A method for the amplification of Paramecium micronuclear DNA by polymerase chain reaction and its application to the central repeats of Paramecium primaurelia G surface antigen genes.
This paper describes a method which allows the amplification of Paramecium micronuclear DNA. Amacronucleate cells are first obtained by an appropriate treatment with nocodazole, a microtubule depolymerizing agent which blocks the elongation of the macronucleus and the distribution of the micronuclei at cell division between the two daughter cells; then, DNA from such cells is amplified by the polymerase chain reaction technique. We have applied this method to the problem of the central repeats of the G surface antigen of P. primaurelia (strain 156). The central repeats consist of a 74 amino acid sequence repeated in tandem. The sequence identity of these repeats is also found in the nucleotide sequence even at silent codon positions, suggesting the existence of a mechanism of identity maintenance acting at the nucleotide level. Mechanisms based on RNA secondary structure which are frequently proposed as an explanation of this phenomenon are unlikely to be valid in this case. One can, therefore, imagine that these repeats might originate from one micronuclear sequence through duplicative processes which could occur during the formation of the macronucleus. We have used the described technique to amplify the micronuclear version of the central repeats and showed that it is identical to the macronuclear version, thus ruling out the above hypothesis. Therefore, intragenic recombination appears to be the most likely explanation of the sequence identity of these central repeats.
Caron
F
F
Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, Paris, France.
Ruiz
F
F
D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000235:genetics*; D002467:Cell Nucleus / Q000187:drug effects; D004261:DNA Replication; D016054:DNA, Protozoan / Q000302:isolation & purification; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D010247:Paramecium / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid
D000954:Antigens, Surface; D016054:DNA, Protozoan; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; C051841:surface protein G, Paramecium; D015739:Nocodazole