The use of Quantitative Agarose Gel Electrophoresis for rapid analysis of the integrity of protein-DNA complexes

Recent biochemical studies evaluated the affinity of histones to DNA in the context of nucleosome core particle (NCP). These have indicated a concentration-dependence for nucleosome stability. However, when studying chromatin the preferred templates are nucleosome arrays (NA) and not the NCP. Biochemical methods are poorly suited for structural analysis of chromatin. To overcome that technical hindrance, and investigate the effect of concentration on stability of the histone-DNA interactions, we have applied the multigel Quantitative Agarose Gel Electrophoresis (QAGE) method to in vitro-assembled nucleosomal arrays. The results demonstrated the method to be extremely valuable for the evaluation of the effect of low concentration on NA. However, QAGE is a fairly time-demanding and complex method. To maximize the efficiency of use of this technology, we devised a protocol that allowed for multiple sets of templates to be analyzed simultaneously. Briefly, samples can be loaded at regular intervals and analyzed individually for their molecular composition. The technique presented in this study describes the calibration steps and proof of concept necessary to validate the use of multiple loading of multigel to evaluate the composition of nucleosomal arrays as a function of concentration.     

An Asian Origin for Subtype IV BK Virus Based on Phylogenetic Analysis

Similarly to other members of the Polyomaviridae family, BK virus (BKV) is thought to have co-evolved with its human host. BKV has four subtypes that are distinguishable by immunological reactivity, with two (subtypes I and IV) being most prevalent in human populations. Subtype I is the major subtype worldwide, whereas subtype IV is prevalent in East Asia and Europe but rare in Africa. The geographic distribution pattern of subtype IV BKV is in apparent disagreement with the hypothesis that BKV co-evolved with humans, since subtype IV rarely occurs in Africa. To elucidate the origin of subtype IV, 53 complete subtype IV sequences derived from East Asians and Europeans were subjected to a detailed phylogenetic analysis using the maximum-likelihood and neighbor-joining methods. We identified six subgroups (a1, a2, b1, b2, c1, and c2) that formed a tree represented by the formula: “(a1, a2), ((b1, b2), (c1, c2)).” Interestingly, we found a close correlation between subtype IV subgroups and population geography; thus, all subgroups except c2 were prevalent in particular East Asian populations, with c2 occurring in both Europe and Northeast Asia. From these findings, we conclude that subtype IV of BKV now prevalent in modern humans is derived from a virus that infected ancestral Asians. We introduce two hypotheses to explain how ancestral Asians became infected with subtype IV BKV. [Reviewing Editor: Dr. Joshua Plotkin] Yuriko Nishimoto and Huai-Ying Zheng are two authors contributed equally to this article.     

A most distant intergeneric hybrid offspring (Larcon) of lesser apes, <Emphasis Type="Italic">Nomascus leucogenys</Emphasis> and <Emphasis Type="Italic">Hylobates lar</Emphasis>

Unlike humans, which are the sole remaining representatives of a once larger group of bipedal apes (hominins), the “lesser apes” (hylobatids) are a diverse radiation with numerous extant species. Consequently, the lesser apes can provide a valuable evolutionary window onto the possible interactions (e.g., interbreeding) of hominin lineages coexisting in the same time and place. In the present work, we employ chromosomal analyses to verify the hybrid ancestry of an individual (Larcon) produced by two of the most distant genera of lesser apes, Hylobates (lar-group gibbons) and Nomascus (concolor-group gibbons). In addition to a mixed pelage pattern, the hybrid animal carries a 48-chromosome karyotype that consists of the haploid complements of each parental species: Hylobates lar (n = 22) and Nomascus leucogenys leucogenys (n = 26). Studies of this animal’s karyotype shed light onto the processes of speciation and genus-level divergence in the lesser apes and, by extension, across the Hominoidea.     

Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana

Receptor-like kinases (RLK) comprise a large gene family within the Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. Here we report that a leucine-rich repeat receptor-like kinase (LRR-RLK), RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), is a key regulator of anther development in Arabidopsis. Two RPK2 T-DNA insertional mutants (rpk2-1 and rpk2-2) displayed enhanced shoot growth and male sterility due to defects in anther dehiscence and pollen maturation. The rpk2 anthers only developed three cell layers surrounding the male gametophyte: the middle layer was not differentiated from inner secondary parietal cells. Pollen mother cells in rpk2 anthers could undergo meiosis, but subsequent differentiation of microspores was inhibited by tapetum hypertrophy, with most resulting pollen grains exhibiting highly aggregated morphologies. The presence of tetrads and microspores in individual anthers was observed during microspore formation, indicating that the developmental homeostasis of rpk2 anther locules was disrupted. Anther locules were finally crushed without stomium breakage, a phenomenon that was possibly caused by inadequate thickening and lignification of the endothecium. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated throughout anther development in rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate by triggering subsequent tapetum degradation, and that mutating RPK2 impairs normal pollen maturation and anther dehiscence due to disruption of key metabolic pathways.     

Relationships between BK virus lineages and human populations

BK polyomavirus (BKV) is ubiquitous in human populations, infecting children asymptomatically and then persisting in the kidney, in which it can cause nephropathy in renal transplant patients. BKV isolates are classified into four subtypes (I-IV) using serological or genotyping methods, and subtype I is further divided into four subgroups, Ia, Ib-1, Ib-2, and Ic, based on DNA sequence variations. To clarify whether there is an association between BK virus lineages and human populations, we examined BKV-positive urine samples collected from immunocompetent individuals at various locations in Europe, Africa, and Asia. Partial BKV DNA sequences (n=299) in these samples were determined and subjected to phylogenetic and single nucleotide polymorphism analysis to classify BKV isolates around the world. The validity of the classification was confirmed by analyses based on complete BKV DNA sequences. Subtype I was the major subtype throughout the studied regions, and subtype IV was prevalent only in Asia and Europe. Subtype-I subgroups showed close relationships to major geographical areas. It has recently been shown that JC virus (a human polyomavirus closely related to BKV) co-evolved with human populations, and the present study thus suggests that host-linked evolution is the general mode of polyomavirus evolution. Additionally, our results indicate certain unique aspects of the relationship between BKV and humans.     

Brain induction in ascidian embryos is dependent on juxtaposition of FGF9/16/20-producing and -receiving cells

Coordinated regulation of inductive events, both spatially and temporally, during animal development ensures that tissues are induced at their specific positions within the embryo. The ascidian brain is induced in cells at the anterior edge of the animal hemisphere by fibroblast growth factor (FGF) signals secreted from vegetal cells. To clarify how this process is spatially regulated, we first identified the sources of the FGF signal by examining the expression of brain markers Hr-Otx and Hr-ETR-1 in embryos in which FGF signaling is locally inhibited by injecting individual blastomeres with morpholino oligonucleotide against Hr-FGF9/16/20, which encodes an endogenous brain inducer. The blastomeres identified as the inducing sources are A5.1 and A5.2 at the 16-cell stage and A6.2 and A6.4 at the 24-cell stage, which are juxtaposed with brain precursors at the anterior periphery of the embryo at the respective stages. We also showed that all the cells of the animal hemisphere are capable of expressing Hr-Otx in response to the FGF signal. These results suggest that the position of inducers, rather than competence, plays an important role in determining which animal cells are induced to become brain tissues during ascidian embryogenesis. This situation in brain induction contrasts with that in mesoderm induction, where the positions at which the notochord and mesenchyme are induced are determined mainly by intrinsic competence factors that are inherited by signal-receiving cells.     

Biochemical investigation and gene expression analysis of the immunostimulatory functions of an edible Salacia extract in rat small intestine

Roots and bark from plants belonging to genus Salacia of the family Hippocrateaceae (Salacia reticulata, Salacia oblonga, etc.) have been used for traditional Ayurvedic medicine, particularly for the treatment of diabetes. In our study, we evaluated the gene expression profiles in the small intestinal epithelium of rats that were given a Salacia plant extract to gain insight into its effects on the small intestine. In detail, DNA microarray analysis was performed to evaluate the gene expression profiles in the rat ileal epithelium. The intestinal bacterial flora was also studied using T-RFLP (Nagashima method) in these rats. Expressions of many immune-related genes, especially Th1-related genes associated with cell-mediated immunity, were found to increase in the small intestinal epithelium and the intestinal bacterial flora became similar to those in the case with Salacia plant extract administration. Our study thus revealed that Salacia plant extract exerts bioregulatory functions by boosting intestinal immunity.