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University of Rochester, Department of Biology, Rochester, New York 14627
Received 1 August 2006/ Accepted 1 September 2006
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies of inserted rDNA units have suggested that both the insertions and the rDNA units they inhabit are not transcribed. Northern blots of total RNA from various tissues of D. melanogaster have revealed only low levels of R1 and R2 transcripts derived from both the full-length and 5' truncated elements (18, 37, 43). These low levels were similar to that derived from the cotranscription of foreign sequences inserted into the 28S gene target site (20). Direct electron microscopic observations of actively transcribing rDNA units revealed few RNA polymerase-nasent RNA complexes longer or shorter than expected for uninserted rDNA units (10, 11, 26, 35). These findings have given rise to the model that the inserted rDNA units are silenced at the chromatin level, with the low levels of transcripts occasionally observed derived from sporadic read-through transcription (18, 27, 43).
That a large fraction of the rDNA units in an organism could be sequestered from the transcriptional machinery is consistent with findings that there is considerable plasticity in the number of rDNA units in any organism (44). Most organisms appear have more than the minimum number of rDNA units needed for full viability. In eukaryotes as diverse as yeast, plants, and mammals, less than half of the units appear to be active at a given time (13-15). The active rDNA units are more accessible to nucleases and cross-linking reagents than the inactive units. While the establishment of these two rDNA chromatin states is not understood, epigenetic factors, such as histone modifications and in some organisms DNA methylation, are either the cause or the consequence of the different chromatin states (review in reference 29).
Previous studies of the chromatin structure of the rRNA genes of D. melanogaster either did not differentiate between the inserted and uninserted units (3, 40, 61) or indicated that the inserted units were less accessible (62). In this report, we compared the chromatin structures of inserted and uninserted rDNA units of D. melanogaster and conducted nuclear run-on experiments to directly measure their rates of transcription. We found the inserted and uninserted rDNA units of embryonic cells cannot be differentiated based on their accessibility to nucleases, psoralen cross-linking, and histone 3 and 4 modifications. The nuclear run-on experiments demonstrated that R1 and R2 elements are transcribed, but these transcripts unusually terminate within the element. These findings are discussed in relationship to the earlier studies as well as the possible mechanisms that regulate the activity of the R1 and R2 elements.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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0.5-g 1- to 16-h or 3- to 20-h embryos of the D. melanogaster line w1118 (64). After homogenization in nuclear buffer A (60 mM KCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris-HCl, pH 7.5, 0.5 mM dithiothreitol [DTT], and 300 mM sucrose), debris was removed by centrifugation at 550 x g for 1 min, and the nuclei were pelleted by centrifugation at 1,300 x g for 10 min. Nuclei were resuspended in nuclear buffer A and further purified by sedimentation through a 1.7 M sucrose cushion (60 mM KCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris-HCl, pH 7.5, 0.5 mM DTT, and 1.7 M sucrose) at 28,000 x g for 20 min. The probes used in this study were prepared by PCR amplification of cloned fragments or genomic DNA and are summarized in Fig. 1. The location of each fragment is as follows: for GenBank accession number M21017, 240 repeat (240), 10081 to 10323; promoter (Pro), 10636 to 10866; ETS, 10872 to 11105; 18S gene, 901 to 1140; ITS-1 (internal transcribed spacer [ITS]), 2202 to 2451 or 1953 to 2452; 28S gene (28S), 5784 to 6031; for GenBank accession number X51968,: R1 5' end, 1 to 255 or 1 to 507; R1 3' end, 5090 to 5343 or 4838 to 5343; for GenBank accession number X51967, R2 5' end, 1 to 250 or 1 to 510; R2 3' end, 3352 to 3589 or 3076 to 3589. The probe for Fig. 3 corresponded to sequence 6254 to 6554 of M21017, and the 18S/ITS probe in Fig. 5A was sequence 901 to 2451.
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ChIP experiments. Chromatin immunoprecipitation (ChIP) assays (9) utilized 1 g of 1- to 16-h dechorionated w1118 embryos, which were cross-linked in 10 ml of 50 mM HEPES, 1 mM EDTA, pH 8.0, 100 mM NaCl, and 1.8% formaldehyde in the presence of 30 ml n-heptane for 15 min. Embryos were gently spun down and washed for 10 min, first with 50 ml of 0.125 M glycine and 0.01% Triton X-100 in phosphate-buffered saline, next with 10 ml of 10 mM HEPES, pH 7.6, 10 mM EDTA, pH 8.0, 0.5 mM EGTA, pH 8.0, and 0.25% Triton X-100, and finally with 10 ml of 10 mM HEPES, pH 7.6, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 0.01% Triton X-100. Embryos were then resuspended in 5.5 ml of sonication buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, and 0.5 mM EGTA, pH 8.0) and homogenized with a type A pestle for three strokes in a 7-ml Dounce homogenizer. The homogenate was transferred to a 15-ml conical tube and sonicated for 30-s intervals four times with a Branson Sonifier 450 at setting 6 of constant power. After spinning at 4,000 x g for 30 min at 4°C, the supernatant was collected and adjusted to low-salt RIPA buffer (10 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA, pH 8.0, and 1x complete protease inhibitor [Roche]). Roughly 70-µg aliquots of chromatin were separately incubated with 10 µl of anti-acetyl H3, anti-acetyl H4, and anti-trimethyl H3K9 antibodies (Upstate Biotechnology) at 4°C overnight. Antibody-chromatin immune complexes were recovered by adding N-protein A beads (Amersham Biosciences) preequilibrated with low-salt RIPA containing 100 µg/ml herring sperm DNA and further incubated for 2 h. Samples were centrifuged at 6,000 rpm for 30 s and the pellets washed five times with low-salt RIPA buffer, two times with high-salt RIPA buffer (as above but with 500 mM NaCl) with 100 µg/ml herring sperm DNA, and once with 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0 and finally resuspended in 100 µl of the same buffer. RNase A was added to a final concentration of 100 µg/ml and incubated at 37°C for 30 min. Proteinase K and SDS were then added to final concentrations of 0.5 mg/ml and 0.5%, respectively, and incubated at 65°C overnight to reverse the cross-linking. Samples were extracted with phenol-chloroform, chloroform, precipitated with ethanol in the presence of 20 µg glycogen, and subjected to PCR analysis. PCR cycles were adjusted to allow the amplifications to remain in the linear range. Primer sequences and coordinates for the amplified fragments are shown in Table 1.
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Nuclear run-on transcription assays.
Nuclear run-on assays (31) were conducted with isolated nuclei in 0.3 ml of 90 mM KCl, 25 mM DTT, 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM (each) ATP, CTP, and GTP, and 150 µCi [
-32P]UTP (3,000 Ci/mmol) with or without 100 µg/ml -amanitin (Sigma). Incubations were at 25°C or 37°C for 15 min, the reactions stopped by the addition of 30 mM EDTA and 2% SDS, and the RNA isolated. RNA from each nuclear run-on reaction was boiled for 5 min and hybridized to 0.5 µg denatured DNA fragments from various regions of the rDNA units bound to nitrocellulose paper (31). Hybridizations were conducted at 65°C in 6x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5x Denhardt's, and 0.5% SDS for 20 h. Membranes were washed twice each in 2x SSC-0.1% SDS and 0.5x SSC-0.1% SDS at 65°C before exposure to a PhosphorImager screen. By using only regions of the rDNA that are processed from the major transcript, it was not necessary to eliminate by RNase treatment the 18S and 28S rRNA present in the nuclei (60).
To determine the relative hybridization efficiency, DNA fragments used in the nuclear run-on experiments were cloned to pCR-Blunt vector (Invitrogen) and sequenced. Clones containing the appropriate insert orientation were picked and digested with a restriction enzyme that cleaved the downstream end of the insertion. From these DNA templates, RNA probes were made by in vitro transcription with T7 RNA polymerase in the presence of[-32P]UTP and purified from a denaturing polyacrylamide gel. Equal counts of these RNA probes were pooled, hybridized to nitrocellulose membranes, and immobilized with identical amounts of DNA fragments under the same hybridization conditions used in the nuclear run-on experiments, and the relative hybridization efficiencies of the different DNA fragments was determined.
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RESULTS |
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Micrococcal digestion of embryonic nuclei for different times (Fig. 2A) revealed that all regions of the rDNA units were packaged into nucleosomal arrays similar to that of bulk DNA (stained DNA in panel at left). Scans of the longest nuclease digestion times from each blot are shown in Fig. 2B, while the fractions of the DNA digested to mono-, di- and trinucleosomes at 1 and 4 min are shown in Fig. 2C and D. The nucleosomal profile and the extent of digestion were similar for the 18S, 28S, R1, and R2 probes, suggesting that most inserted and uninserted units were packaged into nucleosomal arrays with similar accessibility to MNase.
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Inserted and uninserted units are equally accessible to DNase I. ClaI cleaves the 28S gene at sites flanking the R1 and R2 insertion sites but not within either element, while SalI and PstI cleave short distances from the 3' end of each element (Fig. 3A). Using this ability to place inserted and uninserted rDNA units on different-size restriction fragments, the relative nuclease accessibilities of the different types of rDNA units could be compared. DNA extracted from DNase I-treated embryonic nuclei was followed by two sets of restriction digests: (i) ClaI plus SalI, which placed the DNA from R1- and R2-inserted units on larger fragments than those for the uninserted units, and (ii) ClaI plus PstI, which placed the DNA from the inserted units on shorter fragments than those for the uninserted units. After digestion the DNA was blotted and probed with a downstream region of the 28S gene (Fig. 3A). Because R1 elements are inserted downstream of R2 elements, rDNA units with both R1 and R2 insertions appeared as R1-inserted fragments in these blots.
The DNase I sensitivities of the uninserted, R1-, and R2-inserted units are shown in Fig. 3B. To control for variations in the amount of DNA loaded in each lane, a graph of the fraction of the three different rDNA fragments at each time point normalized to the fraction at time zero is shown in Fig. 3C. The restriction fragment derived from the R1-inserted units was most rapidly digested in the left panel, while the fragment from the uninserted units was most rapidly digested in the right panel. Thus, longer DNA fragments were more rapidly digested irrespective of whether they corresponded to inserted or uninserted units. Only in the case of the ClaI-SalI digest were the R2 units somewhat more slowly digested than the uninserted units, even though the R2 units were on slightly larger fragments. These experiments again suggest that most inserted rDNA units are packaged into a chromatin structure similar to that of the uninserted units. While a previous report has suggested that uninserted units are more accessible to DNase I digestion than the inserted units in D. melanogaster, that report did not control for the size of the DNA restriction fragments being monitored (62).
Histone modifications of R1/R2 inserted and uninserted rDNA units. It has been shown that "active" genes are generally packaged with histone H3 and H4 containing hyperacetylated N-terminal tails (AcH3 and AcH4), while "silenced" genes are packaged with histone H3 hypermethylated at lysine 9 (MeH3K9) (36). The chromatin structures of active and inactive rDNA units have also been proposed to contain these differences in histone modifications (3, 52, 59). We therefore carried out ChIP experiments with antibodies to acetyl H3, acetyl H4, and trimethyl H3K9 to test whether the chromatin of R1- and R2-inserted units differed from the chromatin of uninserted units.
The ChIP assays were conducted with chromatin from 1- to 16-h embryos (9). As shown in Fig. 4A, a region of the 28S gene downstream of the R1 and R2 insertion sites was selected for amplification to represent all rDNA units (TOT, for total); the insertion site region of the 28S gene was selected to represent the uninserted units (UN); and the R1- and R2-inserted units were monitored by separate amplification of regions near their 5' and 3' ends (R1-5', etc.). All PCR amplifications were adjusted to be within the linear range and conducted in triplicate. Figure 4B shows the PCR products from one amplification, while the intensities of bands from all experiments were normalized to that of the input band and plotted in Fig. 4C. Confirmation of the approach and the specificity of the three antibodies was demonstrated by the enrichment of AcH3 and AcH4 and the absence of MeH3K9 in the chromatin of the transcriptionally active actin 87E gene (ACT) and a deficit of AcH3 and AcH4 and enrichment of MeH3K9 in the chromatin of a transcriptionally inactive centromeric sequence (CEN) (1).
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Psoralen cross-linking of inserted and uninserted rDNA units. Sogo and colleagues have demonstrated that transcriptionally active rDNA units are more accessible to psoralen and after UV cross-linking can be separated from the inactive units by their slower migration during electrophoresis (14). This psoralen accessibility has been used to detect active rDNA units in many organisms (13, 15, 16, 47). Isolated nuclei from 1- 16-h embryos were incubated with psoralen and UV cross-linked (15, 47, 58). To monitor all rDNA units, the cross-linked DNA was digested with BglII or BglII plus PstI and probed with an 18S/ITS-1 sequence (Fig. 5A, left panel). To separately score uninserted, R1-inserted, and R2-inserted rDNA units (right panel), the cross-linked DNA was digested with ClaI plus PstI and probed with the downstream 28S gene probe (the same digests and probe used for Fig. 3). As a control, psoralen cross-linking of the rDNA units from Saccharomyces cerevisiae was also conducted. After cross-linking, the S. cerevisiae DNA was digested with EcoRI and probed with a gene region spanning parts of the 18S and 25S genes (Fig. 5B).
Consistent with previous findings for S. cerevisiae (15, 16, 58), cross-linked DNA (lane X) migrated as two distinct bands slower than those for uncross-linked DNA (lane C). The more accessible (i.e., active) band corresponded to one-third of the total units (31% for the 18S band and 34% for the 25S band). In contrast to the yeast rDNA units, the cross-linked D. melanogaster rDNA units showed a single band that was slower migrating than that for the uncross-linked fragment. The shift in migration of the cross-linked D. melanogaster units was similar to that of the inactive units in yeast. This single cross-linked product was observed for the fragments in the left panel, representing all rDNA units, and for the fragments in the right panel, representing the individual uninserted or R1- or R2-inserted fractions. The series of faint lower and higher molecular bands seen in both the cross-linked and uncross-linked DNA corresponded to the greater restriction polymorphisms present in the rDNA units of D. melanogaster. Because of these polymorphisms, the lower limit in our ability to detect a second more slowly migrating band was about 10%. Assuming that actively transcribed rDNA units in D. melanogaster embryos are similar in structure to those of other organisms and thus would have been detected by psoralen cross-linking, these data suggest only a minor percentage of the inserted and uninserted units are actively transcribed. Similar results have been obtained with nuclei isolated from embryos of different ages and with different levels of cross-linking (data not shown).
Nuclear run-on transcription assays. As the most direct assay to monitor the fraction of the inserted and uninserted rDNA units that are actively being transcribed, we conducted nuclear run-on experiments. Several arguments suggest that any transcripts detected from R1 and R2 would represent cotranscription with the rRNA genes. First, transcripts initiating at sites within the insertions have not been observed by electron microscopy (10, 35). Second, we have been unable to detect promoters associated with the R2 elements (25). Finally, transcripts can be detected of 5'-truncated copies of the elements or of foreign sequences inserted into the R2 site (20).
In the run-on experiments, 32P-labeled RNAs synthesized during a 15-min incubation of embryonic nuclei were hybridized to immobilized DNA fragments corresponding to different regions of the rDNA unit. Because no sequences are unique to uninserted units, the level of transcription from the R1 and R2 insertions was compared to the total transcription of all units. Three regions of the rDNA repeat were tested to represent the combined transcription of all units: the ETS region, the ITS1 region, and a segment of the 28S gene upstream of the R1 and R2 insertion sites. The ITS1 and 28S levels were similar in the various trial experiments, while the ETS hybridizations were more variable and averaged eightfold lower than the ITS1 or 28S hybridizations (see examples in Fig. 6). Presumably the ETS sequences near the promoter of the rDNA unit are either more rapidly degraded or become depleted of polymerase. We used RNA hybridization to the ITS1 region of the rDNA as the transcription standard in the following experiments, because the low levels of R1 and R2 transcripts detected on Northern blots or by RNA protection assays (20, 37, 43) suggest that if the inserts are transcribed, their RNA is quickly degraded like that of the ITS.
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-amanitin (42). The presence of -amanitin had little effect on the relative transcript levels of R1 and R2, with transcription of the insertions in line SWIFF6 somewhat reduced in the presence of -amanitin but somewhat increased in line w1118. Reducing the size of the DNA fragments bound to the nitrocellulose filters to 250 bp instead of 500 bp (experiment C) and conducting the incubations at 25°C instead of 37°C (experiment D) also did not consistently change the levels of R1 and R2 transcripts compared to that of ITS1. Finally, we also conducted run-on assays with nuclei isolated from adult animals (data not shown). The levels of both R1 and R2 transcripts relative to that of ITS1 were two- to threefold higher in the adult female nuclei; however, the level of ITS1 transcripts was reduced 10- to 20-fold compared to that in embryonic nuclei, making quantitation less accurate. Based on the ITS1 signal, the total transcript levels of the rDNA units in adult male nuclei were even lower. Therefore, the following discussions are based on the transcription levels obtained from only the experiments conducted with embryonic nuclei. As summarized in Table 2, the SWIFF6 line showed higher levels of R1 and R2 transcription than the w1118 line, consistent with the higher fraction of R1 and R2 insertions in SWIFF6 (20). Surprisingly, in both lines transcription of the 3' end of the R1 element was about threefold lower than that of the 5' end. This suggests that most transcription complexes fail to reach the 3' end of the R1 element or that the 3' RNAs are more rapidly degraded. In the case of R2, the levels of 5' and 3' end transcripts were more similar. However, nearly half of the R2 elements in these strains are 5' truncated (i.e., do not contain the region being monitored by the 5' probe) (20). If 5'-truncated insertions are cotranscribed at the same rate as full-length insertions, then many R2 transcription complexes also fail to reach the 3' end of the element.
These run-on experiments indicated that the R1 and R2 elements were transcribed at measurable levels. To estimate how efficiently the inserted units were transcribed relative to transcription of the uninserted units, the transcription values of their 5' ends were divided by the fraction of the rDNA units that contained each insertions. In the case of R1, 38% (w1118) and 45% (SWIFF6) of the rDNA units contained full-length insertions. Thus, full-length R1-inserted units appear to be transcribed at 17% and 24%, or about one fifth, of the efficiency of the uninserted units. In the case of R2, 7.5% (w1118) and 9.5% (SWIFF6) of the rDNA units contained full-length insertions. Thus, full-length R2-inserted units appear to be transcribed at 7% and 11%, or about 1/10 the efficiency of the uninserted units.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The discovery that R1 and R2 insertions in the rDNA units are active retrotransposable elements renewed interest in their expression, because transcription even at low levels could be a critical component of their regulation. The run-on transcription assays reported here indicated that R1- and R2-inserted rDNA units of D. melanogaster are transcribed at significant levels. Once activated, the rates of transcription initiation of inserted and uninserted units are similar, because the same densities of RNA polymerase complexes were observed by microscopy for the rare long (inserted) transcripts as for the shorter (uninserted) transcripts (10). Correcting for the fraction of the rDNA locus containing the insertions, full-length R1-inserted units are activated at about 1/5 and full-length R2-inserted units at about 1/10 of the rate of activation of the uninserted units. Inserted rDNA units may be activated at even higher relative levels if the R1 or R2 transcripts are more rapidly degraded than the ITS1 transcript used for comparison.
What explains the low levels of "long transcripts" in the electron microscopic studies? Our run-on data also suggest that most of the transcription complexes on the inserted units do not extend to the 3' end of the insertion elements. Run-on transcripts from the 5' end of the R1 elements were threefold more abundant than transcripts from the 3' end (Table 2). Because 20% of the R1 insertions in the Drosophila strains used in the assays are 5' truncated, containing 1 kb or less of the 3' end of the element, cotranscription of these 5'-truncated R1 elements could account for most of the 3' transcripts detected in the run-on assays. In a similar manner, while the levels of R2 5' and 3' run-on transcripts were about equal, half of the R2 insertions in the assayed lines do not contain the 5' end of the element, again suggesting that transcription of the 5'-truncated copies could account for most of the 3' R2 transcripts detected.
As additional support for premature terminations within inserted units, histone H3.3, which is associated with transcription (3), has been shown to accumulate in the first 1 kb of the R1 sequences but not in regions further downstream (S. Henikoff, personal communication). If most of the transcription complexes on the inserted units do not extend to the end of the insertion elements, our run-on results do not contradict the earlier electron microscopic studies of rRNA transcription. Transcription complexes that terminate within the first 1 to 2 kb of the R1 and R2 insertions would appear in electron microscopic observations to be of similar length to the complexes derived from uninserted units (the R1 and R2 insertion sites are 1.3 kb from the normal termination site of the rDNA unit; see Fig. 1). Thus, scoring by electron microscopy only transcription complex length significantly underestimated the level of transcription associated with inserted rDNA units. Indeed, direct electron microscopic evidence that transcription complexes were terminating within the insertion elements was obtained in the early studies. Chooi (10) observed two classes of intergenic spacer lengths between consecutive rDNA transcription units of D. melanogaster: a shorter class consistent with the 3- to 5-kb physical lengths of the intergenic spacer and a longer class consistent with transcription truncations within the R1 or R2 elements.
The only apparent conflict that exists between our run-on data and the earlier reports of R1 and R2 transcript levels is that full-length transcripts of R2 elements are more readily detected on Northern blots than full-length R1 transcripts (37, 43), while the run-on transcription data suggested that the R1-inserted units are more frequently activated for transcription than the R2-inserted units. We suggest this difference between the number of units activated for transcription and actual transcript levels is a result of the more-efficient termination of transcription within R1 elements or the greater stability of R2 transcripts.
What fractions of the rDNA units are transcribed? The number is likely to vary among tissues and strains, but our nuclease digestion and histone modification assays suggest that these fractions are quite low (Fig. 2 to 4). The most sensitive assay for detecting the level of active rDNA units is psoralen cross-linking (13, 15, 16, 47). However, our psoralen cross-linking study did not reveal active units, suggesting such units represent less than 10% of the units in any of the bands we detected on Southern blots (Fig. 5). Based on the run-on data, the highest proportion of active units would be the uninserted units. The w1118 line used for this study contained 300 rDNA units on each of the X and Y chromosomes, 40% of which are uninserted (i.e., about 240 uninserted units per cell) (20, 34). If less than 10% of the uninserted units are active, then the absolute number of such units is less than 24. This surprisingly low number is in fact consistent with that estimated from other studies. Only an estimated 50 rDNA units (both inserted and uninserted) are sufficient to avoid the bobbed phenotype (20, 22, 30). Electron microscopic observations of early embryo development suggested an average of 30 transcriptionally active rDNA units in each nucleolus of D. melanogaster (50). The requirement for such low numbers of functional rDNA units in Drosophila could explain why more than 75% of the units can be inserted in strains of D. melanogaster (34) and why more than 90% of the rDNA units can be inserted in strains from other Drosophila species (32, 44, 49).
How are the active units arranged within the nucleolus? Electron microscopic observations suggest that transcriptionally active rDNA units are frequently consecutive, suggesting that in any cell, transcriptional activity is clustered within one or a few regions of the rDNA loci (10, 11, 26, 35). A recent report on the replacement of histone H3.3 in actively transcribed genes also suggested that transcriptionally active and inactive rDNA units are physically separated (3). Because inserted and uninserted rDNA units are extensively interspersed across the rDNA locus in D. melanogaster (10, 63; W. Burke, K. Averbeck, and T. Eickbush, unpublished data), a simple model to explain our R1 and R2 transcription data is that within these "activated domains" all rDNA units are transcriptionally active independently of whether they are inserted or uninserted. The lower levels of transcription of the R1- and R2-inserted units would simply reflect the fraction of these units in the active domains and the advantage to the cells of activating those regions of the locus with the highest fraction of uninserted units. In this model, most control over R1 and R2 activity would occur after transcription initiation. We have recently detected isofemale lines of Drosophila simulans with active R2 retrotransposition (66). This R2 activity is correlated with high levels of stable full-length R2 transcripts on Northern blots (D. G. Eickbush, X. Zhang, J. Ye, and T. H. Eickbush, unpublished data). We should be able to determine if this R2 retrotransposition activity is associated with increased rates of R2 transcription, a reduction in the rate of termination within the element, or an increase in the stability of the R2 transcripts.
R1 and R2 have been stably associated (vertically transmitted) with insect lineages since the origin of arthropods (6, 23, 48). The competition between the drive of these elements to survive by increasing their numbers and that of the hosts to bring about their elimination is but one of the many battlefields in the genomic war that is being fought in all organisms. Just as this war between the genome and mobile elements has been postulated to give rise to new types of gene regulation (e.g., DNA methylation and RNA interference) (55, 65), it can be postulated that the battle between R1 and R2 and the rDNA locus has given rise to new means of regulating the expression of the rDNA units. These new mechanisms could involve the activation of only a small number of genes, mechanisms to promote termination of the transcription apparatus within the insertions, or new means to process the rRNA transcripts.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant GM42790 to T.H.E.
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FOOTNOTES |
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Published ahead of print on 25 September 2006.
Present address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138.
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