Histochemical study of detailed laticifer structure and rubber biosynthesis-related protein localization in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> using spectral confocal laser scanning microscopy

In Hevea brasiliensis, laticifers produce and accumulate rubber particles. Despite observation using histochemical methods, development stage structure and structures with ceasing functions have rarely been described. Spectral confocal laser scanning microscopy with Nile red staining simplifies laticifer structure observation in tangential sections while enhancing the resolution. Laticifer and ray images were extracted from unmixed images and used to monitor changes during growth. A laticifer network structure developed from increased anastomoses between adjoining laticifers outside of the conducting phloem, but because of increased radial division and growth of rays, the network structure ruptured and disintegrated. We also investigated immunohistochemical localization of two rubber particle-associated proteins in the laticifers: small rubber particle protein (SRPP) and rubber elongation factor (REF). Mature bark test results show that SRPP is localized only in the laticifer layers in the conducting phloem; REF is localized in all laticifer layers. Because SRPP plays a positive role in rubber biosynthesis, results show that the rubber biosynthesis capability of laticifers is concentrated where rays and the sieve tube actively transport metabolites.     

A novel application of metabolomics in vertebrate development

Many studies have demonstrated the functions of individual genes associated with embryogenesis and have determined the genome sequences of several organisms. Despite the availability of enormous amount of genetic information, dynamic changes that occur during embryogenesis have not yet been completely understood. In order to understand the dynamic processes involved in embryogenesis, we employed the metabolomic approach. The results of our study indicated that there is a close correlation between metabolomes and developmental stages. Our method enables the identification of embryonic stages using metabolomes as “fingerprints.” In this manner, we could successfully predict embryonic development on the basis of metabolomic fingerprints. This is the first report describing a model for predicting vertebrate development by using metabolomics.     

CrxOS maintains the self-renewal capacity of murine embryonic stem cells

Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiated morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.     

Temperature-sensitive cloning vector for Corynebacterium glutamicum

We constructed a temperature-sensitive form of the Corynebacterium glutamicum ATCC13869 cryptic plasmid, pBL1. The C. glutamicum/Escherichia coli shuttle vector pSFK6, which is composed of pBL1 and the E. coli cloning vector pK1, was mutagenized in vitro by treatment with hydroxylamine, and introduced into C. glutamicum cells. A mutant plasmid, which was stably maintained at 25 degrees C but not at 34 degrees C, was isolated from the cells. Sequencing the plasmid, which was named p48K, revealed four substitutions in the Rep protein coding region. Moreover, site-directed single-nucleotide substitutions showed that a G to A transition at position 2,920, which resulted in a Pro-47 to Ser substitution in the Rep protein, was responsible for its temperature-sensitive replication. Pro-47 is conserved among the Rep proteins of the pIJ101/pJV1 family of plasmids. This temperature-sensitive cloning vector will be useful for disrupting genes in this industrially important bacterium.     

A novel Rad18 function involved in protection of the vertebrate genome after exposure to camptothecin

In Saccharomyces cerevisiae, Rad18 functions in post-replication repair pathways, such as error-free damage bypass involving Rad30 (Poleta) and error-prone damage bypass involving Rev3/7 (Polzeta). Chicken DT40 RAD18(-/-) cells were found to be hypersensitive to camptothecin (CPT), while RAD30(-/-) and REV3(-/-) cells, which are defective in translesion DNA synthesis, were not. RAD18(-/-) cells also showed higher levels of H2AX phosphorylation and chromosomal aberrations, particularly chromosomal gaps and breaks, upon exposure to CPT. Detailed analysis by alkaline sucrose density gradient centrifugation revealed that RAD18(-/-) and wild type cells exhibited similar rates of elongation of newly synthesized DNA in the presence or absence of low concentrations of CPT but that DNA breaks frequently occurred on both parental and nascent strands within 1h after a brief exposure to an elevated concentration of CPT, with more breaks induced in RAD18(-/-) cells than in wild type cells. These data suggest a previously unanticipated role for Rad18 in dealing with replication forks upon encountering DNA lesions induced by CPT.     

Differential usage of non-homologous end-joining and homologous recombination in double strand break repair

Repair of DNA double strand breaks (DSBs) plays a critical role in the maintenance of the genome. DSB arise frequently as a consequence of replication fork stalling and also due to the attack of exogenous agents. Repair of broken DNA is essential for survival. Two major pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to deal with these lesions, and are conserved from yeast to vertebrates. Despite the conservation of these pathways, their relative contribution to DSB repair varies greatly between these two species. HR plays a dominant role in any DSB repair in yeast, whereas NHEJ significantly contributes to DSB repair in vertebrates. This active NHEJ requires a regulatory mechanism to choose HR or NHEJ in vertebrate cells. In this review, we illustrate how HR and NHEJ are differentially regulated depending on the phase of cell cycle and on the nature of the DSB.     

Co-pigmentation and flavonoid glycosyltransferases in blue Veronica persica flowers

Glycosylation is one of the key modification steps for plants to produce a broad spectrum of flavonoids with various structures and colors. A survey of flavonoids in the blue flowers of Veronica persica Poiret (Lamiales, Scrophulariaceae), which is native of Eurasia and now widespread worldwide, led to the identification of highly glycosylated flavonoids, namely delphinidin 3-O-(2-O-(6-O-p-coumaroyl-glucosyl)-6-O-p-coumaroyl-glucoside)-5-O-glucoside (1) and apigenin 7-O-(2-O-glucuronosyl)-glucuronide (2), as two of its main flavonoids. Interestingly, the latter flavone glucuronide (2) caused a bathochromic shift on the anthocyanin (1) toward a blue hue in a dose-dependent manner, showing an intermolecular co-pigment effect. In order to understand the molecular basis for the biosynthesis of this glucuronide, we isolated a cDNA encoding a UDP-dependent glycosyltransferase (UGT88D8), based on the structural similarity to flavonoid 7-O-glucuronosyltransferases (F7GAT) from Lamiales plants. Enzyme assays showed that the recombinant UGT88D8 protein catalyzes the 7-O-glucuronosylation of apigenin and its related flavonoids with preference to UDP-glucuronic acid as a sugar donor. Furthermore, we identified and functionally characterized a cDNA encoding another UGT, UGT94F1, as the anthocyanin 3-O-glucoside-2″-O-glucosyltransferase (A3Glc2″GlcT), according to the structural similarity to sugar-sugar glycosyltransferases classified to the cluster IV of flavonoid UGTs. Preferential expression of UGT88D8 and UGT94F1 genes in the petals supports the idea that these UGTs play an important role in the biosynthesis of key flavonoids responsible for the development of the blue color of V. persica flowers.     

Simultaneous Disruption of Two DNA Polymerases,Polη and Polζ, in Avian DT40 Cells Unmasks the Role of Polη in Cellular Response to Various DNA Lesions

Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase η, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase ζ by generating POLη^−/−/POLζ^−/− cells from the chicken DT40 cell line. POLζ^−/− cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV) only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that Polη plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POLη^−/−/POLζ^−/− cells shows that, unexpectedly, the loss of Polη significantly rescued all mutant phenotypes of POLζ^−/− cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, Polη contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells.     

DNA as a ‘Nanomaterial’

DNA is recognized as a nanomaterial, not as a biological material, in the research field of nanotechnology. This article reviews recent research on nanowires, nanoarchitectures, computing, aptamers, biocatalysts, devices, and machines using DNA. In these works, the characteristics of DNA including facile synthesis by the solid-phase method, self-assembly, electro-conductivity, information elements, amplification, switching, molecular recognition, and catalytic functions, were appropriately applied. Multiple functions of DNA could be used simultaneously, and activated independently, by molecular switching. Therefore, the combinations of functional sequences of DNA can produce unique materials. It is obvious that the DNA molecule is one of the most promising functional nanomaterials. However, the application of DNA molecules is still under study because of the big gap that exists between theory and practice. We eagerly anticipate a ‘coming out’ of DNA due to breakthroughs in nanobiotechnology.