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Advances in DNA sequencing technologies have, for the first time, provided us with enough whole chromosome-level genomes to understand in detail how chromosome number and composition change over time. Here, we use the genomes of butterflies and moths to look at the levels and age of macrosynteny in the Lepidoptera and Trichoptera. We used comparative BUSCO analsysis to define reproducible units of macrosynteny which we term ‘Lepidopteran Synteny Units’ or LSUs. The 31 chromosomes of the model butterflyMelitaea cinxiaserved as a reference point. The results show that chromosome-wide macrosynteny extends from the most basal branches of the Lepidopteran phylogeny to the most distal. This synteny also extends to the order Trichoptera, a sister group of the Lepidoptera. Thus, chromosome-wide macrosynteny has been conserved for a period of >200 My in this group of insects. We found no major interchromosomal translocations, reciprocal or non-reciprocal, in the genomes studied. Intrachromosomal rearrangements, in contrast, were abundant. Beyond its use in defining LSUs, this type of homology-based analysis will be useful in determining the relationships between chromosomal elements in different animals and plants. Further, by more precisely defining the breakpoints of chromosomal rearrangements we can begin to look at their potential roles in chromosomal evolution.StatementThe authors declare no conflicting interestsContributionsConceptualisation: W.T., R.H.f.; data analysis: W.T.; writing & editing: W.T., K.S., R.H.f All authors read and approved the final manuscript.

Neo-sex chromosomes are found in many taxa, but the forces driving their emergence and spread are poorly understood. The female-specific neo-W chromosome of the African monarch (or queen) butterfly Danaus chrysippus presents an intriguing case study because it is restricted to a single ‘contact zone’ population, involves a putative colour patterning supergene, and co-occurs with infection by the male-killing endosymbiont Spiroplasma. We investigated the origin and evolution of this system using whole genome sequencing. We first identify the ‘BC supergene’, a broad region of suppressed recombination across nearly half a chromosome, which links two colour patterning loci. Association analysis suggests that the genes yellow and arrow in this region control the forewing colour pattern differences between D. chrysippus subspecies. We then show that the same chromosome has recently formed a neo-W that has spread through the contact zone within approximately 2,200 years. We also assembled the genome of the male-killing Spiroplasma, and find that it shows perfect genealogical congruence with the neo-W, suggesting that the neo-W has hitchhiked to high frequency as the male-killer has spread through the population. The complete absence of female crossing-over in the Lepidoptera causes whole-chromosome hitchhiking of a single neo-W haplotype, carrying a single allele of the BC supergene and dragging multiple non-synonymous mutations to high frequency. This has created a population of infected females that all carry the same recessive colour patterning allele, making the phenotypes of each successive generation highly dependent on uninfected male immigrants. Our findings show how hitchhiking can occur between the physically unlinked genomes of host and endosymbiont, with dramatic consequences., A chromosome carrying a colour patterning supergene has spread rapidly through a population of African monarch butterflies (Danaus chrysippus) by hitchhiking with a male-killing endosymbiont, Spiroplasma, showing how hitchhiking can occur between the unlinked genomes of host and endosymbiont, with dramatic consequences.

The number of sequenced lepidopteran genomes is increasing rapidly. However, the corresponding assemblies rarely represent whole chromosomes and generally also lack the highly repetitive W sex chromosome. Knowledge of the karyotypes can facilitate genome assembly and further our understanding of sex chromosome evolution in Lepidoptera. Here, we describe the karyotypes of the Glanville fritillary Melitaea cinxia (n = 31), the monarch Danaus plexippus (n = 30), and the African queen D. chrysippus (2n = 60 or 59, depending on the source population). We show by FISH that the telomeres are of the (TTAGG)n type, as found in most insects. M. cinxia and D. plexippus have “conventional” W chromosomes which are heterochromatic in meiotic and somatic cells. In D. chrysippus, the W is inconspicuous. Neither telomeres nor W chromosomes are represented in the published genomes of M. cinxia and D. plexippus. Representation analysis in sequenced female and male D. chrysippus genomes detected an evolutionarily old autosome-Z chromosome fusion in Danaus. Conserved synteny of whole chromosomes, so called “macro synteny”, in Lepidoptera permitted us to identify the chromosomes involved in this fusion. An additional and more recent sex chromosome fusion was found in D. chrysippus by karyotype analysis and classical genetics. In a hybrid population between 2 subspecies, D. c. chrysippus and D. c. dorippus, the W chromosome was fused to an autosome that carries a wing colour locus. Thus, cytogenetics and the present state of genome data complement one another to reveal the evolutionary history of the species.

Moths and butterflies (Lepidoptera) are the most species-rich group of animals with female heterogamety, females mostly having a WZ, males a ZZ sex chromosome constitution. We studied chromatin conformation, activity, and inactivity of the sex chromosomes in the flour moth Ephestia kuehniella and the silkworm Bombyx mori, using immunostaining with anti-H3K9me2/3, anti-RNA polymerase II, and fluoro-uridine (FU) labelling of nascent transcripts, with conventional widefield fluorescence microscopy and 'spatial structured illumination microscopy' (3D-SIM). The Z chromosome is euchromatic in somatic cells and throughout meiosis. It is transcriptionally active in somatic cells and in the postpachaytene stage of meiosis. The W chromosome in contrast is heterochromatic in somatic cells as well as in meiotic cells at pachytene, but euchromatic and transcriptionally active like all other chromosomes at postpachytene. As the W chromosomes are apparently devoid of protein-coding genes, their transcripts must be non-coding. We found no indication of 'meiotic sex chromosome inactivation' (MSCI) in the two species.

The Mediterranean flour moth, Ephestia kuehniella is a widespread pest of stored products and a classical object in experimental biology. In the present study, we determined its complete mitochondrial genome sequence. The genome is circular, consists of 15,327 bp and comprises 13 protein-coding, 2 rRNA- and 22 tRNA-coding genes in an order typical for the Ditrysia clade of the order Lepidoptera. A phylogenetic study of the Lepidoptera based on complete mitochondrial genomes places E. kuehniella correctly in the family Pyralidae and supports major lepidopteran taxa as phylogenetic clades. The W chromosome of E. kuehniella is an exceptionally rich reservoir of originally mitochondrial sequences (numts). Around 0.7% of the W DNA was found to be of mitochondrial origin, 83% of the mitogenome sequence was represented between 1-11 × in the W chromosome. Phylogenetic analysis further revealed that these numts are an evolutionary recent acquisition of the W chromosome.

The number of sequenced lepidopteran genomes is increasing rapidly. However, the corresponding assemblies rarely represent whole chromosomes and generally also lack the highly repetitive W sex chromosome. Knowledge of the karyotypes can facilitate genome assembly and further our understanding of sex chromosome evolution in Lepidoptera. Here, we describe the karyotypes of the Glanville fritillary Melitaea cinxia (n = 31), the monarch Danaus plexippus (n = 30), and the African queen D. chrysippus (2n = 60 or 59, depending on the source population). We show by FISH that the telomeres are of the (TTAGG)n type, as found in most insects. M. cinxia and D. plexippus have "conventional" W chromosomes which are heterochromatic in meiotic and somatic cells. In D. chrysippus, the W is inconspicuous. Neither telomeres nor W chromosomes are represented in the published genomes of M. cinxia and D. plexippus. Representation analysis in sequenced female and male D. chrysippus genomes detected an evolutionarily old autosome-Z chromosome fusion in Danaus. Conserved synteny of whole chromosomes, so called "macro synteny", in Lepidoptera permitted us to identify the chromosomes involved in this fusion. An additional and more recent sex chromosome fusion was found in D. chrysippus by karyotype analysis and classical genetics. In a hybrid population between 2 subspecies, D. c. chrysippus and D. c. dorippus, the W chromosome was fused to an autosome that carries a wing colour locus. Thus, cytogenetics and the present state of genome data complement one another to reveal the evolutionary history of the species.

Organ growth depends on cell division and (or) cell growth. Here, we present a study on two organs whose growth depends entirely on cell growth, once they are formed in the embryo: Malpighian tubules and silk glands of the flour moth, Ephestia kuehniella . Between first and last larval instar, the volume of Malpighian tubule cells increases by a factor of ∼1800 and that of silk gland cells by a factor of ∼3100. We determined the number of endocyles required to reach these stages by Feulgen cytometry. Cells of Malpighian tubules were in the 2C stage in first instar larvae and reached 1024C after 9 endocycles in last instar larvae (1C = 0.45 pg DNA). Silk gland cells already reached a DNA content of 8C–16C in first instar larvae and attained up to 8192C in last instar larvae after a total of 12 endocycles. The nuclei were small and more or less spherical in first instar larvae, but they were huge, flat, and bizarrely branched in last instar larvae. We consider branching as a compensatory adaptation to improve molecular traffic between nucleus and cytoplasm in these excessively large and highly polyploid cells (i) by reducing the mean distance between nucleus and cytoplasm and (ii) by enlarging the surface-to-volume ratio of these nuclei.

We have previously described the paralogous mouse genes Caspr5-1, -2, and -3 of the neurexin gene family. Here we present the cytogenetic and molecular mapping of a null mutation of Caspr5-2 which was caused by reciprocal translocation between chromosomes 1 and 8 with breakpoints at bands 1E2.1 and 8B2.1, respectively. The translocation disrupts Caspr5-2 between exons 1 and 2 and causes stillbirth or early postnatal lethality of homozygous carriers. Because no other candidate genes were found, the disruption of Caspr5-2 is most likely the cause of lethality. Only rarely do homozygotes survive the critical stage, reach fertility, and are then apparently normal. They may be rescued by one of the two other Caspr5 paralogs. Caspr5-2 is expressed in spinal cord and brain tissues. Despite giving special attention to regions where in wild-type fetuses maximum expression was found, no malformation that might have caused death could be detected in fetal homozygous carriers of the translocation. We, therefore, suspect that Caspr5-2 disruption leads to dysfunction at the cellular level rather than at the level of organ development.

The speciose insect order Lepidoptera (moths and butterflies) and their closest relatives, Trichoptera (caddis flies), share a female-heterogametic sex chromosome system. Originally a Z/ZZ (female/male) system, it evolved by chromosome rearrangement to a WZ/ZZ (female/male) system in the most species-rich branch of Lepidoptera, a monophyletic group consisting of Ditrysia and Tischeriina, which together comprise more than 98% of all species. Further sporadic rearrangements created multi-sex chromosome systems; sporadic losses of the W changed the system formally back to Z/ZZ in some species. Primary sex determination depends on a Z-counting mechanism in Z/ZZ species, but on a female-determining gene, Fem, in the W chromosome of the silkworm. The molecular mechanism is unknown in both cases. The silkworm shares the last step, dsx, of the hierarchical sex-determining pathway with Drosophila and other insects investigated, but probably not the intermediate steps between the primary signal and dsx. The W chromosome is heterochromatic in most species. It contains few genes and is flooded with interspersed repetitive elements. In interphase nuclei of females it is readily discernible as a heterochromatic body which grows with increasing degree of polyploidy in somatic cells. It is used as a marker for the genetic sex in studies of intersexes and Wolbachia infections. The sex chromosome system is being exploited in economically important species. Special strains have been devised for mass rearing of male-only broods in the silkworm for higher silk production and in pest species for the release of sterile males in pest management programs.

The W chromosome of the codling moth, Cydia pomonella, like that of most Lepidoptera species, is heterochromatic and forms a female-specific sex chromatin body in somatic cells. We collected chromatin samples by laser microdissection from euchromatin and W-chromatin bodies. DNA from the samples was amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and used to prepare painting probes and start an analysis of the W-chromosome sequence composition. With fluorescence in situ hybridization (FISH), the euchromatin probe labelled all chromosomes, whereas the W-chromatin DNA proved to be a highly specific W-chromosome painting probe. For sequence analysis, DOP-PCR-generated DNA fragments were cloned, sequenced, and tested by Southern hybridization. We recovered single-copy and low-copy W-specific sequences, a sequence that was located only in the W and the Z chromosome, multi-copy sequences that were enriched in the W chromosome but occurred also elsewhere, and ubiquitous multi-copy sequences. Three of the multi-copy sequences were recognized as derived from hitherto unknown retrotransposons. The results show that our approach is feasible and that the W-chromosome composition of C. pomonella is not principally different from that of Bombyx mori or from that of Y chromosomes of several species with an XY sex-determining mechanism. The W chromosome has attracted repetitive sequences during evolution but also contains unique sequences.

We cloned Bm-Sxl, an orthologue of the Drosophila melanogaster Sex-lethal (Sxl) gene from embryos of Bombyx mori. The full-length cDNAs were of 2 sizes, 1528 and 1339 bp, and were named Bm-Sxl-L and Bm-Sxl-S, respectively. Bm-Sxl-L consists of 8 exons and spans more than 20 kb of genomic DNA. The open reading frame (ORF) codes for a protein 336 amino acids in length. Bm-Sxl-S is a splice variant that lacks the second exon. This creates a new translation start 138 nucleotides downstream and an ORF that codes for 46 amino acids fewer at the N-terminus. Linkage analysis using an F2panel mapped Bm-Sxl to linkage group 16 at 69.8 cM. We isolated 2 BACs that include the Bm-Sxl gene. With BAC-FISH we located Bm-Sxl cytogenetically on the chromosome corresponding to linkage group 16 (LG16) at position >68.8 cM.Key words: Sex-lethal, Sxl, Bombyx mori, alternative splicing, BAC-FISH.

The (TTAGG)n sequence is supposed to be an ancestral DNA motif of telomeres in insects. Here we examined the occurrence of TTAGG telomeric repeats in other arthropods and their close relatives by Southern hybridization of genomic DNAs and fluorescence in-situ hybridization (FISH) of chromosomes with (TTAGG)n probes or, alternatively, with the 'vertebrate' telomeric probe, (TTAGGG)n. Our results show that the (TTAGG)n motif is conserved in entognathous hexapods (Diplura and Collembola), crustaceans (Malacostraca, Branchiura, Pentastomida, and Branchiopoda), myriapods (Diplopoda and Chilopoda), pycnogonids, and most chelicerates (Palpigradi, Amblypygi, Acari, Opiliones, Scorpiones, Pseudoscorpiones, and Solifugae) but not in spiders (Araneae). The presence of TTAGG repeats in these groups suggests that the sequence is an ancestral motif of telomeres not only in insects but in Arthropoda. We failed, however, to detect the TTAGG repeats in close relatives of the arthropods, Tardigrada and Onychophora. But while Onychophora had the 'vertebrate' (TTAGGG)n motif instead, the Tardigrada did not. The (TTAGG)n motif probably evolved from the (TTAGGG)n motif. Based on our and compiled data, we presume that the 'vertebrate' motif (TTAGGG)n is an ancestral motif of telomeres in bilaterian animals and possibly also in the superclade including animals, fungi and amoebozoans.

A polymorphism of the central part of chromosome I has been described from natural populations of the house mouse (Mus muscu/us). The region shows up as a C band-positive homogeneously staining region (HSR) under the light microscope. M. rn. domes~icus mice carry single band HSRs, whereas M. m. musculus animals have double band HSRs. HSR size variations have been described in both subspecies. The frequency of the HSR chromosome 1 in populations varies from 4% to 81 %, but none of the large samples examined consisted only of homozygotes. In the subspecies M. m. domesticus, HSRs were found in North Africa and Western Europe, mainly in the hilly regions of Southern Gemany and Switzerland. Localities with double HSRs are distributed all over the area of M. m. musculus. Based on the population data presented and DNA similarity of different HSRs, the origin and distribution of HSR chromosomes in the house mouse are discussed.

Synaptonemal complexes (SCs) of pachytene bivalents from oocytes and spermatocytes of the wax moth, Galleria mellonella, were studied with a microspreading technique. In both sexes 30 bivalents were found. Male SCs in contrast to female SCs contained recombination nodules. No specific bivalent could be recognized in male complements. In female complements, the WZ bivalent was recognized among the three longest bivalents by its delayed pairing and/or unequal lengths of the lateral elements. Pairing of WZ bivalents is complete and lateral elements are nearly equalized in a few cells, presumably at the end of pachytene.

Chromosome numbers vary greatly in Orgyiu, from low numbers, like n = 11 in 0. thyellina and n = 14 in 0. antiqua, to a high number, n = 30, as in 0. recens and 0. ericue. Meiotic synapsis was regular in 0. thyellina and 0. antiqua; 11 and 14 normal bivalents, respectively, were found in meiosis. The paired homologues displayed homologous chromomere patterns. In the species hybrid between antiqua and thyellina, many synapsed chromosome segments were found in meiosis. This indicates sufficient segmental homology between chromosomes of the two species although the paired pachytene chromosome segments rarely displayed similar chromomere patterns. Chromosomes switched pairing partners, thus forming multivalents, linked by chiasmata in males, and long synaptic chains in the achiasmatic females. Multivalent formation is understood as the consequence of a separate evolution of the two species from a species with a high chromosome number. Multiple chromosome fusions resulted in similarly low chromosome numbers but different segmental compositions of the chromosomes in the two species. Chromosome numbers in animals range from n = 1 in the nematode Parascaris univalens and the ant Myrmecia pilosula to n % 220 in the butterfly Lysandra atlantica (BOVERI 1899; CROSLAND and CROZIER 1986; DE LESSE 1970). Thus increases and/or decreases must have taken place during evolution. Dissociation rather than polyploidization is regarded the main source of increases in chromosome number of bisexually reproducing animal species (WHITE 1973). Decreases on the other hand are thought to be caused by chromosome fusions. The crude limitations to increases and decreases are dictated by the dimensions of the spindle. The chromosome arms must be small enough not be cut by the cleavage furrow but the chromosome number must not exceed the number that fits into the equatorial plate and gets attached to the spindle (WHITE 1973). Although it is conceivable that the number of linkage groups is under selective

While most Lepidoptera have numerous small chromosomes, Orgyia thyellina has few (n = 11) and relatively big chromosomes. We exploited this situation in a light microscopic study of chromosome morphology in mitosis and in various stages of male and female meiosis.—There is no indication of a primary constriction in mitotic chromosomes. Meiosis is chiasmatic in males and achiasmatic in females. Bivalents in the late female pachytene stage have a chromomere pattern of an unusually high degree of resolution. It is especially suited to test structural homology of synapsed chromosomes in bivalents. The chromomere pattern is homologous in the synapsed autosomes but partly differential in the equally synapsed sex chromosomes. The female pachytene affords a good basis for cytogenetic investigations in Lepidoptera.

Comparative genomic hybridization (CGH) with a probe mixture of differently labeled genomic DNA from females and males highlighted the W chromosomes in mitotic plates and the W chromatin in polyploid interphase nuclei of the silkworm Bombyx mori, the flour moth Ephestia kuehniella, and the wax moth Galleria mellonella. The overproportionate fluorescence signal indicated an accumulation of repetitive sequences in the respective W chromosomes. Measurements of the fluorescence signals revealed two components, one that is present also in male DNA (non-W chromosomes) and another one that is present only in or preponderantly in female DNA (W chromosomes). While the W chromosomes of E. kuehniella and G. mellonella had both components, that of B. mori appeared to lack the latter component. Our results show that CGH can be applied to obtain a first estimate of the sequence composition of sex chromosomes in species from which otherwise little is known on the molecular level., http://pubs.nrc-cnrc.gc.ca

We used immunolocalization in tissue sections and cytogenetic preparations of female and male gonads to study the distribution of the proliferation marker pKi-67 during meiotic cell cycles of the house mouse, Mus musculus. During male meiosis, pKi-67 was continuously present in nuclei of all stages from the spermatogonium through spermatocytes I and II up to the earliest spermatid stage (early round spermatids) when it appeared to fade out. It was not detected in later spermatid stages or sperm. During female meiosis, pKi-67 was present in prophase I oocytes of fetal ovaries. It was absent in oocytes from newborn mice and most oocytes of primordial follicles from adults. The Ki-67 protein reappeared in oocytes of growing follicles and was continuously present up to metaphase II. Thus, pKi-67 was present in all stages of cell growth and cell division while it was absent from resting oocytes and during the main stages of spermiocytogenesis. Progression through the meiotic cell cycle was associated with extensive intranuclear relocation of pKi-67. In the zygotene and pachytene stages, most of the pKi-67 colocalized with centromeric (centric and pericentric) heterochromatin and adjacent nucleoli; the heterochromatic XY body in male pachytene, however, was free of pKi-67. At early diplotene, pKi-67 was mainly associated with nucleoli. At late diplotene, diakinesis, metaphase I and metaphase II of meiosis, pKi-67 preferentially bound to the perichromosomal layer and was almost absent from the heterochromatic centromeric regions of the chromosomes. After the second division of male meiosis, the protein reappeared at the centromeric heterochromatin and an adjacent region in the earliest spermatid stage and then faded out. The general patterns of pKi-67 distribution were comparable to those in mitotic cell cycles. With respect to the timing, it is interesting to note that relocation from the nucleolus to the perichromosomal layer takes place at the G2/M-phase transition in the mitotic cell cycle but at late diplotene of prophase I in meiosis, suggesting physiological similarity of these stages.

The proliferation-associated nuclear protein pKi-67 relocates from the nucleolus to the chromosome surface during the G2/M transition of the cell cycle and contributes to the formation of the ‘perichromosomal layer’. We investigated the in-vivo binding preferences of pKi-67 for various chromatin blocks of the mitotic chromosomes from the human and two mouse species, Mus musculus and M. caroli. All chromosomes were decorated with pKi-67 but displayed a gap of pKi-67 decoration in the centromere and NOR regions. pKi-67 distribution in a rearranged mouse chromosome showed that the formation of the centromeric gap was controlled by the specific chromatin in that region. While most chromatin served as a substrate for direct or indirect binding of pKi-67, we identified three types of chromatin that bound less or no pKi-67. These were: (1) the centromeric heterochromatin defined by the alpha satellite DNA in the human, by the mouse minor satellite in M. musculus and the 60- and 79-bp satellites in M. caroli; (2) the pericentromeric heterochromatin in M. musculus defined by the mouse major satellite, and (3) NORs in the human and in M. musculus defined by rDNA repeats. In contrast, the conspicuous blocks of pericentromeric heterochromatin in human chromosomes 1, 9 and 16 containing the 5-bp satellite showed intense pKi-67 decoration. The centromeric gap may have a biological significance for the proper attachment of the chromosomes to the mitotic spindle. In this context, our results suggest a new role for centromeric heterochromatin: the control of the centromeric gap in the perichromosomal layer.

We describe SC complements and results from comparative genomic hybridization (CGH) on mitotic and meiotic chromosomes of the zebrafish Danio rerio, the platyfish Xiphophorus maculatus and the guppy Poecilia reticulata. The three fish species represent basic steps of sex chromosome differentiation: (1) the zebrafish with an all-autosome karyotype; (2) the platyfish with genetically defined sex chromosomes but no differentiation between X and Y visible in the SC or with CGH in meiotic and mitotic chromosomes; (3) the guppy with genetically and cytogenetically differentiated sex chromosomes. The acrocentric Y chromosomes of the guppy consists of a proximal homologous and a distal differential segment. The proximal segment pairs in early pachytene with the respective X chromosome segment. The differential segment is unpaired in early pachytene but synapses later in an 'adjustment' or 'equalization' process. The segment includes a postulated sex determining region and a conspicuous variable heterochromatic region whose structure depends on the particular Y chromosome line. CGH differentiates a large block of predominantly male-specific repetitive DNA and a block of common repetitive DNA in that region.

The well-known sex-determining cascade of Drosophila melanogaster serves as a paradigm for the pathway to sexual development in insects. But the primary sex-determining signal and the subsequent step, Sex-lethal (Sxl), have been shown not to be functionally conserved in non-Drosophila flies. We isolated doublesex (dsx), which is a downstream step in the cascade, from the phorid fly Megaselia scalaris, which is a distant relative of D. melanogaster. Conserved properties, e.g., sex-specific splicing, structure of the female-specific 3' splice site, a splicing enhancer region with binding motifs for the TRA2/RBP1/TRA complex that activates female-specific splicing in Drosophila, and conserved domains for DNA-binding and oligomerization in the putative DSX protein, indicate functional conservation of dsx in M. scalaris. Hence, the dsx step of the sex-determining pathway appears to be conserved among flies and probably in an even wider group of insects, as the analysis of a published cDNA from the silkmoth indicates.Key words: sex-determining cascade, splice regulation, DNA-binding domain, oligomerization.

Sex-lethal (Sxl) is Drosophila melanogaster's key regulating gene in the sex-determining cascade. Its homologue in Megaselia scalaris, the chromosome 3 gene Megsxl, codes for a protein with an overall similarity of 77% with the corresponding D. melanogaster sequence. Expression in M. scalaris, however, is very unlike that in D. melanogaster. Megsxl transcripts with a long ORF occur in both sexes. Differential splicing is conserved but not sex-specific. There are several splice variants, among them one is common to gonads and somatic tissues of all developmental stages investigated, one is specific for ovaries and embryos, and a third one is not found in ovaries. In the ovary, Megsxl is heavily transcribed in nurse cells and transported into eggs. These results suggest a non-sex-determining function during early embryogenesis; the presence of Megsxl RNA in testes and somatic tissues calls for other (or more) functions.Key words: sex determination, RNA-binding domain, differential splicing, RNA in situ hybridization.

Comparative genomic hybridization (CGH) was used to identify and probe sex chromosomes in several XY and WZ systems. Chromosomes were hybridized simultaneously with FluorX-labelled DNA of females and Cy3-labelled DNA of males in the presence of an excess of Cot-1 DNA or unlabelled DNA of the homogametic sex. CGH visualized the molecular differentiation of the X and Y in the house mouse, Mus musculus, and in Drosophila melanogaster: while autosomes were stained equally by both probes, the X and Y chromosomes were stained preferentially by the female-derived or the male-derived probe, respectively. There was no differential staining of the X and Y chromosomes in the fly Megaselia scalaris, indicating an early stage of sex chromosome differentiation in this species. In the human and the house mouse, labelled DNA of males in the presence of unlabelled DNA of females was sufficient to highlight Y chromosomes in mitosis and interphase. In WZ sex chromosome systems, the silkworm Bombyx mori, the flour moth Ephestia kuehniella, and the wax moth Galleria mellonella, the W chromosomes were identified by CGH in mitosis and meiosis. They were conspicuously stained by both female- and male-derived probes, unlike the Z chromosomes, which were preferentially stained by the male-derived probe in E. kuehniella only but were otherwise inconspicuous. The ratio of female:male staining and the pattern of staining along the W chromosomes was species specific. CGH shows that W chromosomes in these species are molecularly well differentiated from the Z chromosomes. The conspicuous binding of the male-derived probe to the W chromosomes is presumably due to an accumulation of common interspersed repetitive sequences.

We studied the occurrence of the TTAGG telomere repeats by fluorescence in-situ hybridization (FISH) and Southern hybridization in ten insect species and two other arthropods. (TTAGG)n-containing telomeres were found in three Lepidoptera species, the silkworm Bombyx mori (in which the telomeric sequence was recently discovered), the flour moth Ephestia kuehniella, and the wax moth Galleria mellonella, in one species of Hymenoptera, the honey bee Apis mellifera, in one species of Coleoptera, the bark beetle Ips typographus, in one species of Orthoptera, the locust Locusta migratoria, and in a crustacean, the amphipod Gammarus pulex. They were absent in another species of Coleoptera, the mealworm Tenebrio molitor, two representatives of Diptera, Drosophila melanogaster and Megaselia scalaris, a species of Heteroptera, the bug Pyrrhocoris apterus and a spider, Tegenaria ferruginea. Our results, which confirm and extend earlier observations, suggest that (TTAGG)n was a phylogenetically ancestral telomere motif in the insect lineage but was lost independently in different groups, being replaced probably by other telomere motifs. In the Coleoptera this must have happened rather recently as even members of the same family, Curculionidae, differ with respect to the telomeric DNA.

Satellite DNAs (stDNAs) of four Acomys species (spiny-mice), A. cahirinus, A. cineraceus, A. dimidiatus and A. russatus, belong to closely related sequence families. Monomer sizes range from 338 to 364 bp. Between-species sequence identity was from 81.0% to 97.2%. The molecular phylogeny of the sequences helps to clarify the taxonomy of this ‘difficult’ group. The A. dimidiatus genome contains about 60 000 repeats. According to the restriction patterns, repeats are arranged in tandem. The stDNA maps to the centromeric heterochromatin of most autosomes, both acrocentric and metacentric, but appears to be absent in the centromeric region of Y chromosomes. A well-conserved centromere protein B (CENP-B) box is present in the stDNA of A. russatus while it is degenerated in the other species.

The polymorphic Sp100-rs repeat cluster in chromosome band 1D of the house mouse, Mus musculus, makes up as much as 0.1–5% of the haploid genome. ‘High-copy’ versions of this long-range repeat cluster are cytogenetically apparent as DAPI-negative chromomycin-A3-positive homogeneously staining regions (HSRs). The cluster is a relatively recent acquisition in the genus Mus; the related species M. caroli possesses neither the Sp100-rs cluster nor even the Sp100-rs gene. Except for chromosomes with high-copy clusters, no major rearrangements are visible in chromosomes 1 from M. musculus and M. caroli: they have the same order of G-bands, DAPI-bands and chromomycin A3-bands. Comparative genomic hybridization (CGH) visualizes the cluster in M. musculus and detects a single region of sequence homology to the cluster in M. caroli chromosome band 1D. This indicates that the M. musculus cluster has evolved in situ from sequences originally present in the same chromosome band.

The sex chromosomes of the Megaselia scalaris wild-type strain Wien are homomorphic. We studied a roughly 1.8 kb X/Y DNA segment of this strain. It includes, at one end, the first part of a coding sequence for a protein of the vespid antigen 5 family. Molecular differentiation between the X and Y chromosomes has commenced, but homology, even of short DNA stretches, is still assessable beyond doubt. The most conspicuous differences between the X and the homologous Y segment were insertions/deletions in the noncoding region: among them, deletions, a duplication, and an insertion of a mobile element. These structural changes grossly disrupted homology. In comparison, base substitutions, though more numerous, contributed little to the differentiation of the X/Y DNA segment.Key words: sex determination, molecular differentiation, mobile element, vespid antigen 5.

Sp100 is a single-copy gene in the human and the mouse. A related gene, Sp100-rs, occurs in multiple copies and forms a conspicuous cluster in the mouse chromosome 1. Murine Sp100 and Sp100-rs are homologous from the promoter up to a position in intron 3, but they differ 3′ of that position. In the genus Mus, Sp100-rs is present in one phylogenetic branch, represented by the house mouse, M. musculus, but probably does not exist in another branch, represented by M. caroli. Thus, Sp100-rs arose relatively late in the evolution of the genus Mus, whereas Sp100 existed in the common ancestor of the human and the mouse. The Sp100-rs gene cluster probably evolved by gene fusion followed by amplification and diversification.

The human SP100 gene encodes an autoantigen that colocalizes with two other proteins, PML and NDP52, in distinct nuclear domains, called "nuclear dots" (NDs). NDs do not overlap with other known subnuclear structures, and their function is still unknown. Patients suffering from the autoimmune disease primary biliary cirrhosis often produce antibodies against the SP100 protein. The present study describes the structure and expression of the murine Sp100 gene. In the species Mus caroli, Sp100 consists of 17 exons that are distributed over a range of 52 kb. The human and murine Sp100 promoters are very similar, and both harbor an interferon-stimulated response element. Like its human counterpart, the murine Sp100 gene is responsive to interferon treatment. The house mouse, Mus musculus, harbors the Sp100 gene and a second gene with homology to Sp100, the multicopy Sp100-rs gene. However, in contrast to the genuine mouse homolog, Sp100-rs shares only segmental homology with the human Sp100 gene. Replacement of the murine Sp100 gene by a defective copy is now feasible and should shed light on its function in an animal model.

Sex-lethal (Sxl) and doublesex (dsx) are known to represent parts of the sex-determining cascade in Drosophila melanogaster. We generated cDNA probes of the homologous genes from Megaselia scalaris, a fly species with an epistatic maleness factor as the primary sex determining signal. In Northern blot hybridization of poly(A)+RNA, the M. scalaris dsx probe detected two bands, one of which had a sex-specific size difference, while the Sxl probe bound to RNAs of equal size in females and males. RT-PCR showed Sxl to be transcribed in gonads of adult females and males but not in somatic tissues. Thus, while dsx appears to have a similar function in M. scalaris and D. melanogaster, Sxl does not. The results suggest that the sex-determining pathway of M. scalaris joins that of D. melanogaster between the Sxl and dsx steps.Key words: RNA-binding domain, zinc finger, differential splicing, Drosophila.

Sex chromosome morphology of eight Lepidoptera species was studied, exploiting predominantly the pachytene stage when chromosomes display a remarkable chromomere pattern. Six species had a WZ/ ZZ sex chromosome system, one species a W1W2Z/ ZZ system and one species was of the Z/ZZ type. Much like XY chromosomes in groups with male heterogamety, the lepidopteran sex chromosomes showed various degrees of structural differentiation. Differences between Z and W chromomere patterns ranged from undetectable to obviously non-homologous. A common property of the W chromosomes (the W1 in the W1W2Z/ZZ system) was the possession of a block of heterochromatin. The heterochromatin block comprised a small or a large segment of the W or even the entire W, depending on the species. Segments with apparent structural homology are evolutionarily young parts of the sex chromosomes-recently fused autosomes that have not had sufficient time for differentiation. The 'primitive' lepidopteran species Micropterix calthella had a Z/ZZ sex chromosome system. This supports the hypothesis that the lepidopteran W chromosome came into being at the base of the 'advanced' Lepidoptera; it was presumably an autosome whose homologue fused to the original Z chromosome.

Like mammals, Lepidoptera possess female-specific sex chromatin. In a compilation of new and published data, 81% of the 238 investigated Lepidoptera species display one or more heterochromatin bodies in female somatic interphase cells, but not in male cells. In contrast with the similar phenomenon in mammals, this sex-specific heterochromatin does not function as a dosage compensation mechanism. Most Lepidoptera have a WZ/ZZ sex chromosome mechanism, and the sex chromatin is derived from the univalent W sex chromosome. Sex chromatin is regarded as an indicator of an advanced stage of W chromosome evolution. In species with a Z/ZZ sex chromosome mechanism, loss of the W chromosome is accompanied by loss of the female-specific heterochromatin. Since sex chromatin can be discerned easily in interphase nuclei, and especially so in the highly polyploid somatic cells, it is a useful marker for diagnosing chromosomal sex of embryos and larvae, and of identifying sex chromosome aberrations in mutagenesis screens. All species with sex chromatin belong to the Ditrysia, the main clade of Lepidoptera that contains more than 98% of all extant species. Sex chromatin has not been reported for clades that branched off earlier. The nonditrysian clades share this character with Trichoptera, a sister group of the Lepidoptera. We propose that Lepidoptera originally had a Z/ZZ sex chromosome mechanism like Trichoptera; the WZ/ZZ sex chromosome mechanism evolved later in the ditrysian branch of Lepidoptera. Secondary losses of the W chromosome account for the sporadically occurring Z/ZZ sex chromosome systems in ditrysian families. The lepidopteran sex chromatin, therefore, appears to mirror the full evolutionary life cycle of a univalent sex chromosome from its birth through heterochromatinization to sporadic loss.

A family of closely related genes is a component of the polymorphic long-range repeat cluster D1Lub1 of the house mouse. Members of the gene family have diverged from one another by rearrangements and point mutations. D1Lub1 clusters have low (≈50) or high (≥500) copy numbers. In mice with high-copy clusters five or six poly(A)+ RNAs are found, while in mice with low-copy clusters only a single member of the RNA family is detected. The RNA family is synthesized in a tissue-independent manner. Each member of the RNA family is defined by a set of DNA probes. Cross hybridization with the probes reveals common 5′ regions and variable remaining parts. The RNA variants are probably transcribed from different gene copies.Key words: long-range repeat cluster, long-range repeat derived gene family, mRNA family.

Structure and pairing behavior of sex chromosomes in females of four T(W;Z) lines of the Mediterranean flour moth, Ephestia kuehniella, were investigated using light and electron microscopic techniques and compared with the wild type. In light microscopic preparations of pachytene oocytes of wild-type females, the WZ bivalent stands out by its heterochromatic W chromosome strand. In T(W;Z) females, the part of the Z chromosome that was translated onto the W chromosome was demonstrated as a distal segment of the neo-W chromosome, displaying a characteristic non-W chromosomal chromomere–interchromomere pattern. This segment is homologously paired with the corresponding part of a complete Z chromosome. In contrast with the single ball of heterochromatic W chromatin in highly polyploid somatic nuclei of wild-type females, the translocation causes the formation of deformed or fragmented W chromatin bodies, probably owing to opposing tendencies of the Z and W chromosomal parts of the neo-W. In electron microscopic preparations of microspread nuclei, sex chromosome bivalents were identified by the remnants of electron-dense heterochromatin tangles decorating the W chromosome axis, by the different lengths of the Z and W chromosome axes, and by incomplete pairing. No heterochromatin tangles were attached to the translocated segment of the Z chromosome at one end of the neo-W chromosome. Because of the homologous pairing between the translocation and the structurally normal Z chromosome, pairing affinity of sex chromosomes in T(W;Z) females is significantly improved. Specific differences observed among T(W;Z)1–4 translocations are probably due to the different lengths of the translocated segments.Key words: Mediterranean flour moth, sex chromosomes, sex chromatin, translocations, synaptonemal complexes, microspreading.

The fly Megaselia scalaris Loew possesses three homomorphic chromosome pairs; 2 is the sex chromosome pair in two wild-type laboratory stocks of different geographic origin (designated "original" sex chromosome pair in this paper). The primary male-determining function moves at a very low rate to other chromosomes, thereby creating new Y chromosomes. Random amplified polymorphic DNA markers obtained by polymerase chain reaction with single decamer primers and a few available phenotypic markers were used in testcrosses to localize the sex-determining loci and to define the new sex chromosomes. Four cases are presented in which the primary male-determining function had been transferred from the original Y chromosome to a new locus either on one of the autosomes or on the original X chromosome, presumably by transposition. In these cases, the sex-determining function had moved to a different locus without an obvious cotransfer of other Y chromosome markers. Thus, with Megaselia we are afforded an experimental system to study the otherwise hypothetical primary stages of sex chromosome evolution. An initial molecular differentiation is apparent even in the new sex chromosomes. Molecular differences between the original X and Y chromosomes illustrate a slightly more advanced stage of sex chromosome evolution.

Lepidoptera, i.e. moths and butterflies, have a female heterogametic sex chromosome system, with most females having a WZ constitution while males are ZZ. Besides this predominant WZ/ZZ system, Z/ZZ, WZ(1)Z(2)/Z(1)Z(1)Z(2)Z(2) and W(1)W(2)Z/ZZ systems also occur. Sex is determined by an unknown W-linked gene or genes in Bombyx mori, but by dosage-dependent and equally unknown Z-linked genes in all Z/ZZ species. The female heterogametic sex chromosome system has been conserved for at least 180 MY in the phylogenetic branch that combines Lepidoptera and Trichoptera. The W chromosome, which is present in most lepidopteran species, was incorporated in the sex chromosome system much later, about 90-100 MY ago. The Z chromosomes are highly conserved among Lepidoptera, much like the Z in birds or the X in mammals. The W, on the other hand, is evolving rapidly. It is crammed with repetitive elements which appear to have a high turnover rate but poor in or even devoid of protein-coding genes. It has frequently undergone fusion with autosomes or sporadically lost altogether.

Only a few prominent simple sequence repeat (SSR) loci of the type (GATA)n are found in the genome of the mealmoth Ephestia kuehniella Zeller. Therefore this moth was chosen as a model organism for the genetic and molecular analysis of hypervariability of SSR loci. We characterized alleles of (GATA)n loci in different Ephestia strains by cloning and genomic restriction mapping. Some variants appeared to be mere variable number of tandem repeat (VNTR) alleles, others showed considerable changes in the sequence neighbourhood of the GATA repeats. These may be produced by major rearrangements or by transposition of the (GATA)n block together with flanking sequences into a different sequence environment.

About 50 copies of a long-range repeat DNA family with a repeat size of roughly 100 kb and with sequence homology to mRNAs are clustered in the G-light band D of chromosome 1 of the house mouse, Mus musculus. We studied amplified versions of the cluster which are found in many wild populations of M. musculus. They are cytogenetically conspicuous as one or two C-band positive homogeneously staining regions (single- and double band HSRs) which increase the mitotic length of chromosome 1. The double band HSR was phylogenetically derived from a single band HSR by a paracentric inversion. In homozygous condition, such HSRs contribute, albeit not as much as expected from their mitotic length, to the synaptonemal complex (SC) length of chromosome 1. In HSR heterozygous animals an elongation of the SCs was not noticeable. In single band HSR heterozygous males, synapsis proceeds regularly and continuously from the distal telomere towards the centromeric end without forming buckles. Thus, the single band HSR has no adverse effect on pairing. The same straight pairing behaviour was found in the majority of double band HSR heterozygous spermatocytes. This shows that extensive nonhomologous pairing can take place in the earliest phase of synapsis. Synapsis was discontinuous, leaving the central part of the bivalent 1 asynapsed, in only 14.3% of double band HSR heterozygous cells. In such cells the chromosome 1 SC is completed at a later stage of meiosis. The delay is presumably an effect of the inversion that includes one HSR band and the segment between the two HSR bands.

The phorid fly Megaselia scalaris is a laboratory model for the turnover and early differentiation of sex chromosomes. Isolates from the field have an XY sex-determining mechanism with chromosome pair 2 acting as X and Y chromosomes. The sex chromosomes are homomorphic but display early signs of sex chromosome differentiation: a low level of molecular differences between X and Y. The male-determining function (M), maps to the distal part of the Y chromosome’s short arm. In laboratory cultures, new Y chromosomes with no signs of a molecular differentiation arise at a low rate, probably by transposition of M to these chromosomes. Downstream of the primary signal, the homologue of the Drosophila doublesex (dsx) is part of the sex-determining pathway while Sex-lethal (Sxl), though structurally conserved, is not.

We describe synthesis and testing of a novel type of dye-modified nucleotides which we call macromolecular nucleotides (m-Nucs). Macromolecular nucleotides comprise a nucleotide moiety, a macromolecular linear linker, and a large macromolecular ligand carrying multiple fluorescent dyes. With incorporation of the nucleotide moiety into the growing nucleic acid strand during enzymatic synthesis, the macromolecular ligand together with the coupled dyes is bound to the nucleic acid. By the use of this new class of modified nucleotides, signals from multiple dye molecules can be obtained after a single enzymatic incorporation event. The modified nucleotides are considered especially useful in the fields of nanobiotechnology, where signal stability and intensity is a limiting factor.

We established homogeneous sublines that contained two or three homozygous DNA loci with long (GATA)n tracts from polymorphic Ephestia laboratory strains. Crossbreeding analysis assigned these loci to two or three different chromosomes, respectively. A nonrelated and rather recently isolated strain contained at least three other poly(GATA) loci located on different chromosomes. Germline mutations, visible as restriction fragment size changes between parents and offspring or loss of a poly(GATA) tract, are relatively rare in some strains but unusually frequent in the ml strain. The mutations affect not only GATA repeats but also flanking sequences. In five mutations investigated by crossbreeding, the altered poly(GATA)-containing restriction fragments remained in their original linkage groups.Key words: Bkm DNA, microsatellite, simple sequence repeats.