Kank proteins: a new family of ankyrin-repeat domain-containing proteins

The human Kank gene was found as a candidate tumor suppressor for renal cell carcinoma, and encodes an ankyrin-repeat domain-containing protein, Kank. Here, we report a new family of proteins consisting of three Kank (Kank1)-associated members, Kank2, Kank3 and Kank4, which were found by domain and phylogenetic analyses. Besides the conserved ankyrin-repeat and coiled-coil domains, there was a conserved motif at the N-terminal (KN motif) containing potential motifs for nuclear localization and export signals. Gene expression of these genes was examined by RT-PCR at the mRNA level and by Western blotting and immunostaining at the protein level. Kank family genes showed variations in the expression level among tissues and kidney cell lines. Furthermore, the results of overexpression of these genes in NIH3T3 cells suggest that all of these family proteins have an identical role in the formation of actin stress fibers.     

Dinucleosome DNA of human K562 cells: experimental and computational characterizations

Dinucleosome formation is the first step in the organization of the higher order chromatin structure. With the ultimate aim of elucidating the dinucleosome structure, we constructed a library of human dinucleosome DNA. The library consists of PCR-amplifiable DNA fragments obtained by treatment of nuclei of erythroid K562 cells with micrococcal nuclease followed by extraction of DNA and adaptor ligation to the blunt-ended DNA fragments. The library was then cloned using a plasmid vector and the sequences of the clones were determined. The dominating clones containing the Alu elements were removed. A total of 1002 clones, which comprised a dinucleosome database, contained 84 and 918 clones from the clones before and after removing Alu elements, respectively. Approximately 70% of the clones were between 300 and 400 bp in size and they were distributed to various locations of all chromosomes except the Y chromosome. The clones containing A(2)N(8)A(2)N(8)A(2) or T(2)N(8)T(2)N(8)T(2) sequences were classified into three types, Type I (N shape), Type II (V shape) and Type III (M shape) according to DNA curvature plots. The locations of experimentally determined curved DNA segments matched well with the calculated ones though the clones of Types I and III showed additional curved DNA segments as revealed by the curvature plots. The distributions of complementary dinucleotides in the nucleosome DNA, at the ends of the dinucleosome DNA clones, allowed us to predict the positions of the nucleosome dyad axis, and estimate the size of the nucleosome core DNA, 125nt. The distributions of AA and TT dinucleotides, as well as other RR and YY dinucleotides, showed a periodicity with an average period of 10.4 bases, close to the values observed before. Mapping of nucleosome positions in the dinucleosome database based on the observed periodicity revealed that the nucleosomes were separated by a linker of 7.5+ approximately 10 x n nt. This indicates that the nucleosome-nucleosome orientations are, typically, halfway between parallel and antiparallel. Also an important finding is that the distributions of AA/TT and other RR/YY dinucleotides, apparently, reflect both DNA curvature and DNA bendability, cooperatively contributing to the nucleosome formation.     

Application of Fluolid-Orange-labeled probes for DNA microarray and immunological assays

The usefulness of Fluolid-Orange, a novel fluorescent dye, for DNA microarray and immunological assays has been examined. Fluolid-Orange-labeled probes (DNA and IgG) were stable as examined by laser-photo-bleaching and under heat and dry conditions. Statistical analyses were performed to evaluate the reproducibility of the microarray assay, while stage-specific immunostaining of marker proteins, Kank1 and calretinin, was performed for renal cancers, both giving satisfactory results. The stability of the dye should provide advantages for storing fluorescently labeled probes and re-examining the specimens later in genetic and pathological diagnostics.     

Expression profiling of the estrogen responsive genes in response to phytoestrogens using a customized DNA microarray

Here, we examined phytoestrogens, isoflavones (genistein, daidzein, glycitein, biochanin A and ipriflavone), flavones (chrysin, luteolin and apigenin), flavonols (kaempferol and quercetin), and a coumestan, a flavanone and a chalcone (coumestrol, naringenin and phloretin, respectively) by means of a DNA microarray assay. A total of 172 estrogen responsive genes were monitored with a customized DNA microarray and their expression profiles for the above phytoestrogens were compared with that for 17beta-estradiol (E2) using correlation coefficients, or R values, after a correlation analysis by linear regression. While R values indicate the similarity of the response by the genes, we also examined the genes by cluster analysis and by their specificity to phytoestrogens (specific to genistein, daidzein or glycitein) or gene functions. Several genes were selected from p53-related genes (CDKN1A, TP53I11 and CDC14), Akt2-related genes (PRKCD, BRCA1, TRIB3 and APPL), mitogen-activated protein kinase-related genes (RSK and SH3BP5), Ras superfamily genes (RAP1GA1, RHOC and ARHGDIA) and AP-1 family and related genes (RIP140, FOS, ATF3, JUN and FRA2). We further examined the extracts from two local crops of soy beans (Kuro-daizu or Mochi-daizu) by comparing the gene expression profiles with those of E2 or phytoestrogens as a first step in utilizing the expression profiles for various applications.     

Sequence structure of human nucleosome DNA

Positional distributions of various dinucleotides in experimentally derived human nucleosome DNA sequences are analyzed. Nucleosome positioning in this species is found to depend largely on GG and CC dinucleotides periodically distributed along the nucleosome DNA sequence, with the period of 10.4 bases. The GG and CC dinucleotides oscillate counterphase, i.e., their respective preferred positions are shifted about a half-period from one another, as it was observed earlier for AA and TT dinucleotides. Other purine-purine and pyrimidine-pyrimidine dinucleotides (RR and YY) display the same periodical and counterphase pattern. The dominance of oscillating GG and CC dinucleotides in human nucleosomes and the contribution of AG(CT), GA(TC), and AA(TT) suggest a general nucleosome DNA sequence pattern - counterphase oscillation of RR and YY dinucleotides. AA and TT dinucleotides, commonly accepted as major players, are only weak contributors in the case of human nucleosomes.     

Prevalence of Cardinium Bacteria in Planthoppers and Spider Mites and Taxonomic Revision of “Candidatus Cardinium hertigii” Based on Detection of a New Cardinium Group from Biting Midges

Cardinium bacteria, members of the phylum Cytophaga-Flavobacterium-Bacteroides (CFB), are intracellular bacteria in arthropods that are capable of inducing reproductive abnormalities in their hosts, which include parasitic wasps, mites, and spiders. A high frequency of Cardinium infection was detected in planthoppers (27 out of 57 species were infected). A high frequency of Cardinium infection was also found in spider mites (9 out of 22 species were infected). Frequencies of double infection by Cardinium and Wolbachia bacteria (Alphaproteobacteria capable of manipulating reproduction of their hosts) were disproportionately high in planthoppers but not in spider mites. A new group of bacteria, phylogenetically closely related to but distinct from previously described Cardinium bacteria (based on 16S rRNA and gyrB genes) was found in 4 out of 25 species of Culicoides biting midges. These bacteria possessed a microfilament-like structure that is a morphological feature previously found in Cardinium and Paenicardinium. The bacteria close to the genus Cardinium consist of at least three groups, A, B, and C. Group A is present in various species of arthropods and was previously referred to as “Candidatus Cardinium hertigii,” group B is present in plant parasitic nematodes and was previously referred to as “Candidatus Paenicardinium endonii,” and group C is present in Culicoides biting midges. On the basis of morphological and molecular data, we propose that the nomenclature of these three groups be integrated into a single species, “Candidatus Cardinium hertigii.”Compared to the Wolbachia bacteria, which belong to the alpha subdivision of the phylum Proteobacteria and are known as master manipulators of arthropod reproduction (48), the Cardinium bacteria, which belong to the phylum Cytophaga-Flavobacterium-Bacteroides (CFB), are relatively new to biological study. The phylum CFB includes many other bacteria associated with arthropods, such as symbionts in cockroaches (3) and termites (4) and the male-killing agents of ladybird beetles (21). Cardinium was first observed in tick cell cultures as an unknown intracellular prokaryote that was rod shaped and had an array of tubes extending from the cytoplasmic membrane (22). In 2001, related symbiotic bacteria were reported as manipulators of arthropod reproduction because they caused feminization, by which genetic males were converted into phenotypic females, in the false spider mite Brevipalpas obovatus (45) and parthenogenesis, in which haploid eggs were converted into viable diploid females, in the parasitoid wasp Encarsia pergandiella (50). Since the 16S rRNA gene sequences of these bacteria exhibited 96% to 98% similarity to the tick microorganism, they were classified in the phylum CFB. Subsequently, bacteria in this group were found to induce cytoplasmic incompatibility (CI), in which uninfected female hosts produce few offspring when mated with infected males in parasitic wasps of the genus Encarsia (20) and in two spider mites, Eotetranychus suginamensis and Bryobia sarothamni (14, 35). These bacteria were arbitrarily called CFB or Cytophaga-like organisms in earlier studies until the scientific name of “Candidatus Cardinium hertigii” was proposed by Zchori-Fein et al. (52). Since then, the bacteria have often been referred to as Cardinium for convenience. Recently, a bacterium related to Cardinium was found in plant parasitic nematodes, for which the scientific name “Candidatus Paenicardinium endonii” was proposed (31).Three independent studies have shown that rates of Cardinium infection were consistently low in wide samplings of arthropods, i.e., 7.2% of 223 species (46), 6% of 99 species (51), and 4.4% of 136 species (11). However, the infection frequencies in mites and spiders were 31.6% (46) and 22% (12), respectively. Cardinium has previously been detected only in hymenopteran insects (20, 25, 46, 50, 51), hemipteran insects (6, 24, 37, 46, 51), mites (13, 14, 15, 19, 45, 46), and spiders (11, 12). Infection by Wolbachia, another group of bacteria belonging to the Alphaproteobacteria that are capable of manipulating arthropod reproduction, is more widespread among arthropods. A recent meta-analysis of published data on Wolbachia infection surveys demonstrated that the proportion of insect species with at least one infected individual is around 66% (16). Other arthropods, such as wood lice, spiders, and mites, are also infected with Wolbachia. Outside of arthropods, Wolbachia infection has been detected in filarial nematodes (2, 23). Compared to Wolbachia, Cardinium organisms have been found in more restricted taxonomic groups (11, 46, 51).In this study, we performed PCR-based screening of various species of planthoppers (Hemiptera: Fulgoroidea), spider mites (Acari: Tetranychidae), and Culicoides biting midges (Diptera: Ceratopogonidae) for Cardinium infection by using primers that detect bacteria closely related to Cardinium. The frequencies of Cardinium infection were considerably higher in planthoppers and spider mites. In Culicoides biting midges, which are important vectors of arthropod-borne viruses pathogenic to livestock (27), some species were infected with Cardinium-like bacteria that had lower nucleotide sequence similarity to other Cardinium species, including those previously found in arthropods. Morphological characteristics and molecular phylogenetic analyses of these bacteria are reported, and their taxonomic classification is reconsidered.