Nucleic Acids Research

Strains, transformants and culture methods

Tetrahymena strains were grown at 30°C in 2% PPYS (2% proteose peptone, 0.2% yeast extract, 10 µM FeCl₃), supplemented with 250 µg/ml penicillin, 250 µg/ml streptomycin and 25 µg/ml amphotericin B (24). Tetrahymena pyriformis strain 30039 was obtained from the American Type Culture Collection. For DNA transformation studies, matings between T.thermophila strains CU427 and CU428 were subjected to electroporation and transformants were selected for resistance to the antibiotic paromomycin (Pm) as previously described (25). Plasmid prD4-1/Tp3[prime] was constructed by inserting a 950 bp T.pyriformis 5[prime] NTS fragment (see below) into the 3[prime] NTS polylinker region of NotI+XhoI-digested prD4-1 (26). Plasmid prD1/Tp5[prime] is a derivative of prD1 (26) in which the 5[prime] NTS of the T.thermophila rDNA was replaced with a 950 bp 5[prime] NTS fragment from the T.pyriformis 5[prime] NTS. prD1/[delta]d2 is a prD1 derivative in which domain 2 of the T.thermophila 5[prime] NTS (nt 935-1370) was deleted (27).

DNA isolation and two-dimensional gel electrophoresis

Vegetative replication intermediates were examined in DNA prepared from log phase cultures following restriction enzyme digestion and enrichment for replication intermediates by benzoylated naphthoylated DEAE-cellulose (BND cellulose; Sigma) chromatography as previously described (19,28). Neutral/neutral (29) and neutral/alkaline (30) two-dimensional (2D) gel electrophoresis were performed on 2-20 µg input DNA and hybridized to rDNA-specific probes (19). Short autoradiography exposures were used to accurately measure the position of stalled nascent strands in neutral/alkaline gels. Nascent strand sizes were estimated by linear regression of radiolabeled 123 bp and 1 kb DNA ladder markers (Gibco BRL), with errors calculated based on a measurement accuracy of ±1 mm. Cloned HindIII fragments from the T.thermophila rRNA coding region were used as probes to study replication of the T.pyriformis coding region. Probes for the T.pyriformis 5[prime] and 3[prime] NTS were generated by PCR with the following primers: 5[prime] NTS fragment, 5[prime]-GATGTTATGATAGAGATAAAATGA (forward) and 5[prime]-CCGGAGATGTTTCCCCTTCTTAAATA (reverse) (31); 400 bp 3[prime] NTS fragment, 5[prime]-AATCGTAATTCCAAATTATC (forward) and 5[prime]-TGGAAATGAATGGATCACCC (reverse) (32).

Pulsed field gel electrophoresis

Undigested DNA from T.thermophila transformants was electrophoresed on a BioRad DRIII pulsed field gel electrophoresis unit for 14 h at 6 V/cm in 45 mM Tris-borate, pH 8.3, 1 mM EDTA buffer (initial switch time 1 s, final switch time 6 s). DNA was transferred to Hybond N⁺ and probed with either the 1.9 kb 5[prime] NTS fragment or pBR322 vector sequences.

S100 extracts and electrophoretic mobility shift assays

S100 extracts were prepared from vegetative T.pyriformis and T.thermophila cells harvested at a density of 2 × 10⁵ cells/ml as previously described (18). Cell lysates were centrifuged at50 000 r.p.m. for 1 h at 4°C in a Beckman TLA 100.3 rotor and supernatants were dialyzed against 20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl, 100 mM EDTA, 0.2 mM PMSF and 0.5 mM DTT for 4 h prior to storage at -70°C. Protein concentrations were measured by the Bradford method (33).

Subsaturating amounts of type I element binding activities (typically 3.5 µg S100 extract) were incubated with 0.1 pmol [[gamma]-³²P]ATP-labeled oligonucleotide for 15 min on ice in 17 mM HEPES, pH 7.9, 1 mM EDTA, 200 mM NaCl, 8.7% glycerol (v/v), 1 mM DTT and 0.1 mM PMSF containing 1 µg poly(dI·dC) as a non-specific competitor. For competition assays, unlabeled oligonucleotides were added to the binding reaction. Affinity-purified T.thermophila ssA-TIBF protein (kindly provided by Drena Dobbs) was also examined. Samples were electrophoresed in 8% polyacrylamide gels containing 50 mM Tris-glycine, pH 8.5, with 12% glycerol at 16 V/cm for 2 h at room temperature, dried and exposed to Kodak XAR-5 film or phosphorimaged on a Molecular Dynamics PhosphorImager. A33 oligos contain the entire A-rich strand of type I elements, whereas A37 oligos lack the seven 5[prime]-most nucleotides of the respective type I element, but contain 11 bases immediately downstream. The non-specific oligo (5[prime]-AACAGTACTAGCGTGTTGCG) and an oligo complementary to the T.pyriformis type IB element (5[prime]-GTCCGGACGGTCTACTATATTTTTTTTTTTTTTTTGCC) were also examined. Oligonucleotides were 5[prime]-end-labeled with [[gamma]-³²P]ATP using T4 polynucleotide kinase or used as cold competitor DNAs.

Localization of replication origins by neutral/neutral 2D gel mapping

Informative comparative studies of eukaryotic chromosomal replication origins have been limited due to the lack of sequence conservation and organization of origins in traditionally studied organisms. The rDNA minichromosomes of two closely related ciliates, T.thermophila and T.pyriformis, provide a unique opportunity to address this subject due to the high degree of conservation of sequence elements within the 5[prime] NTS. Phylogenetically conserved type I, II and III elements are present and organized in a similar manner in the respective 5[prime] NTS regions, imbedded in sequences that have otherwise diverged significantly (Fig. 1; 20). EM (21,22) and 2D gel studies (T.thermophila only; 23) previously demonstrated that replication initiates in the 5[prime] NTS in both species. To better understand the relationship of these conserved sequences to replication initiation and the regulation of replication fork movement, we performed structural (2D gel) and functional studies of the T.pyriformis rDNA molecule.

Neutral/neutral 2D gel electrophoresis (29) was first used to localize origins in DNA isolated from vegetatively growing T.pyriformis cultures. The migration pattern of replication intermediates is diagnostic for initiation within or outside a DNA fragment (Fig. 2A). Five restriction fragments spanning the entire rDNA minichromosome were initially examined for origin activity (Fig. 2B, fragments A-E). The 3.2 kb fragment A spans both copies of the 1.1 kb 5[prime] NTS, present at the center of the rDNA palindrome, and an additional ~500 bp downstream of the rRNA promoter. A discontinuous bubble-to-Y arc pattern was detected in this fragment, indicating that replication initiates upstream of the HindIII site (Fig. 2C, fragment A). No complete, simple Y arcs were observed in this or longer exposures, suggesting that initiation does not occur in the downstream coding region or 3[prime] NTS. As bubble-to-Y arcs patterns are diagnostic for origins positioned asymmetrically within a fragment, these data might suggest that the initiation site is asymmetrically positioned in the 5[prime] NTS. However, abundant stalled replication intermediates were detected on both the bubble and Y arc for this palindromic fragment. This indicates that the diverging forks are not moving at equal, uniform rates away from the origin. The previously assigned map position for the origin was inferred based on the assumption that forks move away from the initiation site at constant, equivalent rates (21). Whereas fork stalling invalidates this assumption, the initiation site was still localized by mapping the pausing sites and using them as landmarks (see below). Replication bubbles are `trapped' in the accumulating bubble arc replication intermediates of this palindromic fragment, indicating that initiation must occur upstream of the site(s) that induces pausing.

A    B    C

Figure 2. Neutral/neutral 2D analysis of the T.pyriformis rDNA minichromosome. (A) Schematic of replication intermediates resolved by neutral/neutral 2D gel electrophoresis (29). Simple Y, passive replication by a single fork entering from one end of a restriction fragment. Bubble, bidirectional replication from an origin positioned at the center of a fragment. Bubble-to-Y, bidirectional replication from an asymmetrically positioned origin. Pause, transient arrest of a replication fork at a specific site in a fragment (filled spot). Barrier, replication of a fragment by converging forks, where one fork enters and terminates at a barrier prior to entry of the second fork. Double Y, passive replication by two simultaneously converging replication forks. Diagonal dashed line, migration of linear duplex DNA fragments. Dotted arc, reference pattern for simple Y arc intermediates. (B) Schematic of examined restriction fragments. Palindromic fragments A and F (3.2 kb HindIII and 2.1 kb MspI fragments) span both 5[prime] NTS copies. Non-palindromic fragments B and G (3.2 kb KpnI-PstI and 1.4 kb KpnI-HindIII fragments) contain one 5[prime] NTS copy and adjacent coding sequence. Overlapping fragments C-E (2.4 kb PstI-BamHI, 1.9 kb BglII and 2.1 kb HindIII fragments, respectively) span the coding region and 3[prime] NTS. B, BamHI; Bg, BglII; H, HindIII; K, KpnI; M, MspI; P, PstI restriction sites. (C) Southern blot analysis of vegetative rDNA replication intermediates. Probe for fragments A, F and G, 950 bp T.pyriformis 5[prime] NTS fragment. Probes for fragments B-D, cloned HindIII fragments from the T.thermophila rRNA coding region. Probe for fragment E, 400 bp T.pyriformis 3[prime] NTS fragment. Arrows point to accumulated replication intermediates.

Only simple Y arc intermediates were detected in the non-palindromic, 3.2 kb fragment B, which contains most of the 5[prime] NTS and 1.6 kb of the rRNA coding region (Fig. 2C). A single accumulated intermediate was detected in low molecular weight replication intermediates, consistent with forks stalling at one site in the 5[prime] NTS portion of this fragment. Bubble arc intermediates should be detected if initiation occurs in the centrally positioned promoter region. Their absence suggests that the initiation site is not promoter-proximal, but is much further upstream. Only simple Y arcs were observed in the remainder of the coding region and 3[prime] NTS (Fig. 2B and C, fragments C-E). Examination of a smaller 5[prime] NTS fragment (Fig. 2B, fragment F) verified our conclusion that initiation occurs upstream of the rRNA promoter. MspI cuts just upstream of the rRNA promoter, generating a 2.1 kb palindromic fragment. Bubble-to-Y arc intermediates were detected in this fragment, indicating that initiation occurs at an upstream site (Fig. 2C, fragment F). Finally, only simple Y arcs were detected in the 1.4 kb, 5[prime] NTS fragment G. A single prominent paused intermediate was observed, indicating that the fork arrest site is upstream of the HindIII site. The absence of bubble arc intermediates in fragment G is inconclusive due to the small fragment size of this fragment. From these data, we conclude that vegetative rDNA replication initiates in the 5[prime] NTS only in T.pyriformis. Replication forks pause transiently at one 5[prime] NTS site and initiation occurs upstream of the pausing site.

Mapping the 5[prime] NTS pause site by neutral/alkaline 2D gel electrophoresis

Neutral/alkaline 2D gel electrophoresis was used to map the replication fork arrest site in T.pyriformis rDNA. Replication intermediates were resolved in the first dimension under non-denaturing conditions and nascent DNA strands were released from parental strands prior to electrophoresis in the second dimension. If the fork arrests at a specific site, an accumulated intermediate will be detected on the diagonal arc of nascent strand intermediates (Fig. 3A). One accumulated nascent strand intermediate with a calculated size of 900 nt (±50 nt) was detected in nascent strand replication intermediates derived for the 1.3 kb KpnI+HaeIII 5[prime] NTS fragment (Fig. 3B and C). A nascent strand intermediate of the same size was detected for the 1.6 kb KpnI-HindIII fragment, indicating that pausing occurs in forks moving from the center of the palindrome towards the promoter. The 1.0 kb Sau3A fragment generated an 800 nt (±50 nt) stalled intermediate, corroborating the position of the arrest site. The pausing site maps to a nucleosome-free region upstream of the rRNA transcription initiation site, ~50 bp upstream of the T.pyriformis type IB element. As neutral/neutral 2D gels revealed that replication bubbles are trapped by pausing sites in the rDNA palindrome, we conclude that initiation occurs upstream of pause site 1 (Fig. 1, p1), within the 900 bp segment at the 5[prime]-most end of the T.pyriformis 5[prime] NTS. Similar to T.thermophila, replication initiates well upstream of promoter-proximal type I elements.

A    B    C

Figure 3. Neutral/alkaline 2D gel analysis of the T.pyriformis 5[prime] NTS. (A) Schematic of nascent strand replication intermediates resolved by neutral/alkaline 2D gel electrophoresis. The 1N spot corresponds to non-replicating DNA. The vertical smear is derived from nicked, non-replicating DNA, whereas the horizontal smear represents parental strands in replicating intermediates of different sizes. The diagonal arc contains nascent strand replication intermediates liberated from the parental strand by alkali denaturation prior to electrophoresis in the second dimension. (B) Schematic of restriction fragments analyzed on neutral/alkaline 2D gels. P, rRNA promoter. Restriction sites: Ha, HaeIII; H, HindIII; K, KpnI; S, Sau3A. Other symbols are described in Figure 1. DNA from log phase vegetative cultures was digested with Sau3A, KpnI+HaeII or KpnI+HindIII, generating 1.0, 1.3 and 1.6 kb fragments, respectively. (C) Southern blot analysis of neutral/alkaline 2D gels probed with a radiolabeled 950 bp 5[prime] NTS fragment. Markers, radiolabeled 1 kb and 123 bp ladders (Gibco-BRL).

Conserved sequence elements at replication fork pausing sites

A conserved sequence was previously identified at the three fork arrest sites in T.thermophila rDNA (Fig. 1, p1, p2 and p3; 19). This sequence, termed the PSE, consists of three blocks of homology, 7, 12 and 7 bp in length, separated by 18 and 8 bp spacers (Fig. 4), located 50-100 bp upstream of type IA-C elements. Only one fork pausing site was detected in T.pyriformis rDNA, ~50 bp upstream of the tandemly arrayed type IB and IC elements. Examination of the DNA sequence revealed significant similarity to the T.thermophila PSE element at this position (Fig. 4), but not in the remainder of the T.pyriformis 5[prime] NTS. Sequence identity is highest in the first and third blocks of this tripartite element (block 1, 6/7 bp match; block 3, 7/7 bp match; asterisks mark sites of sequence identity) and is less conserved in block 2, with a perfect match at 6/10 positions that constitute the T.thermophila consensus. The spacer length separating blocks 1 and 2 is absolutely conserved (18 bp), however, blocks 2 and 3 are separated by 18 bp in T.pyriformis and 8 bp in T.thermophila, the difference corresponding to one turn of the helix. No additional similarity was detected in the flanking sequences.

Figure 4. 5[prime] NTS pause site sequence alignments. Boxed regions correspond to the three conserved sequence blocks present at replication fork pausing sites in the T.pyriformis and T.thermophila rDNA minichromosomes (Fig. 1). The length of the spacer regions separating the conserved blocks is indicated. The orientation of the tripartite PSEs at the T.pyriformis pause site 1 and T.thermophila pause site 3 are opposite to those found at pause sites 1 and 2 in T.thermophila.

A single-stranded DNA binding activity that specifically recognizes the conserved type I element in T.pyriformis rDNA

Mutations in or immediately downstream of type I elements cause defects in replication initiation (13,14; reviewed in 10) and arrest of replication forks at upstream pausing sites in T.thermophila rDNA (19). A potential trans-acting factor regulating these processes was previously identified (17). This protein recognizes the A-rich strand of the type I element. ssA-TIBF (single-stranded A strand, type I element binding factor) binds the B rDNA allele with reduced affinity relative to C3 rDNA (18) and the B rDNA allele is impaired for replication when placed in competition with C3 rDNA (13). ssA-TIBF binds DNA in an orientation-dependent manner (17). The type IA-D elements are arranged in the same orientation in the 5[prime] NTS and pausing only occurs when forks move through type I elements in one direction (19).

Using gel mobility shift assays, we looked for a DNA binding activity in T.pyriformis with properties similar to T.thermophila ssA-TIBF. Binding was assayed on oligonucleotide substrates corresponding to the A-rich strand of type I elements (Fig. 5A). A33 oligos contain just the respective type I element sequence, whereas A37 oligos are truncated at their 5[prime]-end, but carry an 11 nt 3[prime] extension. Previous studies showed that the T tract at the 5[prime]-end of the T.thermophila type IB element is not required for binding (17). Several complexes were detected when T.pyriformis and T.thermophila extracts were incubated with the T.pyriformis type IB oligonucleotide T.p. IB-A37 (Fig. 5B). These binding activities will hereafter be referred to as Tp-TIBF and Tt-TIBF, respectively. The T.pyriformis complexes migrate at a similar position to those formed with affinity-purified Tt-TIBF. Competition studies revealed that Tt-TIBF binds to T.p. IB-A37 and T.t. IB-A37 with comparable affinity (data not shown) and that Tp-TIBF DNA binding was enhanced ~30-fold by addition of 11 nt downstream of the type IB element (Fig. 5C, T.p. IB-A33 versus T.p. IB-A37). The addition of downstream sequences increases binding of Tt-TIBF to DNA ~80-fold, in comparison (18). Sequences downstream of the T.pyriformis type IC element, but not the type IA element, increase the binding affinity for Tp-TIBF (Fig. 5C). Neither a random oligonucleotide nor the T-rich strand of the type I element efficiently competed for Tp-TIBF binding to type I element substrates (data not shown). Since all of these properties are indistinguishable between the T.pyriformis and T.thermophila single-stranded type I element binding proteins, it seemed plausible that these binding activities might function in vivo across species boundaries.

A    B    C

Figure 5. Identification and characterization of a type I element binding activity in T.pyriformis cell extracts. (A) Oligonucleotides used in gel shift experiments. T.t. and T.p. designate T.thermophila and T.pyriformis oligonucleotides, respectively. T.t. IB-A33 contains the entire A-rich strand of the T.thermophila type IB element. T.t. IB-A37 has 11 additional downstream bases, but lacks seven 5[prime] T residues previously shown to be dispensable for binding of Tt-TIBF (17). T.p. IB-A33 corresponds to the type IB element of T.pyriformis. T.p. IA/IB/IC-A37 correspond to 5[prime] truncated T.pyriformis type IA-C elements with 11 added downstream bases. (B) In vitro gel shift analysis of type I element oligonucleotides with T.pyriformis and T.thermophila S100 extracts and purified Tt-TIBF. DNA-protein complexes formed with T.p. IB-A37 are shown.-/+, reactions performed with 0.1 pmol radiolabeled DNA substrate in the absence or presence of 1 µg dI·dC. (C) DNA binding competition assays. Binding of T.pyriformis S100 proteins to radiolabeled T.p. IB-A37 oligonucleotide was challenged with increasing concentrations of unlabeled T.p. IB-A37, T.p. IB-A33, T.p. IA-A37 and T.p. IC-A37 oligonucleotides (concentration gradient 0-, 0.1-, 0.3-, 1-, 3-, 10-, 30-, 100-, 300- and 1000-fold unlabeled oligonucleotide substrate).

Functional analysis of the T.pyriformis origin region in T.thermophila cells

In addition to type I elements, conserved type II, type III and promoter-proximal elements are present in the 5[prime] NTS of T.thermophila and T.pyriformis rDNA (Fig. 1). Type I and III elements reside in nucleosome-free regions in both species (34) and the spatial organization of conserved elements is very similar. Type II elements help maintain the rDNA copy number by an unknown mechanism (12) and type III elements are in vivo binding sites for DNA topoisomerase I (35), although a role in rDNA replication has yet to be ascribed. Conserved promoter-proximal sequences are capable of directing in vitro transcription of rRNA genes with heterologous extracts (36,37), suggesting that cross species recognition could occur in vivo.

Two plasmid constructs were generated to test whether the T.pyriformis 5[prime] NTS was competent to initiate replication in T.thermophila cells. In the first construct, prD4-1/Tp3[prime], the T.pyriformis 5[prime] NTS was introduced into the 3[prime] NTS of plasmid prD4-1 (Fig. 6A). DNA from several stable transformants was examined by neutral/neutral 2D gel electrophoresis. Only simple Y arc intermediates were detected in a restriction fragment spanning the T.pyriformis 5[prime] NTS (data not shown), indicating that the T.pyriformis origin was not active in prD4-1/Tp3[prime] transformants. Since the T.pyriformis origin was competing in cis with the T.thermophila 5[prime] NTS in this plasmid, a second construct was examined. In plasmid prD1/Tp5[prime], the T.thermophila 5[prime] NTS was replaced with the T.pyriformis 5[prime] NTS. Whereas the parental plasmid prD1 generated >1800 transformants in seven control transformation experiments, no prD1/Tp5[prime] transformants were obtained. Thus, the T.pyriformis 5[prime] NTS also failed to compete in trans with endogenous T.thermophila rDNA.

Functional analysis of a T.pyriformis-like derivative of the T.thermophila 5[prime] NTS

The T.thermophila 5[prime] NTS contains a tandem 430 bp duplication, encompassing domains 1 and 2, each of which contains one type I and three type III elements. Both domains function as initiation sites for DNA replication (23), suggesting that the tandem domain duplication might provide a selective advantage over the single domain present in T.pyriformis rDNA. By analogy, reiteration of core ARS elements confers an advantage to artificial minichromosomes in S.cerevisiae (38). To test whether a single domain is sufficient for DNA replication in T.thermophila, a prD1 derivative, prD1/[delta]d2, was constructed in which domain 2 was deleted from the T.thermophila 5[prime] NTS (Fig. 6A). In contrast to prD1/Tp5[prime], Pm-resistant transformants were obtained with prD1/[delta]d2. The transformation frequency was ~100-fold lower than the parental prD1 plasmid, suggesting that replication efficiency was diminished.

Pulsed field gel analysis revealed that the entire plasmid sequence had integrated into the endogenous rDNA chromosome, generating a ladder of rDNA molecules containing up to 10 integrated plasmid copies (Fig. 6B). Restriction analysis indicated that all of the mutant 5[prime] NTS copies and adjoining coding and vector sequences were downstream of the endogenous rDNA copy (Fig. 6C, schematic; data not shown). Consequently, mutant origin activity could be assessed by examining the direction of fork movement in expanded rDNA chromosomes. If only the wild-type 5[prime] NTS origins were active in co-integrants, then plasmid vector sequences should only be replicated from the center of the palindrome towards the telomere. Fork movement in the opposite direction would indicate that a downstream mutant origin had fired. Digestion with PvuII+XbaI generates a 4.8 kb fragment containing the terminal 1.5 kb of the 3[prime] NTS, prD1 vector backbone (pBR322) and first 700 bp of the 5[prime] NTS of the integrated plasmid copies. A 500 bp 5[prime] NTS probe was used to examine fork movement in DNAs resolved by neutral/alkaline gel electrophoresis. Hybridization to intermediates along the entire nascent strand arc was observed (Fig. 6D), indicating that the [delta]d2 5[prime] NTS is competent for replication initiation. Hence, the inability of the T.pyriformis 5[prime] NTS plasmid to transform T.thermophila is not due to the absence of a domain 1/domain 2 duplication.

A    B    C    D

Figure 6. Analysis of T.thermophila cells transformed with plasmid prD1/[delta]d2. (A) rDNA derivative plasmids examined by DNA transformation of T.thermophila cells. prD4-1/Tp3[prime], prD4-1 derivative containing two tandem copies of the T.thermophila 5[prime] NTS, one copy of the coding region and 3[prime] NTS and the T.pyriformis 5[prime] NTS fragment inserted into a polylinker cloning site. prD1, T.thermophila rDNA plasmid containing one entire copy of the T.thermophila 5[prime] NTS, coding region and 3[prime] NTS. prD1/Tp5[prime], substitution of the T.pyriformis 5[prime] NTS for the T.thermophila 5[prime] NTS in the plasmid prD1. prD1/[delta]d2, deletion of the 430 bp segment spanning domain 2 in plasmid prD1. Diagrams are not drawn to scale. All of these plasmids are derived from macronuclear rDNA genes and lack chromosome breakage sequences required for processing and palindrome formation. (B) Pulsed field gel analysis of DNA from stable prD1/[delta]d2 transformants propagated for >100 generations in Pm. Uncut DNA from wild-type and prD1/[delta]d2 transformant cell lines was electrophoresed in a BioRad pulsed field gel apparatus and hybridized to a T.thermophila 5[prime] NTS probe. Migration of the input plasmid, prD1/[delta]d2, and endogenous rDNA are also shown. (C) Structure of endogenous rDNA minichromosomes following integration of prD1/[delta]d2 plasmid DNA into endogenous rDNA. The 5[prime] NTS probe will only hybridize to high molecular weight nascent strands in the 4.8 kb PvuII-XbaI fragment if just the wild-type origins are active. (D) Neutral/alkaline 2D gel analysis of DNA from a prD1/[delta]d2 transformant. DNA was digested with PvuII and XbaI and subjected to neutral/alkaline 2D gel electrophoresis. Following transfer to Hybond N⁺ (Amersham), the sample was probed with fragment derived from the first 500 bp of the 5[prime] NTS.

DISCUSSION

The naturally occurring rDNA minichromosomes of tetrahymenid species serve as useful models for examining the structure and evolution of chromosomal origins of replication. Their small size and abundance has facilitated studies of replication origins and genetic determinants for replication control. The sequence and physical organization of conserved type I, II and III elements is remarkably similar in T.thermophila and T.pyriformis rDNA. A genetic approach previously identified cis-acting mutations affecting rDNA replication in T.thermophila. A single, repetitive cis-acting determinant, the type I element, was identified in numerous screens (13-15,39). The dispersed organization of type I elements resembles that of replication determinants in higher eukaryotes more than S.cerevisiae (2,6-9), although the complexity of determinants appears to be lower. Experiments presented here suggest a higher degree of complexity than previously realized.

To examine the role of conserved sequence elements in replication control, we first performed 2D gel studies of the T.pyriformis rDNA minichromosome. Our results demonstrate a high degree of similarity in the replication properties of the T.pyriformis and T.thermophila rDNAs. They refine the basic conclusion of a previous T.pyriformis EM study, that replication initiation only occurs in the 5[prime] NTS (21), and demonstrate that regulated fork movement occurs in this region. Like T.thermophila (22,23), the T.pyriformis initiation site is not promoter-proximal, despite the presence of type I elements at the respective promoters. Thus, type I elements alone are not sufficient to specify the site of initiation (23; this work). Although the T.pyriformis initiation site could not be mapped as precisely as in T.thermophila, the outer limits were readily defined by the 2D approach. The T.pyriformis origin resides in the first 900 bp of the 5[prime] NTS. In the previous T.thermophila study, we showed that replication initiates asymmetrically at two different sites in the 5[prime] NTS, the first residing in the 430 bp segment between pause sites 1 and 2 (which includes domain 1) and the second one being less well demarcated, but clearly proximal of domain 2 (23). Whether the T.pyriformis origin co-localizes to the type I/type III repeat arrays, as in T.thermophila, or further upstream is unclear. As the nearest informative restriction site is >200 bp downstream of the pausing site that we identified, the pausing site proved to be the most useful marker for localizing the T.pyriformis rDNA origin. Experiments with non-palindromic restriction fragments suggest that the T.pyriformis origin is positioned very close to the center of the rDNA palindrome. If so, it would reside in a segment that is stably associated with nucleosomes (34). Whether chromatin organization plays an important role in specifying Tetrahymena replication origins, as proposed for higher eukaryotes (reviewed in 40), remains to be determined.

Regulated fork movement is another shared feature of the rDNA of these related species. Similarly to T.thermophila, transient pausing of replication forks was observed in the T.pyriformis 5[prime] NTS and nowhere else in the rDNA. Previous studies in T.thermophila demonstrated that type I elements are genetic determinants for fork arrest (19). Fork pausing is directional, arresting 50-100 bp upstream of type I elements. The T.pyriformis fork arrest site mapped here is also upstream of a type I element. Strong homology to the tripartite T.thermophila PSE sequence element was observed at this pausing site. Interestingly, no such homology was detected upstream of the T.pyriformis type IA element, which shows no evidence for pausing. As pausing only occurs at PSE-associated type I elements, DNA-protein interactions at these two sites might somehow coordinate fork arrest. Indeed, we recently detected in vitro binding to the PSE element with both T.thermophila and T.pyriformis extracts and identified a putative trans-acting factor that crosslinks specifically to the PSE element (J.Rincon and G.M.Kapler, unpublished results). Although fork arrest sites have been identified in the intergenic regions of the tandemly arrayed rRNA genes of several species (28,41-43) and at other sites in the yeast genome (44,45), no obvious conserved sequence elements have been identified at arrest sites, including fork barrier sites in several related yeast species (46). Fork arrest in the human rDNA has recently been shown to be mediated by the transcription terminator protein TTF-1 (47). TTF-1 binds to a DNA sequence in the intergenic region of the tandemly arrayed rRNA genes. In addition to mediating transcription termination, TTF-1 arrests replication forks that are moving in the opposite direction to that of transcription. The trans-acting factors that mediate fork arrest in Tetrahymena rDNA must be different, since replication forks arrest upstream of the transcription start site rather than downstream of the transcription unit.

As type I elements are the only known determinant for rDNA replication control in T.thermophila, we sought to determine if they could function across species boundaries. A single protein has been identified which binds to type I elements with high specificity (18). The biochemical properties of this protein, ssA-TIBF, make it a plausible candidate for a regulator of both initiation and elongation of replication forks (18,19). Our results demonstrate the presence of a type I element binding activity in T.pyriformis with binding properties indistinguishable from purified T.thermophila ssA-TIBF (Tt-TIBF; 18). Importantly, high affinity DNA binding was observed across species boundaries for both the T.thermophila and T.pyriformis proteins. Thus, conservation between these species extends beyond the cis-acting DNA sequences to include trans-acting factors that bind this important regulatory element. Consequently, T.pyriformis type I elements might function when introduced into T.thermophila cells. Despite the appreciable degree of conservation, which extends to type II, type III and promoter elements, the T.pyriformis 5[prime] NTS failed to support replication in T.thermophila cells. A T.thermophila 5[prime] NTS derivative deleted for domain 2 was still replication competent, indicating that the imperfect tandem domain 1/domain 2 reiteration is not required. This result confirms previous 2D gel mapping studies showing that both domains function as initiation sites and indicates that domain 2 is not absolutely required as a genetic determinant for initiation. By analogy, the B rDNA allele (42 bp domain 2 deletion) has a reduced replication efficiency when placed in competition with C3 rDNA, but this mutation does not differentially alter usage of the domain 1 and domain 2 origins present in B rDNA (23). This is consistent with experiments performed with deletion derivatives of a rearrangement vector that undergoes excision and palindrome formation (12).

ACKNOWLEDGEMENTS

We thank Dorothy Shippen and Swati Saha for critical reading of this manuscript and comments and Drena Dobbs for providing purified T.thermophila ssA-TIBF protein and for discussions. This work was support by NIH grant GM56572.

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*To whom correspondence should be addressed. Tel: +1 409 847 8690; Fax: +1 409 847 9481; Email: gkapler@tamu.edu

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