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Chapter 20 Studying the Telomerase RNA in Tetrahymena
CHAPTER 20
Studying the Telomerase R N A in Tetrahymena E. Blackburn*, D. GilleJ, T. Ware*, A. Bhattacharyyat, K. Kirk$, and H. Wang* ' University of California, San Francisco Departments of Microbiology and Immunology, and Biochemistry and Biophysics San Francisco, California 94143 t University of Chicago Medical Center
Department of Radiation and Cellular Oncology Chicago, Illinois 60637 8 Lake Forest College Department of Biology Lake Forest, Illinois 60045
I. Introduction 11. General Strategy and Overview 111. Basic Methods A. Mutagenesis of Telomerase RNA B. Introducing Mutant Telomerase R N A Genes into Tetrahymena C. Transformation of the Telomerase R N A Gene D. In Vivo Studies E. In W h o Studies References
I. Introduction Telomeres are specialized structures at the ends of eukaryotic chromosomes that are required for chromosome stability and the complete replication of linear chromosomes. In ciliates de novo telomere formation is an essential step in a critical developmental stage-the generation of the new macronucleus (Blackburn, 1995). Telomeric DNA sequences are generally composed of short G-rich METHODS IN CELL BIOLOGY, VOL. 62 Coppght 0 1999 by Academic Press. AU righu of reproducoon in any form reserved 0091-h79X/W $30.00
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tandem repeats, running 5' to 3' toward the distal end of the chromosome. The telomeres found at the ends of Tetrahymena macronuclear chromosomes are composed of GGGGTT repeats (see also Chapter 19). Telomerase is a ribonucleoprotein (RNP) reverse transcriptase responsible for the synthesis of telomeric repeats. Telomerase is the only known reverse transcriptase that contains its own template, which is present in the telomerase RNA moiety as an integral part of the RNP complex (Greider and Blackburn, 1989). The telomerase RNA of Tetrahymena thermophila is a relatively small RNA (159 nucleotides) (Greider and Blackburn, 1989) with a very well-defined secondary structure. Within this telomerase, RNA is a region complementary to the G-rich telomeric strand, 3'-AACCCCAACJ', termed the templating domain (Greider and Blackburn, 1989). Specific residues of the templating domain are copied to produce the G-rich telomeric strand (Yu et al., 1990). The secondary structure of the telomerase RNA was determined by phylogenetic covariation based on sequence input from'over 25 ciliate telomerase RNAs (Romero and Blackburn, 1991; Lingner et al., 1994; McCormick-Graham and Romero, 1995, 1996) (Fig. 1). Additional support for this RNA structure came from both in vitro and in vivo chemical and enzymatic structural analyses (Bhattacharyya and Blackburn, 1994; Zaug and Cech, 1995). The small size of this RNA along with its well-defined secondary structure have made it very amenable for fundamental studies of telomerase and telomere function. Because changes in telomeres and telomerase have been linked to tumorigenesis and aging in humans, there has been an explosion of interest and research in this area (reviewed in de Lange, 1994). Tetrahymena and other ciliates have been invaluable model systems for the study of telomeres and telomerase, due in part to the relative abundance of telomeres and telomerase in these cells. Ciliates devote significant cellular resources to the maintenance and replication of telomeres due to the large number of macronuclear chromosomes and the relatively small size of each macronuclear chromosome. Consequently, each cell contains an unusually large number of chromosome ends. For example, each Tetrahymena macronucleus contains about 20,000-40,000 telomeres, and each Euplotes macronucleus contains about 80 million telomeres; therefore, there is an abundance of telomeric DNA, telomere proteins, and telomerase in these interesting and unusual unicellular organisms. Nearly all the basic knowledge
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Fig. 1 Secondary structure of Tetrahymena telomerase RNA (adapted from Gilley and Blackburn, 1996).
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that has been uncovered about telomeres and telomerase was first discovered using ciliates. The following is a brief listing of some of the major discoveries using ciliates for studying telomeres and telomerase: the first telomeric sequence, the discovery of telomerase, the discovery of the telomerase RNA moiety (reviewed in Gall, 1990),the first telomeric proteins (Gottschling and Zakian, 1986), and the isolation of the catalytic subunit of telomerase (Lingner et ul., 1997) and other telomerase-associated proteins (Collins et al., 1995; Lingner and Cech, 1996). In this chapter, we introduce to the reader how we have studied various functional aspects of the telomerase RNA from Tetruhymena thermophilu. This chapter covers broad descriptions of general research strategies along with more detailed methodologies. Some of the methods and general strategies discussed will be useful for studies with a broad range of organisms. In addition, we include information that will be specifically useful to those who use ciliates to study this fascinating RNA molecule.
11. General Strategy and Overview One general approach that has been extremely useful in the study of the telomerase RNA is to mutate specific nucleotides or regions of the telomerase RNA gene. Mutant telomerase RNA genes are then introduced back into cells on a high copy number vector to observe the effects of these alterations on the in vivo assembled mutant telomerases. This method was first used to demonstrate that specific residues within the template domain are copied and incorporated into telomeres (Yu et al., 1990; Yu and Blackburn, 1991). This experimental approach has proven valuable for studying telomerase RNA because the mutant telomerase is assembled into an RNP complex in the cell-unless the mutation prevents assembly-and the function (or lack of function) of the in vivo assembled mutant telomerase can be analyzed within the environment of the cell. In addition, the in vivo assembled mutant telomerase can be partially purified and studied under a variety of in vitro conditions. Although the endogenous telomerase RNA gene is still present in cells containing the newly introduced mutant telomerase RNA, the mutant telomerase RNA gene is present in the cell on a high copy number vector (-10,000 copies of the mutant gene versus -45 copies of the wild-type gene) (Yu and Blackburn, 1989). This system leads to specificproblems but also has advantages. For example, one potential problem due to the presence of the endogenous wild-type telomerase RNA gene is that cells transformed with a mutant gene can revert, thereby expressing the wildtype gene. (Reversion can be monitored in clonal lines by methods discussed in Section 1II.B.) Reversion to the wild-type telomerase RNA gene can arise in several ways. Either the mutant gene is lost from the cell (selected against), not expressed, or the RNA is unstable andor fails to be assembled into an active telomerase complex. In these cases, the endogenous wild-type RNA is reestab-
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lished, thereby becoming the dominant telomerase complex in these cells. On the other hand, the presence of the endogenous RNA can also be advantageous because it provides useful information about rate-limiting steps at which the mutant RNA must compete with endogenous RNA. Alterations in the telomerase RNA were observable at two basic levels in Tetrahymena: changes in cellular phenotypes and changes of the telomerase RNP and its enzymatic properties. Cellular phenotypes were easily detected in Tetrahymenadue to its large size and readily observable stages of nuclear division. For example, certain mutations within the RNA templating domain can cause synthesis of telomeric repeats that are “toxic” to the cell when added distally to the chromosomal telomeres. Studying the phenotypes of such telomerase mutants has suggested possible functions for telomeres in chromosome segregation (Yu et aL, 1990, Kirk et al., 1997). Mutations outside the RNA template domain can also be deleterious to the cell (e.g., by creating a nonfunctional telomerase RNA capable of competing for limiting telomerase proteins) (Yu et aL, 1990; Kirk et aL, 1997; Gilley et aL, 1995). Because Tetrahymena is a relatively abundant source of telomerase, the RNP complex and the enzymatic properties of telomerase can be studied in several ways. Certain mutations within the telomerase RNA affect telomerase RNP assembly. Others affect basic enzymatic processes of telomerase such as processivity and fidelity. Some enzymatic effects are due to alterations in base-pairing interactions with telomerase and its substrate. However, certain enzymatic alterations caused by specific mutations within the telomerase RNA are not explainable by altered base-pairing interactions and instead suggest that the RNA plays an important role in active site functions that affect telomerase/substrate interactions (Gilley el al., 1995; Yu and Blackburn, 1990; Romero and Blackburn, 1995; Gilley and Blackburn, 1996).
In. Basic Methods In this section we discuss details of methods found useful in making alterations within the telomerase RNA. We also discuss some of the general strategies employed to study this RNA and introduce some of the “tools” that have been useful in these studies.
A. Mutagenesis of Telomerase RNA To generate alterations within the telomerase RNA gene, we have generally relied on a polymerase chain reaction (PCR) based method (Erlich, 1989) except for the following modifications: Twenty cycles of PCR involved denaturation (30 s, 94”C), annealing (30 s, 5OoC), and extension (30 s, 72°C) using conditions supplied by the manufacturer of the DNA Thermal Cycler (Perkin Elmer
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Cetus). Potential mutant genes were then carefully sequenced to detect possible unwanted errors introduced by Taq polymerase. Mutant telomerase RNAs were inserted into the vector prD4-1 (Yu and Blackburn, 1989) at the 2 0 1 site within the vector polylinker. The vector prD4-1 confers paromomycin resistance to transformed Tetrahymena. The transformation efficiency of this vector into E. coli is low due to its large size (14.85 kb). Therefore, it was necessary to perform colony hybridization to detect colonies with the correct insert. B. Introducing Mutant Telomerase RNA Genes into Tetruhymena
For most purposes, the most reliable and useful method for introducing mutant telomerase RNA genes into Tetrahymena has been electroporation of synchronized cells early in development as described by Gaertig and Gorovsky (1992) (see also Chapter 26). Electroporation supplanted the microinjection of vegetatively dividing cells used in earlier analyses of telomerase RNA mutants (Yu et al., 1990;Yu and Blackburn, 1991;Romero and Blackburn, 1995). The electroporation method allows efficient use of the vector prD4-1, which is unstable over time. By electroporating at the start of macronuclear development, it was possible to observe transformants before vector alterations or mutant telomerase RNA gene loss takes place, thereby making it possible to study even mutants that are extremely “toxic” to the cell. Using this method, we have been able to identlfy unique classes of cellular phenotypes caused by telomerase RNA mutations (see Section III.C.l). Cells were electroporated using a GenePulser system (Biorad), generally with 15-25 pg plasmid DNA isolated by the use of Qiagen columns (Qiagen, Chatsworth, CA) in 0.4-cm electroporation-cuvettes (Biorad) under the following conditions: resistance, 200 R capacitance 25 pF;voltage 0.44 kV.Electroporated cells were immediately diluted into 20 ml 2% PPYS culture medium prior to plating them into sterile, 96-well microtiter dishes (200 pl/well) at empirically derived dilutions (1 :500 or 1:1000) in order to obtain potential single or multiple clonal transformant lines. Transformants were selected in the presence of 100 pg/ml paromomycin (available from Parke-Davis or Sigma and used at a final concentration of 120 pg/ml) and added optimally 12-15 h postelectroporation (50 pl/well of 500 pg/ml stock). Paromomycin-selected transformants appeared and were harvested routinely 3-4 days after drug addition. C. Transformation of the Telomerase RNA Gene
1. Clonal Lines The episomal vector prD4-1 is initially maintained in transformed cells at very high copy (>lO,OOO copies per cell), which allows overexpression of the introduced mutant gene. However, one potential problem with this vector is that
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it is not possible to generate a stable transformant line (Yu and Blackburn, 1990; Romero and Blackburn, 1995), due to the unstable nature of the vector and selection against deleterious mutant telomerase RNA genes. The more deleterious the mutant telomerase RNA gene, the fewer fissions before selection against the gene and its loss from the cell population. However, by using clonal lines it was possible to eliminate reversion for up to -50 fissions. A definitive way to monitor reversion was to isolate the telomerase from transformed cells and determine the proportion of mutant relative to wild-type telomerase (see Section III.D.2). In the case of a mutant telomerase RNA that is assembled into an active RNP complex, essentially only the mutant enzyme activity was evident in in v i m telomerase activity assays because the overexpressed mutant RNA is far more abundant than the wild-type RNA.
2. Mass Transformation Mass transformation was used to observe transformants at the earliest possible moment after transformation and to study early-lethal telomerase RNA mutants. This allows thousands of transformants to be pooled so that enough matenalDNA, RNA, and telomerase-can be isolated for study before reversion. For example, it was possible to study mutations such as the telomerase RNA mutation 44G, which is extremely toxic once introduced into cells (Yu et al., 1990; Gilley et al., 1995). Cells transformed with the 44G mutation can undergo only seven to nine fissions before cessation of cell division. By combining transformants, it was possible to isolate enough material to study this severe phenotype. Because many transformants were combined, we found it important to monitor reversion closely by assaying telomerase activity, cessation of cell division, and altered phenotype. Using a pooled transformant population, it was possible to monitor reversion of a telomerase RNA mutant that causes a micronuclear anaphase block phenotype (Kirk et al., 1997) as the population was passaged over time. The micronuclear phenotype was monitored by DAPI staining, and population reversion was indicated by a decrease in the ratio of anaphase micronuclei to interphase micronuclei. For example, in the 43AA mutant (Kirk et aZ., 1997), from 5 to 7 days after transformation, the ratio remained at 6: 10. By day 10, the ratio had decreased to 2:10, and by day 13 the cell population appeared virtually wild type with a ratio of roughly 1 : l O . The effect of a range of paromomycin concentrations (0, 10, 25, 75, 150 pg/ml) on reversion has been tested by placing the cell populations in the new drug concentration 5 days after electroporation. The rate of reversion was unaffected by as little as 10 pg/ml paromomycin and as great as 150 pg/ml paromomycin. However, if the cells were placed in medium lacking paromomycin, population reversion was evident at 7 days and virtually complete by 10 days.
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D. In vivo Studies
1. Phenotype Classification of Telomerase R N A Mutants We have identified four classes of cellular phenotypes caused by telomerase RNA mutations: early lethal, delayed lethal, “stress” response, and wild-typelike phenotype. The early lethal phenotype is characterized by cells capable of undergoing seven to nine fissions before cessation of cellular division. Therefore, each clonal transformant line produces only 200-500 cells before the terminal phenotype is expressed. Mutant cells are about ten times larger than normal wild-type transformed cells and are extremely flat, a phenotype originally termed the monster phenotype. An example of the terminal phenotype of an early lethal mutant, the 44G mutation, is shown in Fig. 2. Mutations that cause a delayed lethal phenotype are essentially wild type until -20 fissions after the mutant telomerase RNA gene is introduced. Then, between -20 to 25 fissions, the population fission rate progressively slows, cells gradually display a monster phenotype, and then cellular divison stops. Mutations have also been observed that display a phenotypic response similar to a stress response. For example, these include mutations that disrupt the pseudoknot structure within the RNA (Fig. 1) under log phase culture conditions. These pseudoknot disruption mutations affect assembly of the active RNP particle (Gilley and Blackburn, 1999). It is possible that these mutations compete for telomerase protein components with the endogenous wild-type telomerase RNA. Finally, certain telomerase RNA mutations display essentially no cellular changes. These mutations fall into two groups-those mutations that change the enzymatic properties of the mutant telomerase but do not detectably affect cells, and those mutations that incorporate mutant telomeric repeats that are tolerated by the cells. 2. Marking the Template An extremely useful tool available for studying the telomerase RNA has been to introduce the sequence changes of interest into a telomerase RNA gene that, in addition, has been “marked” by a specificbase change(s) within the templating residues. The base changes within the template are designed to produce correspondingly mutated telomeric repeats. By adding additional mutations outside the template, the marked gene can then be monitored for function. Such function can be monitored by incorporating the specific marked mutant repeat sequences into telomeres, and any phenotypes caused by the marked mutant repeats, and by analyzing and assaying activity of the mutant telomerase RNP complex in vitro. For example, the 43A mutant, which has a change from a C to an A at residue 43 (the 5’ residue of the template), has been useful for marking the RNA when a relatively “silent” or wild-type-like phenotype is desired (Gilley et al., 1995; Bhattacharyya and Blackburn, 1997). The 43A mutation has a variable penetrance-transformants display either a wild-type-like or a delayed lethal
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Fig. 2 Tetrahyrnena themophila cells, transformed by wild-type (A) or 44G mutant (B, C) telomerase RNA (TER) genes as described in this chapter. Cells were stained with 2,6-diaminido-4phenylindole (DAPI) and viewed by fluorescence light microscopy. See also color insert at back of book.
phenotype. By isolating clonal transformant lines, one can use this mutation to ascertain the effects of altering regions outside the template, hence determining the functionality of the second site mutation. In addition, template mutations are available that synthesize toxic telomeric repeats, causing a lethal phenotype in 100%of clonal lines. Marking the template with a lethal mutation allows one to test whether second site mutations are functional. If second site mutations outside the template allow telomerase function, then a lethal phenotype is dis-
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played. In contrast, if the second site mutation eliminates the function of the telomerase RNA, then the transformant line will be wild type (i.e., the toxic phenotype is suppressed) due to expression and function of the endogenous telomerase RNA (T. Ware and E. H. Blackburn, unpublished results).
3. Detection of Mutant Telomeres In studies of Tetrahymena telomerase RNA function (for example, (Yuet al., 1990;Yu and Blackburn, 1991;Romero and Blackburn, 1995;Bhattacharyya and Blackburn, 1997), mutant telomeric DNA was readily monitored by Southern blotting analysis or by cloning and sequencing telomeric DNA from cells transformed by a mutant telomerase RNA. a. Southern Blots for Mutant Telomeric Sequences
Total genomic DNA was digested with BamHl or PstI and electrophoresed overnight, retaining all DNA fragments above 0.5 kb (25 cm gel, 1%agarose, 1 X TBE). The DNA was depurinated in the gel (0.25 M HCl), transferred to Hybond+ (Amersham) in 0.4 M NaOH, and UV cross-linked. Hybridization and washes were performed essentially as described by Church and Gilbert (1984). [One liter of hybridization solution contains 0.5 liter of 1 M NaH2P04, pH 7.2 (per liter: 71 g Na2HPO4 or 134 g Na2HPO4. 7H20, plus 4 ml H3P04); 0.35 liter of 20% SDS; 2 ml of 0.5 M EDTA, pH 8.0. One liter of wash solution contains 0.2 liter 1M NaH2P04and 0.05 liter of 20% SDS.] Oligonucleotides were designed to hybridize differentially to mutant versus wild-type telomeric repeats, depending on the expected telomeric sequence. For example, the 44G template mutation creates GGGGTC telomeres. An oligonucleotide, 5’ GGGGTCGGGGTC 3’ was end-labeled using T4 polynucleotide kinase (Sambrook et al., 1989). Conditions for this probe were as follows: prehybridization, 5 min; hybridization, 1 h to overnight; washes (10 min each), once at room temperature, then twice at 32°C; followed by autoradiography. When available, a genomic DNA sample known to contain the expected sequence was loaded as a positive control. After detection of mutant repeats as described, the probe was removed from the blot with 0.4 M NaOH treatment. Removal was verified by overnight exposure to X-ray film.The blot was then available for detection of wild-type telomeric sequence using a similar procedure and an oligonucleotide probe, 5’ GGGGTTGGGGTT 3’. Detection of micronuclear-specific telomeres was achieved using the same procedure and a few variations (Kirk and Blackburn, 1995). First, we found it was important to use purified micronuclear DNA as the starting material. Second, restriction enzymes were used that digest the A+T-rich Tetrahymena DNA extremely frequently (MseI, Tsp5091, or DraI, New England Biolabs), thereby separating the short nontelomeric G4T2 tracts (Cherry and Blackburn, 1985) from the longer true telomeric DNA tracts (Kirk and Blackburn, 1995).
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b. Cloning Macronuclear Telomeresfrom Transformants The telomere cloning strategy described here was used to obtain an arbitrary sample of macronuclear telomeres (rather than mostly the more abundant rDNA telomeres). The resulting cloned telomeres contained a relatively short telomereassociated sequence, facilitating cloning and subsquent DNA sequence analysis. The procedure used standard techniques (Sambrook et al., 1989) and followed manufacturers’ suggestions for enzymatic reactions (New England Biolabs). The telomere cloning vector stock was prepared by digesting 5 p g pBluescipt (Stratagene) with ClaI and EcoRV, and the restriction enzymes were heat inactivated. The DNA was methylated by incubating with S-adenosylmethionineand TaqI methylase; then it was agarose gel purified using Geneclean (Bio 101, Inc.). The methylation was verified by testing resistance of an aliquot to TaqI digestion. Genomic DNA was isolated from transformants. The DNA ends were made flush by treating 1.0 pg genomic DNA with T4 DNA polymerase in the presence of all four dNTPs in a total volume of 5 p1 at 14°C for 15 min, and the polymerase was heat inactivated. The entire 1.0-pg transformant genomic DNA was ligated to 0.4-pg prepared vector stock with T4 DNA ligase in 10 pl total volume at room temperature overnight, and the ligase was heat inactivated. The DNA was then digested with TaqI in 100 ml total volume, treated with phenolkhloroform, and ethanol precipitated. The DNA was circularized by T4 DNA ligase in 100pl total volume at room temperature for 2 h, treated with phenolkhloroform,and ethanol precipitated to concentrate to 10 pl. The library was made by using 1 pl DNA to transform E. coli XLI-blue MRF’by electroporation. Colonies were screened initially with a wild-type telomeric oligonucleotide probe, and positive clones were subsequently cross-screened with the mutated telomeric sequence. rDNA telomeric clones were identified by an rDNA-specific probe. The cloned telomeric DNA could be sequenced directly by using vector sequence primers. Therefore, if the telomere-associated sequence was too large to read through, the telomeric tract was subcloned by digesting with the very frequent cutter Tsp5091, which leaves termini complementaryto those produced by EcoRI digestion. Another procedure for cloning transformant telomeres has been described (Bhattacharyya and Blackburn, 1997). By using this method, it was possible to inadvertently repurify the original Tetrahymena transformation vector ( ~ r D 4 - 1 ) ~ and it was therefore found useful to cross-screen the library with a probe specific for this vector, such as a nontemplate region of the telomerase RNA gene. ’
c. Cloning Wild-Type Micronuclear Telomeres
Micronuclear telomeres were cloned as follows (Kirk and Blackburn, 1995) using standard techniques (Sambrook et al., 1989) and following manufacturers’ suggestions for enzymatic reactions (New England Biolabs). To prepare the micronuclear DNA for cloning, contaminating macronuclear telomeres were elimiqated by digesting 40-pg purified micronuclear DNA (the
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micronuclear DNA isolation procedure is described in Chapter 8) with 2 units Ba131 in a total volume of 200 p1 at 30°C for 1 min, and the reaction stopped by adding 1/10 volume 0.2 M EGTA. The extent of telomere shortening was roughly 500 bp, as verified by Southern blot analysis. The DNA ends were made flush by treatment with T4 DNA polymerase in the presence of all four dNTPs in a total volume of 300 pl at 14°C for 15 min, and the reaction was treated with phenoYchloroform and ethanol precipitated. In a total volume of 50 p1, 8 pg NotI linkers (New England Biolabs) were ligated to the micronuclear DNA ends with T4 DNA ligase at 14°C overnight, and the ligase was heat inactivated. The DNA was then digested with NotI, followed by digestion with Tsp5091, which leaves termini complementary to those produced by EcoRI digestion. The DNA fragments were separated by 0.8% agarose electrophoresis. The region of the gel corresponding to 1.0-3.5 kb was cut out, 2.5 pg E. coli DNA was added as carrier, and the DNA was purified using Geneclean (Bio 101, Inc.) by eluting into 5 p1. Lambda Zap I1 (Stratagene) was used as a cloning vector, prepared by digesting 2.5 pg lambda DNA in 200 p1 total volume with NotI and heat inactivating the enzyme. The DNA was dephosphorylated with calf intestinal phosphatase, and the reaction was treated with phenoYchloroform and ethanol precipitated. The DNA was then digested with EcoRI, treated with phenoYchloroform, ethanol precipitated, and resuspended in 4 pl. One-half of the purified micronuclear telomeric DNA was ligated to one-half of the prepared lambda Zap I1 vector for 2 days at 14”C, and the DNA was packaged (these were Stratagene recommendations). The entire unamplified library was screened with a telomeric oligonucleotideprobe. Bluescript plasmids containing positive inserts were excised from the phage DNA following Stratagene’s protocol. Telomeres can be difficult to sequence due to potential secondary structure formation. The most unambiguous telomere sequencing results have been obtained by reading the C-strand using the ThermoSequenaseradiolabeled terminator cycle sequencing kit (Amersham Life Science). d. Northern Blotting Analysis of Telomerase RNAs The Northern blotting method provides a good representation of molecular weights of cellular RNA species and is specifically able to retain small molecular weight RNAs (e.g., the 150/9nucleotide T. thermophilu telomerase RNA) which some other RNA extraction methods fail to do. Fifty milliliter cultures of T. thermophilu transformant cell lines were grown in 250-ml flasks at 30°C with shaking (100 rpm) in selective 2% PPYS medium containing 1 X PSF antibiotic (Gibco, Bethesda, MD) and 100 pg/d paromomycin (with Sigma, “cell culture tested grade” used at a final concentration of 120 pg/d).The transformants cells were grown to midlog phase (approx. 2.5 X lo5cells/ml), harvested (2500 rpm IEC clinical centrifuge) in 50-ml Falcon tubes, washed once in sterile 10 mM Tris-HC1 (pH 7.5), and recentrifuged.
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After aspirating off the supernatant, pelleted cells were resuspended in TRI reagent (Molecular Research Center, Inc., Cincinnati, OH), 1 d 5 - 1 0 X lo6 cells. This reagent is based on an RNA isolation procedure described previously (Chomczynski and Sacchi, 1987; Chomczynski, 1993). A number of other RNA isolation protocols have been employed, but this is by far the fastest and most user friendly. The cells were homogenized in TRI reagent by pipetting up and down several times and then transferred to 1.5-ml Eppendorf tubes. After 5 min at room temperature, the homogenates were treated with 0.2-ml chlorofodml TRI reagent, and the tubes were covered with Parafilm to seal them and vortexed for 15 s; then they were left at room temperature for 215 min. They were centrifuged for 15 min at 12,000g, 4°C. The mixture separated into a clear upper aqueous phase containing the RNA, a whitish interphase and a lower red-colored phenol-chloroform phase. The clear supernatant containing the RNA was carefully removed and transferred to a fresh tube at 4"C, care being taken not to take up any of the interphase and/or phenolic phase which contain proteins and DNA, respectively. The RNA was precipitated by addition of 0.5 volume isopropanoYml of TRI reagent added initially. Samples were left at 4°C for 10 min and centrifuged (12,000 g, 4"C, 10 min). The supernatant was discarded, and the transluscent pellet was washed with 1ml ice-cold 75% ethanol and recentrifuged (as before). All the ethanol supernatant was removed (a brief spin allowed collection and removal of any remaining liquid). The pellet was air dried for 5-10 min (with care not to overdry, which sometimes led to decreased pellet solubility). The RNA was resuspended in water (nuclease-free water from Promega) or TE (10 mM TrisCl, 1 mM EDTA, pH 8.0) and stored at -80°C. Sometimes there were problems encountered in redissolving the RNA pellet; if so, pipetting the RNA solution through a pipette tip and also placing the solution at 55°C for 10-15 min was helpful. We generally employed the Tall Mighty Small SE280 (Hoefer, San Francisco, CA) mini-gel apparatus to size fractionate the total cellular RNA to detect small ciliate telomerase RNA species (approximately 160 nucleotides). Total cell RNAs were loaded onto denaturing 6% polyacrylamide, 7 M urea gels (19: 1 acrylamide:bis ratio; 0.75 mm thickness), and electrophoresed in 0.6 Tris-borate (TBE)buffer. The RNA was lyophilized to reduce the volume and subsequently resuspended in deionized formamide (containingxylene cyanol and bromophenol dyes). Samples were heated to 90°C for 2 min, and immediately placed on ice prior to loading. There were 10-20 pg RNA loaded per lane. Gels were prerun for 30 min prior to loading; samples were electrophoresed at 180-200 V for 1.5-2 h. Good separation of RNAs was achieved in the 100-350 nucleotide size range by running the xylene cyanol dye 2-3 cm from the bottom of the gel. The RNA was transferred onto HyBond N+ (Amersham) membrane by electroblotting using the Biorad mini-gel transfer apparatus, at 4°C in 0.6 TBE buffer for 60-70 min, at 65-70 V (approx, 300-500 mA). The RNA was subsequently
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UV cross-linked to the membrane using a Stratalinker (StrataGene, San Diego, CA). Detection of telomerase RNA species on the blots was achieved using genespecific probes generated by PCR methods. There were 107-108 cpm of "Prandom-primer labeled probes (Multiprime, Amersham) used per blot. Probe hybridization was performed using Church and Gilbert (1984) methodology; blots were washed in 50 mM phosphate buffer. With transformants expressing mutant telomerase RNA genes, detection of the mutant RNA was achieved by differential hybridization. For example, when a telomerase RNA gene from a related species of ciliate, Glaucoma chattoni, was expressed in T. thermophila cells, allele-specificdetection of the G. chattoni telomerase RNA (TERl) using a Glaucoma TERl gene probe (Bhattacharyya and Blackburn, 1997)was achieved. Detection of mutant telomerase RNA genes bearing single-base template changes by Northern blotting analysis have also been determined using differential hybridization schemeswith oligonucleotideprobes (McCormick-Graham and Romero, 1996).
4. Telomerase RNP Complexes a. Visualization of Telomerase RNP Complexes &y Northern Blotting
Northern blotting was used to analyze telomerase RNA-associated R" complexes found in Tetrahymena. Aliquots of S-100 preparations (-20 pl) were separated on native gels composed of 3.5% polyacrylamide (60: 1, acrylamide to bis-acrylamide), 0.45% agarose, 50 mM Tris-acetate (pH 8.0) in running buffer of 50 mM Tris-acetate (pH 8.0). Gels were run at 200 V at 4°C. The gels were then placed in 50% urea at room temperature for 30 min to dissociate the RNP complex. The RNA was transferred by electroblotting onto a nylon filter and then UV cross-linked to the nylon filter. Northern hybridization was performed as described earlier using the Church and Gilbert method (Church and Gilbert, 1984). &. Visualization of Telomerase RNP Complexes by Western Blotting
Telomerase RNP complexes were separated on native gels as already described for Northern bloting analysis and then transferred onto a nitrocellulose membrane by electroblotting. Western blot analysis was performed with an enhanced chemiluminescencedetection kit (Amersham) essentially as outlined in the manufacturer's instructions. E. In Wtro Studies
Studies of mutant telomerases under various in vitro conditions have been used to identify functional aspects of specific residues and regions of the telomerase RNA. In vitro observations of mutant telomerases allow one to study how alterations within the telomerase RNA directly affect enzyme function.
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1. Small-Scale Telomerase Preparations Using this small-scale procedure for partial purification of mutant telomerases has allowed the rapid isolation of several different mutant telomerase at the same time. Enough enzyme was recovered by this method for -200 reactions. A mutant clonal line containing the desired telomerase RNA mutation was cultured in selective paromomycin-containing medium. The cells were directly inoculated into a flask of 50 ml2% PPYS with 100 pg/ml of paromomycin. Cells were cultured at 30°C with shaking (100 rpm) for -20 h. Cells were pelleted by centrifugation and washed once with 10 mM Tris, pH 7.5. Again cells were pelleted by centrifugation, and the cell pellet was resuspended at -2 X 105cells/ ml in 10 mM Tris, pH 7.5. Cell suspensions were placed at 30°C and shaken at 100 rpm for 16-24 h. The starved cells were pelleted by centrifugation and resuspended on ice with 1 ml TMG (20 mM Tris-HC1, pH 8.0, 1 mM MgClB, 10%glycerol). The cell suspension was transferred to a 1.5-ml tube on ice, and the cells were lysed by adding 1/20 volume of 4% NP-40-TMG and vortexing the cell suspension at half of the maximal speed for 30 min (in a rack attached to the vortex vibrator). The cell lysate was spun at 100,OOO g for 1h. Supernatants (S-100) were either stored at -80°C or loaded directly on the following column. All chromatographic procedures were carried out at 4"C, and all buffers contained the protease inhibitor Pefabloc at 0.1 mM (Boehringer Mannheim, Indianapolis, IN). One milliliter of the Sl00 supernatant (from 50- to 100-ml culture) was loaded onto a DEAE-agarose column (Biorad, Hercules, CA) preequilibrated with TMG. The column was washed with 2 column volumes of TMG and TMG supplementedwith 0.2 Msodium acetate (NaOAc). The protein was eluted in 0.3 M NaOAc-TMG. After adjusting the eluded 0.5 M NaOAc, the sample was loaded onto a 0.3-ml Octyl-Sepharose column (Pharmacia,Uppsala, Sweden) preequilibrated in 0.5 M NaOAc-TMG, and the column was washed with TMG. The protein was eluted with 1% Triton-XlW-TMG,and to 0.1- to 0.2-ml fractions were collected. These fractions were assayed directly or stored at -70°C. For further purification, an additional heparin agarose column chromatography step was performed. For heparin-agarose (Biorad) chromatography, 1ml of Sl00 was loaded onto a 1-ml preequilibrated heparin-agarose column with TMG and washed with TMG. The protein was eluted with 0.2 M NaOAc-TMG. This eluted fraction was then loaded onto a 0.5- to 1-ml DEAE-agarose column in 0.2 M NaOAc-TMG. Elution of telomerase from DEAE and Octyl-Sepharosechromatography was as described earlier. 2. The Telomerase Activity of Mutant Telomerases in Ktro Mutant telomerase RNA genes were introduced into cells as described in Section 1II.B. Mutant telomerases were then isolated by the foregoing partial purification scheme. Assays of mutant activity were performed as described previously.
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One of the valuable aspects of this approach is that mutant telomerases can be studied under a variety of conditions.This allows the investigator to determine the exact effect of specific alterations of the telomerase RNA that might not be evident by the in vivo approaches described in Section 1II.C. For example, the 50G mutation showed no in vivo effect (ie., cells were wild-type-like and telomeric DNA was wild type in sequence and length). However, by studying the 50G mutant enzyme under in vitro conditions, it was found that this alteration of the telomerase RNA essentially eliminates second and subsequent rounds of repeat addition. More examples of situations that used this method are described in detail in Gilley et al. (1995). In addition, studying mutant telomerases is the most definitive way to determine whether reversion has taken place, by marking the template of the mutant enzyme as discussed in Section 1II.C. Acknowledgments Research from the laboratory of the authors described in this chapter was supported by National Institutes of Health grants GM26259 and GM32565 to E. H.B.
References Bhattacharyya,A., and Blackbum, E. H. (1994).Architecture of telomerase RNA. EMBOJ. l3,57215723. Bhattacharyya, A., and Blackburn, E. H. (1997). A functional telomerase RNA swap in vivo reveals the importance of nontemplate RNA domains. Proc. NatL Acad Sci. U.S.A. 94,2823-2827. Blackburn, E.H.(1995). Developmentally programmed healing of chromosomes. In “Telomeres” E. H. Blackburn and C. W. Greider. eds.), pp. 193-218. Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY. Cherry, J. M., and Blackbum, E. H. (1985).The internally located telomeric sequences in the germtine chromosomes of Tetrahymena are at the ends of transposon-like elements. Cell (Cambridge, Mass.) 43,747-758. Chornczynski,P. (1993). A single reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. BioTechniques 15,532-537. Chornczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by guanidinium thiocyanate-phenol-chloroformextraction. Anal. Biochem. 162,156-159. Church, G. M.,and Gilbert, W.(1984). Genomic sequencing. Proc. Natl. Acad. Sci. U.S.A. 81,19911995. Collins, K., Kobayashi, R., and Greider, C. W. (1995). Purification of Tetrahymena telomerase and cloning of genes encoding the two protein components of the enzyme. Cell (Cambridge, Mass.) 81,677-686. de Lange, T. (1994). Activation of telomerase in a human tumor. Proc. Natl. Acad Sci. U.S.A. 91,2882-2885. Erlich, H. A. (1989). “PCR Technology.” Stockton Press, New York. Gaertig, J., and Gorovsky, M. A. (1992). Efficient mass transformation of Tetruhymena themophifa by electroporation of conjugants. Proc. Natl. Acad Sci U.S.A. 89,9196-9200. Gall, J. G. (1990). Telomerase R N A Tymg up the loose ends. Nature (London) 344,108-109. Gilley, D., and Blackburn, E. H. (1996). Specific RNA residue interactions required for enzymatic functions of Tetrahymena telomerase. Mol. Cell. Biol. 16, 66-75. Gilley, D., and Blackburn, E. H. (1999). The telomerase RNA pseudoknot is critical for the stable assembly of a catalytically active ribonucleoprotein. Proc. Natl. Acad. Sci. U.S.A. 96 (in press).
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Blackburn et al. Gilley, D., Lee, M. S., and Blackburn, E. H. (1995). Altering specific telomerase RNA template residues affects active site function. Genes Dev. 9,2214-2226. Gottschling,D. E., and Zakian, V. A. (1986).Telomere proteins: Specific recognition and protection of the natural termini of Oxytricha macronuclear DNA. Cell (Cambridge, Mass.)47,195-205. Greider, C.W.,and Blackburn, E. H. (1989). A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nuture (London) 337,331-337. Kirk, K. E.,and Blackburn, E. H. (1995). An unusual sequence arrangement in the telomeres of the germ-line micronucleus in Tetrahymena thennophila. Genes Dev. 9,59-71. Kirk, K. E., Harmon, B. P., Reichardt, I. K., Sedat, J. W.,and Blackburn, E. H. (1997). Block in anaphase chromosome separation caused by a telomerase template mutation. Science 275,14781481. Lingner, J., and Cech, T. R. (19%). Purification of telomerase from Euplofesuediculutus: Requirement of a primer 3’ overhang. Proc. Natl. Acad. Sci U.S.A. 93,10712-10717. Lingner, J., Hendrick, L. L., and Cech, T. R. (1994).Telomerase RNAs of different ciliates have a common secondary structure and a permuted template. Genes Dev. 8,1984-1998. Lingner, J., Hughes, T. R., Shevchenko,A., Mann, M., Lundblad, V., and Cech, T. R. (1997).Reverse transcriptase motifs in the catalytic subunit of telomerase. Science 276,561-567. McCormick-Graham, M., and Romero, D. P. (1995).Ciliate telomerase RNA structural features. Nucleic Acids Res. 23, 1091-1097. McCormick-Graham, M., and Romero, D. P. (1996).A single telomerase RNA is sufficient for the synthesis of variable telomeric DNA repeats in ciliates of the genus Paramecium. Mol. Cell. Biol. 16 1871-1879. Romero, D. P., and Blackburn, E. H. (1991).A conserved secondary structure for telomerase RNA. Cell (Cambridge, Mass.)67,343-353. Romero, D. P., and Blackburn, E. H. (1995). Circular rDNA replicons persist in Tefruhymenu fhemophila transformants synthesizing GGGGTC telomeric repeats. J. Eukuryotic Microbiol. 42,32-43. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). “Molecular Cloning: A Laboratory Manual,” 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, NY. Yu, G. L., and Blackbum, E. H. (1989).Transformation of Tefruhymenuthemophila with a mutated circular ribosomal DNA plasmid vector. Proc. Nutl. Acad Sci. U.S.A. 86,8487-8491. Yu,G. L., and Blackburn, E. H. (1990).Amplification of tandemly repeated origin control sequences confers a replication advantage on rDNA replicons in Tefruhymenathemophila Mol. Cell. Biol. 10,2070-2080. Yu, G. L.. and Blackburn, E. H. (1991).Developmentally programmed healing of chromosomes by telomerase in Tetrahymena. Cell (Cambridge, Mass.)67,823-832. Yu, G. L., Bradley, J. D., Attardi, L. D., and Blackburn, E. H. (1990).In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature (London) 344,126-132. Zaug, A. J., and Cech, T. R. (1995). Analysis of the structure of Tetrahymena nuclear RNAs in vivo: Telomerase RNA, the self-splicingrRNA intron, and U2 snRNA. RNA 1,363-374.
Studying the Telomerase R N A in Tetrahymena E. Blackburn*, D. GilleJ, T. Ware*, A. Bhattacharyyat, K. Kirk$, and H. Wang* ' University of California, San Francisco Departments of Microbiology and Immunology, and Biochemistry and Biophysics San Francisco, California 94143 t University of Chicago Medical Center
Department of Radiation and Cellular Oncology Chicago, Illinois 60637 8 Lake Forest College Department of Biology Lake Forest, Illinois 60045
I. Introduction 11. General Strategy and Overview 111. Basic Methods A. Mutagenesis of Telomerase RNA B. Introducing Mutant Telomerase R N A Genes into Tetrahymena C. Transformation of the Telomerase R N A Gene D. In Vivo Studies E. In W h o Studies References
I. Introduction Telomeres are specialized structures at the ends of eukaryotic chromosomes that are required for chromosome stability and the complete replication of linear chromosomes. In ciliates de novo telomere formation is an essential step in a critical developmental stage-the generation of the new macronucleus (Blackburn, 1995). Telomeric DNA sequences are generally composed of short G-rich METHODS IN CELL BIOLOGY, VOL. 62 Coppght 0 1999 by Academic Press. AU righu of reproducoon in any form reserved 0091-h79X/W $30.00
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tandem repeats, running 5' to 3' toward the distal end of the chromosome. The telomeres found at the ends of Tetrahymena macronuclear chromosomes are composed of GGGGTT repeats (see also Chapter 19). Telomerase is a ribonucleoprotein (RNP) reverse transcriptase responsible for the synthesis of telomeric repeats. Telomerase is the only known reverse transcriptase that contains its own template, which is present in the telomerase RNA moiety as an integral part of the RNP complex (Greider and Blackburn, 1989). The telomerase RNA of Tetrahymena thermophila is a relatively small RNA (159 nucleotides) (Greider and Blackburn, 1989) with a very well-defined secondary structure. Within this telomerase, RNA is a region complementary to the G-rich telomeric strand, 3'-AACCCCAACJ', termed the templating domain (Greider and Blackburn, 1989). Specific residues of the templating domain are copied to produce the G-rich telomeric strand (Yu et al., 1990). The secondary structure of the telomerase RNA was determined by phylogenetic covariation based on sequence input from'over 25 ciliate telomerase RNAs (Romero and Blackburn, 1991; Lingner et al., 1994; McCormick-Graham and Romero, 1995, 1996) (Fig. 1). Additional support for this RNA structure came from both in vitro and in vivo chemical and enzymatic structural analyses (Bhattacharyya and Blackburn, 1994; Zaug and Cech, 1995). The small size of this RNA along with its well-defined secondary structure have made it very amenable for fundamental studies of telomerase and telomere function. Because changes in telomeres and telomerase have been linked to tumorigenesis and aging in humans, there has been an explosion of interest and research in this area (reviewed in de Lange, 1994). Tetrahymena and other ciliates have been invaluable model systems for the study of telomeres and telomerase, due in part to the relative abundance of telomeres and telomerase in these cells. Ciliates devote significant cellular resources to the maintenance and replication of telomeres due to the large number of macronuclear chromosomes and the relatively small size of each macronuclear chromosome. Consequently, each cell contains an unusually large number of chromosome ends. For example, each Tetrahymena macronucleus contains about 20,000-40,000 telomeres, and each Euplotes macronucleus contains about 80 million telomeres; therefore, there is an abundance of telomeric DNA, telomere proteins, and telomerase in these interesting and unusual unicellular organisms. Nearly all the basic knowledge
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Fig. 1 Secondary structure of Tetrahymena telomerase RNA (adapted from Gilley and Blackburn, 1996).
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that has been uncovered about telomeres and telomerase was first discovered using ciliates. The following is a brief listing of some of the major discoveries using ciliates for studying telomeres and telomerase: the first telomeric sequence, the discovery of telomerase, the discovery of the telomerase RNA moiety (reviewed in Gall, 1990),the first telomeric proteins (Gottschling and Zakian, 1986), and the isolation of the catalytic subunit of telomerase (Lingner et ul., 1997) and other telomerase-associated proteins (Collins et al., 1995; Lingner and Cech, 1996). In this chapter, we introduce to the reader how we have studied various functional aspects of the telomerase RNA from Tetruhymena thermophilu. This chapter covers broad descriptions of general research strategies along with more detailed methodologies. Some of the methods and general strategies discussed will be useful for studies with a broad range of organisms. In addition, we include information that will be specifically useful to those who use ciliates to study this fascinating RNA molecule.
11. General Strategy and Overview One general approach that has been extremely useful in the study of the telomerase RNA is to mutate specific nucleotides or regions of the telomerase RNA gene. Mutant telomerase RNA genes are then introduced back into cells on a high copy number vector to observe the effects of these alterations on the in vivo assembled mutant telomerases. This method was first used to demonstrate that specific residues within the template domain are copied and incorporated into telomeres (Yu et al., 1990; Yu and Blackburn, 1991). This experimental approach has proven valuable for studying telomerase RNA because the mutant telomerase is assembled into an RNP complex in the cell-unless the mutation prevents assembly-and the function (or lack of function) of the in vivo assembled mutant telomerase can be analyzed within the environment of the cell. In addition, the in vivo assembled mutant telomerase can be partially purified and studied under a variety of in vitro conditions. Although the endogenous telomerase RNA gene is still present in cells containing the newly introduced mutant telomerase RNA, the mutant telomerase RNA gene is present in the cell on a high copy number vector (-10,000 copies of the mutant gene versus -45 copies of the wild-type gene) (Yu and Blackburn, 1989). This system leads to specificproblems but also has advantages. For example, one potential problem due to the presence of the endogenous wild-type telomerase RNA gene is that cells transformed with a mutant gene can revert, thereby expressing the wildtype gene. (Reversion can be monitored in clonal lines by methods discussed in Section 1II.B.) Reversion to the wild-type telomerase RNA gene can arise in several ways. Either the mutant gene is lost from the cell (selected against), not expressed, or the RNA is unstable andor fails to be assembled into an active telomerase complex. In these cases, the endogenous wild-type RNA is reestab-
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lished, thereby becoming the dominant telomerase complex in these cells. On the other hand, the presence of the endogenous RNA can also be advantageous because it provides useful information about rate-limiting steps at which the mutant RNA must compete with endogenous RNA. Alterations in the telomerase RNA were observable at two basic levels in Tetrahymena: changes in cellular phenotypes and changes of the telomerase RNP and its enzymatic properties. Cellular phenotypes were easily detected in Tetrahymenadue to its large size and readily observable stages of nuclear division. For example, certain mutations within the RNA templating domain can cause synthesis of telomeric repeats that are “toxic” to the cell when added distally to the chromosomal telomeres. Studying the phenotypes of such telomerase mutants has suggested possible functions for telomeres in chromosome segregation (Yu et aL, 1990, Kirk et al., 1997). Mutations outside the RNA template domain can also be deleterious to the cell (e.g., by creating a nonfunctional telomerase RNA capable of competing for limiting telomerase proteins) (Yu et aL, 1990; Kirk et aL, 1997; Gilley et aL, 1995). Because Tetrahymena is a relatively abundant source of telomerase, the RNP complex and the enzymatic properties of telomerase can be studied in several ways. Certain mutations within the telomerase RNA affect telomerase RNP assembly. Others affect basic enzymatic processes of telomerase such as processivity and fidelity. Some enzymatic effects are due to alterations in base-pairing interactions with telomerase and its substrate. However, certain enzymatic alterations caused by specific mutations within the telomerase RNA are not explainable by altered base-pairing interactions and instead suggest that the RNA plays an important role in active site functions that affect telomerase/substrate interactions (Gilley el al., 1995; Yu and Blackburn, 1990; Romero and Blackburn, 1995; Gilley and Blackburn, 1996).
In. Basic Methods In this section we discuss details of methods found useful in making alterations within the telomerase RNA. We also discuss some of the general strategies employed to study this RNA and introduce some of the “tools” that have been useful in these studies.
A. Mutagenesis of Telomerase RNA To generate alterations within the telomerase RNA gene, we have generally relied on a polymerase chain reaction (PCR) based method (Erlich, 1989) except for the following modifications: Twenty cycles of PCR involved denaturation (30 s, 94”C), annealing (30 s, 5OoC), and extension (30 s, 72°C) using conditions supplied by the manufacturer of the DNA Thermal Cycler (Perkin Elmer
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Cetus). Potential mutant genes were then carefully sequenced to detect possible unwanted errors introduced by Taq polymerase. Mutant telomerase RNAs were inserted into the vector prD4-1 (Yu and Blackburn, 1989) at the 2 0 1 site within the vector polylinker. The vector prD4-1 confers paromomycin resistance to transformed Tetrahymena. The transformation efficiency of this vector into E. coli is low due to its large size (14.85 kb). Therefore, it was necessary to perform colony hybridization to detect colonies with the correct insert. B. Introducing Mutant Telomerase RNA Genes into Tetruhymena
For most purposes, the most reliable and useful method for introducing mutant telomerase RNA genes into Tetrahymena has been electroporation of synchronized cells early in development as described by Gaertig and Gorovsky (1992) (see also Chapter 26). Electroporation supplanted the microinjection of vegetatively dividing cells used in earlier analyses of telomerase RNA mutants (Yu et al., 1990;Yu and Blackburn, 1991;Romero and Blackburn, 1995). The electroporation method allows efficient use of the vector prD4-1, which is unstable over time. By electroporating at the start of macronuclear development, it was possible to observe transformants before vector alterations or mutant telomerase RNA gene loss takes place, thereby making it possible to study even mutants that are extremely “toxic” to the cell. Using this method, we have been able to identlfy unique classes of cellular phenotypes caused by telomerase RNA mutations (see Section III.C.l). Cells were electroporated using a GenePulser system (Biorad), generally with 15-25 pg plasmid DNA isolated by the use of Qiagen columns (Qiagen, Chatsworth, CA) in 0.4-cm electroporation-cuvettes (Biorad) under the following conditions: resistance, 200 R capacitance 25 pF;voltage 0.44 kV.Electroporated cells were immediately diluted into 20 ml 2% PPYS culture medium prior to plating them into sterile, 96-well microtiter dishes (200 pl/well) at empirically derived dilutions (1 :500 or 1:1000) in order to obtain potential single or multiple clonal transformant lines. Transformants were selected in the presence of 100 pg/ml paromomycin (available from Parke-Davis or Sigma and used at a final concentration of 120 pg/ml) and added optimally 12-15 h postelectroporation (50 pl/well of 500 pg/ml stock). Paromomycin-selected transformants appeared and were harvested routinely 3-4 days after drug addition. C. Transformation of the Telomerase RNA Gene
1. Clonal Lines The episomal vector prD4-1 is initially maintained in transformed cells at very high copy (>lO,OOO copies per cell), which allows overexpression of the introduced mutant gene. However, one potential problem with this vector is that
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it is not possible to generate a stable transformant line (Yu and Blackburn, 1990; Romero and Blackburn, 1995), due to the unstable nature of the vector and selection against deleterious mutant telomerase RNA genes. The more deleterious the mutant telomerase RNA gene, the fewer fissions before selection against the gene and its loss from the cell population. However, by using clonal lines it was possible to eliminate reversion for up to -50 fissions. A definitive way to monitor reversion was to isolate the telomerase from transformed cells and determine the proportion of mutant relative to wild-type telomerase (see Section III.D.2). In the case of a mutant telomerase RNA that is assembled into an active RNP complex, essentially only the mutant enzyme activity was evident in in v i m telomerase activity assays because the overexpressed mutant RNA is far more abundant than the wild-type RNA.
2. Mass Transformation Mass transformation was used to observe transformants at the earliest possible moment after transformation and to study early-lethal telomerase RNA mutants. This allows thousands of transformants to be pooled so that enough matenalDNA, RNA, and telomerase-can be isolated for study before reversion. For example, it was possible to study mutations such as the telomerase RNA mutation 44G, which is extremely toxic once introduced into cells (Yu et al., 1990; Gilley et al., 1995). Cells transformed with the 44G mutation can undergo only seven to nine fissions before cessation of cell division. By combining transformants, it was possible to isolate enough material to study this severe phenotype. Because many transformants were combined, we found it important to monitor reversion closely by assaying telomerase activity, cessation of cell division, and altered phenotype. Using a pooled transformant population, it was possible to monitor reversion of a telomerase RNA mutant that causes a micronuclear anaphase block phenotype (Kirk et al., 1997) as the population was passaged over time. The micronuclear phenotype was monitored by DAPI staining, and population reversion was indicated by a decrease in the ratio of anaphase micronuclei to interphase micronuclei. For example, in the 43AA mutant (Kirk et aZ., 1997), from 5 to 7 days after transformation, the ratio remained at 6: 10. By day 10, the ratio had decreased to 2:10, and by day 13 the cell population appeared virtually wild type with a ratio of roughly 1 : l O . The effect of a range of paromomycin concentrations (0, 10, 25, 75, 150 pg/ml) on reversion has been tested by placing the cell populations in the new drug concentration 5 days after electroporation. The rate of reversion was unaffected by as little as 10 pg/ml paromomycin and as great as 150 pg/ml paromomycin. However, if the cells were placed in medium lacking paromomycin, population reversion was evident at 7 days and virtually complete by 10 days.
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D. In vivo Studies
1. Phenotype Classification of Telomerase R N A Mutants We have identified four classes of cellular phenotypes caused by telomerase RNA mutations: early lethal, delayed lethal, “stress” response, and wild-typelike phenotype. The early lethal phenotype is characterized by cells capable of undergoing seven to nine fissions before cessation of cellular division. Therefore, each clonal transformant line produces only 200-500 cells before the terminal phenotype is expressed. Mutant cells are about ten times larger than normal wild-type transformed cells and are extremely flat, a phenotype originally termed the monster phenotype. An example of the terminal phenotype of an early lethal mutant, the 44G mutation, is shown in Fig. 2. Mutations that cause a delayed lethal phenotype are essentially wild type until -20 fissions after the mutant telomerase RNA gene is introduced. Then, between -20 to 25 fissions, the population fission rate progressively slows, cells gradually display a monster phenotype, and then cellular divison stops. Mutations have also been observed that display a phenotypic response similar to a stress response. For example, these include mutations that disrupt the pseudoknot structure within the RNA (Fig. 1) under log phase culture conditions. These pseudoknot disruption mutations affect assembly of the active RNP particle (Gilley and Blackburn, 1999). It is possible that these mutations compete for telomerase protein components with the endogenous wild-type telomerase RNA. Finally, certain telomerase RNA mutations display essentially no cellular changes. These mutations fall into two groups-those mutations that change the enzymatic properties of the mutant telomerase but do not detectably affect cells, and those mutations that incorporate mutant telomeric repeats that are tolerated by the cells. 2. Marking the Template An extremely useful tool available for studying the telomerase RNA has been to introduce the sequence changes of interest into a telomerase RNA gene that, in addition, has been “marked” by a specificbase change(s) within the templating residues. The base changes within the template are designed to produce correspondingly mutated telomeric repeats. By adding additional mutations outside the template, the marked gene can then be monitored for function. Such function can be monitored by incorporating the specific marked mutant repeat sequences into telomeres, and any phenotypes caused by the marked mutant repeats, and by analyzing and assaying activity of the mutant telomerase RNP complex in vitro. For example, the 43A mutant, which has a change from a C to an A at residue 43 (the 5’ residue of the template), has been useful for marking the RNA when a relatively “silent” or wild-type-like phenotype is desired (Gilley et al., 1995; Bhattacharyya and Blackburn, 1997). The 43A mutation has a variable penetrance-transformants display either a wild-type-like or a delayed lethal
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Fig. 2 Tetrahyrnena themophila cells, transformed by wild-type (A) or 44G mutant (B, C) telomerase RNA (TER) genes as described in this chapter. Cells were stained with 2,6-diaminido-4phenylindole (DAPI) and viewed by fluorescence light microscopy. See also color insert at back of book.
phenotype. By isolating clonal transformant lines, one can use this mutation to ascertain the effects of altering regions outside the template, hence determining the functionality of the second site mutation. In addition, template mutations are available that synthesize toxic telomeric repeats, causing a lethal phenotype in 100%of clonal lines. Marking the template with a lethal mutation allows one to test whether second site mutations are functional. If second site mutations outside the template allow telomerase function, then a lethal phenotype is dis-
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played. In contrast, if the second site mutation eliminates the function of the telomerase RNA, then the transformant line will be wild type (i.e., the toxic phenotype is suppressed) due to expression and function of the endogenous telomerase RNA (T. Ware and E. H. Blackburn, unpublished results).
3. Detection of Mutant Telomeres In studies of Tetrahymena telomerase RNA function (for example, (Yuet al., 1990;Yu and Blackburn, 1991;Romero and Blackburn, 1995;Bhattacharyya and Blackburn, 1997), mutant telomeric DNA was readily monitored by Southern blotting analysis or by cloning and sequencing telomeric DNA from cells transformed by a mutant telomerase RNA. a. Southern Blots for Mutant Telomeric Sequences
Total genomic DNA was digested with BamHl or PstI and electrophoresed overnight, retaining all DNA fragments above 0.5 kb (25 cm gel, 1%agarose, 1 X TBE). The DNA was depurinated in the gel (0.25 M HCl), transferred to Hybond+ (Amersham) in 0.4 M NaOH, and UV cross-linked. Hybridization and washes were performed essentially as described by Church and Gilbert (1984). [One liter of hybridization solution contains 0.5 liter of 1 M NaH2P04, pH 7.2 (per liter: 71 g Na2HPO4 or 134 g Na2HPO4. 7H20, plus 4 ml H3P04); 0.35 liter of 20% SDS; 2 ml of 0.5 M EDTA, pH 8.0. One liter of wash solution contains 0.2 liter 1M NaH2P04and 0.05 liter of 20% SDS.] Oligonucleotides were designed to hybridize differentially to mutant versus wild-type telomeric repeats, depending on the expected telomeric sequence. For example, the 44G template mutation creates GGGGTC telomeres. An oligonucleotide, 5’ GGGGTCGGGGTC 3’ was end-labeled using T4 polynucleotide kinase (Sambrook et al., 1989). Conditions for this probe were as follows: prehybridization, 5 min; hybridization, 1 h to overnight; washes (10 min each), once at room temperature, then twice at 32°C; followed by autoradiography. When available, a genomic DNA sample known to contain the expected sequence was loaded as a positive control. After detection of mutant repeats as described, the probe was removed from the blot with 0.4 M NaOH treatment. Removal was verified by overnight exposure to X-ray film.The blot was then available for detection of wild-type telomeric sequence using a similar procedure and an oligonucleotide probe, 5’ GGGGTTGGGGTT 3’. Detection of micronuclear-specific telomeres was achieved using the same procedure and a few variations (Kirk and Blackburn, 1995). First, we found it was important to use purified micronuclear DNA as the starting material. Second, restriction enzymes were used that digest the A+T-rich Tetrahymena DNA extremely frequently (MseI, Tsp5091, or DraI, New England Biolabs), thereby separating the short nontelomeric G4T2 tracts (Cherry and Blackburn, 1985) from the longer true telomeric DNA tracts (Kirk and Blackburn, 1995).
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b. Cloning Macronuclear Telomeresfrom Transformants The telomere cloning strategy described here was used to obtain an arbitrary sample of macronuclear telomeres (rather than mostly the more abundant rDNA telomeres). The resulting cloned telomeres contained a relatively short telomereassociated sequence, facilitating cloning and subsquent DNA sequence analysis. The procedure used standard techniques (Sambrook et al., 1989) and followed manufacturers’ suggestions for enzymatic reactions (New England Biolabs). The telomere cloning vector stock was prepared by digesting 5 p g pBluescipt (Stratagene) with ClaI and EcoRV, and the restriction enzymes were heat inactivated. The DNA was methylated by incubating with S-adenosylmethionineand TaqI methylase; then it was agarose gel purified using Geneclean (Bio 101, Inc.). The methylation was verified by testing resistance of an aliquot to TaqI digestion. Genomic DNA was isolated from transformants. The DNA ends were made flush by treating 1.0 pg genomic DNA with T4 DNA polymerase in the presence of all four dNTPs in a total volume of 5 p1 at 14°C for 15 min, and the polymerase was heat inactivated. The entire 1.0-pg transformant genomic DNA was ligated to 0.4-pg prepared vector stock with T4 DNA ligase in 10 pl total volume at room temperature overnight, and the ligase was heat inactivated. The DNA was then digested with TaqI in 100 ml total volume, treated with phenolkhloroform, and ethanol precipitated. The DNA was circularized by T4 DNA ligase in 100pl total volume at room temperature for 2 h, treated with phenolkhloroform,and ethanol precipitated to concentrate to 10 pl. The library was made by using 1 pl DNA to transform E. coli XLI-blue MRF’by electroporation. Colonies were screened initially with a wild-type telomeric oligonucleotide probe, and positive clones were subsequently cross-screened with the mutated telomeric sequence. rDNA telomeric clones were identified by an rDNA-specific probe. The cloned telomeric DNA could be sequenced directly by using vector sequence primers. Therefore, if the telomere-associated sequence was too large to read through, the telomeric tract was subcloned by digesting with the very frequent cutter Tsp5091, which leaves termini complementaryto those produced by EcoRI digestion. Another procedure for cloning transformant telomeres has been described (Bhattacharyya and Blackburn, 1997). By using this method, it was possible to inadvertently repurify the original Tetrahymena transformation vector ( ~ r D 4 - 1 ) ~ and it was therefore found useful to cross-screen the library with a probe specific for this vector, such as a nontemplate region of the telomerase RNA gene. ’
c. Cloning Wild-Type Micronuclear Telomeres
Micronuclear telomeres were cloned as follows (Kirk and Blackburn, 1995) using standard techniques (Sambrook et al., 1989) and following manufacturers’ suggestions for enzymatic reactions (New England Biolabs). To prepare the micronuclear DNA for cloning, contaminating macronuclear telomeres were elimiqated by digesting 40-pg purified micronuclear DNA (the
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micronuclear DNA isolation procedure is described in Chapter 8) with 2 units Ba131 in a total volume of 200 p1 at 30°C for 1 min, and the reaction stopped by adding 1/10 volume 0.2 M EGTA. The extent of telomere shortening was roughly 500 bp, as verified by Southern blot analysis. The DNA ends were made flush by treatment with T4 DNA polymerase in the presence of all four dNTPs in a total volume of 300 pl at 14°C for 15 min, and the reaction was treated with phenoYchloroform and ethanol precipitated. In a total volume of 50 p1, 8 pg NotI linkers (New England Biolabs) were ligated to the micronuclear DNA ends with T4 DNA ligase at 14°C overnight, and the ligase was heat inactivated. The DNA was then digested with NotI, followed by digestion with Tsp5091, which leaves termini complementary to those produced by EcoRI digestion. The DNA fragments were separated by 0.8% agarose electrophoresis. The region of the gel corresponding to 1.0-3.5 kb was cut out, 2.5 pg E. coli DNA was added as carrier, and the DNA was purified using Geneclean (Bio 101, Inc.) by eluting into 5 p1. Lambda Zap I1 (Stratagene) was used as a cloning vector, prepared by digesting 2.5 pg lambda DNA in 200 p1 total volume with NotI and heat inactivating the enzyme. The DNA was dephosphorylated with calf intestinal phosphatase, and the reaction was treated with phenoYchloroform and ethanol precipitated. The DNA was then digested with EcoRI, treated with phenoYchloroform, ethanol precipitated, and resuspended in 4 pl. One-half of the purified micronuclear telomeric DNA was ligated to one-half of the prepared lambda Zap I1 vector for 2 days at 14”C, and the DNA was packaged (these were Stratagene recommendations). The entire unamplified library was screened with a telomeric oligonucleotideprobe. Bluescript plasmids containing positive inserts were excised from the phage DNA following Stratagene’s protocol. Telomeres can be difficult to sequence due to potential secondary structure formation. The most unambiguous telomere sequencing results have been obtained by reading the C-strand using the ThermoSequenaseradiolabeled terminator cycle sequencing kit (Amersham Life Science). d. Northern Blotting Analysis of Telomerase RNAs The Northern blotting method provides a good representation of molecular weights of cellular RNA species and is specifically able to retain small molecular weight RNAs (e.g., the 150/9nucleotide T. thermophilu telomerase RNA) which some other RNA extraction methods fail to do. Fifty milliliter cultures of T. thermophilu transformant cell lines were grown in 250-ml flasks at 30°C with shaking (100 rpm) in selective 2% PPYS medium containing 1 X PSF antibiotic (Gibco, Bethesda, MD) and 100 pg/d paromomycin (with Sigma, “cell culture tested grade” used at a final concentration of 120 pg/d).The transformants cells were grown to midlog phase (approx. 2.5 X lo5cells/ml), harvested (2500 rpm IEC clinical centrifuge) in 50-ml Falcon tubes, washed once in sterile 10 mM Tris-HC1 (pH 7.5), and recentrifuged.
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After aspirating off the supernatant, pelleted cells were resuspended in TRI reagent (Molecular Research Center, Inc., Cincinnati, OH), 1 d 5 - 1 0 X lo6 cells. This reagent is based on an RNA isolation procedure described previously (Chomczynski and Sacchi, 1987; Chomczynski, 1993). A number of other RNA isolation protocols have been employed, but this is by far the fastest and most user friendly. The cells were homogenized in TRI reagent by pipetting up and down several times and then transferred to 1.5-ml Eppendorf tubes. After 5 min at room temperature, the homogenates were treated with 0.2-ml chlorofodml TRI reagent, and the tubes were covered with Parafilm to seal them and vortexed for 15 s; then they were left at room temperature for 215 min. They were centrifuged for 15 min at 12,000g, 4°C. The mixture separated into a clear upper aqueous phase containing the RNA, a whitish interphase and a lower red-colored phenol-chloroform phase. The clear supernatant containing the RNA was carefully removed and transferred to a fresh tube at 4"C, care being taken not to take up any of the interphase and/or phenolic phase which contain proteins and DNA, respectively. The RNA was precipitated by addition of 0.5 volume isopropanoYml of TRI reagent added initially. Samples were left at 4°C for 10 min and centrifuged (12,000 g, 4"C, 10 min). The supernatant was discarded, and the transluscent pellet was washed with 1ml ice-cold 75% ethanol and recentrifuged (as before). All the ethanol supernatant was removed (a brief spin allowed collection and removal of any remaining liquid). The pellet was air dried for 5-10 min (with care not to overdry, which sometimes led to decreased pellet solubility). The RNA was resuspended in water (nuclease-free water from Promega) or TE (10 mM TrisCl, 1 mM EDTA, pH 8.0) and stored at -80°C. Sometimes there were problems encountered in redissolving the RNA pellet; if so, pipetting the RNA solution through a pipette tip and also placing the solution at 55°C for 10-15 min was helpful. We generally employed the Tall Mighty Small SE280 (Hoefer, San Francisco, CA) mini-gel apparatus to size fractionate the total cellular RNA to detect small ciliate telomerase RNA species (approximately 160 nucleotides). Total cell RNAs were loaded onto denaturing 6% polyacrylamide, 7 M urea gels (19: 1 acrylamide:bis ratio; 0.75 mm thickness), and electrophoresed in 0.6 Tris-borate (TBE)buffer. The RNA was lyophilized to reduce the volume and subsequently resuspended in deionized formamide (containingxylene cyanol and bromophenol dyes). Samples were heated to 90°C for 2 min, and immediately placed on ice prior to loading. There were 10-20 pg RNA loaded per lane. Gels were prerun for 30 min prior to loading; samples were electrophoresed at 180-200 V for 1.5-2 h. Good separation of RNAs was achieved in the 100-350 nucleotide size range by running the xylene cyanol dye 2-3 cm from the bottom of the gel. The RNA was transferred onto HyBond N+ (Amersham) membrane by electroblotting using the Biorad mini-gel transfer apparatus, at 4°C in 0.6 TBE buffer for 60-70 min, at 65-70 V (approx, 300-500 mA). The RNA was subsequently
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UV cross-linked to the membrane using a Stratalinker (StrataGene, San Diego, CA). Detection of telomerase RNA species on the blots was achieved using genespecific probes generated by PCR methods. There were 107-108 cpm of "Prandom-primer labeled probes (Multiprime, Amersham) used per blot. Probe hybridization was performed using Church and Gilbert (1984) methodology; blots were washed in 50 mM phosphate buffer. With transformants expressing mutant telomerase RNA genes, detection of the mutant RNA was achieved by differential hybridization. For example, when a telomerase RNA gene from a related species of ciliate, Glaucoma chattoni, was expressed in T. thermophila cells, allele-specificdetection of the G. chattoni telomerase RNA (TERl) using a Glaucoma TERl gene probe (Bhattacharyya and Blackburn, 1997)was achieved. Detection of mutant telomerase RNA genes bearing single-base template changes by Northern blotting analysis have also been determined using differential hybridization schemeswith oligonucleotideprobes (McCormick-Graham and Romero, 1996).
4. Telomerase RNP Complexes a. Visualization of Telomerase RNP Complexes &y Northern Blotting
Northern blotting was used to analyze telomerase RNA-associated R" complexes found in Tetrahymena. Aliquots of S-100 preparations (-20 pl) were separated on native gels composed of 3.5% polyacrylamide (60: 1, acrylamide to bis-acrylamide), 0.45% agarose, 50 mM Tris-acetate (pH 8.0) in running buffer of 50 mM Tris-acetate (pH 8.0). Gels were run at 200 V at 4°C. The gels were then placed in 50% urea at room temperature for 30 min to dissociate the RNP complex. The RNA was transferred by electroblotting onto a nylon filter and then UV cross-linked to the nylon filter. Northern hybridization was performed as described earlier using the Church and Gilbert method (Church and Gilbert, 1984). &. Visualization of Telomerase RNP Complexes by Western Blotting
Telomerase RNP complexes were separated on native gels as already described for Northern bloting analysis and then transferred onto a nitrocellulose membrane by electroblotting. Western blot analysis was performed with an enhanced chemiluminescencedetection kit (Amersham) essentially as outlined in the manufacturer's instructions. E. In Wtro Studies
Studies of mutant telomerases under various in vitro conditions have been used to identify functional aspects of specific residues and regions of the telomerase RNA. In vitro observations of mutant telomerases allow one to study how alterations within the telomerase RNA directly affect enzyme function.
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1. Small-Scale Telomerase Preparations Using this small-scale procedure for partial purification of mutant telomerases has allowed the rapid isolation of several different mutant telomerase at the same time. Enough enzyme was recovered by this method for -200 reactions. A mutant clonal line containing the desired telomerase RNA mutation was cultured in selective paromomycin-containing medium. The cells were directly inoculated into a flask of 50 ml2% PPYS with 100 pg/ml of paromomycin. Cells were cultured at 30°C with shaking (100 rpm) for -20 h. Cells were pelleted by centrifugation and washed once with 10 mM Tris, pH 7.5. Again cells were pelleted by centrifugation, and the cell pellet was resuspended at -2 X 105cells/ ml in 10 mM Tris, pH 7.5. Cell suspensions were placed at 30°C and shaken at 100 rpm for 16-24 h. The starved cells were pelleted by centrifugation and resuspended on ice with 1 ml TMG (20 mM Tris-HC1, pH 8.0, 1 mM MgClB, 10%glycerol). The cell suspension was transferred to a 1.5-ml tube on ice, and the cells were lysed by adding 1/20 volume of 4% NP-40-TMG and vortexing the cell suspension at half of the maximal speed for 30 min (in a rack attached to the vortex vibrator). The cell lysate was spun at 100,OOO g for 1h. Supernatants (S-100) were either stored at -80°C or loaded directly on the following column. All chromatographic procedures were carried out at 4"C, and all buffers contained the protease inhibitor Pefabloc at 0.1 mM (Boehringer Mannheim, Indianapolis, IN). One milliliter of the Sl00 supernatant (from 50- to 100-ml culture) was loaded onto a DEAE-agarose column (Biorad, Hercules, CA) preequilibrated with TMG. The column was washed with 2 column volumes of TMG and TMG supplementedwith 0.2 Msodium acetate (NaOAc). The protein was eluted in 0.3 M NaOAc-TMG. After adjusting the eluded 0.5 M NaOAc, the sample was loaded onto a 0.3-ml Octyl-Sepharose column (Pharmacia,Uppsala, Sweden) preequilibrated in 0.5 M NaOAc-TMG, and the column was washed with TMG. The protein was eluted with 1% Triton-XlW-TMG,and to 0.1- to 0.2-ml fractions were collected. These fractions were assayed directly or stored at -70°C. For further purification, an additional heparin agarose column chromatography step was performed. For heparin-agarose (Biorad) chromatography, 1ml of Sl00 was loaded onto a 1-ml preequilibrated heparin-agarose column with TMG and washed with TMG. The protein was eluted with 0.2 M NaOAc-TMG. This eluted fraction was then loaded onto a 0.5- to 1-ml DEAE-agarose column in 0.2 M NaOAc-TMG. Elution of telomerase from DEAE and Octyl-Sepharosechromatography was as described earlier. 2. The Telomerase Activity of Mutant Telomerases in Ktro Mutant telomerase RNA genes were introduced into cells as described in Section 1II.B. Mutant telomerases were then isolated by the foregoing partial purification scheme. Assays of mutant activity were performed as described previously.
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One of the valuable aspects of this approach is that mutant telomerases can be studied under a variety of conditions.This allows the investigator to determine the exact effect of specific alterations of the telomerase RNA that might not be evident by the in vivo approaches described in Section 1II.C. For example, the 50G mutation showed no in vivo effect (ie., cells were wild-type-like and telomeric DNA was wild type in sequence and length). However, by studying the 50G mutant enzyme under in vitro conditions, it was found that this alteration of the telomerase RNA essentially eliminates second and subsequent rounds of repeat addition. More examples of situations that used this method are described in detail in Gilley et al. (1995). In addition, studying mutant telomerases is the most definitive way to determine whether reversion has taken place, by marking the template of the mutant enzyme as discussed in Section 1II.C. Acknowledgments Research from the laboratory of the authors described in this chapter was supported by National Institutes of Health grants GM26259 and GM32565 to E. H.B.
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