Asato Kuroiwa

Two mouse piwi-related genes: miwi and mili.

Genes belonging to the piwi family are required for stem cell self-renewal in diverse organisms. We cloned mouse homologues of piwi by RT-PCR using degenerative primers. The deduced amino acid sequences of mouse homologues MIWI and MILI showed that each contains a well-conserved C-terminal PIWI domain and that each shares significant homology with PIWI and their human counterparts HIWI. Both miwi and mili were found in germ cells of adult testis by in situ hybridization, suggesting that these genes may function in spermatogenesis. Furthermore, mili was expressed in primordial germ cells (PGCs) of developing mouse embryos and may therefore play a role during germ cell formation. MIWI may be involved in RNA processing or translational regulation, since MIWI was found to possess RNA binding activity. Our data suggest that miwi and mili regulate spermatogenesis and primordial germ cell production.

Molecular analysis of avian circadian clock genes.

Unlike mammals, avian circadian rhythms are regulated by a multiple oscillatory system consisting of the retina, the pineal and the suprachiasmatic nucleus in the hypothalamus. To understand avian circadian system, we have cloned Clock and Period homologs (qClock, qPer2 and qPer3) and characterized these genes in Japanese quail. Overall, qCLOCK, qPER2 and qPER3 showed approximately 79%, approximately 46% and approximately 33% amino acid identity to mCLOCK, mPER2, mPER3, respectively. Clock was mapped to quail chromosome 4 and chicken chromosome 4q1.6-q2.1. Per2 and Per3 genes were both localized to microchromosomes. qClock mRNA was expressed throughout the day, while qPer2 and qPer3 showed robust circadian oscillation in the eye and the pineal gland. All three genes were expressed in various tissues. In addition, qPer2 mRNA was induced by light, but neither qClock nor qPer3 was induced. These results can explain the molecular basis for circadian entrainment in Japanese quail and also provide new avenues for molecular understanding of avian circadian clock and photoperiodism.

Efficient chromosomal transposition of a Tc1/mariner- like transposon Sleeping Beauty in mice

The presence of mouse embryonic stem (ES) cells makes the mouse a powerful model organism for reverse genetics, gene function study through mutagenesis of specific genes. In contrast, forward genetics, identification of mutated genes responsible for specific phenotypes, has an advantage to uncover novel pathways and unknown genes because no a priori assumptions are made about the mutated genes. However, it has been hampered in mice because of the lack of a system in which a large-scale mutagenesis and subsequent isolation of mutated genes can be performed efficiently. Here, we demonstrate the efficient chromosomal transposition of a Tc1/mariner-like transposon, Sleeping Beauty, in mice. This system allows germ-line mutagenesis in vivo and will facilitate certain aspects of phenotype-driven genetic screening in mice.

Paired activating and inhibitory immunoglobulin-like receptors, MAIR-I and MAIR-II, regulate mast cell and macrophage activation.

Immune responses are regulated by opposing positive and negative signals triggered by the interaction of activating and inhibitory cell surface receptors with their ligands. Here, we describe novel paired activating and inhibitory immunoglobulin-like receptors, designated myeloid-associated immunoglobulin-like receptor (MAIR) I and MAIR-II, whose extracellular domains are highly conserved by each other. MAIR-I, expressed on the majority of myeloid cells, including macrophages, granulocytes, mast cells, and dendritic cells, contains the tyrosine-based sorting motif and the immunoreceptor tyrosine-based inhibitory motif-like sequences in the cytoplasmic domain and mediates endocytosis of the receptor and inhibition of IgE-mediated degranulation from mast cells. On the other hand, MAIR-II, expressed on subsets of peritoneal macrophages and B cells, associates with the immunoreceptor tyrosine-based activation motif-bearing adaptor DAP12 and stimulates proinflammatory cytokines and chemokine secretions from macrophages. Thus, MAIR-I and MAIR-II play important regulatory roles in cell signaling and immune responses.

Highly conserved linkage homology between birds and turtles: Bird and turtle chromosomes are precise counterparts of each other

The karyotypes of birds, turtles and snakes are characterized by two distinct chromosomal components, macrochromosomes and microchromosomes. This close karyological relationship between birds and reptiles has long been a topic of speculation among cytogeneticists and evolutionary biologists; however, there is scarcely any evidence for orthology at the molecular level. To define the conserved chromosome synteny among humans, chickens and reptiles and the process of genome evolution in the amniotes, we constructed comparative cytogenetic maps of the Chinese soft-shelled turtle (Pelodiscus sinensis) and the Japanese four-striped rat snake (Elaphe quadrivirgata) using cDNA clones of reptile functional genes. Homology between the turtle and chicken chromosomes is highly conserved, with the six largest chromosomes being almost equivalent to each other. On the other hand, homology to chicken chromosomes is lower in the snake than in the turtle. Turtle chromosome 6q and snake chromosome 2p represent conserved synteny with the chicken Z chromosome. These results suggest that the avian and turtle genomes have been well conserved during the evolution of the Arcosauria. The avian and snake sex Z chromosomes were derived from different autosomes in a common ancestor, indicating that the causative genes of sex determination may be different between birds and snakes.

Molecular cloning of mammalian Spred-3 which suppresses tyrosine kinase-mediated Erk activation.

We have reported on Spred-1 and Spred-2, which inhibit MAP kinase activation by interacting with c-kit and ras/raf. Here, we report the cloning of a third member in this family, Spred-3. Spred-3 is expressed exclusively in the brain and its gene locates in chromosome 19q13.13 in human. Like Spred-1 and -2, Spred-3 contains an EVH1 domain in the N-terminus and a Sprouty-related cysteine-rich region (SPR domain) in the C-terminus that is necessary for membrane localization. However, Spred-3 does not possess a functional c-kit binding domain (KBD), since the critical amino acid Arg residue in this region was replaced with Gly in Spred-3. Although Spred-3 suppressed growth factor-induced MAP kinase (Erk) activation, inhibitory activity of Spred-3 was lower than that of Spred-1 or Spred-2. By the analysis of chimeric molecules between Spred-3 and Spred-1, we found that the SPR domain, rather than KBD, is responsible for efficient Erk suppression. The finding of Spred-3 revealed the presence of a novel family of regulators for the Ras/MAP kinase pathway, each member of which may have different specificities for extracellular signals.

B Chromosomes Have a Functional Effect on Female Sex Determination in Lake Victoria Cichlid Fishes

The endemic cichlid fishes in Lake Victoria are a model system for speciation through adaptive radiation. Although the evolution of the sex-determination system may also play a role in speciation, little is known about the sex-determination system of Lake Victoria cichlids. To understand the evolution of the sex-determination system in these fish, we performed cytogenetic analysis in 11 cichlid species from Lake Victoria. B chromosomes, which are present in addition to standard chromosomes, were found at a high prevalence rate (85%) in these cichlids. In one species, B chromosomes were female-specific. Cross-breeding using females with and without the B chromosomes demonstrated that the presence of the B chromosomes leads to a female-biased sex ratio in this species. Although B chromosomes were believed to be selfish genetic elements with little effect on phenotype and to lack protein-coding genes, the present study provides evidence that B chromosomes have a functional effect on female sex determination. FISH analysis using a BAC clone containing B chromosome DNA suggested that the B chromosomes are derived from sex chromosomes. Determination of the nucleotide sequences of this clone (104.5 kb) revealed the presence of several protein-coding genes in the B chromosome, suggesting that B chromosomes have the potential to contain functional genes. Because some sex chromosomes in amphibians and arthropods are thought to be derived from B chromosomes, the B chromosomes in Lake Victoria cichlids may represent an evolutionary transition toward the generation of sex chromosomes.

Sequence polymorphisms, allelic expression status and chromosome locations of the chicken IGF2 and MPR1 genes

By screening 26 chicken breeds and lines, DNA polymorphisms were identified in the IGF2 and MPR1 genes, of which mammalian homologues are parentally imprinted, and the GAPD gene, a housekeeping control. Using the polymorphisms as genetic markers, we found that all three genes are expressed biallelically in embryonic tissues. IGF2 and MPR1 were mapped on chicken chromosomes 5 and 3, respectively, by fluorescence in situ hybridization, demonstrating conserved linkage homology between mammals and birds.

CPEB2, a novel putative translational regulator in mouse haploid germ cells

Abstract Translational control of specific mRNAs by cytoplasmic polyadenylation has fundamental roles in gametogenesis. The cytoplasmic polyadenylation element binding (CPEB) protein regulates cytoplasmic polyadenylation of mRNAs as a trans factor in oogenesis and spermatogenesis. The CPEB protein contains two RNA recognition motifs and a Zn-finger structure. Proteins (KIAA0940 and KIAA1673) with similar structures are predicted from the genome database, but nothing is known about their expression and function. Here, we report another novel member of the CPEB protein family, CPEB2. Comparison of the amino acid sequences of CPEB family members suggests that the family can be divided structurally and, perhaps, functionally into two groups: the CPEB group, and the CPEB2-KIAA0940-KIAA1673 group. The CPEB2 maps to mouse chromosome distal 5B and is abundantly expressed in testis. However, it was detected by reverse transcription-polymerase chain reaction in all tissues that we examined. It preferentially binds to poly(U) and localizes to the cytoplasm in transfected HeLa cells. The CPEB2 is expressed postmeiotically in mouse spermatogenesis, suggesting a possible role in translational regulation of stored mRNAs in transcriptionally inactive haploid spermatids.

Over-expression of DMRT1 induces the male pathway in embryonic chicken gonads.

DMRT1 encodes a conserved transcription factor with an essential role in gonadal function. In the chicken, DMRT1 in located on the Z sex chromosome and is currently the best candidate master regulator of avian gonadal sex differentiation. We previously showed that knockdown of DMRT1 expression during the period of sexual differentiation induces feminisation of male embryonic chicken gonads. This gene is therefore necessary for proper testis development in the chicken. However, whether it is sufficient to induce testicular differentiation has remained unresolved. We show here that over-expression of DMRT1 induces male pathway genes and antagonises the female pathway in embryonic chicken gonads. Ectopic DMRT1 expression in female gonads induces localised SOX9 and AMH expression. It also induces expression of the recently identified Z-linked male factor, Hemogen (HEMGN). Masculinised gonads show evidence of cord-like structures and retarded female-type cortical development. Furthermore, expression of the critical feminising enzyme, aromatase, is reduced in the presence of over-expressed DMRT1. These data indicate that DMRT1 is an essential sex-linked regulator of gonadal differentiation in avians, and that it likely acts via a dosage mechanism established through the lack of global Z dosage compensation in birds.