Ran GTPase guanine nucleotide exchange factor RCC1 is phosphorylated on serine 11 by cdc2 kinase in vitro

RCC1, a guanine nucleotide exchange factor for Ran GTPase, plays essential roles in the growth and viability of mammalian cells. Here, we examined the phosphorylation of specific serine and threonine residues of RCC1 in vivo and showed that RCC1 is indeed phosphorylated. Analysis by two-dimensional (2D) gel electrophoresis suggested that serine 11 (S11) of hamster RCC1 is phosphorylated in vivo. A point mutation of S11 of hamster RCC1 resulted in a decrease in the number of 2D gel spots, indicating a lack of phosphorylation at the mutant residue. S11 phosphorylation in vitro depended on cyclin B-cdc2 kinase. An RCC1 mutant in which all N-terminal serine and threonine residues were substituted with glutamate residues to mimic phosphorylation at these residues showed decreased binding to the karyopherin, KPNA4, compared with wild type RCC1. We conclude that RCC1 undergoes post-translational phosphorylation.     

Nuclear c-Abl-mediated tyrosine phosphorylation induces chromatin structural changes through histone modifications that include H4K16 hypoacetylation

c-Abl tyrosine kinase, which is ubiquitously expressed, has three nuclear localization signals and one nuclear export signal and can shuttle between the nucleus and the cytoplasm. c-Abl plays important roles in cell proliferation, adhesion, migration, and apoptosis. Recently, we developed a pixel imaging method for quantitating the level of chromatin structural changes and showed that nuclear Src-family tyrosine kinases are involved in chromatin structural changes upon growth factor stimulation. Using this method, we show here that nuclear c-Abl induces chromatin structural changes in a manner dependent on the tyrosine kinase activity. Expression of nuclear-targeted c-Abl drastically increases the levels of chromatin structural changes, compared with that of c-Abl. Intriguingly, nuclear-targeted c-Abl induces heterochromatic profiles of histone methylation and acetylation, including hypoacetylation of histone H4 acetylated on lysine 16 (H4K16Ac). The level of heterochromatic histone modifications correlates with that of chromatin structural changes. Adriamycin-induced DNA damage stimulates translocation of c-Abl into the nucleus and induces chromatin structural changes together with H4K16 hypoacetylation. Treatment with trichostatin A, a histone deacetylase inhibitor, blocks chromatin structural changes but not nuclear tyrosine phosphorylation by c-Abl. These results suggest that nuclear c-Abl plays an important role in chromatin dynamics through nuclear tyrosine phosphorylation-induced heterochromatic histone modifications.     

Correlations between biological activity and the number of functional groups chemically modified

In interpreting the data obtained by chemical modification of biologically active macromolecules, e.g. enzymes, it has been a common practice to plot the remaining activity against the number of residues modified. An extrapolation of the initial, nearly linear portion of the curve generated to the axis giving numbers of residues often has been presumed to specify the actual number of critical groups modified. That this is usually not the case can be shown if one considers treatment of a typical macromolecule containing a number of a given kind of modifiable residues among which some, including those that are essential for activity, react at one rate, and the remainder react at a different rate. The behavior and character of the plots obtained have been analyzed and are presented with numerical and experimental examples to illustrate those features that should be considered.     

Genetic population structure of the eight-barbel loach Lefua echigonia in the Ise Bay region,a single paleo-river basin in central Honshu,Japan

Ichthyological Research - We examined the genetic population structure of Lefua echigonia in the Ise Bay region based on the nucleotide sequences of the mitochondrial DNA cytochrome b region....     

Rediscovery of Celes akitanus (Orthoptera: Acrididae) from semi‐natural grasslands in Japan

Celes akitanus was rediscovered in semi‐natural grasslands in Japan. This species is one of the most endangered insects in Japan; most local populations were thought to be extinct. Reliable records of this rare species had been obtained only from Yamagata, Niigata, Tokyo and Nagano Prefectures. We compared the morphology of the holotype of Oedipoda akitana (=C. akitanus) and newly collected specimens and found that they matched with each other. The species was redescribed based on the holotype and new specimens. A molecular phylogenetic analysis involving other acridid species indicated that our samples were practically identical to C. akitanus from China. We conclude that the specimens we collected are C. akitanus, which has been rediscovered in Japan after about 30 years. Because the discovered populations were restricted to narrow and scattered areas, conservation of these populations should be a high priority. Population monitoring and habitat assessment are required. Because C. akitanus and many other endangered species are found in the grassland areas, these habitats should be protected and maintained.     

Origin of eukaryotic cell nuclei by symbiosis of Archaea in Bacteria supported by the newly clarified origin of functional genes

In the previous report, we demonstrated the origin of eukaryotic cell nuclei as the symbiosis of Archaea in Bacteria by the newly developed "Homology-Hit Analysis". In that case, we counted yeast Open Reading Frames (ORFs) showing the highest similarity to a bacterial ORF as orthologous ORFs (Orthologous ORFs were produced by speciation from a common ancestor, and have the highest similarity to each other.) by comparing whole ORFs of yeast with those of individual bacteria. However, we could not count all yeast ORFs showing the highest similarity to a bacterial ORF in functional categories of yeast. Therefore, the origin of ORFs in the functional categories of yeast could not be inferred strictly. Here, we have improved the method for detecting orthologous ORFs. In this method, we count the numbers of ORF with the highest similarity between individual yeast functional categories and individual bacteria as orthologous ORFs. By this method, it was possible to detect the correct orthologous ORFs and to infer the origins of the functional categories in eukaryotic cells. As a result, two categories, assembly of protein complexes and DNA repair were newly judged to be of Archaeal origin, while five categories, lipid (fatty-acid and isoprenoid) metabolism, protein folding and stabilization, signal transduction, organization of the plasma membrane and organization of the cytoplasm, were newly judged to be of Bacterial origin. On the other hand, the origins of two categories (meiosis and cellular import, which were determined in the previous analysis) could not be judged. It is considered that functional categories related to the nucleus have origins common to Archaea, while those related to the cytoplasm have origins common to Bacteria. From these data including the origin of plasma membrane, it was further clarified that cell nucleus originated by the symbiosis of Archaea in Bacteria.     

Origin of eukaryotic cell nuclei by symbiosis of Archaea in Bacteria is revealed by homology-hit analysis

The origin of eukaryotic cell nuclei by symbiosis of Archaea in Bacteria was proposed on the basis of the phylogenetic topologies of genes. However, it was not possible to conclude whether or not the genes involved were authentic representative genes. Furthermore, using the BLAST and FASTA programs, the similarity of open reading frame (ORF) groups between three domains (Eukarya, Archaea and Bacteria) was estimated at one threshold. Therefore, their similarities at other thresholds could not be clarified. Here we use our newly developed 'homology-hit analysis' method, which uses multiple thresholds, to determine the origin of the nucleus. We removed mitochondria-related ORFs from yeast ORFs, and determined the number of yeast orthologous ORFs in each functional category to the ORFs in six Archaea and nine Bacteria at several thresholds (E-values) using the BLAST. Our results indicate that yeast ORFs related to the nucleus may share their origins with archaeal ORFs, whereas ORFs that are related to the cytoplasm may share their origins with bacterial ORFs. Our results thus strongly support the idea of nucleus symbiosis.     

Mathematical analysis of ligand-induced monomerization and dimerization in the monomer dimer equilibrium of proteins. Application to D-amino acid oxidase.

We have mathematically analyzed ligand-induced monomerization and dimerization in a protein monomer-dimer equilibrium system, in which the monomer has one and the dimer two binding sites. These dimer sites have the same binding constants for the first ligand but may cooperatively interact when one of them is occupied by a ligand molecule. In this system, the apparent dimerization constant and the apparent molecular weight are functions of free ligand concentration, and depend on the intrinsic binding constants of the ligand molecule to the monomer and the dimer. The behavior of these functions is classified into 17 cases according to the values of the three intrinsic binding constants, and some calculated examples are shown graphically for selected parameters. The theory was also applied to D-amino acid oxidase [EC 1.4.3.3], a flavoprotein, and the pH dependence of the apparent dimerization constant and the apparent molecular weight in the presence of ligand, p-aminobenzoate, were studied theoretically using parameters obtained in our previous experiments (5).     

Spermatophore displacement and male fertilization success in the ground beetle Carabus insulicola

Sperm displacement is an effective male mating tactic of spermcompetition to ensure paternity. However, the effectivenessof sperm displacement may depend on defensive mating tacticsemployed by rival males and also may be affected by females.To test this hypothesis, I conducted double-mating experimentswith microsatellite paternity analyses using the ground beetleCarabus insulicola. First, I examined how often spermatophoredisplacement by the second male occurs. Spermatophore depositionsuccess of the second male was high (85%) as was that of thefirst male (100%), in which 80% of the second males displacedthe first male's spermatophore, suggesting that spermatophoredisplacement is a highly effective offensive tactic of spermcompetition in C. insulicola. Second, I examined the effectsof postcopulatory guarding by the first male and remating intervaland sperm utilization pattern of the female on fertilizationsuccess of the second male. P₂ values were moderate on average(0.30–0.57) and negatively correlated with postcopulatoryguarding duration of the first male. Sperm use by the femalewas temporally nonrandom, such that sperm of the second maletended to be used first, although there was no significant effectof female-remating interval. These results suggest that theeffectiveness of spermatophore displacement depends on postcopulatoryguarding by the first male and the sperm utilization patternof the female. These findings provide empirical evidence forthe conflicting nature of selection for male offensive and defensivetactics in sperm competition and significant contribution offemales therein.