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.

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.

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.

Diversity of mitochondrial D-loop haplotypes from ancient Thracian horses in Bulgaria.

The domestication of the horse began possibly more than 5000 years ago in the western part of the Eurasian steppe, and according to the leading hypothesis, horses first spread from the Steppe toward the region of the Thracian culture, starting in the second half of the 2nd millennium BCE and flourished from the fifth to first centuries BCE, mainly located in present-day Bulgaria. We analyzed 17 horse bone remains excavated from Thracian archaeological sites (fourth to first centuries BCE) in Bulgaria and successfully identified 17 sequences representing 14 different haplotypes of the mitochondrial D-loop. Compared with the mtDNA haplotypes of modern horses around the world, ancient Thracian horses in Bulgaria are thought to be more closely related to modern horses of Southern Europe and less related to those of Central Asia. In addition, the haplotypes we obtained represented 11 previously reported modern horse mtDNA haplogroups: A, B, D, E, G, H, I, L, N, P, and Q. All the haplogroups contain modern and regionally predominant haplotypes occurring in Europe, the Middle East, and Central Asia. Our results indicate that Thracian horses in Bulgaria have had relatively high genetic diversity and are closely related to modern horse breeds.

Diversity of the MHC class II DRB gene in the wolverine (Carnivora: Mustelidae: Gulo gulo) in Finland.

The wolverine (Gulo gulo) in Finland has undergone significant population declines in the past. Since major histocompatibility complex (MHC) genes encode proteins involved in pathogen recognition, the diversity of these genes provides insights into the immunological fitness of regional populations. We sequenced 862 amplicons (242 bp) of MHC class II DRB exon 2 from 32 Finnish wolverines and identified 11 functional alleles and three pseudogenes. A molecular phylogenetic analysis indicated trans-species polymorphism, and PAML and MEME analyses indicated positive selection, suggesting that the Finnish wolverine DRB genes have evolved under balancing and positive selection. In contrast to DRB gene analyses in other species, allele frequencies in the Finnish wolverines clearly indicated the existence of two regional subpopulations, congruent with previous studies based on neutral genetic markers. In the Finnish wolverine, rapid population declines in the past have promoted genetic drift, resulting in a lower genetic diversity of DRB loci, including fewer alleles and positively selected sites, than other mustelid species analyzed previously. Our data suggest that the MHC region in the Finnish wolverine population was likely affected by a recent bottleneck.

Evolution of MHC class I genes in Japanese and Russian raccoon dogs, Nyctereutes procyonoides (Carnivora: Canidae).

Major histocompatibility complex (MHC) genes have been widely studied to assess the immunological fitness and evolutionary adaptation of animal populations. Among the Canidae, the raccoon dog's adventurous nature, omnivorous behavior, and high variability of intracellular pathogens make it ideal to study selection on MHC class I in a non-model canid species. Here, we examined allelic diversity and evolutionary patterns of MHC class I genes in the raccoon dog (Nyctereutes procyonoides). We identified 48 novel MHC class I alleles from 31 raccoon dogs from Japan and Russia. Some alleles were geographically restricted, whereas others were widely distributed across the species' range. The rate of non-synonymous substitutions was greater than that of synonymous substitutions for both exon 2 and exon 3 encoding α1 and α2 domains, respectively, in the α chain of the MHC class I protein. Positively selected sites at the amino acid level were evident in both the α1 and α2 domains, and a recombination breakpoint was found in exon 3. Bayesian phylogenetic trees showed no evidence of trans-species polymorphism (TSP) with alleles from carnivoran species in other families but did detect TSP between raccoon dogs and the domestic dog, Canis familiaris, indicative of long-term balancing selection in canids. Our results indicate that the extensive allelic diversity of MHC class I in Japanese and Russian raccoon dogs has been influenced and maintained by pathogen-driven positive selection, recombination, and long-term balancing selection. Supplementary Information: The online version contains supplementary material available at 10.1007/s13364-021-00561-y.

Population genetic structure and diversity of the East Balkan Swine (Sus scrofa) in Bulgaria, revealed by mitochondrial DNA and microsatellite analyses.

The East Balkan Swine (EBS) is the only indigenous pig breed in Bulgaria. We analyzed the mitochondrial DNA (mtDNA) control region and 21 microsatellite loci for 198 individuals from 11 farms in Bulgaria. Obtained 11 mtDNA haplotypes including three novel ones were grouped to two major clades, European clade E1 (146/198 individuals, 73.7%) and Asian clade A (52/198, 26.3%). The mixture of the two clades may have resulted from historical crossbreeding between the European and Asian pig breeds. Clade A was frequent in southeastern Bulgaria (Burgas Province), but less frequent or absent in northeastern Bulgaria (Varna and Shumen Provinces). The distribution of Europe- and Asia-specific haplotypes relative to EBS farm locations could be attributed to regional differences of breeding systems (e.g., crossbreeding with imported commercial pigs). A microsatellite analysis showed high heterozygosities for all the EBS farms, and negative inbreeding coefficients presumably due to crossing with commercial pigs or wild boars and/or efforts to reduce inbreeding by farmers. Bayesian clustering analyses showed that all farm populations are genetically well distinguishable from one another. Although diversity has been maintained by the efforts of farmers and a breeding association, the effective population size remains small, and conservation efforts should be continued.

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.

Differential usage of two promoters of the Broad-Complex gene in the silkworm, Bombyx mori.

The Broad-Complex gene encodes one of the key regulators of the ecdysone signal cascade. We previously isolated part of the genomic DNA and cDNAs of Broad-Complex in Bombyx mori (BmBR-C). Here, we report structures of the entire genomic DNA and 5' untranslated region (5'-UTR) of the cDNAs. BmBR-C was found to span about 158 kbp including 13 exons. In the 5'-UTR, additional alternatively spliced exons were identified. The 5' ends of the cDNAs were mapped to two different positions, the distal promoter (P(dist)) and proximal promoter (P(prox)), separated by 86 kbp. Expression from these promoters was controlled differentially. Semi-quantitative PCR using cDNAs from the carcass, silk glands and fat body revealed that expression from P(prox) was changed moderately and expression from P(dist) was weak and constant during the fourth ecdysis. At the onset of pupation, expression from P(prox) was suppressed in all tissues, but that from P(dist) was induced in the carcass and ASG. In the fat body, expression from both promoters increased in the prepupal stage. A combination of promoters differing in responsiveness to an ecdysone signal may serve to achieve a complex regulation of downstream genes in reply to a simple hormonal signal.

Structure and expression of the gene encoding a Broad-Complex homolog in the silkworm, Bombyx mori.

The steroid hormone ecdysone (20-hydroxyexdysone) initiates metamorphosis and also larval ecdysis in many insects by activating a cascade of genes that includes primary response genes (early genes), most of which encode transcriptional regulators, and secondary response genes (late genes) regulated by the early genes. One of the early genes, Broad-Complex (BR-C), a key regulator of the ecdysone cascade, shares a common amino-terminal BTB domain which is fused by alternative splicing to one of four pairs of C(2)H(2) zinc finger domains (Z1, Z2, Z3, and Z4). cDNAs for BR-C (BmBR-C) were isolated from the silkworm Bombyx mori. These genes showed 90.3% and 98.2% amino acid identity with the Drosophila BR-C and Manduca BR-C in the N-terminal BTB domain; 96.0%, 90.7%, and 85.2% identity with the three zinc finger domains of the Drosophila Z1, Z2, and Z4 isoforms; and 96.3% and 98.1% identity with the two zinc finger domains of the Manduca Z2 and Z4 isoforms, respectively. Partial genomic sequencing (from the 3' region of the core sequence to the 3' region of the Z3 class zinc finger-coding sequence) of the BmBR-C gene showed that four exons coding the zinc finger domains are arranged the same as the BR-C gene in Drosophila. The amino acid sequence predicted from the genomic sequence corresponding to the BmBR-C Z3 class zinc finger domain is 100% identical to the Z3 isoforms of Drosophila and Manduca. We examined expression patterns of the BmBR-C isoforms during late larval to pupal development in the epidermis, fatbody and silk gland. During the metamorphic transformation, the epidermis and silk gland are completely histolyzed; however, the fat body survives into the adult phase. Expression patterns of BmBR-C during development differed extensively between the histolyzed group and the survival group. The BmBR-C expression patterns in silk glands also differed between the anterior and other areas (the middle and posterior silk glands).

Bombyx Y-box protein BYB facilitates specific DNA interaction of various DNA binding proteins independently of the cold shock domain.

A new member of the Y-box protein family of the silkworm Bombyx mori (BYB) was co-purified with the fibroin gene enhancer-binding protein FMBP-1, and stimulated the binding of FMBP-1 to its cognate DNA element. However, the stimulatory effect was not specific to FMBP-1, BYB also enhancing the binding of mammalian transcription factors OTF2, SP1 and AP2 to their specific binding elements. Besides the above transcription regulatory factors, BYB facilitated the binding of basal transcription factor TBP, and enhanced transcription from the adenovirus 2 major late promoter in a reconstituted transcription system. Moreover, BYB stimulated the reactions of some restriction endonucleases under cold conditions. The C-terminal region of BYB was sufficient for these stimulatory effects, and the highly conserved cold shock domain (CSD) in the N-terminal region was dispensable. GST-pull down experiments showed that the C-terminal region could interact with DNA independently of the CSD. The above results suggest that the C-terminal region of BYB causes the active interaction of various DNA binding proteins with their targets. Such a function of the C-terminal region of BYB may partly explain the functional diversity of Y-box proteins.