Yoshinori Nishita

Krüppel homolog 1 inhibits insect metamorphosis via direct transcriptional repression of Broad-complex, a pupal specifier gene

The Broad-Complex gene (BR-C) encodes transcription factors that dictate larval-pupal metamorphosis in insects. The expression of BR-C is induced by molting hormone (20-hydroxyecdysone (20E)), and this induction is repressed by juvenile hormone (JH), which exists during the premature larval stage. Krüppel homolog 1 gene (Kr-h1) has been known as a JH-early inducible gene responsible for repression of metamorphosis; however, the functional relationship between Kr-h1 and repression of BR-C has remained unclear. To elucidate this relationship, we analyzed cis- and trans elements involved in the repression of BR-C using a Bombyx mori cell line. In the cells, as observed in larvae, JH induced the expression of Kr-h1 and concurrently suppressed 20E-induced expression of BR-C. Forced expression of Kr-h1 repressed the 20E-dependent activation of the BR-C promoter in the absence of JH, and Kr-h1 RNAi inhibited the JH-mediated repression, suggesting that Kr-h1 controlled the repression of BR-C. A survey of the upstream sequence of BR-C gene revealed a Kr-h1 binding site (KBS) in the BR-C promoter. When KBS was deleted from the promoter, the repression of BR-C was abolished. Electrophoresis mobility shift demonstrated that two Kr-h1 molecules bound to KBS in the BR-C promoter. Based on these results, we conclude that Kr-h1 protein molecules directly bind to the KBS sequence in the BR-C promoter and thereby repress 20E-dependent activation of the pupal specifier, BR-C. This study has revealed a considerable portion of the picture of JH signaling pathways from the reception of JH to the repression of metamorphosis.

Fibroin-modulator-binding protein-1 (FMBP-1) contains a novel DNA-binding domain, repeats of the score and three amino acid peptide (STP), conserved from Caenorhabditis elegans to humans

The predicted transcriptional regulatory factor for the fibroin gene of the silkworm Bombyx mori, fibroin-modulator-binding protein-1 (FMBP-1), was purified by sequential DNA affinity column chromatography, and cDNA clones corresponding to FMBP-1 were isolated from a library. The N-terminal half of FMBP-1 has a weak similarity to the DNA-binding domain of several transcriptional regulatory factors in higher plants. The C-terminal half contains four tandem repeats of a novel 23 amino acid motif, which we named the score and three amino acid peptide (STP). Other genes containing STP repeats were found in Drosophila, Caenorhabditis elegans, mouse and human. Mutational analysis of FMBP-1 showed that the STP repeats form a novel DNA-binding domain. Sequences flanking STP repeats modulated DNA-binding activity. The FMBP-1 gene was expressed during the fourth to fifth instar. FMBP-1 activity appeared to be regulated at the transcriptional level and by the post-transcriptional modification.

Genetic variation of the MHC class II DRB genes in the Japanese weasel, Mustela itatsi, endemic to Japan, compared with the Siberian weasel, Mustela sibirica

Major histocompatibility complex (MHC) genes encode proteins that play a critical role in vertebrate immune system and are highly polymorphic. To further understand the molecular evolution of the MHC genes, we compared MHC class II DRB genes between the Japanese weasel (Mustela itatsi), a species endemic to Japan, and the Siberian weasel (Mustela sibirica), a closely related species on the continent. We sequenced a 242-bp region of DRB exon 2, which encodes antigen-binding sites (ABS), and found 24 alleles from 31 M. itatsi individuals and 17 alleles from 21 M. sibirica individuals, including broadly distributed, species-specific and/or geographically restricted alleles. Our results suggest that pathogen-driven balancing selection have acted to maintain the diversity in the DRB genes. For predicted ABS, nonsynonymous substitutions exceeded synonymous substitutions, also indicating positive selection, which was not seen at non-ABS. In a Bayesian phylogenetic tree, two M. sibirica DRB alleles were basal to the rest of the sequences from mustelid species and may represent ancestral alleles. Trans-species polymorphism was evident between many mustelid DRB alleles, especially between M. itatsi and M. sibirica. These two Mustela species divided about 1.7 million years ago, but still share many MHC alleles, indicative of their close phylogenetic relationship.

Low genetic variation in the MHC class II DRB gene and MHC-linked microsatellites in endangered island populations of the leopard cat (Prionailurus bengalensis) in Japan

Isolated populations of the leopard cat (Prionailurus bengalensis) on Tsushima and Iriomote islands in Japan are classified as subspecies P. b. euptilurus and P. b. iriomotensis, respectively. Because both populations have decreased to roughly 100, an understanding of their genetic diversity is essential for conservation. We genotyped MHC class II DRB exon 2 and MHC-linked microsatellite loci to evaluate the diversity of MHC genes in the Tsushima and Iriomote cat populations. We detected ten and four DRB alleles in these populations, respectively. A phylogenetic analysis showed DRB alleles from both populations to be closely related to those in other felid DRB lineages, indicating trans-species polymorphism. The MHC-linked microsatellites were more polymorphic in the Tsushima than in the Iriomote population. The MHC diversity of both leopard cat populations is much lower than in the domestic cat populations on these islands, probably due to inbreeding associated with founder effects, geographical isolation, or genetic drift. Our results predict low resistance of the two endangered populations to new pathogens introduced to the islands.

Genetic Diversity of MHC Class II DRB1 Exon 2 in the Red Fox (Vulpes vulpes) on Hokkaido, Japan

To assess the genetic diversity of the red fox (Vulpes vulpes) population on Hokkaido Island, northern Japan, we examined genotypes of exon 2 of the major histocompatibility complex (MHC) class II DRB1 gene for 232 individuals and identified 17 novel alleles. The subpopulation in the Southern area was genetically differentiated from those in all other areas on Hokkaido, suggesting isolation in southern Hokkaido. In addition, the Southern subpopulation is lower in genetic diversity than the other subpopulations, possibly resulting from purifying selection and/or a recent bottleneck. The non-synonymous substitutions exceeded the synonymous substitutions for codons encoding antigen-binding sites (ABSs) in exon 2, indicating that the red fox DRB1 alleles have evolved under positive selection. In a Bayesian phylogenetic tree, although most of the DRB1 alleles from the Hokkaido red fox were contained within a red fox-like canid (Vulpes) clade, some belonged to another canid clade. This means trans-species polymorphism maintained by balancing selection. Our results showed the local variability and the presence of selection on the MHC gene in this population, which contributes to the understanding of the historical background and the molecular evolution.

Evolution of MHC class I genes in Eurasian badgers, genus Meles (Carnivora, Mustelidae)

Because of their role in immune defense against pathogens, major histocompatibility complex (MHC) genes are useful in evolutionary studies on how wild vertebrates adapt to their environments. We investigated the molecular evolution of MHC class I (MHCI) genes in four closely related species of Eurasian badgers, genus Meles. All four species of badgers showed similarly high variation in MHCI sequences compared to other Carnivora. We identified 7−21 putatively functional MHCI sequences in each of the badger species, and 2−7 sequences per individual, indicating the existence of 1−4 loci. MHCI exon 2 and 3 sequences encoding domains α1 and α2 exhibited different clade topologies in phylogenetic networks. Non-synonymous nucleotide substitutions at codons for antigen-binding sites exceeded synonymous substitutions for domain α1 but not for domain α2, suggesting that the domains α1 and α2 likely had different evolutionary histories in these species. Positive selection and recombination seem to have shaped the variation in domain α2, whereas positive selection was dominant in shaping the variation in domain α1. In the separate phylogenetic analyses for exon 2, exon 3, and intron 2, each showed three clades of Meles alleles, with rampant trans-species polymorphism, indicative of the long-term maintenance of ancestral MHCI polymorphism by balancing selection.

Genetic diversity of MHC class II DRB alleles in the marbled polecat, Vormela peregusna, in Bulgaria

The marbled polecat, Vormela peregusna, is a small marten-like mustelid distributed from southeastern Europe to western China. Since the individual numbers of this species have declined, it has been classified as a vulnerable (VU) species in the 2008 IUCN Red List. To better understand the adaptation of V. peregusna, we investigated diversity and selection in the major histocompatibility complex (MHC) class II DRB gene which is reported to be highly polymorphic in many other species. Among 10 V. peregusna individuals from Bulgaria, we detected 9 novel sequences of partial exon 2, including antigen-binding site (ABS) codons, as representatives of DRB allele (Vope-DRBs). Two of the alleles were detected in all individuals analysed, while the others showed limited geographical distributions. Our results provided evidence for positive selection on ABS codons and recombination break points, contributing to maintaining the diversity of Vope-DRBs. In a Bayesian phylogenetic tree, all Vope-DRBs grouped within the mustelid clade, with three of them in the basal group consisted of alleles from Meles, Martes, and Vormela but no Mustela alleles, and the others in the derived group consisted of alleles diverged relatively late in the various mustelid species. The Vope-DRBs showed trans-species polymorphism within the mustelid clade, suggesting that the DRB genes evolved under long-lasting balancing selection.