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A Micronuclear Locus Containing Three Protein-Coding Genes Remains Linked During Macronuclear Development in the Spirotrichous Ciliate Holosticha

Posted on: Thursday, 26 August 2004, 06:00 CDT

We have discovered a three-gene macronuclear chromosome in a spirotrichous ciliate of the genus Holosticha. From 5' to 3', this chromosome contains genes encoding a member of the small G-protein family, an NAD kinase domain-containing protein, and the large subunit of DNA polymerase [alpha]. These three genes are separated by 16 and 38 nucleotides, respectively, and are oriented in the same direction in both the macronuclear and the micronuclear genomes. Probes made to these genes all hybridize to a single, strong band of size 7.0 kbp on a Southern Blot of Holosticha sp. macronuclear DNA, corresponding to the size of the three-gene macronuclear chromosome. Mapping the 5' and 3' ends of each of these genes using RACE showed that the transcripts of these genes exist as discrete mRNAs that are capped and polyadenylated. No nucleotides appeared to be added at the 5' ends of these transcripts, indicating that these transcripts are not generated by alternative or trans-splicing, but rather that each gene is transcribed from its own distinct promoter. Analysis of these linked genes may help define the evolutionary pressures leading to the extensive chromosome fragmentation seen in spirotrichous ciliates.

Introduction

Following conjugation, the sexual cycle of ciliated protozoa, a copy of the zygotic nucleus develops into a transcriptionally active macronucleus. Portions of the zygotic genome are excised and destroyed during this process, while the remaining sequences are joined together or capped with telomeres, then finally amplified to produce the new macronuclear genome (reviewed in Jahn and Klobutcher 2002). The extent of genome fragmentation performed at this stage and the proportion of fragments later capped with telomeres vary widely among different ciliate species (reviewed in Coyne et al. 1996). In concert, high levels of fragmentation and telomere addition create macronuclear genomes comprised of many small linear chromosomes. These levels appear to have reached an extreme in Class Spirotrichea, where most macronuclear chromosomes in these species consist of a single gene (a transcribed region encoding a single polypeptide, plus short, non-coding flanking regions) bounded by telomere sequences (Hoffman et al. 1995).

Few full-length spirotrichous macronuclear chromosomes have been sequenced to date. Currently fewer than 100 are listed in Genbank, from a wide variety of species. Only a small percentage of these chromosomes appear to contain multiple genes. When we began the study described in this paper, only two multi-gene macronuclear chromosomes, both derived from the "81-MAC" locus of Oxytricha fallax, had been confirmed in spirotrichous ciliates ( seegmiller et al. 1997). Since then a study to estimate the percentage of macronuclear chromosomes containing genes in Sterkiella nova (formerly Oxytricha nova) has provided an estimate of the number of multi-gene chromosomes in the macronuclear genome of this species (Prescott et al. 2002). Of 31 completely sequenced chromosomes, 10- 20% contained multiple open reading frames with significant and distinct BLAST hits. Whether this number holds for spirotrich species in general is not known, but it seems reasonable given that the average macronuclear chromosome size is similar in the spirotrichs studied to date (Klobutcher and Herrick 1997; Prescott 1994).

Given the paucity of multi-gene macronuclear chromosomes known in spirotrichs, we were surprised to find what appeared to be a three- gene macronuclear chromosome in a species of Holosticha, a freshwater spirotrich evolutionary distinct from the more commonly studied Euplotids and Oxytrichids. During a survey of spirotrich DNA polymerase alpha (DNA pol-[alpha]) sequences, we discovered that this gene was located on a surprisingly long molecule that appeared to contain two additional genes. Here we demonstrate the presence of this multi-gene macronuclear chromosome in Holosticha sp. and discuss how this finding adds to our knowledge of chromosome fragmentation and transcription in spirotrichs.

Figure 1. Graphical representation of the three-gene macronuclear chromosome. The open box bounded by telomeres represents the DNA molecule drawn to scale. Locations and directions of the three mRNA transcripts produced from this chromosome are shown by arrowed lines, with their names or functional associations shown on top. Grey boxes represent the six introns found in this sequence; the short black lines intersecting the chromosome show the positions of IESs in the corresponding micronuclear DNA, with IES lengths (in bp) shown in parentheses. The three horizontal lines (A-C) represent long micronuclear PCRs to confirm the continuity in the micronuclear sequence. Unlike many other spirotrich DNA pol-[alpha] genes, MDSs in this gene are not scrambled. The phylogeny of DNA pol-[alpha] scrambling will be addressed in a separate publication.

Results

Partial macronuclear DNA polymerase [alpha] (DNA pol-[alpha]) gene sequences from Holosticha sp. were determined by PCR using degenerate primers ( see Methods). The 3' and 5' ends of this gene were amplified using a poly-A anchored PCR approach and Telomere Suppression PCR (Curtis and Landweber 1999), respectively (see Methods). The total length of this macronuclear chromosome is 7,033 bp (Fig. 1). Upstream of the DNA pol-[alpha] gene we found a surprisingly long (2.4 kbp) sequence that contained multiple open reading frames. BLAST results for these putative open reading frames predicted two genes in the 2.4 kbp region. We used 5' and 3' RACE techniques to determine which regions of this 7.0 kbp macronuclear chromosome are transcriptionally active and to determine the location of mRNA cap and polyadenylation sites. We found a total of three distinct, non-overlapping mRNAs, all of which are encoded on the same strand of the macronuclear chromosome. The 5'-most transcript is 862 bp in length and appears to encode a member of the small G protein family. The second transcript is 1,275 bp and encodes a putative protein containing an NAD kinase domain. The 3'- most transcript is 4,521 bp and encodes DNA pol[alpha] (Fig. 1). These data suggested the existence of a three-gene macronuclear chromosome in this species of Holosticha.

Figure 2. Size distribution of Holosticha sp. DNA. Lane L: 1 kb DNA ladder (Invitrogen), Lane M: 2.5 g Macronuclear-enriched DNA. The sequences of the strong bands seen in Lane M are most likely highly amplified macronuclear chromosomes. (EtBr staining).

Figure 3. Sequences surrounding the mRNA cap addition sites. The capped nucleotides are the centered and enlarged adenosine residues. The putative translation start ATG codons are boxed. The polyadenylation sites we observed for the upstream genes are underlined and bold-typed. Dotted hepta-nucleotides represent a motif present near the mRNA cap sites of all three genes.

Macronuclear chromosomes in Holosticha sp. range in size from 0.5 kbp to approximately 8.0 kbp (Fig. 2). This size distribution is similar to that in three other spirotrichous ciliates: Euplotes aediculatus, Stylonychia pustulata and S. nova (Prescott 1994). Our analysis shows that the chromosomes that comprise the Holosticha sp. macronuclear genome are not abnormally long compared to those of other spirotrichous ciliates. In addition, other macronuclear chromosomes we have sequenced from this species (GenBank nos. AY463800-AY463803) contain single genes. Furthermore, phylogenetic analyses using both actin (Croft et al. 2003) and rDNA (Hewitt et al. 2003) sequences show that a member of the genus Holosticha (Holosticha polystylata) and a sister taxon, Urostyla grandis, are positioned between the Oxytrichids and the Euplotids, spirotrich clades in which the majority of macronuclear chromosomes isolated contain single genes.

Seven other macronuclear DNA pol-[alpha] orthologs have been fully sequenced in a broad range of spirotrichs, including Euplotes aediculatus (Landweber et al., unpublished), Urostyla grandis, Uroleptus sp., Paraurostyla weissei (Chang et al., unpublished), and the Oxytrichids S. nova (formerly Oxythcha nova; Mansour et al. 1994), Sterkiella histriomuscorum (formerly Oxytricha trifallax) (Hoffman and Prescott 1996), and Stylonychia lemnae (Landweber et al. 2000). None of the genes isolated from these other spirotrichs was found with another gene on a macronuclear chromosome. Since Holosticha diverged between these seven lineages, we were therefore surprised to discover the DNA pol-[alpha] gene on a three-gene macronuclear chromosome in Holosticha sp.

The three genes of this macronuclear chromosome are tightly packed. The mRNA cap site of the DNA pol-[alpha] gene is 38 bp downstream of the polyadenylation site of the NAD kinase gene, and the distance between the NAD kinase gene and the small GTPase gene is only 16 bp (Fig. 3). The mRNA cap sites of all three genes are found on adenosine residues, which are preceded by a thymine base and followed by a TA dinucleotide. The first ATG codon is found 33 bp, 15 bp and 28 bp downstream from the capped nucleotide of the small G protein gene, the NAD kinase gene and the DNA pol-[alpha] gene, respectively (Fig. 3). The six introns in these genes are flanked with the GT...AG dinucleotides typically found at the boundaries of eu\karyotic class II (spliceosomal) introns. These introns are small (below 50 bp in length), which is consistent with the sizes of introns observed in other ciliates (Dessen et al. 2001; Prescott and DuBois 1996).

Alternative mRNA splicing can generate multiple mRNA isoforms from a single pre-mRNA transcript in eukaryotes and some viruses (Ladd and Cooper 2002). The tight spacing and matching orientation of the three genes initially suggested to us that Holosticha might use this approach to generate the distinct transcripts seen for each gene. However, mRNA assembled by this mechanism would carry some sequences from exons located upstream, and the mRNA and genomic sequences would be expected to diverge at a splice acceptor (AG) dinucleotide. In Holosticha, all three mRNAs are identical to their individual DNA template sequences, save the intron sequences. This observation indicates that all three genes are transcribed separately, presumably through three distinct promoters belonging to each individual gene. We did not find conserved promoter motifs in the regions around the mRNA capping sites of these three genes (Fig. 3). However, promoter regions of Holosticha genes have not been described to date, and no conserved promoter elements have been found common among all spirotrichs.

Figure 4. Panel A. Southern blot of Holosticha sp. macronuclear DNA. Lane L (stained separately): 1 kb DNA ladder (Invitrogen). Lanes 1, 4, and 7: 10 g macronuclear-enriched DNA. Lanes 2, 5, and 8: Plasmid pH[alpha] 13F7000R digested with NotI and SamHI. Lanes 3, 6, and 9: Plasmid pH[alpha]1 13F7000R digested with HindIII. Blot was probed with the DNA pol-[alpha] probe (lanes 1, 2, and 3); stripped and probed with the NAD kinase probe (lanes 4, 5, and 6); stripped and probed with the small GTPase probe (lanes 7, 8, and 9). The band in lane 5 is faint due to an autoradiography problem. Panel B. The plasmid pH[alpha]1 13F7000R contained an insert generated by F3CR amplification of the macronuclear chromosome between positions 113 and 7000. The three genie regions of this insert are shown in different grey scales, with the positions of the three probes shown below each region (hatched lines). The plasmid was enzymatically digested with HindIII (H) or a combination of NotI (N) and BamHI (B) at the sites shown (short, vertical lines) to generate fragments containing portions of different genes. Sizes of the fragments predicted to bind the probes are indicated between the appropriate restriction sites.

Macronuclear molecules containing multiple genes have been found in another spirotrichous ciliate, O. fallax (Williams and Herrick 1991). In this case, one gene, which is a member of the family of mitochondrial solute carrier genes (CR-MSC), is found on macronuclear chromosomes of different sizes produced by alternative chromosome fragmentation during macronuclear development. Consequently, mRNA for this gene may be transcribed from multiple types of macronuclear chromosomes. In order to determine whether Holosticha possesses macronuclear chromosomes containing only one or two of the three genes described here, we probed macronuclear DNA from Holosticha for each of these three genes on a Southern blot (Fig. 4). Each of these three probes detected a single, strong band of size 7.0 kbp that corresponds to the size of the three-gene macronuclear chromosome. Moreover, we detected only one specific amplicon while using a telomere based PCR protocol (Curtis and Landweber 1999) to obtain the 5' end of this three-gene macronuclear chromosome (data not shown). From these results we conclude that the macronuclear copies of these three genes are present together on identical (or nearly identical) 7 kbp molecules. Accordingly, transcripts of the three genes must derive directly from the three- gene macronuclear chromosome.

The signal responsible for chromosomal breakages in Holosticha has not been examined to date. We searched the sequence of the three gene chromosome for the presence of a pentanucleotide, TTGAA, which has been previously identified as the core sequence of the chromosomal breakage signal (Cbs) in two other spirotrichs, Euplotes crassus and S. lemnae (Jonsson et al. 2001; Klobutcher et al. 1998). We did not find a copy of this sequence until 284 bp downstream of the 5' telomere, or 627 bp upstream of the 3' telomere. A copy of the complementary pentamer TTCAA is present 56 bp downstream from the 5' telomere, or 270 bp upstream of the 3' telomere. In total, there are 13 copies of TTGAA and 25 copies of its reverse complement sequence scattered throughout the chromosome, showing that these sequences are not sufficient to specify chromosome breakage in Holosticha. Experiments specifically addressing the Cbs for Holosticha will be necessary to determine if these or any other specific DNA motifs are necessary for chromosome fragmentation in this species.

We characterized the organization of these three genes in the micronucleus and found these three genes to be arranged in the same order on a single micronuclear locus. There are a total of 13 internal eliminated sequences (IESs) separating the coding sequences of the three genes into a total of 14 macronuclear destined sequences (MDSs) (Fig. 1). Three IESs were found in the first gene, two in the next, and eight in the DNA pol-[alpha] gene. None of these IESs is situated in the intergenic region between the stop codon of one gene and the start codon of the next, and only one IES (IES 7) is contained within an intron (Fig. 1). The exceptional length of IES 1 is noteworthy (2,471 bp). Surprisingly, we did not detect any transposable element-like features, such as long inverted repeats, flanking this long IES (Fig. 1). Furthermore, putative translations of this sequence showed no significant similarity to known proteins in Genbank and the A/T content (76%) is typical of spirotrich IESs (Prescott 1999). To our knowledge this is the longest spirotrich IES discovered that does not display these hallmarks of transposable elements (Doak et al. 1997).

The 862 bp mRNA transcript is capable of encoding a protein of 261 aa that shares similarity to the mammalian Rab23 proteins. Rab23 belongs to the small G protein superfamily, which also includes the members of the Ras, Rho, Sar1/Arf and Ran families. Small G proteins bind and hydrolyze GTP to GDP to regulate gene expression, cytoskeleton reorganization, vesicle transport and nuclear transport (Paduch et al. 2001). Human Rab23, to which the putative Holosticha protein shares the greatest similarity (37% sequence identity and 62% similarity to human Rab23), is presumed to be involved in regulation of vesicle transport (Eggenschwiler et al. 2001; Takai et al. 2001). The 1,275 bp mRNA transcript is capable of encoding a protein of 409 aa containing an NAD kinase domain (pfam01513). Although functions and characteristics of this kinase family are not well known (Kawai et al. 2001), the two highly conserved regions of this family, XXX-XGGDG-XL and DGXXX-TPTGSTAY (where X represents a hydrophobic amino acid), are 100% conserved in the putative Holosticha protein. The 4,521 bp mRNA transcript is capable of encoding a protein of 1,614 aa, which contains domains belonging to DNA polymerase family B (pfam00136). This gene shows a high degree of sequence similarity to DNA pol-[alpha] in many other spirotrichous ciliates. The closest match to this protein in Genbank is to DNA pol-[alpha] of S. lemnae (Genbank: AF194338), which shares 63% sequence identity. To our knowledge no direct functional connection between Rab, NAD kinase or DNA pol-[alpha] has been proposed.

Discussion

Few multi-gene macronuclear chromosomes have been described in spirotrichs. The most intensively studied to date derive from the 81- MAC locus of Sterkiella (Oxytricha) spp. (Seegmiller et al. 1997). There, three genes located adjacent to one another in the zygotic genome become alternatively fragmented during development. The relative abundance of the resulting one- or two-gene macronuclear chromosomes remains constant throughout the population during vegetative growth (Herrick et al. 1987). Macronuclear chromosomes containing all three genes are not found, and only the middle gene, which is present on both two-gene molecules, is also found on single- gene molecules in the mature macronucleus. Interestingly, these single-gene chromosomes are amplified to a copy number ten times higher than the two-gene chromosomes. While the flanking genes are necessarily expressed from the two-gene chromosomes, it has not been determined if the middle gene is transcriptionally active on these two chromosomes. In contrast to the genes belonging to the 81-MAC locus, the three genes from Holosticha sp. that we have described here do not appear to be alternatively fragmented. We have not found evidence that chromosomes containing only one or two of these three separate genes exist in vivo during vegetative growth. Rather, we have found that most, if not all, macronuclear copies of these genes are linked to one another on identical multi-gene molecules. Messenger RNA copies of these three genes must therefore be transcribed from the multi-gene chromosomes.

While this study provides evidence for the existence of a multi- gene, transcriptionally active macronuclear chromosome in Holosticha, it also raises two questions: (i) How are transcription and chromosome fragmentation normally regulated in this spirotrich? (ii) How did this three-gene macronuclear chromosome arise?

The nature of transcriptional signals in spirotrichs is currently under investigation in a number of labs. Specifically, whether signals promote transcription in the correct region, inhibit transcription in the incorrect region, or both is not entirely certain. One attractive hypothesis is that telomeres, or the proteins associated with them, may help direct the transcription machinery to the sites of initiation o\n macronuclear chromosomes (Ghosh et al. 1994). However, since telomere sequences exist at both ends of the chromosome, and since transcription of the opposite strand could provoke an "RNAi-like" gene silencing response (Mollenbeck et al. 2003), it is unlikely that the telomere sequence could be the only signal responsible for transcription initiation. Transcription of multiple distinct loci from a single macronuclear chromosome, as seen in this study, suggests the presence of telomere- independent, positive acting promoter elements. Simple inhibition or promotion of transcription at one end of the chromosome cannot explain transcripts initiating specifically at multiple internal sites in the chromosome 976 bp and 2,389 bp downstream. Our study complements one in E. crassus that also suggests the presence of telomere-independent promoter elements in a spirotrich gene (Bender and Klein 1997). In that study, the transcription start site of a chromosome containing additional subtelomeric sequence stayed constant relative to the start codon, not to the telomere, suggesting a telomere-independent signal in this region.

Features of the mechanism that fragments chromosomes during macronuclear development have been described in a number of ciliate species (Jahn and Klobutcher 2002). One aspect common to almost all of these studies has been the discovery of species specific "chromosome breakage sequences" (Cbs), at or near which chromosomes are broken and capped with telomeres. Cbs sequences in the spirotrichous species Euplotes and Stylonychia are generally found in macronuclear sequences, while the sequences in Tetrahymena are found on micronuclear DNA and removed during macronuclear development. If a consensus sequence required for cleavage exists in Holosticha sp., these three genes could fail to fragment in the developing macronucleus as the result of a simple mutation or deletion of this sequence. The phylogenetic position of Holosticha relative to other spirotrichs (e.g. Croft et al. 2003) suggests the Holosticha sp. DNA pol-[alpha] gene described here evolved from an ancestor that was flanked on both sides by functional Cbs, supporting this hypothesis. We found a seven-nucleotide sequence (5'- TTCAATT-3') in the vicinity of all three mRNA cap sites (Fig. 3) that resembles the reverse complement of the E. crassus (5'- HATTGAAaHH-3') and S. lemnae (5'-TGAA-3') chromosome breakage sites (Jonsson et al. 2001; Klobutcher et al. 1998). This sequence may be the remnant of a functional Holosticha sp. Cbs. However, these three genes are packed tightly in both micro- and macronuclear DNA, suggesting that the original Cbs or associated sequences have been deleted. When signals responsible for chromosome breakage are described more thoroughly in this species, it may become possible to determine how this three gene locus escapes fragmentation.

The functional significance of linking genes together on a single spirotrich macronuclear chromosome is not known. In the case of the 81-MAC locus, a number of reasonable hypotheses have been presented (Seegmilleret al. 1997). In short, the authors proposed 1) that a cis-acting transcription regulation sequence present in one gene is required by all three genes, 2) that a replication origin present in one gene is required by all three genes, and 3) that the copy number of these genes in the macronucleus is important and regulated by alternative fragmentation. Unfortunately, our current study cannot distinguish among these possibilities. However, we will add another hypothesis to this list - that this three-gene macronuclear chromosome is an evolutionarily transient state that is neither useful nor detrimental to the cell. The fact that the macronuclear DNA pol-[alpha] gene was not joined to other genes in the other spirotrichs we have studied suggests that this three-gene macronuclear chromosome in Holosticha sp. has evolved recently and independently. As such, the association of these genes is evidently not essential in the majority of spirotrichs, though it is apparently tolerated in this species.

Inefficient breakage at a Cbs can cause some macronuclear copies of its two flanking genes to remain joined together, as is seen for the 81-MAC locus in Sterkiella spp. Here we have described an extreme version of this phenomenon, where chromosome breakage between three genes has been completely abolished, thus causing all macronuclear copies of these genes to remain on the same chromosome. At present, a compelling case for why unions of this sort would normally be selected against has not been established. Our study further complicates this issue by showing that multiple genes can function on the same macronuclear chromosome, from a variety of positions. Studies are needed to define more thoroughly the mechanism and the selective forces normally at work to maintain single-gene macronuclear chromosomes in spirotrichs. Only then can we begin to determine how, and why, this anomalous chromosome arose in Holosticha.

Methods

Cell culture: Holosticha sp. (similar to H. kessleri) was isolated from lawn mosses in Plainsboro, New Jersey. The cells were cultured in inorganic salt medium (100 M CaCO^sub 3^, 80.4 M KCI, 29.4 M CaPO^sub 4^ and 16.6 M MgSO^sub 4^) at room temperature and fed with the green algae Chlorogonium elongatum (ATCC no: 12895) every 2-3 days according to published protocols (Prescott and Greslin 1992). Holosticha sp. cultures or frozen cysts are available from the authors upon request.

Macro- and micronuclear DNA isolation: Approximately 6 | of Holosticha sp. were concentrated using a 10 m sieve (Sefar American, Depew, NY) to filter out excess media and algae. Ciliates were lysed by using ice-cold C1 buffer from the Blood & Cell Culture DNA kit (Qiagen, Valencia, CA) and ice-cold d^sub 2^H^sub 2^O in a 1:1:3 ratio. Nuclei were collected by centrifuging the lysate at 1,300 g for 15 min. Macro- and micronuclei were then separated by using a 5 m sieve (Sefar American) and collected by centrifugation. Macro- and micronuclear DNA were extracted using the Aqua Genomic DNA kit (Biorad, Hercules, CA) following the manufacturer's protocols. The optional RNase A digestion was performed at 37 C for 15 minutes with an RNase A concentration of 6 g per 300 l sample. Micronuclear DNA was further purified by separating micronuclear DNA from residual macronuclear DNA using 0.75% Nusieve(TM) low melting point agarose (FMC, Rockland, ME) gel electrophoresis. The high molecular weight band which corresponds to the micronuclear DNA was then excised from the gel and melted at 70 C before subsequent PCR. Macronuclear DNA was quantified by UV spectrophotometry.

Macronuclear sequences of the three genes: Parts of the Holosticha sp. macronuclear DNA polymerase alpha (DNA pol-[alpha]) gene were PCR amplified using the degenerate primers listed in Table 1 (Hoffman and Prescott 1997; Landweber et al. 2000). The sequences of these primers were designed to conserved regions of previously sequenced ciliate DNA pol-[alpha] genes. We obtained approximately 75% of the DNA pol-[alpha] gene from Holosticha sp. using these primers. The 3'-end of this gene was determined by an approach similar to that used for rapid amplification of cDNA ends (RACE). We first added a stretch of adenosines to the 3'-ends of total macronuclear DNA by incubating 300 ng Holosticha DNA with 10 units of terminal transferase (TdT, NEB, Beverly, MA), 1 l of 10 mM dATP, and supplied buffers (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol (pH 7.9), 0.25 mM CoCl^sub 2^) at 37 C for 15 minutes, followed by a 10 minute, 70 C inactivation step. 1 l of 1:100 diluted TdT-treated macronuclear DNA was then used as the PCR template with a poly-dT anchored primer and a gene specific primer (GSP) to amplify the 3'-end of the DNA pol- [alpha] gene (Frohman et al. 1988). This PCR product was subjected to a second round of PCR using a nested GSP and a primer specific for a sequence engineered at the 5' end of the poly-dT anchored primer (Frohman et al. 1988). This PCR generated a 1.6 kbp product that contained the 3'-end sequences of the DNA pol-[alpha] gene and the telomere repeats G^sub 4^A^sub 4^, confirming the sequence of the telomere. PCR was performed in a volume of 25 l (0.2 M of each primer; 0.2 mM dNTPs; 1 PCR buffer, Roche) with 35 thermal cycles (94 C, 30 sec; 50 C, 1 min; 72 C, 2-3 min) using 1 unit of Taq polymerase (Roche, Indianapolis, IN). PCR products were cloned into Topo-TA Cloning vectors (Invitrogen, CA). Plasmid DNA was extracted using the Ql-Aprep Spin Miniprep Kit (Qiagen), and samples were sequenced using an ABI Prism Automated DNA Sequencer (Princeton University Synseq Facility).

We were unable to amplify the 5'-end of the macronuclear DNA pol- [alpha] gene using the same approach that we used for the 3'-end. Therefore, we used telomere suppression PCR (TSP), a modified PCR protocol designed specially for amplifying unknown 5'- and 3'-ends of gene-sized ciliate macronuclear chromosomes (Curtis and Landweber 1999; Siebert et al. 1995). This approach takes advantage of the repetitive telomeric sequences common to the ends of most spirotrichous ciliate macronuclear chromosomes (Prescott 1994). TSP was performed in 1 PCR buffer (Roche), 0.2 m M dNTPs, and 0.2-0.6 M of each primer, using 2 units of Taq polymerase and one to two nanograms of template in a 50 l reaction volume. For the first amplification with a gene specific primer and primer DCB, cycling conditions were 7 cycles of 94 C 2 seconds and 72 C 4 minutes, followed by 32 cycles of 94 C 2 seconds and 67 C for 4 minutes, followed by a hold at 67 C for 4 minutes. This PCR product was diluted 1:50, and 1 l of this dilution was used in a nested amplification using gene specific primers with the adaptor primers D and then C. Primer sequences are provided in Table 1. Neste\d PCR amplifications were performed for 5 cycles of 94 C 25 seconds and 72 C 4 minutes, followed by 20 cycles of 94 C 25 seconds and 67 C 4 minutes, polished at 67 C for 4 minutes. Sequences obtained from these PCR fragments, which represented multiple alleles, were then assembled to generate a consensus sequence. The Genbank accession number for the three-gene macronuclear chromosome is AY293851.

Table 1. Primer sequences used in this study

Table 1. Primer sequences used in this study

Micronuclear sequences of the three genes: Partial micronuclear DNA pol-[alpha] sequences were initially determined from PCR products amplified from gel-purified micronuclear DNA using primers designed to macronuclear sequences. Upon obtaining some IES sequence, we designed and used IES-specific primers in conjunction with macronuclear-based primers to recover the whole micronuclear DNA pol-[alpha] gene (see Table 1 for primer pairs D-K). Several long-range PCRs were performed to confirm that MDSs and IESs were located on the same locus (Fig. 1, also see primer pairs A-C in Table 1). The Genbank accession number for the consensus micronuclear sequence of the three genes in Holosticha sp. is AY293853. The variability seen in this sequence is due to allelic differences.

Southern blotting: We used 10 Mg of Holosticha macronuclear DNA for Southern blot analysis. To prepare a positive control, we cloned a PCR fragment encompassing almost the entire three-gene macronuclear chromosome (from positions 113 to 7000) into a PCR-XL vector (Invitrogen). This plasmid, pH[alpha]113F7000R, was digested by SamHI and Notl to release the insert, which has a size very close to the three-gene macronuclear chromosome. The pH[alpha]113F7000R plasmid was also digested with HindIII to generate three smaller fragments. These fragments each contained a portion of a different gene; we designed our gene-specific probes such that each probe would hybridize to only one of these fragments (see Fig. 4). These fragments were used to evaluate the probe specificities and the blot stripping efficiency. DNA was separated on a 1% agarose gel (Invitrogen) at 75 volts for 5 hours in TAE buffer, then transferred onto a Nytran SuPerCharge membrane (Schleicher & Schuell, Keene, NH) with the aid of a TurboBlotter (Schleicher & Schuell) according to the manufacturer's protocol. Blotted DNA was immobilized on the membrane by UV crosslinking (120 mJ/cm^sup 2^). We synthesized three separate, gene-specific DIG-labeled PCR fragment probes using the DIG High-Prime Kit (Roche). Each probe was used independently to probe the blot according to the manufacturer's protocol. Primer sequences for generating the probes are listed in Table 1. Hybridized probes were bound with anti-DIG antibodies conjugated to alkaline phosphatase, which were in turn detected by chemiluminescence and autoradiography according to the DIG High- Prime Kit (Roche) protocol. To strip the membrane prior to reprobing, we washed the membrane twice in 0.2 M NaOH containing 0.1% SDS for 15 minutes at 37 C. The membrane was then reprobed according to the manufacturer's protocol.

RNA isolation and RACE: Holosticha sp. cells were fed with green algae one day prior to harvesting for RNA collection. Cells were pelleted by centrifugation at 400 g for 4 minutes. RNA was extracted by using the Trizol reagent (Invitrogen) following the manufacturer's protocol. The quantity and purity of RNA were assessed by measuring the absorbance at 260 nm/280 nm. 5'-RACE and 3'-RACE were carried out by using the FirstChoice RLM-RACE kit (Ambion, Austin, TX) and 3' RACE system (Cat. No. 18373019, Invitrogen), respectively, following manufacturers' protocols. Sequences of the gene specific primers used for RACE are listed in Table 1.

Acknowledgements

This research was supported by NIGMS grant GM59708 and NSF grant EIA0121422. The authors would like to express their gratitude to JingMei Wang for ciliate culture, Dr. Mann-Kyoon Shin for isolating and characterizing Holosticha sp., and Dr. Thomas G. Doak for helpful reading of the manuscript.

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Wei-Jen Chang, Nicholas A. Stover, Victoria M. Addis, and Laura F. Landweber1

Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ 08544, USA

Submitted October 17, 2003; Accepted December 22, 2003

Monitoring Editor: Eric Meyer

1 Corresponding author;

fax 1 609 258 7892

e-mail LFL@Princeton.edu

Copyright Urban & Fischer Verlag Jun 2004

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