Ribosomal RNA gene amplification in tetrahymena may be associated with chromosome breakage and DNA elimination

Cell, Vol. 24, 765-774,

June

1981,

Copyright

0 1981

by MIT

Ribosomal RNA Gene Amplification in Tetrahymetia May Be Associated with Chromosome Breakage and DNA Elimination Meng-Chao Yao Department of Biology Washington University St. Louis, Missouri 63130

Summary The chromosomal DNA sequence adjacent to one end of the single ribosomal RNA gene (rDNA) in the micronucleus of Tetrahymena has been isolated by cloning. Using this sequence as a hybridization probe the organization of the same sequence in the somatic macronucleus has been examined. The restriction enzyme digestion maps of this sequence in the two nuclei are very different. Detailed map ping studies suggest that a chromosome break has occurred near the junction between the rDNA and the neighboring sequence during the formation of the macronucleus. As a result the flanking sequence is located near a free chromosome end in the macronucleus. The existence of such a linear DNA end has also been shown by digestion with the exonuclease Bal 31. In addition to the breakage, some sequences at this junction are found to be eliminated from the macronucleus. This observation has been interpreted in relation to the mechanism of rDNA amplification, which in Tetrahymena generates extrachromosomal rDNA molecules during macronucleus development. Introduction It was first shown more than a decade ago that the genes coding for ribosomal RNA (rDNA) were selectively amplified in the oocytes of amphibians and many other eucaryotes (see Gall, 1969 and Tobler, 1975, for reviews). The process of gene amplification has since been studied rather extensively. It is now clear that in most cases the amplified molecules are extrachromosomal, and that some of them are the products of rolling circle replication (Hourcade et al., 1973; Rochaix et al., 1974). It is also believed that in some cases the extrachromosomal rDNA is derived from chromosomally integrated copies (Brown and Blackler, 1972; Reeder et al., 1976). However, the mechanism by which the first copy of the extrachromosomal genes is generated remains essentially unknown. More recently rDNA amplification has been found in the ciliated protozoan T. thermophila (Gall, 1974; Yao et al., 1974). The phenomenon of gene amplification in Tetrahymena is somewhat different from that in Xenopus and other metazoa. Tetrahymena normally contains two types of nuclei in each cell: a macronucleus and a micronucleus. Although the two nuclei are very different in structure and function, they both share the same genetic origin. During conjugation, the micronuclei of the mating pair go through a series of

events, including meiosis, fertilization and mitosis, to give rise to the new macro- and micronucleus for the subsequent sexual generation. The old macronucleus simply degenerates in this process (see Elliot, 1974 for review). Previous studies have shown that there is only one copy of rDNA integrated in the chromosome of the micronucleus (Yao and Gall, 1977). This single integrated gene is believed to be amplified during conjugation into multiple copies of extrachromosomal, palindromic molecules in the mature macronucleus (Yao et al., 1974; Karrer and Gall, 1976; Engberg et al., 1976; Yao et al., 1978). Thus, by studying the structure of the rDNA and its flanking sequences in both macro- and micronucleus one might obtain important information regarding how the extrachromosomal gene is first generated. I have isolated one of the two junctions between the rDNA and the chromosomal DNA from the micronucleus by cloning and used it as a hybridization probe to study the structure of the same sequence in the macronucleus. The results strongly suggest that a chromosome break exists at this region in the macronucleus to separate the rDNA from its flanking sequence. Moreover, a portion of this flanking sequence is apparently eliminated from the macronucleus. There seems to be an interesting relationship between gene amplification, DNA elimination and chromosome breakage in Tetrahymena. Results Cloning the Integrated Ribosomal RNA Gene Figure 1 summarizes some relevant features of the extrachromosomal and the integrated rDNA of Tetrahymena. The flanking sequences of the integrated rDNA have been characterized to some extent by the Southern hybridization method using the extrachromosomal rDNA as a probe (Yao and Gall, 1977). Although the integrated copy of rDNA could not be found in the macronucleus, it was not known whether and how the flanking sequences might be present in this nucleus. This kind of information should be valuable in understanding how the extrachromosomal rDNA is first generated. For instance, if the integrated rDNA is removed from the chromosome by excision as the DNA of phage X is removed from the Escherichia coli chromosome, the two flanking sequences should become immediately adjacent to each other (Figure 1). Alternatively, if chromosome breakage occurs without reunion of the broken ends, the two flanking sequences should be physically separated. Other processes may also occur, and will probably generate different structures for the flanking se-

Cell 766

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Possibilities in Generating

Extrachromosomal

2. Restriction

Enzyme

Digestion

Map of cTt 220 and cTt 101

The 8.2 kb fragment of the chromosomal rDNA is represented by the horizontal line in clone cTt 220. The vertical bars are cleavage sites of restriction enzyme Eco RI (e), Hind Ill (h), Hae Ill (a) and Bgl II (b). The region hybridized with the extrachromosomal rDNA is indicated by a black bar in the top of the figure. L and R represent the left and the right end of the internal 7.8 kb Eco RI fragment of Charon 4A (open boxes) which was cloned coincidentally with the rDNA. The molecular weights given inside the parentheses are approximate sizes of deletion in the derivatives of cTt 101. The exact positions of the deletion inside the restriction fragments indicated have not been determined. The restriction maps are constructed based on single and double enzyme digestions, and on the known map of Charon 4A and Tetrahymena rDNA.

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In the micronucleus rDNA (thick line) is flanked by chromosomal DNA sequences (thin line). Eco RI cuts at three sites (marked e) in this region and separates it into a 15 kb and an 8.2 kb fragment, each containing one end of the rDNA. The two sites in the flanking regions are 7.3 kb and 5.8 kb away from the two ends of the rDNA. The rDNA is marked at one end with an arrowhead, which is in the same direction of rRNA transcription. In the macronucleus rDNA exists as extrachromosomal. palindromic dimers. The Eco RI sites are located symmetrically in both halves of the molecule and divide the molecule into a 15.4 kb center fragment and two 2.4 kb end fragments. In the excision model the two flanking sequences are jointed together to give a 13.1 kb segment of DNA between the two Eco RI sites. In the breakage model the two flanking sequences remain as separated molecules. In this case the sizes of the Eco RI fragments should be 7.3 kb and 5.8 kb, or may also be shorter if additional events occur to the free chromosome ends generated.

quences. To investigate this problem further, I proceeded to isolate the flanking sequences of the rDNA from the micronucleus by cloning. A clone library containing micronuclear DNA that had been partially digested with Eco RI was constructed using the phage vector Charon 4A (see Experimental Procedures). Eco RI was known to digest the integrated rDNA into a 8.2 kb and a 15 kb frag-

ment, each containing one end of the gene together with the flanking DNA (Figure 1). Clones containing these two segments of DNA were screened for by the plaque hybridization method (Benton and Davis, 19771, using the extrachromosomal rDNA as a probe. The micronuclear DNA isolated by the method used is normally contaminated with 5-15% of macronuclear DNA (Gorovsky et al., 1975). Since the relative abundance of rDNA was at least 200-fold higher in the macronucleus (Yao and Gall, 19771, there could be lo-30 times more contaminating extrachromosomal rDNA than integrated rDNA in the micronuclear DNA preparation. This level of contamination could make the screening extremely difficult. However, since the two free ends of the extrachromosomal rDNA can not be ligated with the Eco RI sites of the vector and be cloned, their presence shall not interfere with the screening for the 8.2 kb fragment of the integrated rDNA. Hence, using a probe specific for the terminal region of the rDNA (the Hind Ill-e fragment of the extrachromosomal rDNA), one can detect specifically the 8.2 kb fragment of the integrated rDNA in the clone library. Two clones of this kind (cTt 101 and cTt 220) have been isolated and analyzed, and both are found to be derived from this region of the micronuclear genome (see next section). The internal fragment of the extrachromosomal rDNA, on the other hand, can be ligated and cloned, and thus its presence would probably interfere with the screening for the 15 kb fragment of the integrated rDNA. Eight clones containing this region of the rDNA have been isolated,

Chromosome 767

Breakage

in Tetrahymena

through successive cloning, and was taken as a suggestion that the clone was somewhat unstable. The 7.8 kb fragment was believed to be derived from a contaminating middle fragment of the vector Charon 4A. This argument was supported by further restriction enzyme digestion and by hybridization with Charon 4A DNA (data not shown). The 8.2 kb fragment has been further characterized by additional restriction enzyme digestion studies. For instance, Hae Ill was known to cut this region of the micronuclear DNA into a 8.2 kb fragment (Yao and Gall, 1977 and Figure 8b). This fragment was in fact detected in Hae Ill-digested cTt 220 (Figures 3E and 3J). Bgl II was found to cut cTt 220 to give a 6.4 kb fragment which hybridized with the rDNA (Figures 3F and 3K). This fragment should contain 1.8 kb of Charon 4A sequence as well as 4.6 kb of the integrated gene, and was verified by double digestion with Eco RI and Bgl II (data not shown). Double digestion of micronuclear DNA with Bgl II and Eco RI also gives a 4.6 kb fragment which hybridized with the rDNA (Figure 5b), and hence the two maps also agree for their Bgl II sites. Barn Hl has been known not to

and as expected, none of them can be shown to be generated from the integrated copy (data not shown). My current study is therefore concentrated on the analysis of the flanking sequence contained in the 8.2 kb segment. Characterization of the Cloned Fragments Both cTt 101 and cTt 220 have been analyzed by restriction enzyme digestion and by Southern hybridization. The results are summarized in Figure 2 and some of the data are presented in Figures 3 and 4. The DNA of cTt 220 was cleaved by Eco RI into a 7.8 kb and a 8.2 kb fragment in addition to the two arms of the vector which give the top three bands in the gel (Figure 3C). The 8.2 kb fragment was hybridized readily with the extrachromosomal rDNA (Figure 3H), and was believed to be the 8.2 kb fragment of the integrated gene. In addition to this fragment, a few minor fragments were seen in the hybridization that were not easily detectable in the stained gel. The presence of these minor bands indicated that the DNA was slightly heterogeneous. The heterogeneity apparently was not caused by contamination, as it persisted

Figure 3. Restriction Southern Hybridization

Enzyme Digestion of cTt 220

and

DNA from recombinant clone cTt 220 was digested with various restriction enzyme and analyzed by electrophoresis in a 1 .O% agarose gel. The DNA was then blotted onto nitrocellulose filter and hybridized with Tetrahymena rDNA labeled in vitro with “P by nick translation. Lanes A-G are ethidium-bromidestained DNA after electrophoresis. Lanes H-L are autoradiograms of the hybridization. Lane A contains Eco RI-digested Charon 4A DNA. Lane S contains Hind Ill-digested A DNA as size markers. Lanes C. D. E. F and G contain cTt 220 DNA digested with Eco RI, Hind Ill. Hae Ill, Bgl II and Sam HI. respectively. Lanes H-L contain the same DNA as in lanes C-G. The black triangles indicate the major bands in the gel which were hybridized with rDNA.

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Cell 766

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cut within the 8.2 kb region of the integrated gene (Yao and Gall, 1977). This was also found to be the case for cTt 220 (Figures 3G and 3L). Although Hind III digestion map of the integrated rDNA is not known, some cleavage sites can be predicted based on the map of the extrachromosomal rDNA. Digestion of cTt 220 with Hind Ill (Figures 30 and 31) and Hind Ill plus Eco RI (data not shown) gave results that agreed with this prediction. Based primarily on this information the map of cTt 220 was constructed (Figure 2). These results suggested strongly that the 8.2 kb fragment of cTt 220 was in fact derived from the integrated rDNA. Structural analysis of cTt 101 is complicated because it is highly unstable. The original clone forms very small plaques and frequently gives rise to genetic variants that form much larger plaques and rapidly outgrow the original clone in both plate and liquid cultures. Figure 4 shows some examples of the DNA isolated from this clone. Like cTt 220, this clone also contains the 7.8 kb internal Eco RI fragment of the vector Charon 4A. The inserts that hybridized with rDNA were extremely heterogeneous in size. The high degree of heterogeneity made it impossible to determine the exact restriction map of the clone. A few faster growing variants, cTt 101-5, cTt 101-6 and cTt 101-l 9, have thus been analyzed (Figure 4). The inserts in these variants were different in length, and in general were shorter than 8.2 kb. Further studies suggested that the variable region is located near the border between the rDNA and the chromosomal DNA (data not shown). The restriction maps were otherwise not unlike that of the integrated gene. The basis for the instability of cTt 101 and cTt 220 is not known. However, due to this complication it was necessary to make certain that the cloned fragments in both cTt 101 and cTt 220 were actually derived from the expected region of the chromosome. In addition to the restriction map presented earlier, two lines of evidence supported this argument. First, the non-rDNA sequence in the cloned fragment hybridized with a single 8.2 kb fragment among Eco RI digested micronuclear DNA, which was indistinguishable from the fragment hybridized with rDNA (Figure 5). This result Figure 4. Restriction Enzyme of cTt 101 and Its Derivatives

Digestion

and Southern

Hybridization

DNA isolated from cTt 101 and its derivatives were digested either with Eco RI (a) or with Hind Ill (b) and analyzed by electrophoresis in a 0.7% agarose gel. The DNA was then blotted onto a nitrocellulose filter and hybridized with total Tetrahymena macronuclear rDNA labeled with 32P by nick translation. In both a and b. lanes A-F are ethidium bromide staining patterns of the DNA after gel electrophoresis and lanes G-K are autoradiograms of hybridization. Lane A contains Charon 4A DNA as a control. Lanes 6 and C contain two different preparations of cTt 101 DNA. Lanes D. E and F contain DNA from the derivatives cTt 101-5, cTt 101-6 and cTt 101-l 9. respectively. Lanes G-K contain the same DNA as in lanes B-F. Great heterogeneity is seen in the two preparations of cTt 101. Some degree of heterogeneity is also seen in the derivative cTt 101-6. The 7.6 kb Eco RI fragment of Charon 4A is also present in this clone and appears to be stable.

Chromosome 769

Breakage

in Tetrahymena

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suggested that the rDNA and the non-rDNA sequences of cTt 220 were colinear in the micronucleus. Second, the non-rDNA sequence in the two clones crosshybridized with each other, indicating that they were both derived from the same segment of the chromosome (data not shown). It should be emphasized that the two clones were indeed independent isolates and not results of crosscontamination. This point was best illustrated by the fact that although both clones contain the same 7.8 kb fragment of Charon 4A, this fragment was inserted differently in these clones (Figure 2). Based on these data one can safely argue that the flanking sequence cloned here is indeed adjacent to the integrated rDNA in vivo, and is not the result of cloning artifacts. rDNA Flanking Sequence in the Macronucleus The flanking sequence of rDNA was prepared from clone cTt 220 or the derivatives of clone cTt 101 by restriction enzyme digestion and gel electrophoresis, and used as a hybridization probe to study the organization of this sequence in the macronucleus. The fragment Hind Ill-b of cTt 101-6 contains 1.6 kb of the distal portion of the flanking sequence, as well as some X DNA (Figure 6). This fragment hybridized with a single 8.2 kb fragment of Eco RI-digested micronuclear DNA, agreeing quite well with the map presented (Figure 5). When macronuclear DNA was hybridized with the same sequence, instead of the 8.2 kb band, a 3 kb band was detected. This result was rather surprising. Three kb is too short for the 13.1 kb size expected if rDNA alone is excised out of the chromosome (Figure 1). Thus, a simple excision model is not sufficient to explain this result. However, 3 kb is also significantly shorter than the 5.8 kb size expected if chromosome breakage occurs right at the junctions of the rDNA (Figure 1). Apparently a more complicated process is involved. For instance, the excised-out Figure 5. Southern Hybridization of Microwith rDNA and Its Flanking Sequences

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N

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and Macronuclear

DNA

Approximately equal amounts of micro- and macronuclear DNA were digested with various restriction enzymes. analyzed by electrophoresis in 0.7% agarose gels and blotted onto nitrocellulose filters for hybridization. !a) The filters were hybridized with the Hind Ill-b fragment of cTt 101-6, which contains a segment of the flanking sequence of rDNA. tb) The filters were hybridized with the Hind Ill-e fragment of the extrachromosomal rDNA. The probes were labeled with 32P by nick translation and the hybridization was detected by autoradiography. For direct comparison each pair of micro- and macronuclear DNA were treated the same way throughout. Only the lanes grouped together were subjected to electrophoresis under the same conditions. Micro- and macronuclear DNA were digested with Eco RI in lanes A and B. and also in lanes K and L; with Hae Ill in lanes C and D, and also in lanes M and N; with Hind Ill in lanes E and F; with Bgl II in lanes I and J and also in lanes 0 and P. In panel a, the black triangles indicate the major band detected. The minor bands are probably the results of contaminating macronuclear DNA in the micronuclear preparations (lanes A and G). and the contaminating rDNA-containing sequences in the probe (lanes B. f-l and J). Black triangles in panel b indicate the bands derived from the chromosomal rDNA.

Cell 770

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III-b

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,

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1Hind111

Digestion Maps of rDNA and its Flankand Macronucleus

This diagram summarizes the results of Southern hybridizations shown in Figure 5. The two nuclear DNAs were digested with the restriction enzymes shown and hybridized with the Hind Ill-e fragment of the extrachromosomal rDNA and the Hind Ill-b fragment of cTt 101-6. The two open boxes indicate the portion of Tetrahymena DNA included in these two fragments. The sizes of the fragments detected are given in kb for each digestion. Each fragment is flanked either by two digestion sites as indicated by the two vertical bars or by one digestion site and one free end of the DNA as shown by the open end of the line. rDNA is represented by a thick line and the flanking sequence by a thin line. The arrow head indicates one end of the rDNA sequence, and is in the direction of rDNA transcription in this sequence.

region may involve more than the rDNA sequence alone. Alternatively, breakage may not occur right at the junction of the rDNA. To further investigate this point, studies with another restriction enzyme, Bgl II, were performed. Bgl II was found to cleave micronuclear DNA to give a 5.3 kb fragment which hybridized with the same rDNA flanking sequence (Figure 5a). The 5.3 kb fragment is roughly 5.5 kb away from the closest Bgl II site in the rDNA (Figure 5b and Figure 6) or 2.2 kb away from the junction between rDNA and the flanking sequences. When macronuclear DNA was digested and hybridized by the same method, again a shorter fragment, 4.7 kb, was detected (Figure 5a). The difference between these two fragments, 0.7 kb, again suggests that the macronuclear DNA is interrupted approximately 2.9 kb (2.2 kb + 0.7 kb) away from the junction. Since the Eco RI site and the Bgl II site flanking the other end of the integrated rDNA are 4 kb apart (data not shown), this result can not be interpreted by any simple excision model. Moreover, it agrees rather well with a breakage model if a break occurs approximately 2.8 kb away from the

junction. Further restriction enzyme digestion studies also supported this argument. For example, double digestion with Bgl II and Eco RI was found to generate fragments 3.5 kb for micronucleus and 3.0 kb for macronucleus as the model would predict. Both Hind Ill and Hae Ill were known to have one site between the probing sequence and the chromosome end and were found to give rise to fragments of the same sizes for the two nuclear DNAs. Based on these results a restriction enzyme digestion map was constructed for this region of the genome in both the macro- and the micronucleus (Figure 6). The map suggests that the chromosome of the macronucleus is not continuous in the region where the integrated rDNA is originally located. Chromosome breakage seems to have occurred near one end of the rDNA and separated it from the neighboring sequences. The map also suggests that a 2.8 kb segment of DNA in this region is missing. The flanking sequence studied here was found in macronuclear DNA of high molecular weight. Without restriction enzyme digestion the sequence could not be detected as any band and probably migrated in the limited mobility region of a 1.0% agarose gel (data not shown). It was also found to sediment much faster in a sucrose gradient than Tetrahymena extrachromosomal rDNA, and probably also faster than the mitochondrial DNA, which was about 45 kb (data not shown). Although the exact molecular weight of this DNA is not known, it is probably not a small extrachromosomal molecule. Digestion with Exonuclease Bal 31 To provide further support for the structure postulated in Figure 6, studies with the exonuclease Bal 31 were conducted. If chromosome breakage indeed occurs, the flanking sequence should be adjacent to a free DNA end. Sensitivity of this sequence to any exonuclease would indicate the presence of such a free DNA end, although the converse may not be true. For this reason an exonuclease with a wide range of specificity was chosen. The nuclease Bal 31 contains exonuclease activities for DNA with single- or doublestranded ends (Gray et al., 1975), and seemed to be most suited for this study. Purified macronuclear DNA, which was normally larger than 40 kb, was treated with Bal 31 for various lengths of time. This DNA was then digested with Bgl II, analyzed by electrophoresis, blotted and hybridized with the rDNA flanking sequence to determine the sensitivity of this sequence to the exonuclease. Figure 7 shows the result of this study. It was apparent that the flanking sequence was progressively digested by Bal 31 from the expected free end. A control of this experiment is also shown in Figure 7, where an equal amount of micronuclear DNA was mixed with the macronuclear DNA before digested with Bal 31. As expected, the micronuclear flanking

Chromosome 771

Breakage

in Tetrahymena

sequence is indeed adjacent to a free DNA end in the macronucleus. Thus the restriction map presented in Figure 6 appears to be correct.

ABCD Figure 7. Sensitivity Bal 31

EFGH of rDNA Flanking

Sequence

to the Exonuclease

Tetrahymena macronuclear DNA was digested with a fixed amount of Bal 31 for various lengths of time. The DNA was extracted with phenol, digested with Bgl II. separated in 1 .O% agarose gel and then blotted for hybridization. The subclone pTt 220b of clone cTt 220 (see Experimental Procedures) was labeled by nick translation and used as a hybridization probe. pTt 220b contains 3.5 kb of the flanking sequence distal to rDNA. The control (lane A) contains DNA not treated with Bal 31. Lanes B. C and D contain DNA treated with Bal 31 for 2.5, 5 and 10 min, respectively. The flanking sequence is shortened progressively by Bal31. Lanes E. F, G and H are the same as lanes A, B, C and D except that equal amount of micronuclear DNA has been mixed with the macronuclear DNA before Bal 31 treatment. The micronuclear rDNA flanking sequence, which is slightly larger in size (5.3 kb versus 4.7 kb), remains unchanged throughout this treatment. The black bars indicate the positions of the molecular weight markers (Hind Ill digested h DNA) in these gels. They are 23.7, 9.5, 6.8, 4.3, 2.3 and 2.0 kb.

sequence was totally insensitive to Bal 31 under the same condition. It is clear that the flanking sequence of rDNA in the macronucleus is preferentially sensitive to Bal 31. In addition to the exonuclease activity, Bal 31 contains an endonuclease activity for singlestranded DNA (Gray et al., 1975, Legerski et al., 1977). Although the presence of a single-stranded gap may also render the flanking sequence sensitive to Bal 31, this structure alone can not explain the restriction map so far obtained. Furthermore, treatment with the single-stranded nuclease DNAase Sl caused no change in the flanking sequence (data not shown). For this reason I conclude that the flanking

DNA Elimination from the Macronucleus According to the restriction map presented in Figure 6 a 2.8 kb segment of the micronuclear rDNA flanking sequence is missing from the same region in the macronucleus. This sequence is not linked directly to the rest of the flanking sequence, nor is it linked to the amplified rDNA. It should be interesting to know whether it existed in any other form in the macronucleus. For this reason probes specific for this region were prepared from cTt 220 for hybridization. Two restriction fragments, Hae Ill-Bgl II-b and Sau 3A-a, each specific for a portion of the 2.8 kb gap, were isolated from two subclones of cTt 220 and used for hybridization. Figure 8 shows the results of this study. The Hae Ill-Bgl II-b fragment contained 0.5 kb of the gap region distal to the rDNA, together with 1.5 kb of the adjacent flanking sequence. It hybridized with a 5.3 kb fragment of the micronuclear DNA and a 4.7 kb fragment of the macronuclear DNA after Bgl II digestion. These two bands had previously been shown to hybridize with the flanking sequence (Figures 5 and 61, and thus were expected to hybridize with this probe. Since no additional band was detected in the macronucleus, it appeared that at least this portion of the 2.8 kb gap sequence is absent from the macronucleus. Hybridization with the Sau 3A-a fragment gave rather interesting results. This probe contains nearly all of the gap sequence not included in the Hae Ill-Bgl II-b fragment. It hybridized with numerous bands in the Bgl II-digested micronuclear DNA, including the 5.5 kb band expected from this region of the genome. Apparently this sequence, or at least part of it, is repetitive in the genome. Surprisingly, most of these bands were not found in the macronucleus. The only band detected in the macronucleus appeared to be the 3.3 kb terminal fragment of the extrachromosomal rDNA. Hybridization with this band was not surprising since the probe usually was contaminated with a small amount of the adjacent rDNA sequence. From these data it is clear that the 2.8 kb segment of DNA immediately adjacent to rDNA in the micronucleus is not present in the same location of the macronucleus, or anywhere else in this genome. It is apparently eliminated. Moreover, other micronuclear DNA sequences that share some homology with this segment of DNA are also eliminated from the macronucleus. Discussion The DNA sequence adjacent to the single integrated ribosomal RNA gene of Tetrahymena micronucleus has been isolated by cloning. Using this sequence as a hybridization probe, I found that the same sequence

Cell 772

Sau I

ahh

3A-a

HaeIII-Bgl 1 b

h I

11-b 1 ah

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A B Figure 8. Southern Hybridization with the rDNA Flanking Sequence nucleus

C

D

of Micro- and Macronuclear DNA That Is Not Present in the Macro-

The 2.8 kb segment of DNA that cannot be found linked with the rest of the macronuclear rDNA flanking sequence (labeled GAP in the diagram) is prepared in two restriction fragments Sau 3A-a and Hae Ill-Bgl h-b from the two subclones pTt 220a and pTt 220b. respectively. They are labeled by nick translation and used for hybridization with Bgl II-digested macro- and micronuclear DNA in a Southern blot. A simple diagram of the clone cTt 220 is given in the top to indicate the locations of the two fragments used, and the gap region of the flanking sequence. rDNA is represented as a heavy line in this diagram, with an arrowhead to indicate the direction of transcription of this sequence. Lanes A and B contain micro- and macronuclear DNA hybridized with the fragment Sau 3A-a. Lanes C and D contain micro- and macronuclear DNA hybridized with the fragment Hae lllBgl II-b. The gap sequence does not exist anywhere in the macronuclear genome.

exists in a different configuration in the macronucleus. Detailed restriction mapping studies suggested that the difference is probably the result of a chromosome break that occurs between the rDNA and its flanking

sequence in the macronucleus. This idea is further supported by the study using the exonuclease Bal31. The linear DNA end predicted from the digestion map is in fact sensitive to this nuclease. There is little doubt that in the macronucleus the flanking sequence is no longer linked to the rDNA, and is instead adjacent to a free DNA end. Such a structure is probably generated by chromosome breakage, although other more complicated processes could also lead to similar structures and should still be considered as formal possibilities. If chromosome breakage in fact occurs to generate the first copy of the extrachromosomal rDNA, one would expect that the sequence flanking the other end of the rDNA also be adjacent to a free DNA end in the macronucleus. Although no direct study has yet been made, the existing data do argue against any direct linkage between this flanking sequence and the rDNA, or between the two flanking sequences. It should not be surprising if this sequence is in fact adjacent to a free DNA end. Based on this information it seems probable that the extrachromosomal rDNA in Tetrahymena is generated from the integrated copy through chromosome breakage. It should be emphasized that the macronucleus of Tetrahymena is a somatic nucleus. Irreversible changes of genetic information in this nucleus do not necessarily interfere with the genetic continuity of this organism. This situation is a drastic contrast to the case of Xenopus and other higher eucaryotes, where rDNA amplification is known to occur in the oocyte. For this reason it is quite improbable that amplification in those systems could also occur by breakage without reunion. By the same argument it is not probable that chromosome breakage is involved in the generation of the extrachromosomal, palindromic rDNA observed in the two protists, Physarum and Dictyostelium Wogt and Braun, 1976; Cockburn et al., 1978). The new chromosome end generated in the macronucleus is slightly heterogeneous in size. As illustrated in Figure 5a, lanes B, H and J, the bands containing the flanking sequence of rDNA in the macronucleus were significantly broader than other bands in the same region. It is probably more than coincidence that similar observations have also been made on the free ends of the extrachromosomal rDNA of this organism (Blackburn and Gall, 1978). The range of the heterogeneity seems to be rather similar for the two cases, which has been estimated to be around 300 bp for rDNA (Blackburn and Gall, 1978). The nature of this heterogeneity is not known. Since the macronuclear DNA studied is derived from a single clone of Tetrahymena, the heterogeneity is probably the result of some somatic event, and perhaps may even be related to how these linear DNA ends are replicated. Compared with the rDNA, the flanking sequence has changed very little in multiplicity during the for-

Chromosome 773

Breakage

in Tetrahymena

mation of the macronucleus. This point is illustrated in Figure 5a where hybridization of the flanking sequence to both the macro- and the micronuclear DNA can be compared directly. Although hybridization with the macronucleus seems to be slightly higher, this difference can not be more than a fewfold if it indeed exists, and certainly is not in the same order of magnitude as that of the rDNA. Whether such a difference has any biological significance remains to be determined. The rDNA flanking sequence in the macronucleus is 2.8 kb shorter than expected. This missing sequence can not be found elsewhere in the macronucleus, and apparently is eliminated from this nucleus. How the elimination takes place is not known. One could postulate that elimination occurs by degrading the DNA from the ends generated by breakage. If this is indeed the case, then the breakage could have occurred anywhere within the 2.8 kb region. Alternatively, the 2.8 kb segment may have been removed as one piece through two breakage points and subsequently eliminated. Studies on developing macronuclei could clarify this point. It is somehow surprising that other sequences sharing homology with this region are also eliminated from the macronucleus. How the elimination of these homologous sequences are related is not known. However, the phenomenon of sequence-associated elimination has also been observed in at least two other families of repetitive DNA, and may be a rather general phenomenon in Tetrahymena (Yao and Gall, 1979; M.-C. Yao, in preparation). Ciliates are perhaps unique in showing extensive genome reorganization during development. In Tetrahymena, elimination of roughly 15% of the genome has been reported (Yao and Gorovsky, 1974; Yao and Gall, 1979). Other reorganization processes involving a simple repeated sequence have also been suggested (Yao et al., 1978; M.-C. Yao, E. H. Blackburn and J. G. Gall, submitted). More extensive reorganization has been observed in another group of ciliates, the hypotrichs, in which the majority of the genome is eliminated, and the remaining part in the macronucleus is broken down into gene-sized molecules (Lawn et al., 1977). With this information in mind it is perhaps less surprising to know that gene amplification may be associated with sequence elimination and chromosome breakage in Tetrahymena. However, chromosome breakage is by no means a unique feature of ciliates. This phenomenon has long been observed at the cytological level in many metazoa, such as Ascaris, as a process leading to chromosome diminution in somatic cells (see Wilson, 1928 for review). It has undoubtedly played an important role in the normal development of certain organisms. Further studies on Tetrahymena rDNA may shed some light on the molecular basis of these processes.

Experimental

Procedures

Cells and Growth Condition T. thermophila (originally known as Tetrahymena pyriformis syngen 1; Nanney and McCoy. 1976) strain B was obtained from P. Bruns and used for this entire study. Cells were maintained and cultured in axenic medium as described previously (Gorovsky et al., 1975). Nuclei, DNA and rDNA isolation Macro- and micronuclei were isolated as previously described (Gorovsky et al., 1975). Tetrahymena whole-cell and macronuclear DNA were isolated by phenol extraction, and the micronuclear DNA by CsCl gradient centrifugation as described (Yao et al., 1974: Gall, 1974). rDNA was purified from whole-ceil DNA or macronuclear DNA by successive centrifugation in CsCl gradients containing Hoechst 33258 dye (Wild and Gall, 1979) in a Sorvall vertical rotor TV865. Restriction Enzyme Digestion, Gel Electrophoresis and Southern Blotting Restriction enzyme Hae ill was purchased from New England BioLabs. Eco RI was either from Boehringer-Mannheim or from Miles. Hind Ill was from New England BioLabs or Bethesda Research Laboratory. Barn HI and Bgl II were gifts from H. Erba. Digestion was done under standard conditions. Twofold excess of enzyme was normally used to ensure complete digestion. Agarose gel electrophoresis was carried out in a horizontal slab gel apparatus following the method of Helling et al. (1974). Agarose was purchased from Seakem or from Sigma. Hind Ill-digested h DNA fragments were used as molecular weight markers, using the estimates of Phillipsen et al. (1978). Agarose of low gel temperature (Bio-Rad) was used when removal of DNA from the gel was desired. The gel containing the DNA was cut off and melted at 65’C. followed by phenol extraction and ether extraction to remove the agarose. The DNA was then precipitated by ethanol before use. The DNA recovered by this method was used successfully for nick translation, restriction enzyme digestion and cloning. DNA was transferred from the gel onto nitrocellulose filter following the method of Southern (1975) with slight modification. Blotting was normally done immediately following electrophoresis. Bai 31 Digestion The exonuclease Bal 31 was obtained from New England BioLabs. Digestion was carried out using the condition recommended by the supplier. Normally 3 pg of DNA was digested with 1 unit of the enzyme for each time point. After incubation, the samples were chilled in ice and EDTA was added to give a final concentration of 20 mM. The samples were extracted with phenol once, followed by ether extraction and then ethanol precipitation and washed before being dissolved for restriction enzyme digestion. DNA-DNA Hybridization DNA used as hybridization probe was labeled in vitro by nick translation (Rigby et al., 1977). s-32P-labeled dATP or dCTP (specific activity around 400 Ci/mmole) used for labeling was purchased from Amersham. Nitrocellulose filters containing DNA were first wetted with the hybridization mixture without the probe before hybridization. The mixture contained 40% formamide. 4x SSC (SSC contained 0.15 M NaCl and 0.015 M sodium citrate [pH 7.01). 0.1 M Tris-HCI (pH 7.4), 0.5% SDS and Denhardt’s solution. Hybridization was normally done at 37°C overnight. After hybridization the filter was washed extensively with 2x SSC at room temperature and then incubated in the same buffer for 30 min at 65°C. In some cases the high temperature wash was done in 0.5 x SSC. The filter was dried and mounted for autoradiography using Kodak RP-5 X-ray film and DuPont lightening plus intensifying screen.

Cloning of the integrated rDNA The procedure described by Blattner et al. (1978) was used for cloning micronuclear DNA. Total micronuclear DNA was partially digested with Eco RI and fractionated by gel electrophoresis. DNA

Cell 774

fragments between 8 kb and 20 kb in size were extracted from the gel and ligated with the vector Charon 4A DNA. The vector DNA was previously digested with Eco RI. and the two internal fragments were removed by gel electrophoresis before being used for ligation. Ligation was carried out using T4 DNA ligase obtained from W. Barnes. The ligated DNA was packaged into phage particle by the in vitro packaging procedure of Blattner et al. (1978). A total of about 5 x IO5 clones was obtained by this method and over 90% of them contained an insert. If the average size of the inserts is 15 kb, a total of 1.5 x IO’ clones would be needed to cover the entire micronuclear genome. The library was screened by the plaque hybridization method (Benton and Davies, 1977) for clones containing rDNA sequences. The rDNA probes used for the screening were prepared from the clone pTt 122 constructed by E. Stevenson. pTt 122 contains two Hind Ill fragments, the Hind Ill-b (1.95 kb) and the Hind Ill-e (0.9 kb), of the extrachromosomal rDNA of Tetrahymena in the plasmid vector pBA322. The two fragments were separated by Hind Ill digestion and gel electrophoresis before being used for hybridization. Approximately 3 x 1 O4 pfu were screened each time for the integrated gene. The clones cTt 101 and cTt 220 were obtained from two independent screens. Plaques showing positive hybridization were further purified by two more rounds of hybridization to eliminate possible contaminations. Propagation of the phage was normally done on agar plates or in liquid cultures in the host DP50supF as described by Blattner et al. (1978). Phages were normally purified in CsCl gradients before DNA isolation. For subcloning. the Tetrahymena DNA inserted in cTt 220 was removed by Eco RI digestion and gel electrophoresis. It was cleaved into two fragments by Bgl II and cloned in the large Eco RIBarn HI fragment of the vector PBR 322. The two subclones. pTt 220a and pTt 220b were isolated and verified. pTt 220a contains the rDNA and the immediate flanking sequence. pTt 220b contains the distal portion of the flanking sequence. Experiments involving recombinant DNA were carried out in a P2 facility prior to February 1980 and in a PI facility afterward in accordance with the NIH guidelines. Acknowledgments

August

19. 1980;

revised

March

27, 1981

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I thank M. Sharaf and C. H. Chang for technical assistance and D. Lazner for preparing the in vitro packaging system. This work was supported by a grant from the NIH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

Helling, 1235-I

Proc. Cold

Nat.

Spring