RESUMEN

The genus Ctenomys comprises about 70 species with great chromosome diversity. The Corrientes group is one of the most chromosomally variable lineages in the genus, where the diploid number (2n) varies from 41 to 70. In this group, three nominal species and numerous polymorphic and polytypic populations have been described. In order to get insight into the chromosomal evolution of this species complex, we applied different banding and molecular cytogenetic techniques. The results were interpreted in an evolutionary context, based on mitochondrial cytochrome b analyses. Studied samples are representative of the broad chromosomal variability in the group, including specimens with 2n = 42 to 2n = 70. Heterochromatin was scarce but concentrated in a few chromosomes. Centromeric DAPI-negative heterochromatin was observed in some autosomal pairs, which differed among populations. Location and amount of DAPI-neutral heterochromatin within the Y chromosome varied among populations. The variable distribution of heterochromatin indicates its dynamic behavior. NORs were detected in one pair of autosomes, which also differed among some populations. Telomeric FISH signals were observed in all complements only at the chromosome ends. The Corrientes group belongs to a clade that also includes C. pearsoni, C. lami, C. minutus, C. ibicuiensis and C. torquatus. Almost all of these species are variable at the chromosomal level, suggesting that this is the ancestral condition of the clade. Within the Corrientes group, the observed low genetic divergence, in contrast with its high chromosomal variability, is indicative of decoupling between the rates of chromosomal and mitochondrial evolution.

Asunto(s)

Citocromos b/genética , Octodon/genética , Animales , Bandeo Cromosómico/métodos , Cromosomas de los Mamíferos/genética , Análisis Citogenético/métodos , Evolución Molecular , Variación Genética/genética , Cariotipificación/métodos , Filogenia , Roedores/genética , Telómero/genética

Asunto(s)

Cromosomas de los Mamíferos , ADN Satélite , Evolución Molecular , Roedores/genética , Animales

RESUMEN

We investigated the relationship between satellite copy number and chromosomal evolution in tuco-tucos (genus Ctenomys), a karyotypically diverse clade of rodents. To explore phylogenetic relationships among 23 species and 5 undescribed forms, we sequenced the complete mitochondrial cytochrome b genes of 27 specimens and incorporated 27 previously published sequences. We then used quantitative dot-blot techniques to assess changes in the copy number of the major Ctenomys satellite DNA (satDNA), named RPCS. Our analysis of the relationship between variation in copy number of RPCS and chromosomal changes employed a maximum-likelihood approach to infer the copy number of the satellite RPCS in the ancestors of each clade. We found that amplifications and deletions of RPCS were associated with extensive chromosomal rearrangements even among closely related species. In contrast, RPCS copy number stability was observed within clades characterized by chromosomal stability. This example reinforces the suspected role of amplification, deletion, and intragenomic movement of satDNA in promoting extensive chromosomal evolution.

Asunto(s)

Cromosomas/genética , ADN Satélite/genética , Roedores/genética , Animales , Aberraciones Cromosómicas , Grupo Citocromo b/genética , ADN/química , ADN/genética , ADN Mitocondrial/química , ADN Mitocondrial/genética , Evolución Molecular , Amplificación de Genes , Eliminación de Gen , Dosificación de Gen , Datos de Secuencia Molecular , Filogenia , Roedores/clasificación , Análisis de Secuencia de ADN , Especificidad de la Especie

RESUMEN

Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands.

Asunto(s)

Genes Protozoarios , ARN Helicasas/genética , Trypanosoma cruzi/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , ADN Complementario , Perfilación de la Expresión Génica , Biblioteca Genómica , Datos de Secuencia Molecular , ARN Helicasas/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/crecimiento & desarrollo , Regulación hacia Arriba

RESUMEN

Splitting and apparent splicing of ribosomal RNA, both previously unknown in vertebrates, were found in rodents of the genus Ctenomys. Instead of being formed by a single molecule of 4.4 kb, 28S rRNA is split in two molecules of 2.6 and 1.8 kb. A hidden break, mapping within a 106 bp 'intron' located in the D6 divergent region, is expressed in mature ribosomes of liver, lung, heart and spleen, as well as in primary fibroblast cultures. Testis-specific processing eliminates the intron and concomitantly the break site, producing non-split 28S rRNA molecules exclusively in this organ. The intron is flanked by two 9 bp direct repeats, revealing the acquisition by insertion of a novel rRNA processing strategy in the evolution of higher organisms.

Asunto(s)

Intrones/genética , Precursores del ARN/genética , Empalme del ARN/genética , ARN Ribosómico 28S/genética , Testículo/metabolismo , Animales , Secuencia de Bases , Northern Blotting , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Evolución Molecular , Masculino , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Peso Molecular , Conformación de Ácido Nucleico , Especificidad de Órganos , Precursores del ARN/química , Precursores del ARN/metabolismo , ARN Ribosómico 28S/química , ARN Ribosómico 28S/metabolismo , Ratas , Secuencias Repetitivas de Ácidos Nucleicos , Roedores/genética , Testículo/citología , Termodinámica

RESUMEN

Both original and colonizer populations of Drosophila buzzatii have been analyzed for mtDNA restriction polymorphisms. Most of the mtDNA nucleotide variation in original populations of NW Argentina can be explained by intrapopulation diversity and only a small fraction can be accounted for by between-population diversity. Similar results are obtained using either the estimated number of nucleotide substitutions per site or considering each restriction site as a locus. Colonizer populations of the Iberian Peninsula are monomorphic and show only the most common haplotype from the original populations. Under the infinite island model and assuming that populations are in equilibrium, fixation indices indicate enough gene flow to explain why the populations are not structured. Yet, the possibility exists that populations have not reached an equilibrium after a founder event at the end of the last Pleistocene glaciation. Tajima's test suggests that directional selection and/or a recent bottleneck could explain the present mtDNA differentiation. Considering the significant population structure found for the chromosomal and some allozyme polymorphisms, the among-population uniformity for mtDNA variability argues in favor of the chromosomal and some allozyme polymorphisms being adaptive.

Asunto(s)

ADN Mitocondrial/genética , Drosophila/genética , Evolución Molecular , Frecuencia de los Genes , Polimorfismo de Longitud del Fragmento de Restricción , Animales , Argentina , Femenino , Haplotipos , Masculino , Mapeo Restrictivo , España

RESUMEN

The chromosomal distribution of the major satellite DNA of South American rodents of the genus Ctenomys was analyzed in eight species by in situ hybridization, using a probe isolated from C. porteousi. The hybridization patterns showed different numbers of chromosomes with positive pericentromeric regions and/or complete short arms. In some species, a positive signal was scarce (or not detectable, as in C. opimus), and was usually located in the pericentromeric areas (C. occultus and C. latro). In those species where the satellite was highly amplified, its chromosomal localization tended to encompass the entire length of the short arms. These patterns were compared with C-band distribution patterns in the same species. We discuss the putative evolutionary trend of this satellite DNA in the genus Ctenomys and suggest that it evolved from a strictly pericentromeric localization to comprising the whole short arms of some chromosomes.

Asunto(s)

ADN Satélite/análisis , Roedores/genética , Animales , Bandeo Cromosómico/veterinaria , Hibridación in Situ/veterinaria , Cariotipificación/veterinaria , América del Sur

RESUMEN

The major satellite DNA of the subterranean rodent Ctenomys, named RPCS, contains several consensus sequences characteristic of the U3 region of retroviral long terminal repeats (LTRs), such as a polypurine tract, CCAAT boxes, binding sites for the CCAAT/enhancer-binding protein (C/EBP), a TATA box and putative polyadenylation signals. RPCS presents an enormous variation in abundance between species of the same genus: while C. australis or C. talarum have approximately 3 x 10(6) copies per genome, C. opimus has none. A sequence (RPCS-I) with identity to the SV40-enhancer core element, present in all the repeating units of the satellite is specifically protected in DNase I footprintings. Competitions of band-shift assays with different transcription factor binding sites indicate that binding to RPCS-I is specific and involves CCAAT proteins related to NF-1, but not to C/EBP. By the use of quantitative protein/DNA binding assays we determined that, despite of their conspicuous difference in RPCS copy number, C. talarum and C. opimus have equivalent amounts and identical quality of RPCS-binding proteins. These results are consistent with the observation, by in situ hybridization, that RPCS is clustered in heterochromatic regions, where it might have restricted accessibility to transcription factors in vivo. This is the first report of the binding of transcription factors to a satellite DNA of retroviral origin.

Asunto(s)

ADN Satélite/metabolismo , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Nucleares/metabolismo , Retroviridae/genética , Animales , Secuencia de Bases , Elementos de Facilitación Genéticos , Genoma , Datos de Secuencia Molecular , Roedores , Sensibilidad y Especificidad

RESUMEN

It is well known that uninfected mammalian cells contain DNA sequences which are closely related to retroviral genomic segments. However, these sequences seldom (if ever) have been found associated to highly repetitive (satellite) DNA. RPCS is a 348 bp monomer of a major satellite DNA from the South American rodents of the genus Ctenomys. It was found that RPCS contains several elements which are typical of the U3 region of retroviral LTRs. These elements are: a) a polypurine tract; b) two enhancer core sequences; c) two NF1 binding sites; d) two C/EBP binding sites; e) two CCAAT-motifs; f) a TATA box, and g) two putative polyadenylation motifs. Furthermore, the relative positions of these elements are as in the U3 retroviral regions.

Asunto(s)

ADN Satélite/genética , Retroviridae/genética , Roedores/genética , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , ADN Satélite/metabolismo , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Factores de Transcripción NFI , Proteínas Nucleares/metabolismo , TATA Box , Factores de Transcripción/metabolismo

RESUMEN

A major PvuII satellite DNA has been cloned from a South American octodontid rodent of the genus Ctenomys (C. porteousi). The satellite monomer, termed RPCS, is 337 bp in size and 42% G + C. Analysis of the nucleotide sequence demonstrates that RPCS is not composed of a series of shorter repeats. RPCS-related sequences were found in 11 of 12 Ctenomys species analyzed by hybridization under high-stringency conditions. The only negative species, C. opimus, was reactive under low-stringency conditions. RPCS-related sequences were not found under high- or low-stringency conditions in Calomys musculinus and Mus musculus. However, under low-stringency conditions, RPCS-related sequences were found in the octodontid Octodontomys gliroides, which is thought to have diverged from the genus Ctenomys more than 10 Myr ago. The pattern of periodicities observed, by restriction analysis, between Ctenomys species in the satellite array can be mainly accounted for by a rolling-circle amplification mechanism but cannot be solely accounted for by unequal crossing-over.

Asunto(s)

Replicación del ADN , ADN Satélite/genética , Roedores/genética , Animales , Secuencia de Bases , Southern Blotting , Humanos , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Roedores/clasificación , Homología de Secuencia de Ácido Nucleico , América del Sur , Especificidad de la Especie

RESUMEN

Infectivity and dot-blot hybridization techniques were compared for the detection of FMDV in esophageal-pharyngeal fluids from experimentally infected cows. The probe used includes the viral polymerase sequence which allows the detection of the three types of virus (A, O, and C) with equivalent sensitivity. Virus was detected by dot-blot hybridization as well as by infectivity, according to sample analysis of esophageal-pharyngeal fluids extracted seven days post-infection. It was not possible to recover infective virus from some samples extracted at 180 and 560 days post-infection, although specific viral RNA was detected by dot-blot hybridization. This could indicate the presence of a high ratio of non-infective viral mutants in FMDV carrier cattle. These results emphasize the usefulness of molecular hybridization techniques for FMDV carrier-state detection.

Asunto(s)

Aphthovirus/análisis , ADN Viral/análisis , Fiebre Aftosa/diagnóstico , Animales , Bovinos , ADN/genética , Esófago/microbiología , Espacio Extracelular/microbiología , Hibridación de Ácido Nucleico , Faringe/microbiología

RESUMEN

Los rotavirus son los principales agentes responsables de las gastroenteritis virales humanas y animales. La identificación y caracterización de su genoma es necesaria para la comprensión de esta patología así como para el desarrollo de nuevos métodos de diagnóstico y, eventualmente, para la preparación de antígenos virales utilizando técnicas de DNA recombinante. Estos virus poseen un genoma formado por once fragmentos de RNA doble cadena (cd). Aquí se describe la construcción de bancos de cDNA para rotavirus bovino y humano, ambos purificados de materia fecal. Los cDNA fueron preparados por síntesis in vitro utilizando transcriptasa reversa sobre los RNAs genómicos virales, previamente poliadenilados en sus extremos 3. Los cDNAs fueron ligados a un vector plasmídico y propagados en E. coli. Se obtuvieron genotecas correspondientes a los virus bovino y humano con 500 y 100 recombinantes respectivamente. Análisis de restricción de algunos clones permitieron establecer el tamaño de los insertos correspondientes a los distintos segmentos genómicos virales. Dos de estos clones fueron caracterizados, determinándose que contienen las secuencias completas de los fragmentos 10 y 8 del virus bovino. La utilización de estos clones como sondas radioactivas nos permitió diagnosticar la presencia de rotavirus en muestras de materia fecal mediante la detección de los correspondientes RNAs. Este ensayo pudo ser utilizado para la detección viral en muestras infectadas provenientes de distintas especies

Asunto(s)

Bovinos , Animales , Humanos , Clonación Molecular/métodos , ADN Recombinante , Genes Virales , Técnicas In Vitro , ARN Viral/genética , Rotavirus/genética , Antígenos Virales/aislamiento & purificación , Gastroenteritis/diagnóstico

RESUMEN

Los rotavirus son los principales agentes responsables de las gastroenteritis virales humanas y animales. La identificación y caracterización de su genoma es necesaria para la comprensión de esta patología así como para el desarrollo de nuevos métodos de diagnóstico y, eventualmente, para la preparación de antígenos virales utilizando técnicas de DNA recombinante. Estos virus poseen un genoma formado por once fragmentos de RNA doble cadena (cd). Aquí se describe la construcción de bancos de cDNA para rotavirus bovino y humano, ambos purificados de materia fecal. Los cDNA fueron preparados por síntesis in vitro utilizando transcriptasa reversa sobre los RNAs genómicos virales, previamente poliadenilados en sus extremos 3. Los cDNAs fueron ligados a un vector plasmídico y propagados en E. coli. Se obtuvieron genotecas correspondientes a los virus bovino y humano con 500 y 100 recombinantes respectivamente. Análisis de restricción de algunos clones permitieron establecer el tamaño de los insertos correspondientes a los distintos segmentos genómicos virales. Dos de estos clones fueron caracterizados, determinándose que contienen las secuencias completas de los fragmentos 10 y 8 del virus bovino. La utilización de estos clones como sondas radioactivas nos permitió diagnosticar la presencia de rotavirus en muestras de materia fecal mediante la detección de los correspondientes RNAs. Este ensayo pudo ser utilizado para la detección viral en muestras infectadas provenientes de distintas especies (AU)

Asunto(s)

Bovinos , Animales , Humanos , Técnicas In Vitro , ADN Recombinante , ARN Viral/genética , Clonación Molecular/métodos , Genes Virales , Rotavirus/genética , Antígenos Virales/aislamiento & purificación , Gastroenteritis/diagnóstico

Asunto(s)

Clonación Molecular/métodos , ADN Recombinante , Genes Virales , ARN Viral/genética , Rotavirus/genética , Animales , Bovinos , ADN Circular , Electroforesis en Gel de Poliacrilamida , Humanos