RESUMEN
The chromosomes of the rare South American marsupial frogs Gastrotheca walkeri and G. ovifera were extensively reexamined with various banding techniques. The karyotypes of both species are distinguished by a new category of XY female symbol /XX male symbol female sex chromosomes. The unusual Y chromosomes are characterized by containing the least amount of constitutive heterochromatin in the karyotypes. This is in contrast to all previously known amphibian Y chromosomes and does not fit the evolutionary model of early XY differentiation in vertebrates. In male meiosis, the heteromorphic XY chromosomes of both species still exhibit the same pairing configurations as the autosomes. DNA flow cytometric measurements show the nuclear DNA amount of G. walkeri to be 10.90 pg. The significance of the XY/XX sex chromosomes of these marsupial frogs, the various classes of constitutive heterochromatin detected, and the data obtained from meiotic analyses are discussed in detail.
Asunto(s)
Anuros/clasificación , Anuros/genética , Bandeo Cromosómico/métodos , Cromosoma Y/genética , Animales , ADN/análisis , ADN/genética , Embrión no Mamífero/fisiología , Femenino , Citometría de Flujo , Cariotipificación , Masculino , Meiosis , Metafase , Cromosoma X/genéticaRESUMEN
An understanding of the functional organization of nucleoli, the sites of ribosome biosynthesis, is limited by the present uncertainty about the topological arrangement of the transcribing rRNA genes. Since studies with "standard" nucleoli from somatic cells produced conflicting results, we have examined the amplified nucleoli of Xenopus oocytes. These nucleoli are unique in that they contain high copy numbers of rRNA genes, are not attached to chromosomes, lack non-ribosomal DNA and can be examined in light microscopic spread preparations of nuclear contents. By immunostaining and confocal microscopy we show that in growing stage IV oocytes the sites of rDNA are surrounded by the dense fibrillar component. The rDNA is actively transcribed as revealed by BrUTP injection into oocytes and localization of components of the nucleolar transcription machinery (RNA polymerase I and the transcription factor UBF). At the ultrastructural level, the rDNA sites correlate with the fibrillar centers of amplified nucleoli fixed in situ. The results provide clear evidence that the transcriptionally active rRNA genes are confined to the fibrillar centers of the oocyte nucleoli and open the possibility to analyze the protein composition of almost native, transcriptionally highly active nucleolar chromatin by immunofluorescence microscopy.
Asunto(s)
Nucléolo Celular/ultraestructura , Oocitos/ultraestructura , Animales , Femenino , Microscopía Confocal , Microscopía Electrónica , ARN Ribosómico/genética , ARN Ribosómico/ultraestructura , Xenopus laevisRESUMEN
When nuclei (pronuclei) were assembled from sperm chromatin in Xenopus egg extract and examined by immunofluorescence microscopy, UBF was concentrated at a single intranuclear dot-like or more extended necklace-like structure. These UBF-foci contained rDNA as demonstrated by in situ hybridization and hence represent the chromosomal nucleolus organizing regions (NORs). Besides UBF, other components of the transcription machinery such as the TATA-box binding protein (TBP) and RNA polymerase I (pol I) as well as several nucleolar proteins could not be detected at the NORs. Immuno-depletion experiments indicated the UBF is maternally provided and taken up by the pronuclei. Essentially the same results were obtained when we examined the NORs of early Xenopus embryos up to the midblastula stage. After this stage, when transcription of the rRNA genes has begun, nucleoli developed and the NORs acquired TBP and pol I. Our results support the hypothesis that UBF is an architectural element which converts the rDNA chromatin into a transcriptionally competent form.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Región Organizadora del Nucléolo/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Operón de ARNr/fisiología , Animales , Especificidad de Anticuerpos , Blastocisto/química , Núcleo Celular/química , Núcleo Celular/genética , Cromatina/química , ADN Ribosómico/análisis , ADN Ribosómico/metabolismo , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Región Organizadora del Nucléolo/metabolismo , ARN Polimerasa I/análisis , ARN Polimerasa I/inmunología , Espermatozoides/química , Proteína de Unión a TATA-Box , Factores de Transcripción/análisis , Factores de Transcripción/inmunología , Transcripción Genética/fisiología , Xenopus laevisRESUMEN
Xenopus oocytes express a 165 kDa variant of DNA topoisomerase I (topo I) as opposed to the canonical 110 kDa form of somatic cells (Richard and Bogenhagen, Dev. Biol. 146: 4-11, 1991). By immunofluorescence microscopy using variant-specific antibodies we show that this high molecular weight form is associated with lampbrush chromosome loops and the inner regions of the amplified nucleoli. Inhibition of topo I-activity by either Camptothecin-treatment or microinjection of neutralizing antibodies resulted in loop retraction and the condensation of chromosomes and amplified nucleoli. These data indicate that the oocyte-specific 165 kDa form of topo I is involved in transcriptional processes mediated by RNA polymerase I and II and is therefore functionally equivalent to the somatic cell 110 kDa counterpart.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Oocitos/enzimología , Xenopus laevis/metabolismo , Animales , Nucléolo Celular/enzimología , Cromosomas/enzimología , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/inmunología , Femenino , Microscopía Fluorescente , Peso MolecularRESUMEN
Cytogenetic analyses were performed on several populations of the Central American tree frog Agalychnis callidryas, using conventional methods and banding techniques. The karyotype of this species is distinguished by an inversion polymorphism in chromosome 9, which is either submetacentric or telocentric. The populations examined are in Hardy-Weinberg equilibrium with respect to the two alternative morphs of chromosome 9. This is the first report of the occurrence of an intrapopulational chromosomal inversion polymorphism in the order Anura. In male meiosis, the two chromosomes 9 form a bivalent exhibiting a ring-like pairing configuration with terminal chiasmata in both arms, regardless of whether the paired homologs are heteromorphic or homomorphic. Furthermore, individual specimens of A. callidryas exhibit one or two unexpected 18S + 28S ribosomal RNA gene clusters, in addition to the standard nucleolus organizers. The chromosomal localization of these extra nucleolus organizers is identical in all metaphases from the same specimen and shows a specific intraindividual pattern. The karyotype evolution in the phyllomedusine hylids, the structure of the various classes of heterochromatin, and the occurrence and possible origin of the rare inversion polymorphisms and multiple nucleolus organizers in A. callidryas and a few other amphibian species are discussed.