Walther Traut

TTAGG Telomeric Repeats in Chromosomes of Some Insects and Other Arthropods

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.

Meiotic chromosomes and stages of sex chromosome evolution in fish: zebrafish, platyfish and guppy.

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.

Sex Chromosomes and Sex Determination in Lepidoptera

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 evolutionary origin of insect telomeric repeats, (TTAGG) n

RNA polymerase II is responsible for transcription of most eukaryotic genes, but, despite exhaustive analysis, little is known about how it transcribes natural templates in vivo. We studied polymerase dynamics in living Chinese hamster ovary cells using an established line that expresses the largest (catalytic) subunit of the polymerase (RPB1) tagged with the green fluorescent protein (GFP). Genetic complementation has shown this tagged polymerase to be fully functional. Fluorescence loss in photobleaching (FLIP) reveals the existence of at least three kinetic populations of tagged polymerase: a large rapidly-exchanging population, a small fraction resistant to 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) but sensitive to a different inhibitor of transcription (i.e. heat shock), and a third fraction sensitive to both inhibitors. Quantitative immunoblotting shows the largest fraction to be the inactive hypophosphorylated form of the polymerase (i.e. IIA). Results are consistent with the second (DRB-insensitive but heat-shock-sensitive) fraction being bound but not engaged, while the third (sensitive to both DRB and heat shock) is the elongating hyperphosphorylated form (i.e. IIO).

Sex chromatin in lepidoptera.

Like mammals, Lepidoptera possess female-specific sex chromatin. In a compilation of new and published data, 81% 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 sporatic loss.

Molecular differentiation of sex chromosomes probed by comparative genomic hybridization.

Abstract. 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.

The telomere repeat motif of basal Metazoa

In most eukaryotes the telomeres consist of short DNA tandem repeats and associated proteins. Telomeric repeats are added to the chromosome ends by telomerase, a specialized reverse transcriptase. We examined telomerase activity and telomere repeat sequences in representatives of basal metazoan groups. Our results show that the ‘vertebrate’ telomere motif (TTAGGG)n is present in all basal metazoan groups, i.e. sponges, Cnidaria, Ctenophora, and Placozoa, and also in the unicellular metazoan sister group, the Choanozoa. Thus it can be considered the ancestral telomere repeat motif of Metazoa. It has been conserved from the metazoan radiation in most animal phylogenetic lineages, and replaced by other motifs–according to our present knowledge–only in two major lineages, Arthropoda and Nematoda.

A jumping sex determining factor in the flyMegaselia scalaris

To test earlier reports on changes of the sex determining linkage group inMegaselia scalaris, we used a combination of a cytogenetic and a phenotypic marker in a laboratory strain as a detection system for rare cases in which linkage of the marker and the Y chromosome was disrupted. From some of the exceptional males detected in this system we established strains with a different chromosome pair acting as the sex chromosome pair. Cytogenetic analysis, crossbreeding tests with a phenotypic marker and DNA blot hybridization with cloned DNA probes indicated that an epistatic male determining factor had moved to one of the former autosomes at a frequency of at least 0.06%.

Geschlechtschromatin bei Lepidopteren

Occurence of sex chromatin is widespread amongLepidoptera. Somatic interphase nuclei of female larvae and adults contain a distinct heteropycnotic body which is missing in males. Two types of exceptions exist: one type in which heteropycnotic bodies not discernable from the sex chromatin in other species are formed in both sexes while in another type such chromatin bodies are absent in either sex. Absence of sex chromatin is not linked to a certain systematic entity within the Lepidoptera. In species possessing sex chromatin, the structural aspect of the heteropycnotic body varies within certain limits between different species and tissues. Chromosome analysis of 4 species, 3 of which exhibit normal chromatin bodies in females, while the 4th has no chromatin bodies in both sexes, agree with the interpretation of sex chromatin in the heterogametic females ofLepidoptera as a heteropycnotic Y-chromosome. This interpretation does not apply to those exceptional species which contain heteropycnotic bodies in both sexes.Zusammenfassung1.Bei 70 von 83 in beiden Geschlechtern untersuchten Lepidopterenarten lassen sich geschlechtsspezifische Unterschiede in den somatischen Interphasekernen nachweisen. Bei weiblichen Larven und bzw. oder Imagines dieser Arten wurden in den Zellkernen somatischer Gewebe deutlich abgegrenzte, rundliche, feulgenpositive Körper gefunden, die in den Kernen der zugehörigen Männchen fehlen. 3 Arten,Papilio machaon, Coenonympha arcania undLaothoe populi, zeigen in beiden Geschlechtern solche Chromatinkörper, die beiden ersten in gleicher,Laothoe populi in verschiedener Größe. Bei 10 zu verschiedenen Familien zählenden Arten konnten weder im männlichen noch im weiblichen Geschlecht Chromatinkörper nachgewiesen werden.2.Das Geschlechtschromatin tritt bei den beiden eingehender untersuchten Arten,Lymantria dispar undEphestia kühniella, in allen geprüften weiblichen Geweben larvaler und imaginaler Entwicklungsstadien auf, mit Ausnahme der Oogonien und Oocyten. BeiLymantria dispar fehlen die Chromatinkörper auch in den Nährzellen. Mit dem Ploidiegrad der Zellkerne erhöht sich in der Regel das Volumen, gelegentlich auch die Zahl der Chromatinkörper.3.Die drei auf ihren Karyotyp untersuchten Arten mit typischem Geschlechtschromatin,Philosamia cynthia, Pieris brassicae undEphestia kühniella, besitzen im weiblichen Geschlecht jeweils eine gerade Zahl von somatischen Chromosomen und gehören aller Wahrscheinlichkeit nach zum XY-Typ der Geschlechtsbestimmung. Die NoctuideOrthosia gracilis, die kein Geschlechtschromatin ausbildet, gehört zum XO-Typ. BeiPhilosamia cynthia undOrthosia gracilis konnten die X-Chromosomen identifiziert werden.4.Das in der Mehrzahl der Fälle nachgewiesene Geschlechtschromatin der ♀♀ wird als heteropyknotisches Y-Chromosom interpretiert. Die bei (♂♂ und ♀♀ vonPapilio machaon undCoenonympha arcania auftretenden Chromatinkörper werden als heteropyknotische Autosomen gedeutet.

Probing the W chromosome of the codling moth, Cydia pomonella, with sequences from microdissected sex chromatin

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.