Telomere formation on macronuclear chromosomes of Oxytricha trifallax and O. fallax: alternatively processed regions have multiple telomere addition sites

Abstract

Background: Ciliates employ massive chromatid breakage and de novo telomere formation during generation of the somatic macronucleus. Positions flanking the 81-MAC locus are reproducibly cut. But those flanking the Common Region are proposed to often escape cutting, generating three nested macronuclear chromosomes, two retaining "arms" still appended to the Common Region. Arm-distal positions must differ (in cis) from the Common Region flanks.

Results: The Common-Region-flanking positions also differ from the arm-distal positions in that they are "multi-TAS" regions: anchored PCR shows heterogeneous patterns of telomere addition sites, but arm-distal sites do not. The multi-TAS patterns are reproducible, but are sensitive to the sequence of the allele being processed. Thus, random degradation following chromatid cutting does not create this heterogeneity; these telomere addition sites also must be dictated by cis-acting sequences.

Conclusions: Most ciliates show such micro-heterogeneity in the precise positions of telomere addition sites. Telomerase is believed to be tightly associated with, and act in concert with, the chromatid-cutting nuclease: heterogeneity must be the result of intervening erosion activity. Our "weak-sites" hypothesis explains the correlation between alternative chromatid cutting at the Common Region boundaries and their multi-TAS character: when the chromatid-breakage machine encounters either a weak binding site or a weak cut site at these regions, then telomerase dissociates prematurely, leaving the new end subject to erosion by an exonuclease, which pauses at cis-acting sequences; telomerase eventually heals these resected termini. Finally, we observe TAS positioning influenced by trans-allelic interactions, reminiscent of transvection.

Figure 1

Alternative processing of the 81-MAC locus. In the center are maps of the three MAC chromosomes generated from the 81-MAC locus (after [25]), with their genes' exons indicated as blocks separated by gaps representing introns; the 3' of each gene is indicated by an arrowhead. Above, indicated by arrows and blocks, are the four Telomere Addition Site regions of the locus. Below is a diagram illustrating the "partial-digest" model, in which only some chromatids are cut at the CR-L and CR-R regions, and telomeres (signified by terminal blocks) are added to each chromosome carrying a Common Region (signified by a thick line; see text).

Figure 2

Telomere Addition Site mapping by LM-PCR. 2A. LM-PCR strategy, described in the Materials and Methods, and in Results. The native 5'P at the end of the MAC DNA telomere is indicated in red, and the telomere G₄T₄ repeats are indicated in green. Short, thick arrows indicate primers. Ligation to the 3'OH of the anchor linker is indicated by an inverted caret; anchor primer 7A is indicated by hollow arrows. The asterisk at the end of the display primer indicates its 5' ³²P label. 2B. Autoradiogram of electrophoresis gel displaying end-labeled display products derived from a multi-TAS region. TAS positions are determined as described in the text, by running the display chains (central lane) next to the lanes of sequencing reactions derived from the MIC DNA that is processed. Four display bands and corresponding TAS positions on the MIC DNA sequence lanes are marked with black diamonds. This early CR-R result has not been further analyzed: analysed results are shown in Figures 3 and 4. Sequencing lanes derived from a cloned P1-anchor PCR product are run on the other side of the central display lane. This clone carries a product with telomere repeats added to its dot-marked dT, represented by the second-slowest, marked display band. Along side these lanes are diagrammed the extent of the 7A anchor primer, ligated to the 5'P of the telomere G₄T₄ repeats.

Figure 3

LM-PCR evidence that cis-acting sequences determine TAS positions. 3A. Karyonidal reproducibility of CR-Right TAS display patterns. Two display lanes represent CR-R TASs in two karyonides with identical heterozygous 81-locus genotypes, 310/510b. Also shown are two lanes representing two karyonides with 310/510a genotypes. Arrows indicate the positions of distinct allele-specific bands. Specific oligomers used: Extend = ST2; P1 = PEB; Display = VHO. 3B. Allele-specificity of TAS positions, and "verification" of one set of display bands. DNAs from Oxytricha strains with various 81-locus allele compositions were used as templates for LM-PCR and display (see text). Arrows indicate the positions of distinct allele-specific bands. Specific oligomers used: Extend = ST2; P1 = PEB; Display = VHO. A verification PCR reaction was performed with the P1-7A PCR product from O. fallax 3.5, and display lanes are run along side (see text for explanation of this verification procedure). Because short chains ran especially rapidly, the bottoms of the "fal 3.5" lanes are presented to the side of the main panel. Four corresponding bands in the P1-anchor PCR display and the verification display are marked. 3C. Allele-specificity of TAS display pattern for the multi-TAS region bounding the Left end of Common Region. DNAs from strains with various 81 locus genotypes were used as templates for LM-PCR and display (see text). Arrows indicate the positions of distinct allele-specific bands. Specific oligomers used: Extend = PEB-prime; P1 = oRG; P2 primer = T1target; Display = 180L-.

Figure 4

Map positions of TASs of the Right End of the 81-MAC Common Region. Sequences of three O. trifallax alleles and three O. fallax 3.5 alleles across the multi-TAS region right of the Common Region are aligned. TAS positions, defined as the nucleotide 5' of the telomere repeats block, dGGGGTTTTGGGGTTTTGGGG, were inferred from mobilities of display bands (see Fig. 2), and are shaded in this figure. Three mapped O. fallax TASs are known from sequences of cloned chromosomes [27]; they are indicated by white letters with black backgrounds. The two start sites of transcription of the CR gene, mapped by Williams and Herrick [54], are indicated below the beginning of the alignment, in a region of nearly perfect conservation, excepting a bold-faced T in the O. fallax vA sequence. It is tightly associated with a dA TAS in this allele, which is indicated with a "/" below it. The stop codon of the Right Arm gene [25] is underlined. Asterisks above the alignment mark positions with a TAS unique to one allele; daggers mark positions where some, but not all alleles have a TAS. In 5 cases, the inferred position is followed directly by a dG. Because telomerase can complete GGGG runs, it is possible (discussed by [38]) that in each of these cases, the TAS was not at the inferred dT or dA, but instead could have been at the following dG; to indicate these ambiguous cases, the T or A and its 3' G are shown in lower case.

Figure 5

Single TAS regions bounding the 81-MAC locus. Three pairs of display lanes are shown. Display reactions were performed from 7A-anchored products and from verification, tel-anchored templates. Left Arm-distal TAS region: displays and verifications are shown representing the O. trifallax 310 homozygote and O. fallax 3.5. The latter products show three bands, representing the strain's three alleles vA, vB, and vC (not shown, see text). Specific oligomers used: Extend = -123-; P1 = -387-; P2 primer = -903-; Display = LA4. Right Arm-distal TAS region of O. fallax 3.5, specific oligomers used: Extend = RA1.1; P1 = 3239; P2 primer = -894; Display = 247+.