Developmentally regulated chromosome fragmentation linked to imprecise elimination of repeated sequences in paramecia

Abstract

The chromosomes of ciliates are fragmented at reproducible sites during the development of the polyploid somatic macronucleus, but the mechanisms involved appear to be quite diverse in different species. In Paramecium aurelia, the process is imprecise and results in de novo telomere addition at locally heterogeneous positions. To search for possible determinants of chromosome fragmentation, we have studied an approximately 21-kb fragmentation region from the germ line genome of P. primaurelia. The mapping and sequencing of alternative macronuclear versions of the region show that two distinct multicopy elements, a minisatellite and a degenerate transposon copy, are eliminated by an imprecise mechanism leading either to chromosome fragmentation and the formation of new telomeres or to the rejoining of flanking sequences. Heterogeneous internal deletions occur between short direct repeats containing TA dinucleotides. The complex rearrangement patterns produced vary slightly among genetically identical cell lines, show non-Mendelian inheritance during sexual reproduction, and can be experimentally modified by transformation of the maternal macronucleus with homologous sequences. These results suggest that chromosome fragmentation in Paramecium is the consequence of imprecise DNA elimination events that are distinct from the precise excision of single-copy internal eliminated sequences and that target multicopy germ line sequences by homology-dependent epigenetic mechanisms.

FIG. 1.

Restriction maps of macronuclear chromosomes 1, 2, and 3 (mac 1, mac 2, and mac 3, respectively). A1 to A9 are Asp718 sites. The G surface antigen gene is shown as a white box near the right ends of chromosomes 1 and 2. Gray boxes at chromosome ends represent telomere addition regions. Alternative processing at the left ends of chromosomes 2 and 3 results in multiple telomere addition regions distributed over >50 kb. Variable internal deletions were mapped in the middle of chromosome 2, in a region corresponding to the alternative telomere addition regions at the left end of chromosome 1. The broken line at the top represents the unknown micronuclear (mic) sequence. Allelic restriction fragment length polymorphisms were identified in sequences 7R, 5L, 2L, and 1L (black boxes). The numbers of F₂ meiotic recombinants and the macronuclear distances between these sequences are indicated.

FIG. 2.

Alternative versions of chromosomes 2, 3, and 1. (A) Southern blot of a pulsed-field electrophoresis gel of Asp718-digested total DNA, hybridized successively with probe 6R to reveal alternative versions of chromosomes 2 and 3 and with probe 5L to reveal alternative versions of chromosomes 2 and 1. (B) Maps of main alternative versions of macronuclear chromosomes 2, 3, and 1 (mac 2, mac 3, and mac 1, respectively) between Asp718 sites A5 and A6. Almost all of the alternative rearrangements occur between BamHI site B1 and SacI site S. The lengths of the B1-S fragments of the different chromosome 2 versions and of the telomeric fragments of the different versions of chromosomes 3 and 1 are represented by broken lines. Gray boxes show the positions of alternative telomere addition regions. Probes 5L and 6R are shown as black boxes.

FIG. 3.

Germ line sequence of the chromosome fragmentation region. (A) PCR amplification of micronuclear DNA. The positions of primers are indicated by arrowheads. Primers o1 and o1a and primers o4 and o4a are located within the PY and PX sequences, repectively (gray boxes). Micronuclear specificity was achieved by using DNA from ΔPY clones for the o1-o2 and o1a-o5 PCRs and DNA from ΔPX clones for the o3-o4 and o4a-o6 PCRs. In addition, primer o3 was derived from one of the six IESs, 26 to 468 bp long (black boxes), that were identified in the o1-o2 micronuclear product. (B) Features of the germ line sequence. The 20,816-bp sequenced portion (solid line) begins 1.7 kb after BamHI site B1 and ends 0.8 kb after SacI site S. White boxes indicate the positions of the minisatellite and GC-rich sequence, WD40-related sequence, and truncated transposon. Thin vertical lines in the minisatellite represent individual repeats. The dot plot shows a self-comparison matrix of the minisatellite and GC-rich sequence (DNA Strider stringency 15; window 23). The 616-bp sequence of the truncated Tennessee copy is compared with the 3,043-bp consensus sequence derived from the alignment of eight different copies. ORFs are indicated by filled arrows, and terminal 31-bp IRs are indicated by open arrowheads. The Southern blot at the bottom was hybridized successively with a 677-bp probe from the right end of the Tennessee transposon (left panel) and, as a control, with a 1.6-kb EcoRI fragment from the G surface antigen gene (right panel). Lanes t5 and t1 contain 150 and 30 pg of a 3-kb complete Tennessee copy, respectively; these amounts correspond to five copies and one copy per haploid genome in 1 μg of the ∼100-Mb macronuclear genome. Lanes m contain 1 μg of EcoRI-digested DNA from the reference clone. Only two faint bands were revealed by the Tennessee probe in lane m (arrowheads), indicating that two copies of the transposon are partially maintained in the macronucleus, at much less than one copy per haploid genome. Lanes g5 and g1 contain 80 and 16 pg of the 1.6-kb EcoRI fragment from the G surface antigen gene, respectively; these amounts correspond to five copies and one copy per haploid genome. The 1.6-kb fragment revealed by the G surface antigen gene probe in lane m (arrowhead) indicates that the G surface antigen gene is present at one copy per haploid genome in macronuclear DNA; weaker bands arise from paralogous genes that cross-hybridize with the probe under the low-stringency conditions used.

FIG. 4.

PCR mapping of internal deletions. (A) PCR amplification of alternative macronuclear versions. PCR products corresponding to heterogeneous versions 2a and 2b were amplified from total DNA with primers o4 and o5 (see map in panel C) and analyzed on a 0.9% agarose gel. Lane M, markers. The sizes indicated were more precisely measured on different types of gels after the purification of 2a and 2b products. (B) Nested PCRs of o4-o5 products. The principle of the method is illustrated by three primer pairs (a-b, c-d, and c-e) overlapping a deletion observed in version 2a (gray box). For each of the PCRs, the agarose gel shows the amplification products obtained from three different templates: a purified PCR product representing the micronuclear sequence (mic), total cell DNA (tot), and a purified PCR product representing heterogeneous macronuclear version 2a. Lanes M, molecular size markers. (C) Map of the germ line sequence showing the positions of primers o4 and o5 (filled arrowheads). These primers lie outside the rearrangement region, since no macronuclear heterogeneity was detected between B1 and o4 on Southern blots (data not shown). Horizontal lines below the map represent the micronuclear amplification products for the entire set of overlapping nested PCRs.

FIG. 5.

Maps of main alternative rearrangements. The major macronuclear versions are represented by labeled lines (2a, 2b, 2c, 1a, and so forth) below the map of the germ line sequence (top line). Gray boxes in the macronuclear versions show the approximate positions of imprecise internal deletions of the minisatellite and truncated Tennessee copy, as determined by mass PCR mapping. The right end of the large deletion in version 2c is uncertain, as indicated by the disconnected gray boxes. Thick black lines below gray boxes indicate the exact extents of the deletions in individual molecules sequenced from cloned PCR products; thin lines on both sides show the regions covered by the PCRs. The six invariant IESs are shown as black boxes in the germ line sequence; gray boxes in the macronuclear versions indicate the positions of the corresponding precise deletions of the IESs. Hatched regions at the ends of fragmented chromosomes 1 and 3 represent telomere addition regions. The graph at the bottom shows the local density of TA dinucleotides along the germ line sequence (percentage of total dinucleotides computed in a 100-bp window); the short vertical lines above the graph mark the positions of the boundaries of internal deletions for all sequenced molecules (excluding the IESs).

FIG. 6.

Direct repeats at boundaries of imprecise internal deletions. The top line shows the sequence of one example of a deletion junction; the single repeat maintained in the macronuclear sequence is in bold type. Line 1 shows the germ line sequence around the direct repeats at the right and left boundaries of this deletion; the deleted sequence is shown in lowercase letters. The entire set of sequenced deletions is represented in the same way. Deletions 1 to 8 and 9 to 11 remove the minisatellite in versions 2a and 2b, respectively; deletions 12 to 16, 17 to 29, and 30 to 41 occur around the truncated Tennessee copy in versions 2a, 1a, and 2b, respectively.

FIG. 7.

Varibility and experimental modifications of rearrangement patterns. A Southern blot of a pulsed-field electrophoresis gel of Asp718-digested total DNAs from different clones was hybridized successively with probes 6R, 5L, PX, and PY. Lanes 1 to 5, wild-type (WT) caryonidal clones; lanes 6 and 7, ΔPX clones; lanes 8 and 9, ΔPY clones. The different fragments indicated on the sides of the blots are those of the reference clone, which is in lane 2. The map shows the positions of the probes (black boxes) between Asp718 sites A5 and A6 in the main macronuclear versions of the reference clone. Gray boxes represent the average extents of the heterogeneous internal deletions; hatched boxes represent telomere addition regions.