. (1 983) J . Biochem. (Tokyo) 94,735 -7441. The protein has been purified to approximately 80 from FM3A cells by using histone-Sepharose column chromatography. It sedimented at 4.6 S and had a molecular mass of 53kDa. A preincubation of core histones with the 53-kDa peptide before DNA addition was necessary for the nucleosome assembly. The 53-kDa peptide bound to core histones and formed a 12-S complex. This complex contained stoichiometrical amounts of the 53-kDa peptide and core histones, and the core histones in this complex were composed of equal amounts of H2A, H2B, H3 and H4 histones. The nucleosomes were assembled by adding pBR322 DNA to the 12-S complex. When mononucleosome DNA and core histones were mixed in the presence of the 53-kDa peptide, formation of a 10.5-S complex was observed. The complex contained DNA and core histones in equal amounts, while no 53-kDa peptide was detected in the complex.From above results it is suggested that the 53-kDa peptide facilitates nucleosome assembly by mediating formation of histone octarner and transferring it to DNA. Rat antibody against the 53-kDa peptide did not bind to nucleoplasmin from Xenopus eggs. The relationship between the 53-kDa peptide and nucleoplasmin is discussed.In eukaryotic cells, chromatin consists of a repeating unit called a 'nucleosome', which contains about 200 base-pairs of DNA, two molecules each of H2A, H2B, H3 and H4 histones, and one molecule of HI histone [1,2]. In many mammalian cells almost all core histones (H2A, H2B, H3, H4) are synthesized at S phase and their synthesis is coupled with DNA synthesis [3 -51. However, basal histone synthesis is observed also at GI phase in some cells [6], and mouse lymphoma cell synthesizes core histones at G I phase at the same level as S phase [7,8].As to the mode of deposition of newly synthesized histone on DNA, several confusing results have been presented. From the observation of the group of Weintraub [9-111, it is suggested that new histones are bound to the lagging strand of replicated DNA and they are assembled conservatively into an octamer. On the other hand, Jackson et al. [12,13] indicated that new H3 and H4 histones were bound to replicating DNA, while new H2A and H2B histones were deposited on nonreplicating DNA rather than the replicating DNA. There are several reports indicating that new histones are distributed equally to both daughter strands [14,15]. In any case it is not well examined whether new histones are bound to DNA by a single step as an octamer or by multiple steps.There are several factors which facilitatc nucleosome assembly in t'itro at physiological ionic strength. Nucleoplasmin (29 kDa), which has been discovered in Xenopus eggs by Laskey et al. [I 6,171, facilitate nucleosome assembly by the interaction with core histones. Experiments using its fluorescent antibody suggested that it exists in mammalian cells [...

Studies were made on the binding mode of the nucleosome-assembly protein AP-I with histones H2A + H2B and/or H3 + H4. Histones H2A + H2B bound with AP-I to form a 7-S complex which contained twice as much AP-I (by weight) as histones. Histone H3 + H4 formed an 8-S complex with AP-I. The 7-S and 8-S complexes did not form a new complex when mixed, but significant amounts of two histone pairs were assembled into a 12-S complex on mixing the (H2A + H2B)-AP-I complex (7-S) with free H3 + H4. In contrast, when the (H3 + H4) -AP-I complex ( 8 4 was incubated with free H2A + H2B, almost no assembly occurred, but the 7-S complex of H2A + H2B was newly formed. Binding studies by enzyme-linked immunosorbent assay showed that AP-I bound with H2A + H2B faster than with H3 + H4.From these results, it is suggested that AP-I has a higher binding affinity for histone H2A + H2B than for H3 + H4, and that the 7-S complex is an intermediate in formation of the 12-S octamer complex (H2A + H2BIn eukaryotic cells, DNA is bound with histones and organized in the nucleosome structure [I]. The four core histones are present in an octamer complex (H2A + H2B + H3 + H4)2 [2] around which 145 -165 base pairs of DNA are wrapped. In 2 M NaCl at neutral pH, the hstone octamers separate from DNA and are stable in these conditions [3]. When the salt concentration is decreased to 0.6M, the octamer dissociates into two H2A+H2B dimers and a (H3 + H4)2 tetramer. At lower ionic strength or low pH, these complexes dissociate further into histone monomers [3].Nucleosomes can be reconstituted in vitro in physiological ionic conditions by several methods. They can be assembled by direct mixing of histone and DNA under suitable conditions [4, 51, but several factors are known to facilitate their assemly [6, 71. Nucleoplasmin (29 kDa), which was first found in Xenopus eggs by Laskey et al. [S], facilitates nucleosome assembly in vitro by its interaction with core histones. Recently, Kleinschmidt et al. [9] demonstrated the existence of a histone-nucleoplasmin complex in nuclei of Xenopus oocytes. They also discovered another histone complex containing H3 and/or H4 tightly bound to one of a pair of very acidic peptides, designated as N1 and N2.We have purified a novel protein (AP-I, 53 kDa) from mammalian cells that facilitates nucleosome assembly. AP-I binds with core histones forming 6 -7-S and 12-S complexes [lo]. The 6 -7-S complex contains histone H2A + H2B and the 12-S complex consists of one histone octamer bound with a stoichiometric amount of AP-I. Nucleosomes could be formed by mixing the 12-S complex with DNA. In experiments using monoclonal antibodies against AP-I, we found Correspondence to F. Hanaoka, Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Tokyo, Japan 113Abbreviations. AP-I, nucleosome-assembly protein; SDS, sodium dodecyl sulfate ; ELISA, enzyme-linked immunosorbent assay.that AP-I exists as a 58-kDa peptide in vivo, not as a 53-kDa peptide, and that it is localized in the nucl...