RESUMO
DNA molecules injected into the macronucleus of Paramecium primaurelia replicate either as free linear telomerized or chromosome integrated molecules. In the present study we show that when a 1.77 kb BamHI DNA fragment harbouring the his3 gene of Saccharomyces cerevisiae was microinjected into the macronucleus, a fraction of the molecules are integrated into the chromosome via an illegitimate recombination process. The injected molecules were mostly inserted at their extremities at multiple points in the genome by replacing the Paramecium sequences. However, insertion sites were not totally at random. Roughly 30% of the molecules were integrated next to or in telomeric repeats. These telomeric repeats were not at the extremities of chromosomes but occupy an internal or interstitial position. We argue that such sites are hotspots for integration as the probability of random insertion near or in an interstitial telomeric site, of which there are 25-60 in a macronucleus is between 5 x 10(-4) and 3 x 10(-5).
Assuntos
Núcleo Celular/fisiologia , Elementos de DNA Transponíveis , DNA Fúngico/genética , DNA/genética , Paramecium/fisiologia , Recombinação Genética , Saccharomyces cerevisiae/genética , Telômero/fisiologia , Animais , Sequência de Bases , DNA Fúngico/administração & dosagem , Histonas/genética , Microinjeções , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Paramecium/genética , Probabilidade , Sequências Repetitivas de Ácido Nucleico , Mapeamento por RestriçãoRESUMO
Direct injection into the macronucleus of Paramecium tetraurelia of DNA molecules coding for the A-antigen leads to expression of the gene and autonomous replication. When injected into Paramecium primaurelia DNA from probably any origin, procaryote or eucaryote, can replicate as linear telomerized molecules and the number of copies maintained can be very high (up to 20000 copies). We present here evidence that if the injected linear DNA molecules harbour preexisting telomeres at both extremities they are protected from degradation, the number of DNA molecules maintained being 15- to 30-fold higher than if the molecules are injected without telomeres. Some of the injected molecules replicate as multimers, but, only when the fused ends are devoid of preexisting telomeric repeats.