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Eucaryotic transposable genetic elements with inverted terminal repeats
Cell, Vol. 20. 639-647,
July 1980.
Copyright
0 1980 by MIT
Eucaryotic Transposable Genetic Inverted Terminal Repeats
Steven Potter, Martha Truett, Mark Phillips Andrew Maher Department of Biology Wesleyan University Middletown, Connecticut 06457
and
Summary DNA carrying inverted repeats was tested for transposition within the Drosophila genome. Five Barn HI segments containing related inverted repeats were isolated from D. melanogaster and analyzed by electron microscopy and restriction mapping. Southern blot experiments using single-copy flanking sequences as probes allowed the study of DNA arrangements at specific sites in the genomes of five closely related strains. We found that in some genomes the sequences with inverted repeats were present at a particular site, whereas in other genomes they were absent from this site. These results indicated that three of the sequences are transposable genetic elements. In one case we have purified the two corresponding DNA segments, with and without the sequence containing inverted repeats, thereby confirming the mobility of this sequence. These DNA elements were found to be distinct in two ways from copia and others previously described: first, they contain inverted terminal repeats, and second, they have a more heterogeneous construction. Introduction The existence of mobile DNA elements in Drosophila is well documented. In situ hybridizations to salivary gland polytene chromosomes, using cloned DNA fragments as probes, have shown that the number and chromosomal locations of certain DNA sequences are quite variable when the genomes of different D. melanogaster strains are compared (Ilyin et al., 1978; Strobel, Dunsmuir and Rubin, 1979). Furthermore, there is suggestive genetic evidence for mobile DNA resulting from the study of mutations with very high reversion rates (Green, 1977), and there is molecular evidence indicating that the elements of the dispersed repeated gene families 297, 4 12 and copia can transpose to many alternative sites in Drosophila cells grown in tissue culture (Potter et al., 1979). The previously characterized Drosophila transposable genetic elements exhibit two common structural features. First, the elements in each family have direct terminal repeats (Finnegan et al., 1978). Copia elements, for instance, carry direct repeats of 0.3 kb while 472 elements have direct repeats of 0.5 kb. Second, although the elements of a family are widely scattered throughout the genome, the sequences of
Elements
with
the elements are very closely conserved at each chromosomal site. For example, restriction mapping and filter hybridization experiments have shown that all copia elements, regardless of chromosomal location, are extremely similar if not identical (Potter et al., 1979). In this paper we describe a new class of Drosophila transposable genetic elements containing inverted terminal repeats and a heterogeneous construction. The mobility of these sequences was first indicated by Southern filter hybridizations to restriction digests of six closely related D. melanogaster genomes, using either the complete cloned segments or single-copy flanking sequences as radioactive probes. Both interand intrastrain differences in the chromosomal positions of these sequences were detected. Moreover, in one case we isolated two corresponding DNA segments, both from the strain Oregon R, that were identical except for the presence of a mobile DNA element in one of them. Results
and Discussion
A Family of Drosophila DNA Segments with Inverted Repeats In our search for new classes of transposable genetic elements we decided to isolate Drosophila DNA segments with inverted repeats (foldback DNA) and then to test these DNA sequences for mobility. A single cloned DNA segment with inverted repeats was identified in the following manner. A library of Drosophila melanogaster Oregon R Barn HI segments (Potter et al., 1979) was screened by colony filter hybridization (Grunstein and Hogness, 1975) using as a probe foldback DNA that had been purified from D. melanogaster Oregon R embryos (Baker and Thomas, 1977) and 32P-labeled in vitro by nick translation (Rigby et al., 1977). The resulting hybridization pattern was compared to the pattern generated when total genome 32P-labeled D. melanogaster DNA was used as a probe. DNA was then purified from clones giving a relatively more intense response to the foldback probe and tested for the presence of large inverted repeats by electron microscopy. To visualize the inverted repeats, the hybrid DNA molecules were cleaved with a restriction endonuclease and then denatured with alkali. The dilute DNA solution was neutralized to allow the intramolecular reannealing of the self-complementary inverted repeat sequences. The resulting structures were examined with the electron microscope, and the third clone tested was found to carry an inverted repeat (Figure 1, pDm FBl). This cloned DNA sequence, designated Dm FBl, was labeled by nick translation and used to re-screen the library of D. melanogaster Oregon R segments. (In this paper Dm FBl refers to the cloned Drosophila
Cell 640
restriction segment, and pDm FBl refers to the plasmid-Drosophila hybrid DNA molecule.) In addition, a library of Barn HI segments from Schneider tissue culture cell line 2 was screened. [Schneider cell line 2 was started with D. melanogaster Oregon Fi cells (Schneider, 19721.1 DNA was prepared from six randomly selected positives, four from the embryo DNA library and two from the Schneider cell line 2 library. (The capital S in the designation identifies clones of Schneider line 2 DNA.) These were tested by electron microscopy for the presence of foldback DNA, and four of the six did contain inverted repeat sequences (Figure 1). Restriction maps of the five DNA segments with foldbacks are shown in Figure 2. The lengths and
positions of the inverted repeats were determined by electron microscopic examination of at least two separate restriction digests of each cloned segment. For example, analysis of 67 stem-loop structures of Eco RI-digested pDm FB4 established a mean inverted repeat length of 0.90 f 0.22 kb and a mean length for the complete stem-loop structure of 3.56 f 0.44 kb. Analysis of 33 molecules of Sal l-digested pDm FB4 gave an inverted repeat length of 0.92 f 0.20 kb and a total stem-loop size of 3.66 * 0.35 kb. The lengths of the other inverted repeats and stem-loop structures are listed in Table 1. Of course the presence of inverted repeats does not prove the absence of direct repeats. We are currently examining these structures further by base
pDmS FB2
pDm FB4
,
,
pDmS FB3
pDm
FB5
5Kb Figure
1. Inverted
Repeats
in Cloned
Drosophila
DNA
After restriction cleavage the hybrid plasmids containing inserted Drosophila DNA were denatured and then allowed conditions favoring intramolecular hybridization. Inverted repeat sequences result in double-stranded stem structures were cleaved with Sal I (pDm FE1 and pDm FS4); Barn HI (pDmS FB2); Hind Ill (pDmS FB3); Xho I (pDm FB5).
to reanneal under dilute DNA (see arrows). The mclecules
Transposable 641
Genetic
Elements
Figure 2. Restriction Enzyme Barn HI Fragments of Drosophila ing Inverted Repeats
Dm FBl
The horizontal lines represent the Drosophila DNA. The cleavage sites for Barn HI (t). Kpn (V). Sal I (11, Xho I (4 1. Eco Ri (A). Xba I ( 7 ), Sac I ( A), Sst I ( & 1. Pvu II ( 4 ). Hind Ill ( t ), Bgl II (T) and Hinf I(I) are shown. Only Pvu II sites in the region of the inverted repeats of Dm FB4 and Hinf I sites in the region of the inverted repeats of Dm FB5 are shown. The horizqntal arrows indicate the DNA sequences with inverted repeats.
(G I A Aa
f -
A
t t
DmS FB2
-
DmS FB3
ffttt
Maps of Five DNA Contain-
Dm FB4
Dm FB5
sequence analysis. In the following sections we discuss blot filter hybridization experiments and purification of the corresponding DNA segment lacking the mobile DNA element. The results indicate that some of the sequences with inverted repeats are transposable genetic elements. Southern Blot Experiments Using the Entire Cloned DNA Fragment as a Probe These experiments were designed to test the genomic constancy of the DNA arrangements of the sequences homologous to those cloned. Five different geographic isolates of the D. melanogaster species were used. These strains-Oregon R, Canton S, Amherst, Swedish C and Samarkand-are morphologically indistinguishable and fully reproductively compatible, with homosequential polytene chromosome banding patterns. Hind Ill digests of each total genome DNA were electrophoresed on 0.8% agarose gels, transferred to nitrocellulose and hybridized to total cloned segments that were “P-labeled by nick translation. Examples of the resulting autoradiographs are shown in Figure 3. The pattern in a given lane can be used as a fingerprint, with each band signaling the presence of a certain size class of genomic DNA segment with homology to the probe. The significant strain differences in the patterns of hybridization shown in Figure 3 may reflect DNA rearrangements such as transpositions. If the sequences homologous to a probe were found in different genomic locations in the different strains, then the flanking restriction sites would vary and result in distinctive banding patterns. Alternatively, the pattern differences could be the result of simple base changes generating or eliminating restric-
Table 1. Sizes of Inverted Inverted Repeat
Repeat
Structures
(kb) Complete Structure
Loop
Dm FBl
0.41
f 0.07
0.82
DmS FB2
0.93
f 0.11
1.86 f 0.22
k 0.15
DmS FB3
1.31 f 0.09
1.63 + 0.18
4.25
f 0.21
Dm FB4
0.91
f 0.21
1.78
3.60
f 0.41
Dm FB5
0.19
f 0.05
0.38
f
? 0.36
0.11
tion sites, or other sequence changes not related to DNA transpositions. Therefore, although this type of experiment provides evidence suggesting DNA changes, other experiments are necessary to prove the involvement of DNA transpositions. It is interesting to note that the patterns generated by the different probes are generally similar but not identical. For example, compare the Samarkand (Sa) lanes in Figure 3 with the Dm FBl and Dm FB4 probes. Several bands are clearly common to both patterns. This indicates that many, but not all, repetitive sequences found in one cloned segment are also found in the others. The exception was Dm FB5, which under similar conditions of hybridization uniformly generated a more intense pattern with more bands, suggesting that this cloned segment contains many repetitive sequences not found in the others. Southern Blot Experiments Using Single-Copy Flanking Sequences as Hybridization Probes By using a single-copy segment of DNA (found once per haploid genome) as a hybridization probe, in conjunction with Southern blots of total genome DNA, it is possible to analyze the organization of DNA sequences at a specific chromosomal location. This
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Figure 3. Total Genome Blots Using the Entire Cloned DNA Fragments as Probes
SW A C So 0 SW A C Sa 0 SW A C So 0
Total genome DNA was cleaved with Hind III, fractionated on 0.8% agarose gels and transferred to nitrocellulose. Hybridization was to nick-translated Dm FBl, Dm FB4 or Dm FB5. The D. melanogaster strains tested ware Swedish C (SW). Amherst (A), Canton S 0. Samarkand (Sa) and Oregon R (0). Size markers are bacteriophage lambda DNA segments resulting from Hind Ill digestion (Fiandt et al., 1977).
Dm FBI
Dm FB4
strategy has previously been used in the study of globin genes (Jeffreys and Flavell, 1977), and we have used this approach to test further the hypothesis that some of these cloned DNA segments contain mobile DNA elements (mobile element hypothesis; see Figure 4). The rationale for these experiments is illustrated in Figure 4, using Dm FBl as an example. If the sequences with inverted repeats are transposable genetic elements, then they might be located at different genomic positions in the various strains tested, and, conversely, a specific chromosomal site might contain the mobile sequence in one strain and lack it in another strain. The presence or absence of the putative transposable DNA element at a particular site can be determined from the size of the corresponding restriction segment generated, as shown in Figure 4. “Filled” sites, when digested with Barn HI, will result in larger DNA segments than “empty” sites. The resulting Barn HI segment was sized by the following experiment. Total genome DNA was cleaved with the restriction endonuclease Barn HI, fractionated on agarose gels, blotted to nitrocellulose and hybridized to 32P-labeled single-copy flanking sequence (for examples see SCF sequences in Figure 5). Following autoradiography, the position of the band of hybridization reveals the size of the homologous genomic Barn HI segment. When Dm FBl was tested in this manner the result was negative. A single band of hybridization was observed for each of the genomes tested (five strains and Schneider cells). In each instance the size of the homologous Barn HI DNA segment was identical to that of the cloned Dm FBl segment, indicating that the inverted repeats were uniformly present at this site (data not shown). Dm FB5 was not tested because we
Dm FB5 Dm FE1 “Filled”
PTj Figure
AA*dr
4. Transposable
Genetic
Predicted ” Empty ” Element
Hypothesis
If the inverted repeats constitute a mobile DNA element, then at this genomic site some strains might contain the sequences while other strains would be “empty” (not have the inverted repeats). The presence or absence of the putative jumping gene at this chromosomal location can be determined by sizing the resulting Barn HI segment. Restriction endonuclease cleavage sites are marked as in Figure 2.
have not yet identified a restriction segment containing only single-copy DNA. When a similar experiment was performed using DmS FB2, the result predicted by the mobile element hypothesis was obtained. In this case, the total genome DNAs were digested with a combination of Barn HI and Eco RI in order to produce smaller restriction segments (that could be sized more accurately) with homology to the unique sequence probe (see Figures 5a and 2). The Schneider cell line 2 genome, from which DmS FB2 was cloned, gave a single band of hybridization corresponding in size to the restriction segment of DmS FB2 (with the inverted repeats). Three other genomes-Samarkand, Canton S and Swedish C-also gave single bands, but in these cases the hybridizing DNA segments were smaller by approximately the size of the inverted repeat sequences. This result matched well the prediction of our mobile element hypothesis and indicated that these three genomes were empty at this site, while the Schneider 2 genome was filled. The Oregon R strain
Transposable 643
Kb
Genetic
SwAC~aScO
Elements
b. SaScO
KbSwAC
KbSwA
Figure 5. Total Genome Copy Flanking Sequences
C kaSc0
Blots Using as Probes
Single-
Total genome DNAs were digested with Eco RI and Barn HI (a). Barn HI only(b) or Sal I and Barn HI(c). fractionated on 0.6% agarose gels and transferred to nitrocellulose. Hybridization was with the nick-translated restriction segments labeled SCF (single-copy flanker). Restriction endonuclease recognition sites are identified as in Figure 2, but only those used in cleaving the total genome or in purifying the probes are shown. The autoradiograph in (c) was exposed five times longer than those in (a) or fb) in order to locate possible faint bands. In each experiment we detect homologous DNA fragments that are smaller than the cloned segment by approximately the size of the inverted repeat structures.
5.6,
DmS FB2
DmS
FB3
SCF
contained hybridizing restriction segments of both sizes, suggesting the presence of genomic heterogeneity at this chromosomal location. Some Oregon R genomes have the mobile element at this position whereas others do not. Since the Schneider cell line 2 genomes derived from Oregon Fl are uniformly filled, this suggests the loss of genomes empty at this site during passage in tissue culture. This loss may have been random or due to the selective pressures of growth in tissue culture. In evaluating the results of three experiments of this sort we found that about one third of the DNA segments with homology to the probes were not equal in size to either filled or empty sites, and therefore were not interpretable solely on the basis of the mobile DNA element hypothesis (see Table 2). Perhaps some of these segments result from simple nucleotide sequence polymorphisms: single base pair differences can alter restriction sites. Other segments might result from undiscovered mobile DNA elements that generate length heterogeneities at the sites analyzed. Alternative explanations, such as normal insertions and deletions, are also possible. One of these bands is found in the Amherst pattern of Figure 5a. Although there is a faint band at the same mobility as found in Samarkand, Canton S and Swedish C, there is a darker band of hybridization at a slower mobility, corresponding to a slightly larger DNA segment. The results of a similar experiment with DmS FB3 are shown in Figure 5b. Once again the Schneider cell line 2 genome contains filled DNA segments and the Oregon R strain has a mixture of filled and empty sites at this chromosomal location. The three genomes-Samarkand, Canton S and Swedish Cagain show empty sites, but in addition these three strains and the Amherst strain contain a homologous DNA segment of intermediate size that is not appar-
Dm FB4 WF
Table 2. Tabulation
of Data From SW
Figure A
5 C
Sa
DmS FB2 1.86 k 0.22
3.5
DmS FB3
10.5
4.25
0.4
f 0.21 kb
Inverted repeat structure
3.60
3.5
6.4
3.5
5.6
3.5
3.5
10.5
10.5
8.4 6.9
6.9
8.3
7.35
9.7
6.8
5.45
7.8
6.5
6.5
4.35
6.7
6.4
5.4
f 0.41 kb
Inverted repeat structure
3.5
6.9
8.3
Dm FB4
0
5.6 3.6
kb
Inverted repeat structure
SC
6.9
8.3
8.3
6.5
6.5
5.4
5.4
Each number represents the size (in kb) of a homologous DNA segment. Sizes corresponding to cloned segments with inverted repeat structures are in italic. Sizes corresponding to predicted empty segments are in boldface. Genome DNAs are abbreviated as in Figure 5.
ently filled or empty. The possible significance of this DNA segment is not yet understood. However, the multiple appearance of DNA segments of the same size as empties strongly suggests that the structure in DmS FB3 with inverted terminal repeats is indeed a mobile DNA element. The most interesting results have been obtained with Dm FB4. In this case the total genome DNAs were cleaved with a combination of Barn HI and Sal I, and the unique sequence probe was the restriction segment shown in Figure 5c (SCF). The resulting hybridization pattern was similar for the Schneider cell line
Cell 644
2, Oregon R, Swedish C and Amherst genomes (with the Amherst pattern again somewhat aberrant). The Oregon R pattern, for example, contained three bands, indicating the presence of three size classes of homologous DNA segments. The largest DNA segment (faintest band) was identical in size to the cloned restriction segment, and the smallest homologous DNA segment (most intense band) had the predicted size of an empty site restriction segment. The molecular structure of the intermediate-sized DNA segment with homology is not yet known. The low intensity of the top band of hybridization suggested that only a small fraction of the genomes contained the inverted repeat sequences at this chromosomal site. To confirm and quantitate this conclusion, we performed a Southern blot reconstruction experiment. In one lane we loaded 2 pg of total Oregon R genome DNA digested with Barn HI and Sal I. Of this amount only 6.7 x 10e5 pg will be the Barn HI, Sal I segment with the inverted repeat sequences if it is present in one copy per haploid genome [(size of segment/size of genome) x 2 pg]. To serve as standards we loaded in adjacent lanes amounts of Barn HI, Sal l-digested pDm FB4 and pDm EFB4 (see below) corresponding to various copy numbers per haploid genome in the total genome lane. Both pDm FB4 and pDm EFB4 were used to monitor possible differences in transfer efficiencies. After electrophoresis, blotting, hybridization (to the SCF probe of Figure 5c) and autoradiography, the pattern shown in Figure 6 was obtained. The intensity of hybridization for the empty segment in the whole genome lane corresponded to one copy per haploid genome, and in this autoradiograph the fainter bands were not even visible (this exposure time was kept short to allow better quantitation of the empty segment). These results, including longer exposures, allowed us to estimate that fewer than one haploid genome in ten contains the structure with inverted repeats at this site. Two conclusions can be drawn from this reconstruction experiment. First, the single-copy flanking sequence used as a probe is indeed single-copy, and second, there is intergenomic heterogeneity at this site. The inverted repeat sequences are found at this chromosomal position in fewer than one genome in ten. The results with the pDm FB4 clone are particularly intriguing. In all the D. melanogaster strains examined a similar heterogeneity was found at this site, although the faint bands are not as clear in the reproductions. The Samarkand and Canton S strains have intense bands of hybridization at distinct mobilities, but in each instance there are two fainter bands at slower mobilities corresponding to DNA segments 1 .l and 3.0 kb larger (the same as for the other genomes). Perhaps in these two strains the length heterogeneity at this chromosomal site resulting from the occasional transposition(s) of a mobile DNA element(s) persists,
hblGl248
Figure
6. Gene Counting
Experiment
For this Southern blot reconstruction experiment all DNAs were cleaved with both Barn HI and Sal I. In the lanes we loaded (from left to right) 2.3, 5.7 and 11.4 X 1O-5 pg of pDm EFB4 DNA, 2 rrg of total genome D. melanogaster Oregon Fl DNA (G lane) and 14.7, 29.4, 58.8 and 117.6 x 1Oe5 pg of pDm FB4 DNA. After electrophoresis. transfer to nitrocellulose. hybridization to the flanking sequence probe (SCF of Figure SC) and autoradiography. the pattern shown resulted. The numbers above the lanes signify the equivalent copy number per haploid genome in the G lane. For example, in the lane marked 4 there is the same amount of the Barn HI. Sal I restriction segment of interest as there is in the G lane if this segment is found four times per haploid genome. The band of hybridization in the G lane most closely matches in intensity the standard lanes marked I. indicating that the genome segment without the inverted repeat structure is present in one copy per haploid genome. For this exposure time there is no visible band in the G lane corresponding to the “filled” restriction segment (containing the inverted repeats), indicating that it is present in fewer than one copy per haploid genome. For this experiment DNA concentrations were determined by the diphenylamine reaction (Burton, 1956).
but the basic length of the empty Barn HI, Sal I restriction segment has been altered. This was investigated further by repeating the whole genome Southern blot experiment of Figure 5C, using different restriction endonucleases to cleave the strain DNAs. If the strain differences were the result of random nucleotide sequence polymorphism (that is, base changes altering restriction recognition sites), then other restriction enzymes might reveal further variations which would presumably be unrelated to those shown in Figure 5C. However, if the strain differences were caused by insertions or deletions in this region of DNA, then other restriction endonucleases would reveal differences similar to those of Figure 5C, and this was in fact the result (data not shown). When Sac I was used, the Samarkand homologous restriction segments were still all 1.3 kb larger than the Oregon R segments, and the Canton S homologous segments were still 1.05 kb smaller. Therefore, at this chromosomal location, the strain differences appear to be generated by the loss or gain of DNA in this region. The experiments shown in Figure 5 were repeated three times with different batches of restriction endo-
Transposable 645
Genetic
Elements
nuclease and with different levels of enzyme excess (3-10 fold); the patterns shown were completely reproducible and therefore not the result of incomplete digestion. These results demonstrate a significant and consistent genomic variability at the Dm FB4 chromosomal site. In the Oregon R strain, for example, a few genomes contain the inverted repeat structure at this position, but most do not. We do not yet know whether this represents a population-level polymorphism, with only a small percentage of the flies containing the mobile sequences at this chromosomal location, or whether it represents a cellular-level heterogeneity, with just a few cells in each fly containing the mobile element at this site. In any event, to confirm our conclusions further it was necessary to demonstrate that the putative empty sites indeed lacked the structure with inverted repeats. Molecular Cloning of Corresponding DNA Segments Lacking the Transposable Genetic Element In the case of Dm FB4 our interpretation of the Southern blot experiments was confirmed by the isolation and characterization of the corresponding DNA segment lacking the mobile element. We used a singlecopy flanking sequence of Dm FB4 (see Figure 5c) to screen the libraries of embryo Oregon R and Schneider cell line 2 Barn HI DNA segments (a total of 45,000 clones). Only three clones with homology were found (all in the Oregon R library), again indicating that the restriction segment from Dm FB4 used as a probe was in fact a unique sequence. The three molecules identified by the library screening process were designated Dm EFB4 (E for empty) and characterized by restriction analysis. All three were the same. The results of a series of restriction digestion comparisons of Dm EFB4 and Dm FB4 are summarized in Figure 7, which illustrates that both cloned segments are identical except for the presence of 2.8 kb of DNA in Dm FB4 in the region with inverted repeats. The transposable genetic element is present in Dm FB4 and absent in Dm EFB4. It is interesting to note that the measured size of the stem-loop structure in Dm FB4 (3.5 kb) is somewhat larger than the amount of DNA present in this segment and missing in Dm EFB4 (2.8 kb). The nature of this discrepancy was analyzed by forming heteroduplexes between Dm FB4 and Dm EFB4 and examining these with the electron microscope. Figure 8 shows typical -
tit \I
Heterogeneous Construction Electron microscopic, restriction endonuclease and cross hybridization studies indicated that the inverted repeat sequences we studied have a heterogeneous construction very different from the uniform “single element” construction of the Drosophila dispersed repeated gene families such as copia. Figures 1 and 2 show that the sizes of the inverted repeats varied from one cloned segment to another, and while some inverted repeats are separated, generating the stemloop structures, others are contiguous, resulting in stems without detectable loops. The regions of cross homology were determined by cleaving the pDm FBl -pDm FB5 DNAs with restriction enzymes, fractionating on agarose gels, transferring to nitrocellulose and hybridizing to nick-translated DNA segments purified from the other clones. The notable result was that restriction segments containing inverted repeats cross-hybridized in all pairwise combinations, while restriction segments without inverted repeats did not cross-hybridize. It therefore appears probable that each inverted repeat shares a common sequence. In the case of Dm FB5 the restriction segment exhibiting cross homology (and containing the entire inverted repeat structure) was a relatively small 500 bp, but the restriction mapping for the other cloned segments is not yet as detailed. Concluding Remarks To identify a new class of transposable genetic elements, we have purified by molecular cloning a family of restriction segments containing inverted repeat sequences. When tested for mobility by filter hybridiza-
c
t f
structures. The two molecules appear to be perfectly complementary except in the region with the inverted repeat sequences; a single-stranded loop of 640 -+ 200 bp is found opposite the Dm FB4 stem-loop structure. This observation confirms our previous measurements and localizes the area of mismatch. At present, however, one can only speculate on the sequence of events that generated these differences. If the inverted repeat structure is analogous to bacterial transposons, then perhaps the transposition event placing the mobile element at this site was coupled with or followed by a deletion of sequences. It is well established that bacterial transposons are capable of causing deletions extending from their termini (Kleckner, 1977). The various possibilities are currently under further investigation.
\
tt 1
+‘+tt
I
I
a tf l-L A
A
t.
Df nt d I + Dm FE34
Figure 7. Restriction Dm EFB4
Maps
of Dm FB4 and
The two maw are identical exceot for the 2.8 kb of DNA absent in Dm EFB4. Restriction sites are identified as in Figure 2.
Dm EFB4
Cell 646
Figure EFB4
8. Heteroduplex
of Dm FB4 and
Dm
Dm FB4 and Dm EFB4 DNAs were mixed, denatured and allowed to reanneal. Two of the resulting heteroduplexes are shown. The DNAs appear to be perfectly complementary except in the region with the inverted repeats. Opposite the typical Dm FB4 stem-loop structure, as shown in Figure 1, there is a singlestranded DNA loop of 643 f 207 bp (see arrows). We speculate that the transposition of the mobile element to this site was coupled with or followed by the deletion of about 700 bp of DNA.
tion experiments using single-copy flanking sequences as probes, Dm FB1 scored as negative, but corresponding DNA segments without mobile DNA elements were found for DmS FB2, DmS FB3 and Dm FB4. In the case of Dm FB4 we have cloned the corresponding empty DNA segment and we have shown that its difference lies in the absence of the transposable genetic element. The rate of transposition is variable. For Dm FBl no transposition was observed among the six genomes examined, but for Dm FB4 there are interesting intrastrain heterogeneities, with only a small percentage of Oregon R genomes, for example, containing the mobile element at the site tested. This could represent tissue-specific differences or a population-level heterogeneity, with single flies remaining homogeneous in DNA sequence. It is intriguing to note that the heterogeneity is maintained in Schneider line 2 tissue culture cells. The eucaryotic transposable genetic elements studied in this paper are quite different from those previously described. First, these elements have inverted terminal repeats similar to those found on many bacterial transposons, and second, these elements have a variable construction very unlike the uniform construction of the dispersed repeated gene families such as copia, 412 and 297. The relative abundance of these two types of mobile sequence, as well as the mechanism(s) for transposition and the purpose(s) of the mobility, remain to be determined. Experimental
Procedures
D. melanogaster Strains The Canton S, Swedish C, Samarkand and Amherst strains were from the laboratory of B. Kiefer. The Oregon R stock came from the laboratory of M. Meselson. Enzymes Restriction endonucleases were purchased from New England labs or Bethesda Research Laboratories. E. coli DNA ligase prepared as described by Panasenko (1977).
Biowas
Nucleic Acid Preparations and Hybridizations D. melanogaster (Oregon R) embryonic DNA and plasmid DNAs were purified as described by Wensink et al. (1974). and Drosophila cell culture DNA was isolated as described by Potter et al. (1979).
DNA from adult flies was extracted in the following manner. Flies frozen with liquid nitrogen were ground to a fine powder with a mortar and pestle. 1 g of powder was mixed with 9 ml of homogenization buffer lo.1 M NaCI. 0.03 M Tris fpH 8.0). 0.01 M EDTA. 0.01 M 2mercaptoethanol. 0.5% Triton X-100] and stirred for 3 min. then dounced (on ice) and filtered through Nitex. The homogenate was centrifuged at 5000 rpm (SS34 rotor) for 10 min. and the crude nuclear pellet was resuspended in homogenization buffer without Triton X-100 and re-pelleted. This pellet was resuspended in 4.5 ml of extraction buffer [O.l M Tris (pH 8.4). 0.1 M EDTA], and 0.5 ml of 10% sodium dodecylsulfate was added followed by 500 pg of proteinase K. After incubating at 37°C for 4 hr. an equal volume of phenol [saturated with 1 M Tris (pH 7.411 was added. After two phenol extractions, one chloroform extraction and one ethanol precipitation, the nuclei acid was treated with RNAase A (100 pg/ml. 37’C. 30 min). phenol-extracted again, chloroform-extracted and ethanol-precipitated. Individual restriction segments were purified by the method of Thuring, Sanders and Borst (1975). Agarose gel electrophoresis and restriction endonuclease digestion were carried out as described by Finnegan et al. (1978). The libraries of cloned DNA restriction segments were constructed as previously described (Potter et al., 1979). The procedures used for nick-translating DNA (Rigby et al., 1977). DNA blotting (Southern. 1975) and filter hybridization (Potter et al., 1979) have also been described previously. Restriction Maps The positions of restriction endonuclease recognition sites were located by determining the lengths of DNA segments generated by single and double digests. Electron Microscopy Restriction digests of DNA were chloroform-extracted once, denatured in 0.1 M NaOH. 20 mM EDTA for 10 min at 25’C (5 pg DNA/ ml) and neutralized with one tenth volume of 1 M Tris (PH 7.0). An equal volume of formamide was added and the DNA was incubated at 25°C for 5 min. The homoduplexed DNA was then spread and stained from a hypophase containing 50% formamide as described by Davis. Simon and Davidson (1971). Micrographs were taken with a Philips 300 electron microscope and the negatives were projected onto a sheet of drawing paper. Contours of individual molecules were traced and lengths were measured from the drawings with a Numonics Digitizer Model 237. Single- and double-stranded @Xl 74 DNAs were used as length standards. (We found a 20% difference in their lengths under our conditions.) Heteroduplexes were formed as previously described (Potter et al., 1979). Acknowledgments We thank Joseph Gall for the use of his digitizer; M. E. Whitlock and Margi Goldstein for their participation in various phases of the work: and Mark Schiro for excellent technical assistance. This work was supported by a grant from the NIH.
Transposable 647
Genetic
Elements
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
December
6. 1979:
revised
April 25, 1980
References Baker, Ft. F. and Thomas, C. A. (1977). The origin and complexity of inverted repeat DNA sequences in Drosophila. In DNA Insertion Elements, Plasmids and Episomes. A. I. Bukhari. J. A. Shapiro and S. L. Adhya. eds. (New York: Cold Spring Harbor Laboratory, pp. 463489. Burton, K. (1956). A study of the conditions diphenylamine reaction for the calorimetric nucleic acid. Biochem. J. 62, 315-323.
and mechanisms of the estimation of deoxyribo-
Davis, Ft. W.. Simon, M. and Davidson, N. (1971). Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. In Methods in Enzymology, 2 1, L. Grossman and K. Moldave. eds. (New York: Academic Press), pp. 413428. Fiandt, M., Honigman. Precise measurement 2, 289-293.
A., Rosenweld. E. C. and Szybalski, W. (1977). of the b2 deletion in coliphage lambda. Gene
Finnegan, D. J.. Rubin. G. M.. Young, M. W. and Hogness. (1978). Repeated gene families in Drosophila melanogaster. Spring Harbor Symp. Quant. Biol. 42, 1053-l 063.
D. S. Cold
Green, M. M. (1977). A case for DNA insertion mutations in Drosophila. In DNA Insertion Elements, Plasmids and Episomes, A. I, Bukhari. J. A. Shapiro and S. L. Adhya, eds. (New York: Cold Spring Harbor Laboratory), pp. 437-445. Grunstein. M. and Hogness. D. S. (1975). Colony method for the isolation of cloned DNAs that contain Proc. Nat. Acad. Sci. USA 72, 3961-3965.
hybridization: a a specific gene.
Ilyin, Y. V.. Tchurikov, N. A.. Ananiev, E. V., Ryskov. A. P., Yenikolopov, G. N.. Limborska, S. A.. Maleeva. N. E.. Gvozdev, V. A. and Georgiev. G. P. (1978). Studies on the DNA fragments of mammals and Drosophila containing structural genes and adjacent sequences. Cold Spring Harbor Symp. @ant. Biol. 42, 959-969. Jeffreys, A. J. and Flavell, R. A. (1977). A physical map of the DNA regions flanking the rabbit fi-globin gene. Cell 12. 429-439. Kleckner. N. (1977). II, 11-23. Panasenko. California. Potter, (1979). persed
Translocatable
S. M. (1977).
Ph.D. thesis,
elements
in procaryotes.
Stanford
University,
Cell
Stanford,
S. S., Brorein. W. J.. Jr., Dunsmuir. P. and Rubin. G. M. Transposition of elements of the 412. copiaand 297 disrepeated gene families in Drosophila. Cell 17. 415-427.
Rigby. P. W. J.. Dieckmann. M.. Rhodes, C. and Berg, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase 1. J. Mol. Biol. 7 73. 237-251. Schneider, Drosophila Southern, fragments 51 7.
I. (1972). Cell lines derived melanogaster. J. Embryol.
from late embryonic stages Exp. Morphol. 27, 353-365.
E. M. (1975). Detection of specific separated by gel electrophoresis.
of
sequences among DNA J. Mol. Biol. 98. 503-
Strobel. E.. Dunsmuir. P. and Rubin. G. M. (1979). Polymorphisms in the chromosomal locations of elements of the 412, copiaand 297 dispersed repeated gene families in Drosophila. Cell 17, 429-439. Thuring, R. W. J.. Sanders, J. P. M. and Borst. P. (1975). A freezesqueeze method for recovering long DNA from agarose gels. Anal. Biochem. 66, 213-220. Wensink. P. C.. Finnegan, D. J., Donelson. J. E. and Hogness, D. S. (1974). A system for mappmg DNA sequences in the chromosomes of Drosophila melanogaster. Cell 3. 315-325.
July 1980.
Copyright
0 1980 by MIT
Eucaryotic Transposable Genetic Inverted Terminal Repeats
Steven Potter, Martha Truett, Mark Phillips Andrew Maher Department of Biology Wesleyan University Middletown, Connecticut 06457
and
Summary DNA carrying inverted repeats was tested for transposition within the Drosophila genome. Five Barn HI segments containing related inverted repeats were isolated from D. melanogaster and analyzed by electron microscopy and restriction mapping. Southern blot experiments using single-copy flanking sequences as probes allowed the study of DNA arrangements at specific sites in the genomes of five closely related strains. We found that in some genomes the sequences with inverted repeats were present at a particular site, whereas in other genomes they were absent from this site. These results indicated that three of the sequences are transposable genetic elements. In one case we have purified the two corresponding DNA segments, with and without the sequence containing inverted repeats, thereby confirming the mobility of this sequence. These DNA elements were found to be distinct in two ways from copia and others previously described: first, they contain inverted terminal repeats, and second, they have a more heterogeneous construction. Introduction The existence of mobile DNA elements in Drosophila is well documented. In situ hybridizations to salivary gland polytene chromosomes, using cloned DNA fragments as probes, have shown that the number and chromosomal locations of certain DNA sequences are quite variable when the genomes of different D. melanogaster strains are compared (Ilyin et al., 1978; Strobel, Dunsmuir and Rubin, 1979). Furthermore, there is suggestive genetic evidence for mobile DNA resulting from the study of mutations with very high reversion rates (Green, 1977), and there is molecular evidence indicating that the elements of the dispersed repeated gene families 297, 4 12 and copia can transpose to many alternative sites in Drosophila cells grown in tissue culture (Potter et al., 1979). The previously characterized Drosophila transposable genetic elements exhibit two common structural features. First, the elements in each family have direct terminal repeats (Finnegan et al., 1978). Copia elements, for instance, carry direct repeats of 0.3 kb while 472 elements have direct repeats of 0.5 kb. Second, although the elements of a family are widely scattered throughout the genome, the sequences of
Elements
with
the elements are very closely conserved at each chromosomal site. For example, restriction mapping and filter hybridization experiments have shown that all copia elements, regardless of chromosomal location, are extremely similar if not identical (Potter et al., 1979). In this paper we describe a new class of Drosophila transposable genetic elements containing inverted terminal repeats and a heterogeneous construction. The mobility of these sequences was first indicated by Southern filter hybridizations to restriction digests of six closely related D. melanogaster genomes, using either the complete cloned segments or single-copy flanking sequences as radioactive probes. Both interand intrastrain differences in the chromosomal positions of these sequences were detected. Moreover, in one case we isolated two corresponding DNA segments, both from the strain Oregon R, that were identical except for the presence of a mobile DNA element in one of them. Results
and Discussion
A Family of Drosophila DNA Segments with Inverted Repeats In our search for new classes of transposable genetic elements we decided to isolate Drosophila DNA segments with inverted repeats (foldback DNA) and then to test these DNA sequences for mobility. A single cloned DNA segment with inverted repeats was identified in the following manner. A library of Drosophila melanogaster Oregon R Barn HI segments (Potter et al., 1979) was screened by colony filter hybridization (Grunstein and Hogness, 1975) using as a probe foldback DNA that had been purified from D. melanogaster Oregon R embryos (Baker and Thomas, 1977) and 32P-labeled in vitro by nick translation (Rigby et al., 1977). The resulting hybridization pattern was compared to the pattern generated when total genome 32P-labeled D. melanogaster DNA was used as a probe. DNA was then purified from clones giving a relatively more intense response to the foldback probe and tested for the presence of large inverted repeats by electron microscopy. To visualize the inverted repeats, the hybrid DNA molecules were cleaved with a restriction endonuclease and then denatured with alkali. The dilute DNA solution was neutralized to allow the intramolecular reannealing of the self-complementary inverted repeat sequences. The resulting structures were examined with the electron microscope, and the third clone tested was found to carry an inverted repeat (Figure 1, pDm FBl). This cloned DNA sequence, designated Dm FBl, was labeled by nick translation and used to re-screen the library of D. melanogaster Oregon R segments. (In this paper Dm FBl refers to the cloned Drosophila
Cell 640
restriction segment, and pDm FBl refers to the plasmid-Drosophila hybrid DNA molecule.) In addition, a library of Barn HI segments from Schneider tissue culture cell line 2 was screened. [Schneider cell line 2 was started with D. melanogaster Oregon Fi cells (Schneider, 19721.1 DNA was prepared from six randomly selected positives, four from the embryo DNA library and two from the Schneider cell line 2 library. (The capital S in the designation identifies clones of Schneider line 2 DNA.) These were tested by electron microscopy for the presence of foldback DNA, and four of the six did contain inverted repeat sequences (Figure 1). Restriction maps of the five DNA segments with foldbacks are shown in Figure 2. The lengths and
positions of the inverted repeats were determined by electron microscopic examination of at least two separate restriction digests of each cloned segment. For example, analysis of 67 stem-loop structures of Eco RI-digested pDm FB4 established a mean inverted repeat length of 0.90 f 0.22 kb and a mean length for the complete stem-loop structure of 3.56 f 0.44 kb. Analysis of 33 molecules of Sal l-digested pDm FB4 gave an inverted repeat length of 0.92 f 0.20 kb and a total stem-loop size of 3.66 * 0.35 kb. The lengths of the other inverted repeats and stem-loop structures are listed in Table 1. Of course the presence of inverted repeats does not prove the absence of direct repeats. We are currently examining these structures further by base
pDmS FB2
pDm FB4
,
,
pDmS FB3
pDm
FB5
5Kb Figure
1. Inverted
Repeats
in Cloned
Drosophila
DNA
After restriction cleavage the hybrid plasmids containing inserted Drosophila DNA were denatured and then allowed conditions favoring intramolecular hybridization. Inverted repeat sequences result in double-stranded stem structures were cleaved with Sal I (pDm FE1 and pDm FS4); Barn HI (pDmS FB2); Hind Ill (pDmS FB3); Xho I (pDm FB5).
to reanneal under dilute DNA (see arrows). The mclecules
Transposable 641
Genetic
Elements
Figure 2. Restriction Enzyme Barn HI Fragments of Drosophila ing Inverted Repeats
Dm FBl
The horizontal lines represent the Drosophila DNA. The cleavage sites for Barn HI (t). Kpn (V). Sal I (11, Xho I (4 1. Eco Ri (A). Xba I ( 7 ), Sac I ( A), Sst I ( & 1. Pvu II ( 4 ). Hind Ill ( t ), Bgl II (T) and Hinf I(I) are shown. Only Pvu II sites in the region of the inverted repeats of Dm FB4 and Hinf I sites in the region of the inverted repeats of Dm FB5 are shown. The horizqntal arrows indicate the DNA sequences with inverted repeats.
(G I A Aa
f -
A
t t
DmS FB2
-
DmS FB3
ffttt
Maps of Five DNA Contain-
Dm FB4
Dm FB5
sequence analysis. In the following sections we discuss blot filter hybridization experiments and purification of the corresponding DNA segment lacking the mobile DNA element. The results indicate that some of the sequences with inverted repeats are transposable genetic elements. Southern Blot Experiments Using the Entire Cloned DNA Fragment as a Probe These experiments were designed to test the genomic constancy of the DNA arrangements of the sequences homologous to those cloned. Five different geographic isolates of the D. melanogaster species were used. These strains-Oregon R, Canton S, Amherst, Swedish C and Samarkand-are morphologically indistinguishable and fully reproductively compatible, with homosequential polytene chromosome banding patterns. Hind Ill digests of each total genome DNA were electrophoresed on 0.8% agarose gels, transferred to nitrocellulose and hybridized to total cloned segments that were “P-labeled by nick translation. Examples of the resulting autoradiographs are shown in Figure 3. The pattern in a given lane can be used as a fingerprint, with each band signaling the presence of a certain size class of genomic DNA segment with homology to the probe. The significant strain differences in the patterns of hybridization shown in Figure 3 may reflect DNA rearrangements such as transpositions. If the sequences homologous to a probe were found in different genomic locations in the different strains, then the flanking restriction sites would vary and result in distinctive banding patterns. Alternatively, the pattern differences could be the result of simple base changes generating or eliminating restric-
Table 1. Sizes of Inverted Inverted Repeat
Repeat
Structures
(kb) Complete Structure
Loop
Dm FBl
0.41
f 0.07
0.82
DmS FB2
0.93
f 0.11
1.86 f 0.22
k 0.15
DmS FB3
1.31 f 0.09
1.63 + 0.18
4.25
f 0.21
Dm FB4
0.91
f 0.21
1.78
3.60
f 0.41
Dm FB5
0.19
f 0.05
0.38
f
? 0.36
0.11
tion sites, or other sequence changes not related to DNA transpositions. Therefore, although this type of experiment provides evidence suggesting DNA changes, other experiments are necessary to prove the involvement of DNA transpositions. It is interesting to note that the patterns generated by the different probes are generally similar but not identical. For example, compare the Samarkand (Sa) lanes in Figure 3 with the Dm FBl and Dm FB4 probes. Several bands are clearly common to both patterns. This indicates that many, but not all, repetitive sequences found in one cloned segment are also found in the others. The exception was Dm FB5, which under similar conditions of hybridization uniformly generated a more intense pattern with more bands, suggesting that this cloned segment contains many repetitive sequences not found in the others. Southern Blot Experiments Using Single-Copy Flanking Sequences as Hybridization Probes By using a single-copy segment of DNA (found once per haploid genome) as a hybridization probe, in conjunction with Southern blots of total genome DNA, it is possible to analyze the organization of DNA sequences at a specific chromosomal location. This
Cell 642
Kb
Figure 3. Total Genome Blots Using the Entire Cloned DNA Fragments as Probes
SW A C So 0 SW A C Sa 0 SW A C So 0
Total genome DNA was cleaved with Hind III, fractionated on 0.8% agarose gels and transferred to nitrocellulose. Hybridization was to nick-translated Dm FBl, Dm FB4 or Dm FB5. The D. melanogaster strains tested ware Swedish C (SW). Amherst (A), Canton S 0. Samarkand (Sa) and Oregon R (0). Size markers are bacteriophage lambda DNA segments resulting from Hind Ill digestion (Fiandt et al., 1977).
Dm FBI
Dm FB4
strategy has previously been used in the study of globin genes (Jeffreys and Flavell, 1977), and we have used this approach to test further the hypothesis that some of these cloned DNA segments contain mobile DNA elements (mobile element hypothesis; see Figure 4). The rationale for these experiments is illustrated in Figure 4, using Dm FBl as an example. If the sequences with inverted repeats are transposable genetic elements, then they might be located at different genomic positions in the various strains tested, and, conversely, a specific chromosomal site might contain the mobile sequence in one strain and lack it in another strain. The presence or absence of the putative transposable DNA element at a particular site can be determined from the size of the corresponding restriction segment generated, as shown in Figure 4. “Filled” sites, when digested with Barn HI, will result in larger DNA segments than “empty” sites. The resulting Barn HI segment was sized by the following experiment. Total genome DNA was cleaved with the restriction endonuclease Barn HI, fractionated on agarose gels, blotted to nitrocellulose and hybridized to 32P-labeled single-copy flanking sequence (for examples see SCF sequences in Figure 5). Following autoradiography, the position of the band of hybridization reveals the size of the homologous genomic Barn HI segment. When Dm FBl was tested in this manner the result was negative. A single band of hybridization was observed for each of the genomes tested (five strains and Schneider cells). In each instance the size of the homologous Barn HI DNA segment was identical to that of the cloned Dm FBl segment, indicating that the inverted repeats were uniformly present at this site (data not shown). Dm FB5 was not tested because we
Dm FB5 Dm FE1 “Filled”
PTj Figure
AA*dr
4. Transposable
Genetic
Predicted ” Empty ” Element
Hypothesis
If the inverted repeats constitute a mobile DNA element, then at this genomic site some strains might contain the sequences while other strains would be “empty” (not have the inverted repeats). The presence or absence of the putative jumping gene at this chromosomal location can be determined by sizing the resulting Barn HI segment. Restriction endonuclease cleavage sites are marked as in Figure 2.
have not yet identified a restriction segment containing only single-copy DNA. When a similar experiment was performed using DmS FB2, the result predicted by the mobile element hypothesis was obtained. In this case, the total genome DNAs were digested with a combination of Barn HI and Eco RI in order to produce smaller restriction segments (that could be sized more accurately) with homology to the unique sequence probe (see Figures 5a and 2). The Schneider cell line 2 genome, from which DmS FB2 was cloned, gave a single band of hybridization corresponding in size to the restriction segment of DmS FB2 (with the inverted repeats). Three other genomes-Samarkand, Canton S and Swedish C-also gave single bands, but in these cases the hybridizing DNA segments were smaller by approximately the size of the inverted repeat sequences. This result matched well the prediction of our mobile element hypothesis and indicated that these three genomes were empty at this site, while the Schneider 2 genome was filled. The Oregon R strain
Transposable 643
Kb
Genetic
SwAC~aScO
Elements
b. SaScO
KbSwAC
KbSwA
Figure 5. Total Genome Copy Flanking Sequences
C kaSc0
Blots Using as Probes
Single-
Total genome DNAs were digested with Eco RI and Barn HI (a). Barn HI only(b) or Sal I and Barn HI(c). fractionated on 0.6% agarose gels and transferred to nitrocellulose. Hybridization was with the nick-translated restriction segments labeled SCF (single-copy flanker). Restriction endonuclease recognition sites are identified as in Figure 2, but only those used in cleaving the total genome or in purifying the probes are shown. The autoradiograph in (c) was exposed five times longer than those in (a) or fb) in order to locate possible faint bands. In each experiment we detect homologous DNA fragments that are smaller than the cloned segment by approximately the size of the inverted repeat structures.
5.6,
DmS FB2
DmS
FB3
SCF
contained hybridizing restriction segments of both sizes, suggesting the presence of genomic heterogeneity at this chromosomal location. Some Oregon R genomes have the mobile element at this position whereas others do not. Since the Schneider cell line 2 genomes derived from Oregon Fl are uniformly filled, this suggests the loss of genomes empty at this site during passage in tissue culture. This loss may have been random or due to the selective pressures of growth in tissue culture. In evaluating the results of three experiments of this sort we found that about one third of the DNA segments with homology to the probes were not equal in size to either filled or empty sites, and therefore were not interpretable solely on the basis of the mobile DNA element hypothesis (see Table 2). Perhaps some of these segments result from simple nucleotide sequence polymorphisms: single base pair differences can alter restriction sites. Other segments might result from undiscovered mobile DNA elements that generate length heterogeneities at the sites analyzed. Alternative explanations, such as normal insertions and deletions, are also possible. One of these bands is found in the Amherst pattern of Figure 5a. Although there is a faint band at the same mobility as found in Samarkand, Canton S and Swedish C, there is a darker band of hybridization at a slower mobility, corresponding to a slightly larger DNA segment. The results of a similar experiment with DmS FB3 are shown in Figure 5b. Once again the Schneider cell line 2 genome contains filled DNA segments and the Oregon R strain has a mixture of filled and empty sites at this chromosomal location. The three genomes-Samarkand, Canton S and Swedish Cagain show empty sites, but in addition these three strains and the Amherst strain contain a homologous DNA segment of intermediate size that is not appar-
Dm FB4 WF
Table 2. Tabulation
of Data From SW
Figure A
5 C
Sa
DmS FB2 1.86 k 0.22
3.5
DmS FB3
10.5
4.25
0.4
f 0.21 kb
Inverted repeat structure
3.60
3.5
6.4
3.5
5.6
3.5
3.5
10.5
10.5
8.4 6.9
6.9
8.3
7.35
9.7
6.8
5.45
7.8
6.5
6.5
4.35
6.7
6.4
5.4
f 0.41 kb
Inverted repeat structure
3.5
6.9
8.3
Dm FB4
0
5.6 3.6
kb
Inverted repeat structure
SC
6.9
8.3
8.3
6.5
6.5
5.4
5.4
Each number represents the size (in kb) of a homologous DNA segment. Sizes corresponding to cloned segments with inverted repeat structures are in italic. Sizes corresponding to predicted empty segments are in boldface. Genome DNAs are abbreviated as in Figure 5.
ently filled or empty. The possible significance of this DNA segment is not yet understood. However, the multiple appearance of DNA segments of the same size as empties strongly suggests that the structure in DmS FB3 with inverted terminal repeats is indeed a mobile DNA element. The most interesting results have been obtained with Dm FB4. In this case the total genome DNAs were cleaved with a combination of Barn HI and Sal I, and the unique sequence probe was the restriction segment shown in Figure 5c (SCF). The resulting hybridization pattern was similar for the Schneider cell line
Cell 644
2, Oregon R, Swedish C and Amherst genomes (with the Amherst pattern again somewhat aberrant). The Oregon R pattern, for example, contained three bands, indicating the presence of three size classes of homologous DNA segments. The largest DNA segment (faintest band) was identical in size to the cloned restriction segment, and the smallest homologous DNA segment (most intense band) had the predicted size of an empty site restriction segment. The molecular structure of the intermediate-sized DNA segment with homology is not yet known. The low intensity of the top band of hybridization suggested that only a small fraction of the genomes contained the inverted repeat sequences at this chromosomal site. To confirm and quantitate this conclusion, we performed a Southern blot reconstruction experiment. In one lane we loaded 2 pg of total Oregon R genome DNA digested with Barn HI and Sal I. Of this amount only 6.7 x 10e5 pg will be the Barn HI, Sal I segment with the inverted repeat sequences if it is present in one copy per haploid genome [(size of segment/size of genome) x 2 pg]. To serve as standards we loaded in adjacent lanes amounts of Barn HI, Sal l-digested pDm FB4 and pDm EFB4 (see below) corresponding to various copy numbers per haploid genome in the total genome lane. Both pDm FB4 and pDm EFB4 were used to monitor possible differences in transfer efficiencies. After electrophoresis, blotting, hybridization (to the SCF probe of Figure 5c) and autoradiography, the pattern shown in Figure 6 was obtained. The intensity of hybridization for the empty segment in the whole genome lane corresponded to one copy per haploid genome, and in this autoradiograph the fainter bands were not even visible (this exposure time was kept short to allow better quantitation of the empty segment). These results, including longer exposures, allowed us to estimate that fewer than one haploid genome in ten contains the structure with inverted repeats at this site. Two conclusions can be drawn from this reconstruction experiment. First, the single-copy flanking sequence used as a probe is indeed single-copy, and second, there is intergenomic heterogeneity at this site. The inverted repeat sequences are found at this chromosomal position in fewer than one genome in ten. The results with the pDm FB4 clone are particularly intriguing. In all the D. melanogaster strains examined a similar heterogeneity was found at this site, although the faint bands are not as clear in the reproductions. The Samarkand and Canton S strains have intense bands of hybridization at distinct mobilities, but in each instance there are two fainter bands at slower mobilities corresponding to DNA segments 1 .l and 3.0 kb larger (the same as for the other genomes). Perhaps in these two strains the length heterogeneity at this chromosomal site resulting from the occasional transposition(s) of a mobile DNA element(s) persists,
hblGl248
Figure
6. Gene Counting
Experiment
For this Southern blot reconstruction experiment all DNAs were cleaved with both Barn HI and Sal I. In the lanes we loaded (from left to right) 2.3, 5.7 and 11.4 X 1O-5 pg of pDm EFB4 DNA, 2 rrg of total genome D. melanogaster Oregon Fl DNA (G lane) and 14.7, 29.4, 58.8 and 117.6 x 1Oe5 pg of pDm FB4 DNA. After electrophoresis. transfer to nitrocellulose. hybridization to the flanking sequence probe (SCF of Figure SC) and autoradiography. the pattern shown resulted. The numbers above the lanes signify the equivalent copy number per haploid genome in the G lane. For example, in the lane marked 4 there is the same amount of the Barn HI. Sal I restriction segment of interest as there is in the G lane if this segment is found four times per haploid genome. The band of hybridization in the G lane most closely matches in intensity the standard lanes marked I. indicating that the genome segment without the inverted repeat structure is present in one copy per haploid genome. For this exposure time there is no visible band in the G lane corresponding to the “filled” restriction segment (containing the inverted repeats), indicating that it is present in fewer than one copy per haploid genome. For this experiment DNA concentrations were determined by the diphenylamine reaction (Burton, 1956).
but the basic length of the empty Barn HI, Sal I restriction segment has been altered. This was investigated further by repeating the whole genome Southern blot experiment of Figure 5C, using different restriction endonucleases to cleave the strain DNAs. If the strain differences were the result of random nucleotide sequence polymorphism (that is, base changes altering restriction recognition sites), then other restriction enzymes might reveal further variations which would presumably be unrelated to those shown in Figure 5C. However, if the strain differences were caused by insertions or deletions in this region of DNA, then other restriction endonucleases would reveal differences similar to those of Figure 5C, and this was in fact the result (data not shown). When Sac I was used, the Samarkand homologous restriction segments were still all 1.3 kb larger than the Oregon R segments, and the Canton S homologous segments were still 1.05 kb smaller. Therefore, at this chromosomal location, the strain differences appear to be generated by the loss or gain of DNA in this region. The experiments shown in Figure 5 were repeated three times with different batches of restriction endo-
Transposable 645
Genetic
Elements
nuclease and with different levels of enzyme excess (3-10 fold); the patterns shown were completely reproducible and therefore not the result of incomplete digestion. These results demonstrate a significant and consistent genomic variability at the Dm FB4 chromosomal site. In the Oregon R strain, for example, a few genomes contain the inverted repeat structure at this position, but most do not. We do not yet know whether this represents a population-level polymorphism, with only a small percentage of the flies containing the mobile sequences at this chromosomal location, or whether it represents a cellular-level heterogeneity, with just a few cells in each fly containing the mobile element at this site. In any event, to confirm our conclusions further it was necessary to demonstrate that the putative empty sites indeed lacked the structure with inverted repeats. Molecular Cloning of Corresponding DNA Segments Lacking the Transposable Genetic Element In the case of Dm FB4 our interpretation of the Southern blot experiments was confirmed by the isolation and characterization of the corresponding DNA segment lacking the mobile element. We used a singlecopy flanking sequence of Dm FB4 (see Figure 5c) to screen the libraries of embryo Oregon R and Schneider cell line 2 Barn HI DNA segments (a total of 45,000 clones). Only three clones with homology were found (all in the Oregon R library), again indicating that the restriction segment from Dm FB4 used as a probe was in fact a unique sequence. The three molecules identified by the library screening process were designated Dm EFB4 (E for empty) and characterized by restriction analysis. All three were the same. The results of a series of restriction digestion comparisons of Dm EFB4 and Dm FB4 are summarized in Figure 7, which illustrates that both cloned segments are identical except for the presence of 2.8 kb of DNA in Dm FB4 in the region with inverted repeats. The transposable genetic element is present in Dm FB4 and absent in Dm EFB4. It is interesting to note that the measured size of the stem-loop structure in Dm FB4 (3.5 kb) is somewhat larger than the amount of DNA present in this segment and missing in Dm EFB4 (2.8 kb). The nature of this discrepancy was analyzed by forming heteroduplexes between Dm FB4 and Dm EFB4 and examining these with the electron microscope. Figure 8 shows typical -
tit \I
Heterogeneous Construction Electron microscopic, restriction endonuclease and cross hybridization studies indicated that the inverted repeat sequences we studied have a heterogeneous construction very different from the uniform “single element” construction of the Drosophila dispersed repeated gene families such as copia. Figures 1 and 2 show that the sizes of the inverted repeats varied from one cloned segment to another, and while some inverted repeats are separated, generating the stemloop structures, others are contiguous, resulting in stems without detectable loops. The regions of cross homology were determined by cleaving the pDm FBl -pDm FB5 DNAs with restriction enzymes, fractionating on agarose gels, transferring to nitrocellulose and hybridizing to nick-translated DNA segments purified from the other clones. The notable result was that restriction segments containing inverted repeats cross-hybridized in all pairwise combinations, while restriction segments without inverted repeats did not cross-hybridize. It therefore appears probable that each inverted repeat shares a common sequence. In the case of Dm FB5 the restriction segment exhibiting cross homology (and containing the entire inverted repeat structure) was a relatively small 500 bp, but the restriction mapping for the other cloned segments is not yet as detailed. Concluding Remarks To identify a new class of transposable genetic elements, we have purified by molecular cloning a family of restriction segments containing inverted repeat sequences. When tested for mobility by filter hybridiza-
c
t f
structures. The two molecules appear to be perfectly complementary except in the region with the inverted repeat sequences; a single-stranded loop of 640 -+ 200 bp is found opposite the Dm FB4 stem-loop structure. This observation confirms our previous measurements and localizes the area of mismatch. At present, however, one can only speculate on the sequence of events that generated these differences. If the inverted repeat structure is analogous to bacterial transposons, then perhaps the transposition event placing the mobile element at this site was coupled with or followed by a deletion of sequences. It is well established that bacterial transposons are capable of causing deletions extending from their termini (Kleckner, 1977). The various possibilities are currently under further investigation.
\
tt 1
+‘+tt
I
I
a tf l-L A
A
t.
Df nt d I + Dm FE34
Figure 7. Restriction Dm EFB4
Maps
of Dm FB4 and
The two maw are identical exceot for the 2.8 kb of DNA absent in Dm EFB4. Restriction sites are identified as in Figure 2.
Dm EFB4
Cell 646
Figure EFB4
8. Heteroduplex
of Dm FB4 and
Dm
Dm FB4 and Dm EFB4 DNAs were mixed, denatured and allowed to reanneal. Two of the resulting heteroduplexes are shown. The DNAs appear to be perfectly complementary except in the region with the inverted repeats. Opposite the typical Dm FB4 stem-loop structure, as shown in Figure 1, there is a singlestranded DNA loop of 643 f 207 bp (see arrows). We speculate that the transposition of the mobile element to this site was coupled with or followed by the deletion of about 700 bp of DNA.
tion experiments using single-copy flanking sequences as probes, Dm FB1 scored as negative, but corresponding DNA segments without mobile DNA elements were found for DmS FB2, DmS FB3 and Dm FB4. In the case of Dm FB4 we have cloned the corresponding empty DNA segment and we have shown that its difference lies in the absence of the transposable genetic element. The rate of transposition is variable. For Dm FBl no transposition was observed among the six genomes examined, but for Dm FB4 there are interesting intrastrain heterogeneities, with only a small percentage of Oregon R genomes, for example, containing the mobile element at the site tested. This could represent tissue-specific differences or a population-level heterogeneity, with single flies remaining homogeneous in DNA sequence. It is intriguing to note that the heterogeneity is maintained in Schneider line 2 tissue culture cells. The eucaryotic transposable genetic elements studied in this paper are quite different from those previously described. First, these elements have inverted terminal repeats similar to those found on many bacterial transposons, and second, these elements have a variable construction very unlike the uniform construction of the dispersed repeated gene families such as copia, 412 and 297. The relative abundance of these two types of mobile sequence, as well as the mechanism(s) for transposition and the purpose(s) of the mobility, remain to be determined. Experimental
Procedures
D. melanogaster Strains The Canton S, Swedish C, Samarkand and Amherst strains were from the laboratory of B. Kiefer. The Oregon R stock came from the laboratory of M. Meselson. Enzymes Restriction endonucleases were purchased from New England labs or Bethesda Research Laboratories. E. coli DNA ligase prepared as described by Panasenko (1977).
Biowas
Nucleic Acid Preparations and Hybridizations D. melanogaster (Oregon R) embryonic DNA and plasmid DNAs were purified as described by Wensink et al. (1974). and Drosophila cell culture DNA was isolated as described by Potter et al. (1979).
DNA from adult flies was extracted in the following manner. Flies frozen with liquid nitrogen were ground to a fine powder with a mortar and pestle. 1 g of powder was mixed with 9 ml of homogenization buffer lo.1 M NaCI. 0.03 M Tris fpH 8.0). 0.01 M EDTA. 0.01 M 2mercaptoethanol. 0.5% Triton X-100] and stirred for 3 min. then dounced (on ice) and filtered through Nitex. The homogenate was centrifuged at 5000 rpm (SS34 rotor) for 10 min. and the crude nuclear pellet was resuspended in homogenization buffer without Triton X-100 and re-pelleted. This pellet was resuspended in 4.5 ml of extraction buffer [O.l M Tris (pH 8.4). 0.1 M EDTA], and 0.5 ml of 10% sodium dodecylsulfate was added followed by 500 pg of proteinase K. After incubating at 37°C for 4 hr. an equal volume of phenol [saturated with 1 M Tris (pH 7.411 was added. After two phenol extractions, one chloroform extraction and one ethanol precipitation, the nuclei acid was treated with RNAase A (100 pg/ml. 37’C. 30 min). phenol-extracted again, chloroform-extracted and ethanol-precipitated. Individual restriction segments were purified by the method of Thuring, Sanders and Borst (1975). Agarose gel electrophoresis and restriction endonuclease digestion were carried out as described by Finnegan et al. (1978). The libraries of cloned DNA restriction segments were constructed as previously described (Potter et al., 1979). The procedures used for nick-translating DNA (Rigby et al., 1977). DNA blotting (Southern. 1975) and filter hybridization (Potter et al., 1979) have also been described previously. Restriction Maps The positions of restriction endonuclease recognition sites were located by determining the lengths of DNA segments generated by single and double digests. Electron Microscopy Restriction digests of DNA were chloroform-extracted once, denatured in 0.1 M NaOH. 20 mM EDTA for 10 min at 25’C (5 pg DNA/ ml) and neutralized with one tenth volume of 1 M Tris (PH 7.0). An equal volume of formamide was added and the DNA was incubated at 25°C for 5 min. The homoduplexed DNA was then spread and stained from a hypophase containing 50% formamide as described by Davis. Simon and Davidson (1971). Micrographs were taken with a Philips 300 electron microscope and the negatives were projected onto a sheet of drawing paper. Contours of individual molecules were traced and lengths were measured from the drawings with a Numonics Digitizer Model 237. Single- and double-stranded @Xl 74 DNAs were used as length standards. (We found a 20% difference in their lengths under our conditions.) Heteroduplexes were formed as previously described (Potter et al., 1979). Acknowledgments We thank Joseph Gall for the use of his digitizer; M. E. Whitlock and Margi Goldstein for their participation in various phases of the work: and Mark Schiro for excellent technical assistance. This work was supported by a grant from the NIH.
Transposable 647
Genetic
Elements
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
December
6. 1979:
revised
April 25, 1980
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