Characterization of a Thermostable 8-Oxoguanine DNA Glycosylase Specific for GO/N Mismatches from the Thermoacidophilic Archaeon Thermoplasma volcanium.

The oxidation of guanine (G) to 7,8-dihydro-8-oxoguanine (GO) forms one of the major DNA lesions generated by reactive oxygen species (ROS). The GO can be corrected by GO DNA glycosylases (Ogg), enzymes involved in base excision repair (BER). Unrepaired GO induces mismatched base pairing with adenine (A); as a result, the mismatch causes a point mutation, from G paired with cytosine (C) to thymine (T) paired with adenine (A), during DNA replication. Here, we report the characterization of a putative Ogg from the thermoacidophilic archaeon Thermoplasma volcanium. The 204-amino acid sequence of the putative Ogg (TVG_RS00315) shares significant sequence homology with the DNA glycosylases of Methanocaldococcus jannaschii (MjaOgg) and Sulfolobus solfataricus (SsoOgg). The six histidine-tagged recombinant TVG_RS00315 protein gene was expressed in Escherichia coli and purified. The Ogg protein is thermostable, with optimal activity near a pH of 7.5 and a temperature of 60°C. The enzyme displays DNA glycosylase, and apurinic/apyrimidinic (AP) lyase activities on GO/N (where N is A, T, G, or C) mismatch; yet it cannot eliminate U from U/G or T from T/G, as mismatch glycosylase (MIG) can. These results indicate that TvoOgg-encoding TVG_RS00315 is a member of the Ogg2 family of T. volcanium.

Apoptosis occurs during early development of the bursa of Fabricius in chicken embryos.

The bursa of Fabricius (BF) is a unique primary lymphoid organ, and among vertebrates is unique to birds. Despite its importance to the immune systems of various avian species, little is known of the molecular mechanisms underlying early BF development. In the present study, we demonstrated that apoptosis occurs during early development of the bursa of Fabricius in chicken embryos. Initial histological analyses of BF morphogenesis in chicken embryos led to the hypothesis that formation of the bursal lumen correlates with fusion of vacuoles, which appear in the cloacal epithelial bud. Using terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) analysis and immunostaining with an anti-cleaved (activated) caspase-3 antibody, we detected multiple apoptotic cells around these vacuoles. In further experiments, treatments with a caspase inhibitor caused abnormal bursal lumen in vivo. The present data indicate that apoptosis may play important roles in BF morphogenesis in chickens.

Specificity of Fur binding to the oxidative stress response gene promoter in the facultative anaerobic archaeon Thermoplasma volcanium.

The genome of the facultative anaerobic thermoacidophilic archaeon Thermoplasma volcanium contains the open-reading frames (ORFs) tvsod and tvogg, which are predicted to encode a putative superoxide dismutase and an 8-oxoguanine DNA glycosylase, respectively. Tvsod is immediately upstream of tvogg, and these two ORFs are aligned in a head-to-tail manner in a single operon. A previous study showed that T. volcanium contains an ORF (TVN0292) encoding the ferric uptake regulator (Fur) and that the T. volcanium Fur protein (TvFur) binds to its own promoter in a metal-dependent manner in vitro. Here, we demonstrated that TvFur also binds to the tvsod-tvogg promoter and determined the TvFur-binding sequences in the tvsod-tvogg promoter by DNaseI footprinting analysis. These results suggest that Fur is required for resistance against reactive oxygen species in this facultative anaerobic archaeon.

Age-related changes of forkhead transcription factor FOXO1 in the liver of senescence-accelerated mouse SAMP8.

SAMP8 exhibits accelerated aging and a short lifespan. Insulin-like growth factor-1 receptor (IGF-1R)/FOXO pathway is associated with aging. Phosphorylation of IGF-1R, Akt, and FOXO1 was found to be increased during aging in the liver of SAMR1 normal aging mice. However, significant decreases in the phosphorylation of IGF-1R and Akt were observed in the liver of SAMP8 during aging compared with that in SAMR1, whereas phosphorylation of FOXO1 was markedly increased with age in SAMP8. In addition, the protein level of FOXO1 was decreased with age in SAMP8. Protein phosphatase 2A (PP2A) directly dephosphorylates FOXO1. Significant reduction of PP2A activity was observed in the liver nucleus of SAMP8. These results suggest the possibility that the increased FOXO1 phosphorylation might occur by the decreased activity of PP2A, resulting in the decrease in the protein level of FOXO1 in SAMP8. Furthermore, FOXO1 regulates longevity and the expression of antioxidant enzymes such as Mn-SOD and catalase. The expression of Mn-SOD and catalase was significantly decreased in the liver of SAMP8. Therefore, it is possible that the elevation of phosphorylated FOXO1 level with age causes a short lifespan in SAMP8.

Transcriptional factor fur from Thermoplasma volcanium binds its own promoter DNA in a divalent cation-dependent manner.

Because archaea possess many respiratory enzymes or radical scavengers with catalytic domains that contain iron, the expression of the genes encoding these enzymes might be regulated by iron acquisition. The genome of an archaeon, Thermoplasma volcanium contains a gene that encodes Fur (TVN0292). The fur gene of T. volcanium was amplified by PCR, and cloned into plasmid pET28a. TvFur (T. volcanium Fur protein) was expressed in E. coli cells and then purified. EMSA revealed that TvFur binds to its own promoter DNA. The binding to its own promoter was in an Mn(2+)-, Zn(2+)-, and Ni(2+)-dependent manner. DNase I footprinting analysis revealed that the binding sequence of tvfur promoter was 5'-G TTATTAT G TTTATAT A TTAATTA G-3'. An analysis utilizing oligonucleotides in TvFur-binding sequences revealed that TvFur binds to the TATA-box or regions in the vicinity of the TATA-box in the promoter. These results indicated that TvFur regulates transcription depending on the availability of environmental divalent cations.

Body plan of turtles: an anatomical, developmental and evolutionary perspective.

The evolution of the turtle shell has long been one of the central debates in comparative anatomy. The turtle shell consists of dorsal and ventral parts: the carapace and plastron, respectively. The basic structure of the carapace comprises vertebrae and ribs. The pectoral girdle of turtles sits inside the carapace or the rib cage, in striking contrast to the body plan of other tetrapods. Due to this topological change in the arrangement of skeletal elements, the carapace has been regarded as an example of evolutionary novelty that violates the ancestral body plan of tetrapods. Comparing the spatial relationships of anatomical structures in the embryos of turtles and other amniotes, we have shown that the topology of the musculoskeletal system is largely conserved even in turtles. The positional changes seen in the ribs and pectoral girdle can be ascribed to turtle-specific folding of the lateral body wall in the late developmental stages. Whereas the ribs of other amniotes grow from the axial domain to the lateral body wall, turtle ribs remain arrested axially. Marginal growth of the axial domain in turtle embryos brings the morphologically short ribs in to cover the scapula dorsocaudally. This concentric growth appears to be induced by the margin of the carapace, which involves an ancestral gene expression cascade in a new location. These comparative developmental data allow us to hypothesize the gradual evolution of turtles, which is consistent with the recent finding of a transitional fossil animal, Odontochelys, which did not have the carapace but already possessed the plastron.

Hepatocyte growth factor is crucial for development of the carapace in turtles.

Turtles are characterized by their shell, composed of a dorsal carapace and a ventral plastron. The carapace first appears as the turtle-specific carapacial ridge (CR) on the lateral aspect of the embryonic flank. Accompanying the acquisition of the shell, unlike in other amniotes, hypaxial muscles in turtle embryos appear as thin threads of fibrous tissue. To understand carapacial evolution from the perspective of muscle development, we compared the development of the muscle plate, the anlage of hypaxial muscles, between the Chinese soft-shelled turtle, Pelodiscus sinensis, and chicken embryos. We found that the ventrolateral lip (VLL) of the thoracic dermomyotome of P. sinensis delaminates early and produces sparse muscle plate in the lateral body wall. Expression patterns of the regulatory genes for myotome differentiation, such as Myf5, myogenin, Pax3, and Pax7 have been conserved among amniotes, including turtles. However, in P. sinensis embryos, the gene hepatocyte growth factor (HGF), encoding a regulatory factor for delamination of the dermomyotomal VLL, was uniquely expressed in sclerotome and the lateral body wall at the interlimb level. Implantation of COS-7 cells expressing a HGF antagonist into the turtle embryo inhibited CR formation. We conclude that the de novo expression of HGF in the turtle mesoderm would have played an innovative role resulting in the acquisition of the turtle-specific body plan.

Evolution of the turtle body plan by the folding and creation of new muscle connections.

The turtle shell offers a fascinating case study of vertebrate evolution, based on the modification of a common body plan. The carapace is formed from ribs, which encapsulate the scapula; this stands in contrast to the typical amniote body plan and serves as a key to understanding turtle evolution. Comparative analyses of musculoskeletal development between the Chinese soft-shelled turtle and other amniotes revealed that initial turtle development conforms to the amniote pattern; however, during embryogenesis, lateral rib growth results in a shift of elements. In addition, some limb muscles establish new turtle-specific attachments associated with carapace formation. We propose that the evolutionary origin of the turtle body plan results from heterotopy based on folding and novel connectivities.

Origins of protein stability revealed by comparing crystal structures of TATA binding proteins.

The crystal structure of TATA binding protein (TBP) from a mesothermophilic archaeon, Sulfolobus acidocaldarius, has been determined at a resolution of 2.0 A with an R factor of 20.9%. By comparing this structure with the structures of TBPs from a hyperthermophilic archaeon and mesophilic eukaryotes, as well as by comparing amino acid sequences of TBPs from archaea, covering a wide range of optimum growth temperatures, two significant determinants of the stability of TBP have been identified: increasing the interior hydrophobicity by interaction between three residues, Val, Leu, and Ile, with further differentiation of the surface, and increasing its hydrophilicity and raising the cost of unfolding. These findings suggest directions along which the stability of TBP can be engineered.