Telomeric DNA at the ends of chromosomes consist of short, tandem repeat sequences. The telomeres of Paramecium tetraurelia are made up of variable repeats, whereas Paramecium caudatum telomeric repeats are largely invariant. To investigate variable repeat synthesis in P.tetraurelia, mutated telomerase RNA genes were expressed in vivo. We demonstrate that the P.caudatum telomerase RNA can participate in telomere synthesis when expressed in the P.tetraurelia macronucleus, despite 24% primary sequence divergence of the RNAs between the two species. De novo telomeric repeats from transformants indicate that P.tetraurelia telomerase fidelity is dramatically affected by template substitutions and that misincorporation at a single templating position is likely to account for the majority of P.tetraurelia telomeric DNA variability. Furthermore, we show that fidelity is not solely a function of the RNA moiety, as the P.caudatum telomerase RNA does not impart high fidelity to the chimeric enzyme.
Telomeres, the specialized structures found at the ends of chromosomes, consist of simple, tandem DNA repeats and the proteins which bind them (reviewed in Zakian, 1989; Biessmann and Mason, 1992). These structures function as a chromosome ‘cap’, protecting the termini from exonucleolytic cleavage and preventing illegitimate end‐to‐end chromosome fusions. Telomeres also ensure the complete replication of chromosomes and in this way prevent the loss of genetic information at the subtelomere. In addition, telomeres have been proposed to help direct chromosome attachment to the nuclear envelope (Agard and Sedat, 1983; de Lange, 1992).
Telomeric DNA generally consists of short G‐rich tandem repeats on the strand oriented 5′→3′ toward the end of the chromosome. The ribonucleoprotein enzyme telomerase synthesizes the G‐rich strand of telomeric DNA. The RNA moiety of telomerase was first identified in Tetrahymena thermophila (Greider and Blackburn, 1989) and has subsequently been identified in over 20 ciliates (Shippen‐Lentz and Blackburn, 1990; Romero and Blackburn, 1991; Lingner et al., 1994; McCormick‐Graham and Romero, 1995, 1996), yeast (Singer and Gottschling, 1994; McEachern and Blackburn, 1995), mouse (Blasco et al., 1995) and human (Feng et al., 1995). All telomerase RNAs include a templating domain that dictates the telomeric repeat sequence added to the 3′‐end of the chromosome. Two putative telomerase proteins from T.thermophila (Collins et al., 1995) and Euplotes aediculatus (Lingner and Cech, 1996) have been identified and partially purified. The apparent stoichiometry of telomerase components is a 1:1:1 ratio of the RNA and protein subunits. It has been suggested that the 80 kDa protein subunit from the T.thermophila enzyme binds the telomerase RNA, whereas the 95 kDa protein binds the telomeric primer.
Telomerase RNA genes from four Paramecium species were cloned and sequenced in an earlier study (McCormick‐Graham and Romero, 1996). The telomeres from three of the four Paramecium species consist of variable repeats, composed primarily of a random mixture of G4T2 and G3T3 (Baroin et al., 1987; Forney and Blackburn, 1988; McCormick‐Graham and Romero, 1996). In contrast, the telomeric DNA from the fourth species, P.caudatum, is composed primarily of G4T2 repeats (>95%). A single telomerase RNA gene is transcribed in all four species, with templating nucleotides consistent with the synthesis of G4T2 repeats. In addition, there is no RNA editing of the telomerase RNA templating nucleotides that might account for the conventional synthesis of G3T3 repeats by the Paramecium enzyme (McCormick‐Graham and Romero, 1996). These data suggest that Paramecium telomerase is inherently imprecise, with the notable exception of Paramecium caudatum telomerase, which synthesizes G4T2 repeats with reasonably high fidelity.
The mechanism by which G3T3 repeats are synthesized by Paramecium telomerase is not known. It is possible that the observed variability is due to misincorporation by telomerase of dTTP instead of dGTP at one of two positions along the template. Another possiblility is that telomerase ‘stutters’ in the middle of synthesizing a telomeric repeat, by translocating back one position on the template so that the telomere is extended by three sequential dT residues before the addition of dG. Such a stuttering mechanism would be similar to that documented in vitro for telomerase from the yeast Saccharomyces castelli (Cohn and Blackburn, 1995) and demonstrated both in vivo and in vitro for the T.thermophila telomerase utilizing a mutated telomerase RNA (Yu and Blackburn, 1991; Gilley et al., 1995).
We have utilized an in vivo approach to investigate telomere synthesis in Paramecium. Linearized plasmids containing the telomerase RNA gene mutated at various positions along the template were microinjected into the macronucleus of P.tetraurelia. The effect of the mutations on telomerase fidelity was determined by sequencing de novo telomeric repeats. Similarly, the P.caudatum telomerase RNA was expressed in the P.tetraurelia macronucleus to establish if the P.caudatum RNA could function in a heterologous system and whether it could impart high fidelity to the P.tetraurelia telomerase. The sequence of de novo telomeric repeats from the various transformants and the implication of these results for Paramecium telomere synthesis are discussed.
Telomerase RNA mutations
Mutagenic studies have been useful in delineating the telomerase RNA templating domain in ciliates and yeast, both in vivo (Yu et al., 1990; Singer and Gottschling, 1994; McEachern and Blackburn, 1995) and in vitro (Autexier and Greider, 1994; Gilley et al., 1995). To investigate how variable telomeric repeats are synthesized in P.tetraurelia, the telomerase RNA gene sequence was altered at the four nucleotide positions shown in Figure 1A. Two of the four nucleotide substitutions (C49A and A50G) are identical to template mutations expressed in T.thermophila (Yu et al., 1990; Gilley et al., 1995). A prediction for a third template mutation, C52A, is the synthesis of G3T3 repeats. The fourth is the substitution of G57 with an A residue, a nucleotide position which is conserved among all other ciliate telomerase RNAs as an A residue.
Transformation of P.tetraurelia by co‐microinjection
A plasmid suitable for the stable transformation of P.tetraurelia by microinjection has been described (Haynes et al., 1995). This plasmid, pPXV‐NEO, confers resistance to the antibiotic G‐418 through the activity of aminoglycoside 3′‐phosphotransferase II (APH‐3′‐II). The expression of APH‐3′‐II is driven by the constitutive P.tetraurelia calmodulin promoter (Kanabrocki et al., 1991). A second plasmid, pPXVI, was derived from pPXV‐NEO by replacement of the APH‐3′‐II cassette with polycloning sites. Paramecium tetraurelia telomerase RNA genes, with and without various template mutations, were cloned into the polycloning site of pPXVI as described in Materials and methods and illustrated in Figure 1.
Prior to co‐microinjection, both plasmids were linearized by digestion with the restriction enzyme SfiI. The ends of the plasmids include a linker sequence ∼30 bp long, located distal to a tract of 45 G4T2 repeats (Haynes et al., 1995). The telomeric sequence helps protect the plasmids from degradation and also increases the efficiency of transformation in Paramecium (Bourgain and Katinka, 1991). Following co‐microinjection of ∼2×106 copies of each linearized plasmid into the macronucleus of post‐autogamous P.tetraurelia, the division of individual cells was monitored until cultures had expanded to ∼100 cells (3–4 days). Clonal cell lines resistant to G‐418 were expanded and harvested prior to autogamy (between 20 and 22 generations following microinjection). The generation times of transformants were similar to uninjected controls and cells microinjected with pPXV‐NEO alone (∼13 h) and there were no obvious morphological differences between transformants and controls when examined in a phase contrast microscope. Examination of transformants stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) did not reveal any abnormal nuclei or other unusual phenotypic differences between transformants and controls (data not shown).
Plasmid‐encoded versus endogenous telomerase RNA expression
The degree to which microinjected telomerase RNA genes were retained and replicated in transformants was determined by Southern blot analysis. A double digestion of total DNA from P.tetraurelia transformants with SacI and XbaI results in 0.75 and 4.0 kb fragments that include the plasmid‐borne and endogenous telomerase RNA genes respectively. Southern blots were probed with the radiolabeled telomerase RNA gene and the ratio of plasmid‐encoded to endogenous telomerase RNA genes was quantitated by phosphorimaging (Figure 2A and Table I). The amount of the plasmid telomerase RNA gene relative to the endogenous gene was somewhat variable between microinjections, with a range between 1 and 68 (Table I).
Allele‐specific hybridization was used to determine if plasmid‐encoded telomerase RNA genes mutated at the template were transcribed (it was not possible to differentiate the plasmid‐encoded wild‐type telomerase RNA from the endogenous RNA by this method). Oligonucleotides complementary to each mutated RNA were used to sequentially probe a series of Northern blots of total RNA isolated from transformants. Quantitation by phosphorimaging indicated that the ratios of mutated telomerase RNA to endogenous telomerase RNA transcribed ranged from 1 to 57 (Table I and Figure 2B). The transcription of plasmid‐encoded telomerase RNA relative to the endogenous RNA correlates roughly with the gene dosage of the introduced gene relative to that of the endogenous gene (Table I).
Extension of telomeres in transformants
Having established that microinjected telomerase RNA genes were both maintained and transcribed in Paramecium, we then determined what effect, if any, the transcripts had on telomere synthesis. The length of the microinjected plasmid termini was known (Figure 1B), which afforded a convenient reference point in measuring de novo telomere addition in transformants. Southern blot analyses of total DNA from four cell lines transformed with each plasmid construct, harvested 20–22 generations after microinjection, were digested with SacI and probed with the telomerase RNA gene. Plasmid telomere extension varied between the different classes of transformants (Figure 3). Cells microinjected with the G57A construct extended their telomeres to the same extent as cells expressing the plasmid‐encoded wild‐type RNA (an average value of 100 bp or 17 telomeric repeats). Telomeres in the A50G construct were approximately the same length as the input and G4TC repeats were undetected by Southern blot analysis (data not shown). Cells expressing the C49A and C52A template mutations had longer telomeres than any of the other transformants (average increased length of 300 and 250 bp respectively). The same relative increase or decrease in telomere extension was also detected when telomeres from pPXV‐NEO were similarly analyzed (data not shown). It should be noted that the endogenous telomerase RNA is still present and is assumed to also contribute to transformant de novo telomere addition.
De novo telomere sequence analysis
Extension of the microinjected plasmid telomeres is representative of de novo telomere addition in transformed cells (Figure 3). The presence of an ampicillin resistance marker in these linear molecules made it possible to routinely isolate de novo telomeric repeats for more detailed sequence analysis. Briefly, total DNA from transformants was digested with the restriction enzyme XbaI to effectively remove one telomeric end from the microinjected plasmids (Figure 1B). The XbaI‐treated DNA was incubated with T4 DNA polymerase and dNTPs to generate blunt ends, in order to facilitate circularization of the linear molecules with DNA ligase. Transformation of Escherichia coli with the ligation products followed by selection for ampicillin resistance resulted in the isolation of ‘rescued’ circular plasmid clones containing de novo telomeres. Due to the exonucleolytic activity associated with T4 DNA polymerase, the most distal repeats may be lost during the generation of blunt ends.
A compilation of de novo telomeric repeats from the various transformants is shown in Table II. Cells microinjected with either pPXVI or the plasmid expressing wild‐type telomerase RNA had ∼70% G4T2 repeats, consistent with the composition of previously reported Paramecium telomeres (Baroin et al., 1987).
The C52A template substitution leads to a dramatic increase in G3T3 repeat synthesis in transformants. When 97% of telomerase RNAs in transformants included the C52A mutation, de novo telomeric repeats synthesized in vivo were 99% G3T3. A duplicate experiment demonstrated the dosage dependence of this phenomenon. When 88% of telomerase RNAs in transformants were C52A, 85% of de novo telomeric repeats synthesized were G3T3.
De novo telomeric DNA in C49A transformants included a high percentage of the 7 nt repeat G4T3 (∼74%), as well as two novel 8 bp repeats (G4T4 and G5T3). It has been previously reported that the analogous template substitution in the T.thermophila telomerase RNA (C43A) leads to G4T3 telomeric repeat synthesis (Gilley et al., 1995). The effect of the G57A substitution was the somewhat unexpected increase in G3T3 repeat synthesis, from ∼30% in the wild‐type controls to nearly 50%. Finally, the A50G mutated RNA contributed to telomeric repeat synthesis, albeit very poorly. Only two repeats (G4TC and G3TC) were detected from 10 rescued plasmids (data not shown). There was an apparent dominant effect with the A50G template mutation, as the contribution of the endogenous telomerase RNA (up to 13% of the total complement of telomerase RNA in transformants) to de novo telomere addition was much reduced (see Table I and Figure 3).
Only a fraction of the 30 bp distal to the ‘seed’ telomeric repeats of linearized plasmids was detected in ‘rescued’ plasmids, if at all. The junction between the pre‐existing and de novo telomeric repeats ranged between 0 and 23 bp for the >70 transformant telomeres sequenced, consistent with degradation of non‐telomeric DNA ends prior to de novo telomere addition (data not shown). In contrast, a previous study demonstrated that the ends of microinjected, linearized plasmids were generally not removed prior to telomere addition, as measured by the retention of restriction sites very near the linearized plasmid ends (Gilley et al., 1988). Nucleolytic trimming of the distal 30 bp, perhaps by a nuclease activity associated with telomerase (Collins and Greider, 1993; Melek et al., 1996), can apparently precede telomere addition to the linearized plasmids used in this study. It has been shown that developmentally controlled telomere addition at a single locus in P.tetraurelia is specific to a region rather than to a specific site (Forney and Blackburn, 1988). It would appear that the determinants for Paramecium telomerase substrate recognition, during either vegetative growth or macronuclear development, are ill defined at this time.
P.caudatum telomerase RNA expression in P.tetraurelia
Given the effect of various template mutations on P.tetraurelia telomerase fidelity, it is conceivable that a region of the telomerase RNA other than the template accounts for the apparent high fidelity of the P.caudatum enzyme. To test this possibility, a chimeric gene was constructed, consisting of the wild‐type P.caudatum telomerase RNA coding sequence flanked by the upstream and downstream non‐transcribed regions of the P.tetraurelia gene. The chimeric gene was cloned into the expression vector pPXVI and subsequently microinjected into P.tetraurelia as described above. The C52A template mutation, which directs the efficient synthesis of G3T3 repeats in P.tetraurelia, was introduced as a marker to determine if the P.caudatum telomerase RNA can contribute to P.tetraurelia telomere synthesis in vivo. Such a telomerase would have to function despite a primary sequence divergence of 24% between the two species' telomerase RNAs (McCormick‐Graham and Romero, 1996).
Stable maintenance and transcription of the P.caudatum plasmid constructs following microinjection were confirmed and quantitated by Southern and Northern analyses as described earlier (Table III). Figure 4 indicates that telomere extension was similar in transformants expressing either P.tetraurelia or P.caudatum telomerase RNA. As shown previously, de novo telomeres from transformants expressing the C52A telomerase RNA were consistently longer than those expressing the wild‐type RNA.
The sequence of de novo telomeric repeats from the P.caudatum C52A transformants demonstrates that the P.caudatum telomerase RNA can form a functional telomerase enzyme with the P.tetraurelia telomerase proteins. Consistent with the result from the P.tetraurelia C52A telomerase RNA, 94% of de novo telomeric repeats from cells expressing the P.caudatum homolog were G3T3 (compare Tables II and III). However, the G4T2 content of telomeres from transformants expressing the P.caudatum wild‐type RNA (76%) was similar to that seen for the P.tetraurelia wild‐type controls (70%). These data suggest that the high fidelity of the P.caudatum telomerase does not reside solely in the telomerase RNA from that species.
Paramecium tetraurelia telomerase is inherently imprecise, in contrast to telomerase activities from other ciliates that synthesize telomeric repeats with a very low error frequency. In addition to the G4T2 repeats predicted from Paramecium spp. telomerase RNA templates (McCormick‐Graham and Romero, 1996), G4T3 and G3T2 repeats are also detected, with G3T3 constituting the vast majority of variable repeats (Baroin et al., 1987; Forney and Blackburn, 1988).
How are variable telomeric repeats synthesized by Paramecium telomerase? Three error mechanisms, stuttering, stalling and misincorporation, can account for variable telomeric repeats in other organisms. In vitro characterization of telomerase activities from the yeasts Saccharomyces cerevisiae and S.castelli suggests that the normal variations seen in their telomeres are due to premature dissociation from and stuttering along the template, respectively (Cohn and Blackburn, 1995). Insertion of an additional C residue in the T.thermophila telomerase RNA template can lead to dramatic stuttering errors, with multiple dG residues added sequentially, both in vivo and in vitro (Yu and Blackburn, 1991; Gilley et al., 1995). Misincorporation errors (addition of a non‐cognate dNTP opposite a templating nucleotide) have been documented for a C→U transition in the T.thermophila telomerase RNA. A consequence of the C48U mutation is misincorporation of dA at adjacent templating rC residues in vitro (Gilley et al., 1995). These data indicate that the templating nucleotides are involved in the active site of the enzyme, with contributions to telomerase activity beyond that of a passive template. We have shown that Paramecium spp. telomerase RNA template mutations also affect the fidelity of the enzyme in vivo. However, in contrast to the Tetrahymena enzyme, template substitutions in Paramecium can dramatically increase telomerase fidelity.
Based on the sequence of de novo telomeric repeats presented in Table II, we propose that the primary mechanism for variable telomeric repeat synthesis by wild‐type P.tetraurelia telomerase is via misincorporation of dT residues, with the majority of errors occurring at templating nucleotide C52. Such errors should result in a de novo telomeric repeat of 5′‐GGGTTTG‐3′. This free 3′‐end can be properly positioned at the alignment region immediately 3′ of the template for subsequent telomeric repeat synthesis (Figure 5). Misincorporation of dT at C52 would account for the high frequency of G3T3 in Paramecium telomeres (Baroin et al., 1987; Forney and Blackburn, 1988) and in our transformant controls (an average of 45 and 30% respectively). The C52A substitution apparently decreases the incidence of errors by accommodating a presumed tendency of P.tetraurelia telomerase to incorporate dT at this templating position. Alternatively, the three T residues in G3T3 could be generated by stuttering at template positions A50 and/or A51. A minor set of variable repeats, such as G5T2 and G5T3 (Table II), are best explained by stuttering. However, we consider misincorporation rather than stuttering as a more likely mechanism for the majority of G3T3 repeat synthesis by wild‐type telomerase, given our results with the C49A template mutation (see below).
The P.tetraurelia C49A template substitution is analogous to the C43A mutation described previously for the T.thermophila telomerase RNA (Gilley et al., 1995). A consequence of the T.thermophila C43A substitution was the synthesis of G4T3 telomeric repeats, a phenomenon also seen in vivo with the Paramecium C49A substitution (Table II). The most straightforward interpretation of these data is that nucleotide C55, normally a member of the alignment region (Autexier and Greider, 1995), is ‘recruited’ as a templating nucleotide. Alignment of a de novo 3′‐end from the C49A enzyme (5′‐TTT‐3′) for a subsequent round of polymerization would occur at nucleotide positions 3′‐A58GA56−5′, allowing the 7 nt from positions 55 to 49 to serve as the template (Figure 5). Our interpretation is in concordance with that drawn by Gilley and Blackburn (1995) with regard to the Tetrahymena mutation. Infrequent dT misincorporation at template position C49 may account for the occasional G4T3 repeats detected in Paramecium telomeres (Baroin et al., 1987; Forney and Blackburn, 1988) and in the de novo telomeres from wild‐type transformants described in this study (1.5 and 1.4% respectively). An alternative explanation for the occurrence of 5′‐TTT‐3′ at the end of a telomeric repeat is an occasional stutter by the wild‐type telomerase at templating nucleotides A50 or A51, followed by dissociation from the enzyme prior to dG addition at C49.
Another consequence of the C49A template change is an increase in the Paramecium telomerase fidelity in vivo. When 72% of the P.tetraurelia telomerase RNA molecules in transformants include this substitution, 74% of de novo telomeric repeats synthesized are G4T3 (Table II). Continued misincorporation of dT at C52 in the C49A transformants should result in up to 30% of the de novo repeats consisting of G4T4. However, only 3.7% of de novo telomeric repeats in these transformants are G4T4 (Table II). It is not clear how the C49A mutation increases telomerase fidelity. Perhaps the C49A mutation itself, or the utilization of C55 as a templating nucleotide instead of participation in 3′‐end alignment, alters the configuration of the active site such that dT misincorporation at C52 is much reduced.
The putative alignment region of wild‐type Paramecium telomerase RNA includes a G residue (G57), with the potential to form a rG.dT wobble base pair during continued telomeric repeat synthesis (Figure 5). This is in contrast to all other ciliate RNAs, which include an A residue at the homologous position. We explored the effect of a G57A substitution on the error rate of Paramecium telomerase. Somewhat suprisingly, we found that the G57A mutation leads to a 20% increase in G3T3 errors, from 30 to 50%. Chemical and enzymatic probing of the Tetrahymena telomerase RNA indicate that both the template and alignment regions are in an accessible and highly ordered structure (Bhattacharyya and Blackburn, 1994; Zaug and Cech, 1995). The 20% increase in G3T3 errors with the G57A mutation may be due to a subtle perturbation of this structure, consequently increasing dT misincorporation at C52. Altering the geometry of the enzyme's templating domain with the G57A substitution paradoxically has the opposite effect of the C49A mutation on fidelity. Interestingly, substitution of the homologous nucleotide in the Tetrahymena RNA (A51) with a G residue leads to a different sort of error during polymerization: premature product dissociation in vitro (Gilley and Blackburn, 1996).
Telomeres extended primarily by the addition of G4T3 or G3T3 repeats (in the C49A and C52A transformants respectively) were consistently longer than those from wild‐type controls (Figures 3 and 4). This loss of telomere length regulation resembles a similar phenomenon in T.thermophila and Kluvermyces lactis transformants synthesizing mutated telomeres, although not to the same degree (Yu et al., 1990; McEachern and Blackburn, 1995; Romero and Blackburn, 1995). It has been determined that diminished binding of the sequence‐specific DNA‐binding protein RAP1 to mutated telomeres is responsible for the loss of telomere length control in K.lactis (Krauskopf and Blackburn, 1996). A similar defect in the recognition of altered telomeric DNA is the most likely mechanism for the apparent loss of telomere length regulation in this study.
Inefficient telomere addition in cells expressing the A50G telomerase RNA (Figure 3) appears to be a dominant effect, as de novo telomere synthesis was reduced in these transformants, despite the presence of the endogenous, wild‐type RNA (up to 13%; Table II). This phenotype may be due to diminished binding and/or recognition of the de novo 3′‐end (5′‐GTCG‐3′) by telomerase. A critical Watson–Crick base pair between the 3′‐end of a telomeric primer and nucleotide A50 in the Tetrahymena RNA (the homolog to the Paramecium A56 alignment nucleotide) has been demonstrated in vitro (Gilley and Blackburn, 1996). Successful extension of a 5′‐GTCG‐3′ telomeric end by either wild‐type or A50G Paramecium telomerase would necessarily have to tolerate a rA.dC mismatch at alignment nucleotide A56 (Figure 5). Given our results with A50G transformants, this is an apparently inefficient process in vivo in Paramecium. Somewhat paradoxically, in vivo synthesis of G4TC repeats by T.thermophila is quite efficient when the A44G telomerase RNA gene (analogous to Paramecium A50G) is introduced into cells by microinjection (Yu et al., 1990; Romero and Blackburn, 1995).
It is remarkable that wild‐type P.tetraurelia telomerase misincorporates dT residues at the same template position at a fairly high frequency and to the exclusion of non‐cognate dA or dC residues. It is conceivable that a post‐transcriptional modification of C52 could promote pairing to dTTP rather than dGTP at that templating position during telomere synthesis. Alternatively, the observed bias for dT incorporation at C52 may indicate greater binding efficiency for dTTP than the other dNTPs (excluding the cognate dGTP) at that point in the polymerization cycle. Competition and chain termination studies with nucleoside analogs indicate that the nucleotide‐binding properties of Tetrahymena telomerase differ at various positions along the template (Strahl and Blackburn, 1994). X‐ray crystallographic studies of nucleic acid structures have demonstrated the potential for pyrimidine–pyrimidine base pair formation (reviewed by Wahl and Sundaralingam, 1995). A mismatched C.U pair can form with a single base–base hydrogen bond with a water molecule bridging the bases, accommodated by minor distortion of the helix (Cruse et al., 1994). A novel Hoogsteen‐like U.U base pair has been described, demonstrating the possibility of unusual pyrimidine–pyrimidine interactions (Wahl et al., 1996). On the other hand, misincorporation of dT juxtaposed to C52 may be more a function of the P.tetraurelia enzyme's active site conformation than the ability of a C residue to form a base pair with dTTP. It is conceivable that substitution of C52 with either a G or U residue may result in continued dT misincorporation at that position.
What factors determine the error rate of Paramecium telomerase? We have established the involvement of the RNA moiety in fidelity, as a variety of nucleotide substitutions can either increase or decrease the percentage of de novo G3T3 repeats. Mutagenic experiments with Tetrahymena telomerase also indicate that the templating nucleotides contribute more to the active site than simply serving as a template (Gilley et al., 1995). However, our data indicate that the telomerase RNA is not solely responsible for the observed variablilty.
Greater than 95% of P.caudatum telomeres are comprised of G4T2 repeats (McCormick‐Graham and Romero, 1996), consistent with a low error rate for telomerase from this species. Despite the substantial primary sequence divergence of the RNAs from the two species, a chimeric enzyme composed of C52A P.caudatum telomerase RNA and P.tetraurelia proteins functions in vivo, synthesizing a high percentage of G3T3 repeats (Figure 4 and Table III). However, the parallel experiment with wild‐type P.caudatum telomerase RNA expressed in transformants does not lead to a chimeric telomerase with high fidelity (compare Tables II and III). Our data indicate that additional factors, most likely telomerase proteins, contribute to telomerase fidelity. Our data also support the contention of Gilley and Blackburn (1996) that telomerase RNA is a component of a telomerase active site, working in concert with telomerase protein(s) to catalyze the synthesis of telomeric repeats.
Altering the sequence of telomeric DNA in organisms that normally synthesize invariant telomeric repeats can have drastic effects on cell division and morphology, ultimately leading to senescence (Yu et al., 1990; McEachern and Blackburn, 1995). In contrast, the only phenotype detected in P.tetraurelia transformants synthesizing a reduced percentage of G4T2 repeats is a moderate loss of telomere length regulation. Perhaps this is not too suprising, given the naturally occurring variability in wild‐type P.tetraurelia telomeres. It would be interesting to determine whether telomere sequence variability is so readily tolerated in P.caudatum, a species whose telomerase has evolved to precisely synthesize G4T2 repeats. A comparison of in vitro telomerase activities from these two species may reveal whether P.caudatum telomerase is inherently more precise at templating nucleotide C52 or if there is a specific proof‐reading mechanism during telomere addition, perhaps involving the exonucleolytic activity associated with telomerase, that can correct the mistakes of an error‐prone enzyme.
Materials and methods
Paramecium tetraurelia (nd6/nd6 and cam2/cam2; Lefort‐Tran et al., 1981; Kanabrocki et al., 1991) were maintained at room temperature in a monoxenic wheat grass infusion, containing Enterobacter aerogines (Sonneborn, 1970). Restriction enzymes and other molecular biology reagents were purchased from New England Biolabs (Beverly, MA). The antibiotic G‐418 was purchased from Sigma (St Louis, MO).
Genomic DNA was isolated and total RNA extracted P.tetraurelia as previously described (McCormick‐Graham and Romero, 1996). Oligonucleotides were radiolabeled at the 5′‐end with T4 polynucleotide kinase and [γ‐32P]ATP (7000 Ci/mmol; ICN) as described (Sambrook, 1989). Radiolabeled telomerase RNA gene probe was generated by a PCR strategy as previously described (McCormick‐Graham and Romero, 1996). Conventional PCR protocols (Saiki et al., 1988) and molecular techniques (Sambrook et al., 1989) were used for plasmid construction. DNA sequencing was performed with either Sequenase™ (US Biochemical, Cleveland, OH) or the CircumVent Thermal Cycle DNA sequencing kit (New England Biolabs, Beverly, MA).
Utilization of the following oligonucleotides (Amitof Biotech, Boston, MA) is included in the text. PCR primers (P), mutagenic oligonucleotides (M), antisense probes (A) and sequencing primers (S) are as indicated. All oligonucleotides are written 5′→3′: P1T, CGC CAG CTG CTT TGG AAC ATG GGC AC; P2T, CGC CAG CTG CTT GAA TAT TTA CGA; P1C, CGC GTA TAC TGA CAA TTG CTG TAT TCA; P2C, CCG CAG ATC TAA AAA TAA CCA TTC AGA G; M1, TTT CTG GGG TCG TAT GAC TT; M2, GTA ATG GTT TTT GGG GTT GT; M3, GGT TTC TGG GTT TGT ATG AC; M4, TTC TGG GGT TTT ATG ACT TCC; M5, ATT AAT GAA AAA GAT CTA AAA ATA AC; M6, ATT AAT GAA AAA AAT ATA AAA ATA AC; S1, TAA CTT TTA CTC AAT GTC AAA G; S2, GCG TCT AGA AAT AAC TAT TTA GA GC; A1, GGT TTC TGG GGT TGT ATG AC; A2, GGT TTC TGG GGT TTT ATG AC; A3, CAC TTA TGA CTC GTC; A4, TAC TTT TAG GTT CGT.
Transformation vector modification
Plasmid pPXVI was constructed by modification of pPXV‐NEO (Haynes et al., 1995). The APH‐3′‐II expression cassette (2.0 kB) was removed from pPXV‐NEO by digestion with PvuII. A 260 bp PvuII–SmaI fragment from the cloning vector pBluescript II SK (Stratagene, La Jolla, CA) was ligated to the large 3.5 kb fragment from the pPXV‐NEO PvuII digest. Restriction digests confirmed the orientation of pPXVI polylinker sites shown in Figure 1B.
Paramecium tetraurelia telomerase RNA gene mutagenesis
The P.tetraurelia telomerase RNA gene was amplified from genomic DNA by a standard PCR reaction, utilizing oligonucleotides P1T and P2T. The resultant 565 bp amplified product includes 240 bp upstream and 110 bp downstream of the transcribed RNA (McCormick‐Graham and Romero, 1996). This PCR product was cloned into the SmaI site of pUC118 and screened by restriction digests for the correct orientation (designated pPTER). Oligonucleotide, site‐directed mutagenesis of the telomerase RNA gene in pPTER was by the method of Kunkel et al. (1987). Mutagenic oligonucleotides M1, M2, M3 and M4 were used to introduce A50G, G57A, C52A and C49A substitutions respectively (Figure 1A), which were subsequently confirmed by single‐strand DNA sequencing. The wild‐type and mutated telomerase RNA genes were cloned into the vector pPXVI by virtue of unique SacI and XbaI restriction sites. The telomerase RNA genes of the resultant pPXVI‐TER constructs (Figure 1B) were confirmed by double‐stranded DNA sequencing.
Chimeric P.caudatum/P.tetraurelia telomerase RNA gene construct
A unique BglII restriction site was introduced at nucleotide position +209 of the P.tetraurelia telomerase RNA gene in pPTER by site‐directed mutagenesis (oligonucleotide M5). The resultant plasmid (pPTERb) includes a unique Bst1109I site at nucleotide position −4 (McCormick‐Graham and Romero, 1996) and a singular BglII site at nucleotide position +209. The cloned P.caudatum telomerase RNA gene was amplified by PCR with oligonucleotides P1C and P2C, which were designed to ensure unique Bst1109I and BglII restriction sites at the termini of the 218 bp PCR product. Both pPTERb and the P.caudatum PCR product were digested with Bst1109I and BglII, ligated and recombinant molecules screened for an EcoRI site present in the P.caudatum RNA gene sequence (pPCTERb). Site‐directed mutagenesis with oligonucleotide M6 removed the unique BglII site, restoring nucleotide positions +209 to +214 of pPCTERb to the wild‐type sequence (pPCTER). Further mutagenesis of pPCTER with M3 introduced a C52A templating nucleotide substitution (pPCTER‐C52A). The chimeric wild‐type and C52A telomerase RNA genes were cloned into the vector pPXVI at SacI and XbaI restriction sites as described above. Telomerase RNA gene sequences of the resultant pPXVI‐WTC and pPXVI‐C52AC constructs were confirmed by direct sequencing.
Transformation of P.tetraurelia by microinjection
Plasmids suitable for microinjection were prepared with the Wizard™ DNA purification system (Promega, Madison, WI). The plasmids pPXV‐NEO, pPXVI and various pPXVI‐TER constructs were linearized by SfiI digestion (Figure 1B). Preparation and monitoring of post‐autogamous cells prior to microinjection were as previously described (Haynes et al., 1995). Macronuclei were injected with 10–20 pl each DNA sample (1 μg/μl, ∼2×106 molecules/pl) betweeen the third and sixth post‐autogamous division (Haynes et al., 1995). Following microinjection, clonal lines were allowed to expand to 100 cells (3–4 days at room temperature). Approximately 20 cells of potentially transformed cultures were challenged with G‐418 (50 μg/ml). Successfully transformed cells were resistant to the antibiotic 2 days after the initial challenge. Transformant cultures were carefully maintained at a density of 200 cells/ml and expanded for 20–22 generations following microinjection.
In vitro transcription of mutated telomerase RNAs
A plasmid (pPTER‐T7) designed for in vitro transcription of the P.tetraurelia telomerase RNA has been described (McCormick‐Graham and Romero, 1996). Mutagenic oligonucleotides M1–M4 were used to introduce A50G, G57A, C52A and C49A substitutions into pPTER‐T7 as described above. Linearization of these plasmids with XbaI followed by in vitro transcription with T7 RNA polymerase and rNTPs (Promega Transcription in vitro System) results in run‐off transcription of both wild‐type and mutant P.tetraurelia telomerase RNAs.
Northern blot allele‐specific hybridizations
Total RNA (0.2–2 μg) isolated from transformants and telomerase RNA in vitro transcripts (50 ng) were electrophoresed through an 8% polyacrylamide–7.5 M urea gel and electroblotted to Nytran filters (Schleicher & Schuell). The specificity of 32P‐labeled oligonucleotide binding to Northern blots was determined empirically by varying the washing temperatures (52–58°C) and monitoring hybridization to the in vitro transcripts as previously described (McCormick‐Graham and Romero, 1996).
Northern blots were probed sequentially with the appropriate oligonucleotides radiolabeled to a specific activity of 7000 Ci/mmol. Wash temperatures, buffer volumes and specific activities of hybridization solutions (0.4 μCi/ml) were carefully monitored to ensure an accurate, quantitative comparison. A Molecular Dynamics PhosphorImager was used to quantitate the relative ratio of the plasmid‐encoded to endogenous telomerase RNA isolated from transformants. Exposure times to the phosphorimager were adjusted to correct for decay of the 32P radiolabel.
Southern blot hybridizations
DNA transfer from agarose gels onto Nytran membranes, hybridization of radiolabeled DNA probes and washing conditions were as previously described (McCormick‐Graham and Romero, 1996). A Molecular Dynamics PhosphorImager was used to quantitate the relative ratio of the plasmid‐encoded to endogenous telomerase RNA genes present in total DNA from transformants.
Cloning de novo telomeres from P.tetraurelia transformants
Total DNA (5 μg) extracted from transformants 20–22 fissions after microinjection was digested with the restriction enzyme XbaI in a 50 μl reaction, effectively removing one telomeric end from the microinjected pPXVI‐TER plasmids (Figure 1B). The XbaI‐digested DNA was treated with 6 U T4 DNA polymerase (New England Biolabs) in a standard reaction buffer (0.1 mM dNTPs) at 12°C for 15 min to generate blunt ends suitable for ligation. Following phenol:chloroform extraction and ethanol precipitation, 0.5 μg DNA were incubated overnight at 16°C with T4 DNA ligase (100 μl reaction volume). Competent E.coli (strain DH10B) were transformed by electroporation with the ligation products. DNA prepared from ampicillin‐resistant clones was screened for the telomerase RNA gene by Southern blot hybridization. The C‐strands of cloned, de novo telomeric repeats were sequenced, utilizing radiolabeled oligonucleotide S1 as a sequencing primer. The identities of telomerase RNA genes in ‘rescued’ plasmids were also confirmed by sequencing through the plasmid‐encoded genes with oligonucleotide primer S2 (complementary to nucleotide postions +209 to +191 inclusive of the telomerase RNA gene).
We thank Karin Musier‐Forsyth and Greg Connell for critical reading of and helpful comments on the manuscript. This research was supported by NIH grant GM50861.
- Copyright © 1997 European Molecular Biology Organization