RESUMEN
The germline-soma divide is a fundamental distinction in developmental biology, and different genes are expressed in germline and somatic cells throughout metazoan life cycles. Ciliates, a group of microbial eukaryotes, exhibit germline-somatic nuclear dimorphism within a single cell with two different genomes. The ciliate Oxytricha trifallax undergoes massive RNA-guided DNA elimination and genome rearrangement to produce a new somatic macronucleus (MAC) from a copy of the germline micronucleus (MIC). This process eliminates noncoding DNA sequences that interrupt genes and also deletes hundreds of germline-limited open reading frames (ORFs) that are transcribed during genome rearrangement. Here, we update the set of transcribed germline-limited ORFs (TGLOs) in O. trifallax. We show that TGLOs tend to be expressed during nuclear development and then are absent from the somatic MAC. We also demonstrate that exposure to synthetic RNA can reprogram TGLO retention in the somatic MAC and that TGLO retention leads to transcription outside the normal developmental program. These data suggest that TGLOs represent a group of developmentally regulated protein-coding sequences whose gene expression is terminated by DNA elimination.
Asunto(s)
Oxytricha , Animales , Oxytricha/genética , Reordenamiento Génico , Células Germinativas , ADN/metabolismo , ARN/metabolismoRESUMEN
Extrachromosomal circular DNA (eccDNA) is both a driver of eukaryotic genome instability and a product of programmed genome rearrangements, but its extent had not been surveyed in Oxytricha, a ciliate with elaborate DNA elimination and translocation during development. Here, we captured rearrangement-specific circular DNA molecules across the genome to gain insight into its processes of programmed genome rearrangement. We recovered thousands of circularly excised Tc1/mariner-type transposable elements and high confidence non-repetitive germline-limited loci. We verified their bona fide circular topology using circular DNA deep-sequencing, 2D gel electrophoresis and inverse polymerase chain reaction. In contrast to the precise circular excision of transposable elements, we report widespread heterogeneity in the circular excision of non-repetitive germline-limited loci. We also demonstrate that circular DNAs are transcribed in Oxytricha, producing rearrangement-specific long non-coding RNAs. The programmed formation of thousands of eccDNA molecules makes Oxytricha a model system for studying nucleic acid topology. It also suggests involvement of eccDNA in programmed genome rearrangement.
Asunto(s)
ADN Circular/genética , Reordenamiento Génico/genética , Oxytricha/genética , Recombinación Genética , Citoplasma/genética , Elementos Transponibles de ADN/genética , ADN Protozoario/genética , Células Eucariotas , Genoma de Protozoos/genética , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
The antimicrobial properties of nitric oxide (NOâ) have motivated the design of NOâ-releasing materials for the treatment and prevention of infection. The biological activity of NOâ is dependent on its delivery rate, suggesting that variable antimicrobial effects can result from identical NOâ payloads dosed at different rates. Using a kinetic model of the Escherichia coli NOâ biochemical network, we investigated the relationship between NOâ delivery rate, payload, and cytotoxicity, as indicated by the duration of respiratory inhibition. At low NOâ payloads, the model predicted greater toxicity with rapid delivery, while slower delivery was more effective at higher payloads. These predictions were confirmed experimentally, and exhibited quantitative agreement with measured O2 and NOâ concentrations, and durations of respiratory inhibition. These results provide important information on key design parameters in the formulation of NOâ-based therapeutics, and highlight the utility of a model-based approach for the analysis of dosing regimens.