Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei.

Abstract

Paramecium tetraurelia can be transformed by microinjection of cloned serotype A gene sequences into the macronucleus. Transformants are detected by their ability to express serotype A surface antigen from the injected templates. After injection, the DNA is converted from a supercoiled form to a linear form by cleavage at nonrandom sites. The linear form appears to replicate autonomously as a unit-length molecule and is present in transformants at high copy number. The injected DNA is further processed by the addition of parameciumtype telomeric sequences to the termini of the linear DNA. To examine the fate of injected linear DNA molecules, plasmid pSA14SB DNA containing the A gene was cleaved into two linear pieces, a 14-kilobase (kb) piece containing the A gene and flanking sequences and a 2.2-kb piece consisting of the procaryotic vector. In transformants expressing the A gene, we observed that two linear DNA species were present which correspond to the two species injected. Both species had Paramecium telomerelike sequences added to their termini. For the 2.2-kb DNA, we show that the site of addition of the telomerelike sequences is directly at one terminus and within one nucleotide of the other terminus. These results indicate that injected procaryotic DNA is capable of autonomous replication in Paramecium macronuclei and that telomeric addition in the macronucleus does not require specific recognition sequences.Foreign DNA introduced into eucaryotic cells can suffer a variety of fates depending on the sequences introduced and the system used. In mouse L cells in culture, injection of multiple copies of either supercoiled or linear DNA molecules into nuclei can result in integration into host chromosomes at a limited number of sites of head-to-tail concatamers of the input DNA (7, 18). These concatamers result from highly efficient homologous recombination mediated by host recombinational machinery. Alternatively, microinjection of a variety of different supercoiled DNA species into unfertilized Xenopus laevis eggs leads to autonomous replication of the injected DNA as supercoiled forms, apparently regardless of sequence (13,16). In the yeast Saccharomyces cerevisiae, foreign supercoiled DNA introduced by transformation replicates autonomously only if it contains distinctive sequences called autonomously replicating sequences (21), presumably reflecting specific recognition of certain sequences by host replication machinery.We describe here another fate for DNA introduced into the ciliated protozoan Paramecium tetraurelia. We have previously described a transformation system in which exogenous DNA is introduced into the macronuclei of recipients by microinjection (10). Our assay for transformation involves the synthesis of specific cell surface antigens called immobilization antigens (for a review, see reference 19). Paramecia can alter their surface antigens under different environmental conditions. In general, they express only one type at a time. Eleven different serotypes have been described for stock ...

Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA

“…Genomic instability due to eventual excision of introduced DNA has been proposed as the apparent mechanism in other fungi (24,29,33). From these previous experiments, it could be inferred that C. neoformans lacks a gene replacement mechanism yet is receptive to ectopic integration which eventually produces rearrangements and/or addition of telomeric sequences with a telomerase, as seen in Paramecium tetraurelia (11). It was important to determine whether this was a general characteristic of C. neoformans or unique to the recipient strain, gene locus, or DNA delivery system.…”

Functional diversification of centrins and cell morphological complexity

“…Transformation of Paramecium is obtained by micro-injecting the filtered and concentrated plasmid DNA of interest (5 g/l) into the macronucleus (Gilley et al, 1988). Microinjection was made under an inverted Nikon phase-contrast microscope, using a Narishige micromanipulation device and an Eppendorf air pressure microinjector.…”

Interstitial telomeres are hotspots for illegitimate recombination with DNA molecules injected into the macronucleus of Paramecium primaurelia.

“…When circular or linear DNA molecules from different biological origins (bacteria, yeast, paramecia or polyoma virus) are microinjected directly into the Paramecium macronucleus, they are stably replicated as linear telomerized molecules throughout the vegetative cycle (Gilley et al, 1988;Bourgain and Katinka, 1991). We have recently shown (Bourgain and Katinka, 1991) that not all of the injected DNA molecules are maintained as free linear molecules and Oxford University Press some are integrated into the macronuclear chromosomes.…”