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The phylogeography of the Altai weasel (Mustela altaica) based on mitochondrial control-region sequences was investigated using samples from individuals obtained widely across the species range, including Central Asia, Tibet and southern Siberia. Thirty-four haplotypes were identified among 53 individuals from 32 localities. A maximum-likelihood phylogenetic tree for haplotypes showed two major clades, clades I and II. Clade I contained haplotypes from northern Eurasia, including the continental Far East, Altai Mountains and eastern Kazakhstan. Clade II comprised two subclades: group IIa, with haplotypes from around the Pamir Mountains, and the poorly supported group IIb, with haplotypes from the Tibetan Plateau. High haplotype diversity and low nucleotide diversity for clade I suggest that the range of M. altaica expanded over a relatively short period in northern Eurasia. High haplotype and nucleotide diversity for clade II indicate a longer-term, more stable population resulting from geographical isolation of individuals on the Tibetan Plateau by surrounding mountain ranges. Our study suggests that the ancestral population of M. altaica migrated into Tibet from the outside and has diversified in Tibet, seemingly not supporting the ‘out-of-Tibet’ hypothesis; however, we could not completely refute this hypothesis because an individual having a group IIb haplotype was detected in Mongolia.

The European hedgehog (Erinaceus europaeus) is widely distributed in Europe, from Iberia and Italy to Scandinavia, and in New Zealand. They inhabit a wide range of environments, from natural habitats to urban areas. Finland is the northern edge of the current distribution area, which has expanded from Pleistocene refugia in Europe. Finnish hedgehogs are not only found in the suburbs, but also in urban areas such as the capital city, Helsinki. However, the phylogenetic relationships to other populations and the population structure were unknown for the urban hedgehogs in Helsinki. We reconstructed a molecular phylogeny and analyzed the population structure and genetic diversity from samples of 106 hedgehogs collected around Helsinki in 2017–2019, based on partial sequences of the mitochondrial control region and cytochrome b gene as well as 11 microsatellite loci. Our phylogenetic data indicate that the Helsinki population is closely related to the Estonian population. This means that at least part of the Helsinki population originates from Estonia, due to natural expansion from glacial refugia and anthropogenic introduction for vermin control, as shown in previous reports. The microsatellite data indicated that the Helsinki population is genetically homogeneous, with low genetic diversity. This may be due to the absence of effective geographic barriers to gene flow at present, but it is also possible that the subpopulations formed by migration are concealed by the genetic uniformity resulting from founder effect of natural expansion to the northward or artificial introduction. Further study is necessary to evaluate the effect of urban fragmentation in Helsinki.

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.

To investigate the phylogenetic relationships of brown bears (Ursus arctos) on Sakhalin Island in the Far East, mitochondrial DNA (mtDNA) sequences were analyzed for 27 ancient and five contemporary specimens of brown bears obtained from Sakhalin. We successfully determined partial sequences of the mtDNA control region (254–394 base-pairs) and identified six novel haplotypes. All sequences from bears on Sakhalin grouped phylogenetically with clade 3a but were not clearly distinguishable as to subclades 3a1 and 3a2. However, by application of APLP method for detecting single-nucleotide polymorphisms (SNPs) specific to clades, the samples from Sakhalin were all identified to clade 3a1, which is currently widespread in eastern continental Eurasia. Although the ancestors of brown bears on Hokkaido Island, Japan, migrated from continental Eurasia through Sakhalin, none of the clades previously found on Hokkaido (clades 3a2, 3b, and 4) were detected on Sakhalin. Our results suggest that an ancestral bear population from clade 3a1 migrated from continental Eurasia to Sakhalin, independently and after the migration of clade 3a2 to Hokkaido. Alternatively, clade 3a2 could have evolved from phylogenetically closely related clade 3a1 due to geographical isolation on Hokkaido. Our data confirm that bear skull remains from an Okhotsk Cultural archaeological site on small Rebun Island off the northwest coast of Hokkaido were transported from Hokkaido, rather than from Sakhalin.

High polymorphism in major histocompatibility complex (MHC) genes plays an essential role in the adaptive immune system of vertebrates for defense against various pathogens. To further understand the evolution of MHC in wildlife, partial sequences of exon 2 in MHC class II DRB genes were determined and analyzed for the Japanese marten (Martes melampus), which is endemic to Japan. From 26 individuals, 17 DRB alleles (Mame-DRBs) and four presumed pseudogenes were identified, some of them geographically widespread and others of limited distribution. Mixed-effect model evolution (MEME) and single breakpoint recombination (SBP) analyses indicated six amino acid positions under positive selection and one recombination site, respectively, that are implicated in maintaining the diversity of Mame-DRBs. In a Bayesian phylogenetic tree, Mame-DRBs did not group monophyletically, but were scattered among seven sub-clades, each containing DRBs from one or more other mustelid species. This trans-species polymorphism of Mame-DRBs has resulted from long-term balancing selection.

In order to investigate the detailed zoogeographical history of brown bears (Ursus arctos) in Bulgaria and their relationships with populations in neighboring regions of Europe and Asia, the amplified product length polymorphism (APLP) method for the mitochondrial DNA (mtDNA) haplogrouping was applied to ancient bone remains. The results showed that haplogroups for 12 of 31 samples (38.7%) were successfully classified using the APLP method, although partial sequences of the mtDNA control region were determined with PCR product-direct sequencing for only 6 samples (19.4%). Even among 25 samples, of which nucleotide sequences could not be determined, the APLP method successfully classified haplogroups of 6 samples (6/25, 24.0%), indicating an advantage of the APLP method. In Bulgaria, although both the Balkan/Italian lineage of mtDNA (clade 1b) and the Eastern European lineage (clade 3a1) have been identified from modern bears as reported in other studies, all bear remains examined in the present study had APLP haplogroup W. The mtDNA phylogenetic analysis showed that the ancient Bulgarian brown bears had clade 1b. This indicates that clade 1b was originally distributed in Bulgaria, whereas those with clade 3a1 could have entered from Romania to Bulgaria. In addition, the APLP and phylogenetic analysis of recent skin samples from Turkey showed that they have mtDNA of the Middle Eastern/Turkish lineage (referred to clade 7a). Therefore, Bulgaria on the Balkan Peninsula might have been located between two distribution borders: one is between clades 1b and 3a1, and the other is between clades 1b and 7.

Although the present-day distributional boundary between the European badger (Meles meles) and the Asian badger (Meles leucurus) is around the Volga River, studies of ancient bone remains have indicated changes in the distribution of M. meles and M. leucurus in the Urals–Volga region during the Holocene. To examine past changes in distribution using genetic data, changes in genetic diversity, and the relationships of Holocene to modern populations, we sequenced ~150 bp of the mitochondrial DNA control region from the 44 ancient badger remains excavated from European Russian, Ural and Western Siberian sites, and we detected 12 haplotypes. Our study revealed Holocene changes in the distributional boundary between these badger species. Meles meles inhabited the Ural Mountains east of the Volga River in the Early Holocene, whereas M. leucurus expanded its distribution westwards, starting ~2500 years ago. Thereafter, M. leucurus rapidly replaced M. meles in the region between the Urals and the Volga, resulting in the present-day boundary in the Volga–Kama region. Among the 12 haplotypes detected, three for M. leucurus and four for M. meles were identical to partial sequences of haplotypes detected in modern populations, indicating considerable genetic continuity between Holocene and modern populations.

We investigated the genetic diversity and distribution pattern of mitochondrial DNA control-region haplotypes across the distributional range of the Siberian weasel (Mustela sibirica) in Eastern Eurasia. We identified 23 haplotypes from 65 individuals sampled from 21 localities. Our analyses showed two major phylogeographical groups: group I comprised continental Russia, Tsushima and Korea, and group II comprised China, Taiwan and Korea. Two novel haplotypes found in the Amur area and one from Gansu Province were closely related to the Tsushima and Taiwan clades, respectively. Phylogeographical and demographic analyses indicated a recent population expansion for group I, whereas no clear evidence for expansion was obtained for group II. The recent expansion of group I is also supported by historical records. Closely related haplotypes were found between the continental populations and the insular populations on Tsushima and Taiwan, suggesting that the ancestors of the insular populations immigrated from the continent via land bridges. The two groups could have evolved allopatrically in parts of eastern Asia differing in climate and vegetation.

Raccoon dogs, Nyctereutes procyonoides, are native to East Asia, but have been introduced into western Russia and eastern Europe. To determine allelic diversity and elucidate the evolution of major histocompatibility complex (MHC) class II genes in the raccoon dog, we analysed a 237-bp region of DRB exon 2 from 36 individuals of native and introduced populations from Japan and Russia. We detected 23 DRB alleles (Nypr-DRBs), 22 of which were novel. Some alleles were found across the species’ range, while others were geographically restricted. For both native and introduced populations, the ratio of non-synonymous to synonymous substitution rates for codons at predicted antigen-binding sites was significantly greater than 2, indicating that Nypr-DRBs have evolved under positive selection. Mixed effect model evolution analysis and an algorithm to detect recombination showed five positively selected codons and one recombination breakpoint, respectively. Overall, our results suggest that the diversity of MHC class II DRB in N. procyonoides was influenced and maintained by recombination, pathogen-driven positive selection, geographical barriers and the founder effect. A Bayesian phylogenetic tree revealed no evidence of trans-species polymorphism (TSP), but instead showed monophyly for the Nypr-DRB alleles within a larger clade of canid sequences. The lack of TSP may have been due to long-term divergence of raccoon dogs from other canids, or to their having encountered different sets of pathogens due to occupying a different ecological niche.

Two closely related species of Meles (Carnivora), the European badger (M. meles) and the Asian badger (M. leucurus), are distributed allopatrically in continental Eurasia but show a narrow contact zone around the Volga and Kama Rivers, Russia. We analyzed maternally (mitochondrial DNA), paternally (SRY gene and CAN-SINEs on the Y chromosome), and biparentally (CFTR gene and nine microsatellite loci) inherited genes for evidence of hybridization between the two species in the contact zone. Of 71 badgers examined, we identified 17 individuals as hybrids with mixed genotypes for the first time. Some hybrids appeared to have resulted from repeated backcrossing with the parental species. In addition, the hybridization was symmetric between the two species. Compared with previous palaeontological data, the hybridization between the two species could have resulted from secondary contact due to western expansion in distribution by the Asian badgers.

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 bee...

The sable (Martes zibellina) is a medium-sized mustelid inhabiting forest environments in Siberia, northern China, the Korean Peninsula, and Hokkaido Island, Japan. To further understand the molecular evolution of the major histocompatibility complex (MHC), we sequenced part of exon 2 in MHC class II DRB genes, including codons encoding the antigen binding site, from 33 individuals from continental Eurasia and Japan. We identified 16 MHC class II DRB alleles (Mazi-DRBs), some of which were geographically restricted and others broadly distributed, and eight putative pseudogenes. A single-breakpoint recombination analysis detected a recombination site in the middle of exon 2. A mixed effects model of evolution analysis identified five amino acid sites presumably under positive selection. These sites were all located in the region 3′ to the recombination site, suggesting that positive selection and recombination could be committed to the diversity of the M. zibellina DRB gene. In a Bayesian phylogenetic tree, all Mazi-DRBs and the presumed pseudogenes grouped within a Mustelidae clade. The Mazi-DRBs showed trans-species polymorphism, with some alleles most closely related to alleles from other mustelid species. This result suggests that the sable DRBs have evolved under long-lasting balancing selection.

In this study, we investigated molecular phylogenetic status of the marbled polecat (Vormela peregusna) from Bulgaria, using sequences of two Y-chromosomal genes (SRY and ZFY). The phylogenetic tree inferred using combined sequences of both genes indicated that the marbled polecat was split from genera Lutra, Neovison and Mustela after genus Martes was diverged in family Mustelidae. In addition, we analyzed molecular phylogeography of the Bulgarian population of the marbled polecat, using cytochrome b and control region sequences of mitochondrial DNA (mtDNA). The phylogenetic tree of cytochrome b indicated that the haplotypes of the Bulgarian population comprised two haplogroups, which were the most ancestral clades. Additionally, the control region phylogeny showed that the haplotypes of Bulgaria formed two haplogroups: one was the most ancestral clade, and the other was the derivative clade. One individual with the most ancestral cytochrome b clade had a control region haplotype of the derivative clade. Thus, this study revealed that the most ancestral lineages of the marbled polecat are included in the population of Bulgaria. The Bulgarian population could be a remnant lineage from a basal for the species, which in Pleistocene occupied a relatively large area related to the Balkan-Caucasian.

Nishita, Yoshinori, Kosintsev, Pavel A., Haukisalmi, Voitto, Väinölä, Risto, Raichev, Evgeniy G., Murakami, Takahiro, Abramov, Alexei V., Kaneko, Yayoi, Masuda, Ryuichi (2017): Diversity of MHC class II DRB alleles in the Eurasian population of the least weasel, Mustela nivalis (Mustelidae: Mammalia). Biological Journal of the Linnean Society 121: 28-37, DOI: 10.1093/biolinnean/blw028

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.

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.

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.

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. Kruppel 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.

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.

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).

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.

We previously reported isolation of the mouse gene, Mest (mesoderm-specific transcripts), which is mapped to the proximal part of chromosome 6 and predominantly expressed in the mesoderm and its derivatives during development. Peg1 , a paternally expressed gene isolated by a systematic screening of imprinted genes, was recently demonstrated to be identical to Mest . We and others have shown that the human homolog ( MEST ) of Mest is also imprinted so as to be expressed from the paternal copy and maps to 7q32. To study transcriptional regulation of Mest / Peg 1, we examined the effect of DNA methylation on its expression. In the embryonal carcinoma (EC) cell line, MC12, from which Mest was originally isolated, the 5′-region harboring presumptive promoter of the gene was undermethylated. On the other hand, C4XX, a subclone of MC12 which had lost expression of Mest , was characterized by extremely high levels of methylation in the 5′-region, demethylation of which resulted in activation of Mest . Furthermore, a methylated reporter construct with the luciferase gene under the control of the putative promoter region of Mest was not competent to produce luciferase activity in MC12 cells. These results suggest a suppressive role for DNA methylation in Mest expression. However, neither methylated nor unmethylated reporter constructs showed luciferase activity in a primary culture from the adult kidney, in which Mest is down-regulated despite apparent unmethylation of the paternal allele. Taken together, the data suggest that there are probably two modes of regulation for the Mest gene; one being a methylation-dependent mechanism that regulates imprinted expression of Mest during development, and the other being a methylation-independent mechanism that is involved in down-regulation of Mest in adult tissues.

An inactive human X chromosome was introduced by microcell fusion into two mouse embryonal carcinoma cell lines, PSA1-TG8 and OTF9-63, each of which has a single X chromosome. The donor cell line was a mouse-human somatic cell hybrid, CF150, retaining one or more inactive human X chromosome(s) per cell as its only human element. Twenty hybrid clones isolated retained EC morphology and contained the intact human X chromosome(s) or its truncated derivative(s). Replication banding analysis showed that the introduced human X chromosome(s) or its derivative(s) replicated synchronously with other mouse chromosomes, suggesting reactivation of the human X chromosomal elements after transfer. Reversal of inactivation was further confirmed by the expression of five human X-linked genes repressed in CF150, although the XIST (X inactive specific transcript) gene continued to be active. The level of XIST expression in our hybrid cells was almost identical to that of parental CF150 cells. Methylation status of 5' end of the active XIST gene varied considerably from almost full methylation to unmethylation in these hybrids. Thus, mouse EC cells used in this study were capable of altering methylation status of the human XIST gene in a manner lacking consistency and unable to repress its transcription. Furthermore, we failed to obtain any positive evidence for the occurrence of X chromosome inactivation in differentiating monochromosome EC hybrids. Taken together, these findings suggest that the human X chromosome inactivation center including the XIST gene is unable to function effectively in mouse cells.

We have previously reported the human cDNA encoding MSSP-1, a sequence-specific double- and single-stranded DNA binding protein [Negishi, Nishita, Saëgusa, Kakizaki, Galli, Kihara, Tamai, Miyajima, Iguchi-Ariga and Ariga (1994) Oncogene, 9, 1133-1143]. MSSP-1 binds to a DNA replication origin/transcriptional enhancer of the human c-myc gene and has turned out to be identical with Scr2, a human protein which complements the defect of cdc2 kinase in S.pombe [Kataoka and Nojima (1994) Nucleic Acid Res., 22, 2687-2693]. We have cloned the cDNA for MSSP-2, another member of the MSSP family of proteins. The MSSP-2 cDNA shares highly homologous sequences with MSSP-1 cDNA, except for the insertion of 48 bp coding 16 amino acids near the C-terminus. Like MSSP-1, MSSP-2 has RNP-1 consensus sequences. The results of the experiments using bacterially expressed MSSP-2, and its deletion mutants, as histidine fusion proteins suggested that the binding specificity of MSSP-2 to double- and single-stranded DNA is the same as that of MSSP-1, and that the RNP consensus sequences are required for the DNA binding of the protein. MSSP-2 stimulated the DNA replication of an SV40-derived plasmid containing the binding sequence for MSSP-1 or -2. MSSP-2 is hence suggested to play an important role in regulation of DNA replication.

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.

We have isolated a human homologue (MEST) of the mouse mesoderm-specific transcript (Mest) gene that shares about 70% nucleotide sequence homology. Northern blot analysis showed that the MEST gene was expressed in all major fetal organs and tissues so far examined, i.e., amnion, brain, heart, lung, stomach, gut, adrenal, kidney, muscle, and liver, which does not contradict with mesoderm-specific expression. MEST was abundantly expressed in hydatidiform moles of androgenetic origin, whereas it was barely detectable in dermoid cysts of parthenogenetic origin. Thus, it seems likely that the MEST gene, mapped to 7q32 by fluorescence in situ hybridization, is maternally repressed as the mouse homologue.