Molecular phylogeny and evolution of prosimians based on complete sequences of mitochondrial DNAs

Prosimians (tarsiers and strepsirrhini) represent the basal lineages in primates and have a close bearing on the origin of primates. Although major lineages among anthropoidea (humans, apes and monkeys) are well represented by complete mitochondrial DNA (mtDNA) sequence data, only one complete mtDNA sequence from a representative of each of the infraorders in prosimians has been described until quite recently, and therefore we newly determined complete mtDNA sequences from 5 lemurs, 4 lorises, one tarsier and one platyrrhini. These sequences were provided to phylogenetic analyses in combination with the sequences from the 15 primates species reported to the database. The position of tarsiers among primates could not be resolved by the maximum likelihood (ML) and neighbor-joining (NJ) analyses with several data sets. As to the position of tarsiers, any of the three alternative topologies (monophyly of haplorhini, monophyly of prosimians, and tarsiers being basal in primates) was not rejected at the significance level of 5%, neither at the nucleotide nor at the amino acid level. In addition, the significant variations of C and T compositions were observed across primates species. Furthermore, we used AGY data sets for phylogenetic analyses in order to remove the effect of different C/T composition bias across species. The analyses of AGY data sets provided a medium support for the monophyly of haplorhini, which might have been screened by the variation in base composition of mtDNA across species. To estimates the speciation dates within primates, we analyzed the amino acid sequences of mt-proteins with a Bayesian method of Thorne and Kishino. Divergence dates were estimated as follows for the crown groups: about 35.4 million years ago (mya) for lorisiformes, 55.3 mya for lemuriformes, 64.5 mya for strepsirrhini, 70.1 mya for haplorhini and 76.0 mya for primates. Furthermore, we reexamined the biogeographic scenarios which have been proposed for the origin of strepsirrhini (lemuriformes and lorisiformes) and for the dispersal of the lemuriformes and lorisiformes.     

A method for the analysis of domain movements in large biomolecular complexes

A new method for the analysis of domain movements in large, multichain, biomolecular complexes is presented. The method is applicable to any molecule for which two atomic structures are available that represent a conformational change indicating a possible domain movement. The method is blind to atomic bonding and atom type and can, therefore, be applied to biomolecular complexes containing different constituent molecules such as protein, RNA, or DNA. At the heart of the method is the use of blocks located at grid points spanning the whole molecule. The rotation vector for the rotation of atoms from each block between the two conformations is calculated. Treating components of these vectors as coordinates means that each block is associated with a point in a “rotation space” and that blocks with atoms that rotate together, perhaps as part of the same rigid domain, will have colocated points. Thus a domain can be identified from the clustering of points from blocks that span it. Domain pairs are accepted for analysis of their relative movements in terms of screw axes based upon a set of reasonable criteria. Here, we report on the application of the method to biomolecules covering a considerable size range: hemoglobin, liver alcohol dehydrogenase, S‐Adenosylhomocysteine hydrolase, aspartate transcarbamylase, and the 70S ribosome. The results provide a depiction of the conformational change within each molecule that is easily understood, giving a perspective that is expected to lead to new insights. Of particular interest is the allosteric mechanism in some of these molecules. Results indicate that common boundaries between subunits and domains are good regions to focus on as movement in one subunit can be transmitted to another subunit through such interfaces. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Identification of the mouse paternally expressed imprinted gene Zdbf2 on chromosome 1 and its imprinted human homolog ZDBF2 on chromosome 2

In mammals, both the maternal and paternal genomes are necessary for normal embryogenesis due to parent-specific epigenetic modification of the genome during gametogenesis, which leads to non-equivalent expression of imprinted genes from the maternal and paternal alleles. In this study, we identified a paternally expressed imprinted gene, Zdbf2, by microarray-based screening using parthenogenetic and normal embryos. Expression analyses showed that Zdbf2 was paternally expressed in various embryonic and adult tissues, except for the placenta and adult testis, which showed biallelic expression of the gene. We also identified a differentially methylated region (DMR) at 10 kb upstream of exon 1 of the Zdbf2 gene and this differential methylation was derived from the germline. Furthermore, we also identified that the human homolog (ZDBF2) of the mouse Zdbf2 gene showed paternal allele-specific expression in human lymphocytes but not in the human placenta. Thus, our findings defined mouse chromosome 1 and human chromosome 2 as the loci for imprinted genes.     

Establishment of germline-competent embryonic stem cell lines from the MSM/Ms strain

MSM/Ms is an inbred mouse strain established from the Japanese wild mouse, Mus musculus molossinus, which has been phylogenetically distinct from common laboratory mouse strains for about 1 million years. The nucleotide substitution rate between MSM/Ms and C57BL/6 is estimated to be 0.96%. MSM/Ms mice display unique characteristics not observed in the commonly used laboratory strains, including an extremely low incidence of tumor development, high locomotor activity, and resistance to high-fat-diet-induced diabetes. Thus, functional genomic analyses using MSM/Ms should provide a powerful tool for the identification of novel phenotypes and gene functions. We report here the derivation of germline-competent embryonic stem (ES) cell lines from MSM/Ms blastocysts, allowing genetic manipulation of the M. m. molossinus genome. Fifteen blastocysts were cultured in ES cell medium and three ES lines, Mol/MSM-1, -2, and -3, were established. They were tested for germline competency by aggregation with ICR morulae and germline chimeras were obtained from all three lines. We also injected Mol/MSM-1 ES cells into blastocysts of ICR or C57BL/6 × BDF1 mice and found that blastocyst injection resulted in a higher production rate of chimeric mice than did aggregation. Furthermore, Mol/MSM-1 subclones electroporated with a gene trap vector were also highly efficient at producing germline chimeras using C57BL/6 × BDF1 blastocyst injection. This Mol/MSM-1 ES line should provide an excellent new tool allowing the genetic manipulation of the MSM/Ms genome.     

Systematic approaches to using the FOX hunting system to identify useful rice genes

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis . This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.     

Synthesis of 2α-substituted-14-epi-previtamin D3 and its genomic activity

We synthesized and isolated 2α-substituted analogs of 14-epi-previtamin D₃ after thermal isomerization at 80 °C for the first time. The VDR binding affinity and transactivation activity of osteocalcin promoter in HOS cells were evaluated, and the 2α-methyl-substituted analog was found to have greater genomic activity than 14-epi-previtamin D₃.