Micronuclear DNA from Paramecium tetraurelia: Serotype 51 A Gene Has Internally Eliminated Sequences

Abstract

A method for the isolation of micronuclear DNA from Paramecium tetraurelia has been developed. After cell lysis, a low speed centrifugation at 1,000 g is used to remove all of the unbroken cells and macronuclei and approximately two thirds of the macronuclear fragments. Next a higher speed centrifugation of 9,000 g sediments the micronuclei and frees them from small particulates and soluble constituents. Advantage is then taken of the fact that micronuclei have a lower density than do macronuclear fragments in 45%-60% Percoll. Micronuclei float to the top during centrifugation at 24,000 g, while macronuclear fragments sediment. After several cycles of centrifugation in Percoll, the micronuclei, although heavily contaminated with cytoplasmic components, are essentially free of macronuclei and macronuclear fragments. Micronuclear DNA can then be extracted from the suspension. The whole procedure is very rapid and in about an hour micronuclear and macronuclear DNA can be separated. About 2 micrograms of micronuclear DNA can be obtained from 6 x 10(7) paramecia. We find that there are internal sequences in the micronuclear A gene DNA in wild type cells which are eliminated when the micronuclei develop into macronuclei. They yield unique restriction fragments for micronuclei and macronuclei. Therefore the purity of the preparations is easily monitored by probing Southern blots of restriction enzyme-digested DNA with the cloned A gene. No differences have been found between the micronuclear A gene in wild type and the d48 mutant.

Processing of Double-Strand Breaks Is Involved in the Precise Excision ofParameciumInternal Eliminated Sequences

“…Nested primer extensions allowed high-resolution analysis of the PCR products. The results obtained for IES 51A6649 and the left end of 51G4404, located in surface antigen genes A 51 and G 51 , respectively (23,25), are displayed in Fig. 1C.…”

Developmentally Regulated Chromosome Fragmentation Linked to Imprecise Elimination of Repeated Sequences in Paramecia

“…Cultures (400 ml) of exponentially growing cells (1,000 cells/ml) were centrifuged. After being washed in 10 mM Tris-HCl (pH 7.0), the pellet was resuspended in a volume of the same buffer equal to the volume of the cell pellet; the suspension was quickly added to 4 (51) and further purified from contaminating low-molecular-weight macronuclear DNA by agarose gel electrophoresis. This DNA was used to amplify some of the Tennessee copies, but its average molecular weight was not high enough to allow efficient long-range amplification of the micronuclear fragmentation region, which was carried out by using total DNA samples (see Results).…”

Timing of Developmentally Programmed Excision and Circularization of Paramecium Internal Eliminated Sequences

“…1A) and 51G4404 (222 bp) ( Fig. 2A), identified in the micronuclear versions of P. tetraurelia surface antigen genes A 51 and G 51 , respectively (28,32). The physical association of the two IESs with their flanking sequences was monitored in the same time course experiment, using synchronized cell samples taken every 2 h from the time of exconjugant separation.…”

“…Macronuclear development extends over two cell cycles (4) and is accompanied by intensive replication to reach the final ploidy of the mature macronucleus, estimated to be between 800 and 1,700N according to previous studies (33,38). It involves extensive rearrangements of the germ line genome: fragmentation of micronuclear chromosomes coupled to telomere addition to form the macronuclear chromosome ends (13) and precise deletion of internal eliminated sequences (IESs) that interrupt coding and non-coding DNA (reviewed in reference 22).Paramecium IESs are single-copy sequences of AT-rich noncoding DNA and are flanked by two 5Ј-TA-3Ј repeats, one of which is retained in the macronuclear genome after excision (1,7,9,12,21,24,28,32,37,40,44). Since they were initially identified by sequence comparison of macronuclear and micronuclear versions of a given locus, their size-ranging between 26 and 882 bp-represents, by convention, the whole length of the deleted sequence and formally includes one copy of the flanking 5Ј-TA-3Ј.…”

“…Paramecium IESs are single-copy sequences of AT-rich noncoding DNA and are flanked by two 5Ј-TA-3Ј repeats, one of which is retained in the macronuclear genome after excision (1,7,9,12,21,24,28,32,37,40,44). Since they were initially identified by sequence comparison of macronuclear and micronuclear versions of a given locus, their size-ranging between 26 and 882 bp-represents, by convention, the whole length of the deleted sequence and formally includes one copy of the flanking 5Ј-TA-3Ј.…”