MOLECULAR AND CELLULAR BIOLOGY, Apr. 1984, p. 671-680 Vol. 4, No. 4 0270-7306/84/040671-10$02.00/0 Copyright C 1984, American Society for Microbiology Genomic Instability and in Regions Surrounding Two Discoidin I of Dictyostelium discoideum STEPHEN J. POOLEt AND RICHARD A. FIRTEL* Department of Biology, University of California, San Diego, La Jolla, California 92093 Received 29 August 1983/Accepted 22 December 1983

We have found that the genomic regions surrounding the linked discoidin I genes of various Dictyostelium discoideum strains have undergone rapid changes. Wild-type strain NC-4 has three complete discoidin I genes; its axenic derivative strain Ax-3L has duplicated a region starting -1 kilobase upstream from the two linked genes and extending for at least 8 kilobases past the genes. A separately maintained stock, strain Ax- 3K, does not have this duplication but has undergone a different rearrangement -3 kilobases farther upstream. We show that there are repeat elements in these rapidly changing regions. At least two of these elements, Tdd-2 and Tdd-3, have characteristics associated with mobile genetic elements. The Tdd-3 element is found in different locations in related strains and causes a 9- to 10-base-pair duplication of the target site DNA. The Tdd-2 and Tdd-3 elements do not cross-hybridize, but they share a 22-base-pair homology near one end. At two separate sites, the Tdd-3 element has transposed into the Tdd-2 element, directly adjacent to the 22-base-pair homology. The Tdd-3 element may use this 22-base-pair region as a preferential site of insertion.

Transposable genetic elements constitute a fluid compo- sequences. In addition, we show in this paper that strain NC- nent of a number of eucaryotic (31), including those 4 has three full discoidin I genes, but strain Ax-3L, the of yeasts (1), nematodes (4), and Drosophila melanogaster source of DNA for our previously published reports (20, 24), (28), and can be associated with a variety of chromosomal has duplicated a region containing the two linked discoidin I rearrangements (5, 6, 14, 25). We have been investigating genes. To reflect this we are adopting a nomenclature system chromosomal changes around the discoidin I genes of Dic- consistent with that of Devine et al. (3). To avoid confusion tyostelium discoideum. Discoidin I is a developmentally with previous reports from our laboratory (20), we designate regulated carbohydrate-binding protein encoded by a small different genes with Greek letters rather than with the capital multigene family (3, 20, 21-24). Previously we showed that a letters used by Devine et al. (3). In this system, the that particular strain of D. discoideum (Ax-3L) has a discrepancy we formerly designated Discl-A is now Discl-oa, the former in the number of genomic fragments hybridizing probes Discl-D gene is now Discl-1, and the Discl-C gene is now specific for 5' halves of discoidin I genes versus 3' halves Discl--y. These are the three complete genes of strain NC-4. (20). To investigate this we have been examining the organi- This strain also contains a gene fragment which we designate zation of the discoidin I genes in other strains, and in this DiscI-k,. The region containing the Discl-y, Discl-1, and paper we show that the regions around two of the discoidin I DiscI-ql genes has duplicated in strain Ax-3L, yielding the genes have undergone rapid changes, including duplications DiscI-y', DiscI-,', and Discl-gi' genes (20; this paper). Thus, and rearrangements. Within these regions are several repeti- the gene that we formerly designated Discl-B is now DiscI- tive elements whose patterns of hybridization differ among y'. The Discl-a, DiscI-, and Discl-y genes encode the different strains of D. discoideum. Two of these repeats discoidin Ia, lb, and Ic polypeptides, respectively, as desig- share many of the characteristics associated with mobile nated by Devine et al. (3). genetic elements, including a conserved structure, different D. discoideum strains. Strain Ax-3 is an axenic derivative sites of insertion among different strains, and movement. of wild-type strain NC-4 and can grow in liquid culture in the Unlike other eucaryotic transposable elements, these two D. absence of any (15, 16). Two stocks of this strain are discoideum elements do not have terminal repeats. The two used in this report. Strain Ax-3L is a stock of Ax-3 obtained elements do not cross-hybridize but do share a 22-base-pair from William F. Loomis (University of California, San (bp) region of homology near one end. One of the elements Diego, Calif.). Strain Ax-3K is a separately maintained stock appears to use this region of homology as a preferential site of Ax-3 obtained from Richard Kessin (Harvard University, of insertion and causes a 9- to 10-bp duplication of the target Boston, Mass.). Strains MF-B1, MF-B2, MF-D, MF-G, site DNA upon insertion. We suggest that the fluidity around DdK1O, and Dd-Ohio are strains newly isolated from the the discoidin I genes is caused by these mobile genetic wild type and were provided to us by David Francis (Univer- elements. sity of Delaware, Newark, Del.). These were grown under the same conditions as was strain NC-4 (12). Vegetatively MATERIALS AND METHODS growing cells were washed free of bacteria, and DNA was Genetic nomenclature. Devine et al. (3) have correlated the isolated as described previously (12). Growth of strain Ax-3 three isoelectric forms of discoidin I with three mRNA and isolation of DNA have been described previously (12). General methods. Most methods used were essentially as described in Maniatis et al. (17). Construction and screening * Corresponding author. of a strain Ax-3L genomic BamHI library in X BF101 was t Present address: Department of Biochemistry and Biophysics, done as described in Maniatis et al. (18). After identification, University of California, San Francisco, CA 94143. the BamHI insert was cloned into the vehicle pXF3, yielding

671 672 POOLE AND FIRTEL MOL. . BIOL.

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3.0- * - S 3g3313 -5£6 *;*t3; ,3w -4.6 _~ 1.8 - 1.4- go d ib ...

FIG. 1. Genomic EcoRI blots of D. discoideum strains. DNA from the various strains was digested with EcoRI, separated on a 0.8% agarose gel, transferred to nitrocellulose, and hybridized with various nick-translated probes. The strains used were: MF, MF-G (strains MF- B1, MF-B2, and MF-D looked identical); K, K10; Oh, Ohio; NC, NC-4; AL, Ax-3L; AK, Ax-3K. Sizes in kb pairs are indicated. The probes used were: (A) purified insert of pcDdI8, a discoidin I cDNA clone spanning most of the discoidin I coding region (24); (B) fragment A of Fig. 2; and (C) an actin cDNA clone (pcDd actin B1) (8).

pDdB14. Construction, screening, and subcloning of a strain strains recently isolated from the wild type. It is apparent Ax-3K EcoRI library in X Charon 13 was done in a similar that these strains show discoidin I hybridization patterns manner to yield pDdDiscK5.1. that differ from those of strain Ax-3L (lane AL), although Purified fragments to be used as probes were either nick there are certain bands that are conserved in size between translated or subcloned into M13mp8 of M13mp9 (19) and most of the strains. Most strains show the 1.8- and 10- labeled with the M13 hybridization probe primer from New kilobase (kb) bands of hybridization containing the DiscI-a England Biolabs, Beverly, Mass., following instructions gene. All strains have a band of hybridization at 1.4 kb and provided by the supplier. M13 strains were a gift of K. thus have the two tandemly linked Discl--y and DiscI-, genes Buckley. Southern (27) blot filters were hybridized at 37°C in in a similar arrangement (see Fig. 2). However, the remain- 50% formamide-3x SSC (lx SSC is 0.15 M NaCl plus 0.015 ing fragments which contain the front half of the DiscI-y M sodium citrate)-0.12 M sodium phosphate (pH 7.2)-10 gene and the back half of the DiscI-P gene and flanking mM EDTA-1% Sarkosyl-2x Denhardt solution (2). sequences show considerable variation in size among the DNA sequencing. DNA sequencing (26) was done with the different strains. Similar results are seen when other en- M13 sequencing kit of Bethesda Research Laboratories, zymes are used (data not shown). This variation is not Gaithersburg, Md., following the instructions provided by typical of all D. discoideum genomic sequences since the the supplier but with modifications as detailed elsewhere sequences surrounding the 17 to 20 actin genes do not exhibit (S. J. Poole and R. A. Firtel, J. Mol. Biol., in press). For a high degree of restriction site polymorphism (Fig. 1C). The regions of extremely high A+T composition, the 24-bp two linked discoidin I genes are, therefore, in a region that primer (Bethesda Research Laboratories) was used, and has an unusually high degree of strain differences. reactions were carried out at 43°C. Regions beyond homo- These differences are seen not only among the newly polymer deoxyribosyladenine or deoxyribosylthymine isolated wild-type D. discoideum strains, but also among stretches greater than -20 were difficult or impossible to related laboratory strains. Figure 1 shows Southern blots of read reliably. DNA isolated from three closely related strains: wild-type strain NC-4 (lane NC) and two separately maintained stocks of its axenic derivative, strain Ax-3 (lanes AL and AK). The RESULTS pattern of discoidin I hybridization clearly differs among the Regions surrounding two discoidin I genes rapidly change. three isolates, whereas the actin pattern is the same. The We previously studied the organization of the discoidin I discoidin I pattern of strain Ax-3L (lane AL) is the same as genes in strain Ax-3L (20), an axenic strain derived from the NC-4 pattern (lane NC) but with an extra 3.0-kb band. wild-type strain NC-4. We showed that this strain has at The genomic region containing this newly appearing discoi- least four discoidin I genes, two of which are tandemly din I gene (the DiscI--y' gene) has been cloned and described linked with -500 bp separating the two genes (20; Poole and (20) (see map, Fig. 2) and results from a duplication of the Firtel, in press). We have now examined the organization of genomic region containing the two linked discoidin I genes. the genes in other strains ofD. discoideum. Figure 1A shows The duplicated region in strain Ax-3L starts -1 kb 5' to the a genomic Southern blot of EcoRI-digested DNA isolated two tandemly linked genes and likely extends leftward for at from various strains of D. discoideum and hybridized with a least 8 kb. The duplication results in only one new EcoRI discoidin I cDNA probe (see above for origins of strains). band of hybridization in strain Ax-3L, since the remaining The first three lanes contain DNA of several D. discoideum fragments within the duplication are identical in size with the VOL. 4. 1984 MOBILE ELEMENTS AND DISCOIDIN I GENES 673

LL 0 z I Y LL U -j> parental fragments. This results in the discrepancy between A 2 te ° z < <: 2 z < the discoidin I 5' and 3' hybridizations noted above. B 0o 4 Thus, strain Ax-3L, one isolate of an axenic derivative of NC-4, has at least five full discoidin I genes (and two ), with the extra genes resulting from the dupli- cation event. This duplication has occurred relatively recent- 10.- ly since a sibling axenic stock of Ax-3, strain Ax-3K, does not have the 3-kb EcoRI fragment indicative of the duplica- - _ tion (Fig. 1A). This strain has the same number of EcoRI _ 40 * |4 -3.6 bands of hybridization as does NC-4 and a similar pattern of hybridization for four of the bands. Note, however, that in strain Ax-3K the 7.2-kb EcoRI fragment, representing the region 5' to the Discl-y gene, is not present and has been 1.8- replaced by a new band of 5.1 kb (cf. Fig. 1A, lanes NC and AK). As will be shown below, this new band results from the insertion of a mobile genetic element 4.5 kb upstream from the Discl-y gene. Thus, in the time since the separation of these two sibling strains, both have undergone some type of FIG. 3. A conserved repeat element lies upstream from the DNA rearrangements involving regions 5' to the Discl-y Discl-y gene. DNA from the various strains was digested with either gene. EcoRI or BgIII, separated on a 0.8% agarose gel, transferred to Linked discoidin I genes are surrounded by repeats. We nitrocellulose, and hybridized with a nick-translated probe made wished to determine whether there were unusual aspects of from the rightmost PstI-EcoRl fragment of pDdDiscI-CD. See the the DNA surrounding the discoidin I genes which could legend to Fig. 1 for strains used. (A) Southern blot of EcoRI- digested DNA. (B) Southern blot of BglII-digested DNA. DISCOIDIN I GENE ORGANIZATION

Tdd-2 account for the high degree of variation in these regions. E Ev Xb He H E H E H XHeH B B BH HeXB PHe E Previously we showed that there are numerous repetitive L- Ir Ir ')Ir )IrII 1 elements in these regions (20). A region between the Discl-,B

I gene and the fragment shares a moderately A. STRAIN NC-4 kb- with a region upstream from the DiscI--y' duplication (fragments A and B, Fig. 2B). There is a region A Tdd-2 encompassing the XhoI-HaeIII sites -1 kb upstream from E Ev Xb He H E H E H XHe H B B H HeXB P He E w I r the Discl--y gene that is inversely repeated 4 kb farther pDdDisc 56 / pDdDiscl-CD upstream (short horizontal arrows, Fig. 2) (20; Poole and B Firtel, in press). We have identified at least three other E Ev Xb He H E H E H X He He E B H repetitive elements in the region upstream from the Discl--y L I_lllI 4'I /3' ~'' - gene, including one at the distal end of the 7.2-kb EcoRI pDdDisc I-B fragment cloned in pDdDiscl-CD (20; unpublished data). B. STRAIN Ax-3L We have examined the hybridization patterns of these repeats in the different strains and have found that the patterns differ among the strains. For example, when the 0.9- Tdd-3 kb XbaI-HaeIII fragment from the region 3' to the Discl--y- E Ev Xb He I1H 1E HA1f E 1H 1AX HeIArK~~~~~~B B B H He X BE Discl-1 gene pair (fragment A, Fig. 2B) is hybridized to a Southern blot ofEcoRI-digested DNA (Fig. 1B), the patterns pDdDiscK 51 of hybridization are quite different among the various strains, although NC-4 and its two derived Ax-3 strains show C. STRAN Ax-3K similar patterns. Since both actin (Fig. 1C) and the M4 FIG. 2.~~~~~~~~~ICLinked discoidin I genes of NC-4 and derivative strains. repeat, a short (A. Kimmel, personal The restriction maps of the regions containing the linked X and y communication), show little restriction site polymorphism in genes. This region also contains a 139-bp pseudogene fragment (Poole and Firtel, in press) (see text for gene nomenclature). To these strains, the different repeat patterns (e.g., Fig. 1B) emphasize similarities, the regions that are homologous among the most likely indicate DNA rearrangements of some type, strains have been drawn with identical sites, based on the restriction including the possible mobility of the repeats. sites of strain Ax-3L. However, not all of these sites have been Repeat upstream from Disc-Iy gene behaves as mobile checked on genomic Southern blots in strains NC-4 and Ax-3K. The genetic element. The pattern of hybridization of a repeat cloned fragments of strains Ax-3L and Ax-3K that were used to upstream from the Discl--y gene is of particular interest. generate the maps are shown below the maps. pDdDisc5.6 has been When the rightmost PstI-EcoRI fragment of the cloned drawn as containing the Discl-1-Discl-tl region. Since by genomic region 5' to the Discl-y gene (see map, Fig. 2B) is used to restriction mapping the 1B and 1' regions are identical, we cannot say probe a Southern blot of EcoRI-digested DNA, multiple which was the source of pDdDisc5.6. Also shown are the short, inverted repeats upstream from the Discl-y gene (short horizontal bands of hybridization are seen which are not conserved arrows) and the locations of the Tdd-2 and Tdd-3 mobile elements. between the various strains (Fig. 3A). However, when a Fragments A and B above the Ax-3L map indicate the restriction BglII digest is probed, an intense 3.6-kb band of hybridiza- fragments discussed in the text. Restriction site symbols used are: tion is seen in all strains (Fig. 3B), indicating that for each B, BglII; E, EcoRI; Ev, EcoRV; H, Hindlll; He, HaeIII; P, PstI; X, strain most repeat family members have two conserved BglII XhoI; and Xb, XbaI. sites 3.6 kb apart. The less intense bands probably represent 674 POOLE AND FIRTEL MOL. CELL. BIOL.

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Bh B'Ev Ev H XbB" B"' P X BB H Bh -"A I I1 r l 3.6-- _ ^ ^ X ~ -~ ,*0 ,I,lo f ., 11 v- V1-Vl---.-I VII I US 1 Vlll- ,,

map FIG. 4. Restriction of clone pDdB14. A BamHI library of 2.0 - Ax-3L DNA in A BF101 was screened with the PstI-EcoRI fragment of the Tdd-2 element in pDdDiscl-CD. The insert of one positive recombinant phage was subcloned into a vehicle to yield pDdB14 (see text). The restriction map of the 12-kb BamHI insert of pDdB14 is shown. The locations of the various regions of repeat elements Tdd-2 and Tdd-3 are shown above. The dotted arrow FIG. 5. Characterization of the Tdd-2 element of pDdB14. DNA indicates that the exact location of the end of the Tdd-2 element from the various strains were digested with BamHI plus BglII, within fragment VI is not known. Fragments I through VIII are separated on a 0.8% agarose gel, transferred to nitrocellulose, and restriction fragments referred to in the text. Restriction site sym- hybridized with probe IV (A) or VI (B). Strains are as in the legend bols: Bh, BamHI; other sites are as in the legend to Fig. 2. B', B", to Fig. 1, except that both MF-D and MF-G are shown. From the and B"' designate specific BglII sites referred to in Fig. 10. photographs of ethidium bromide-stained gels, the lane containing NC-4 DNA was overloaded with respect to the others. various individual family members that have diverged by losing a BglII site or by gaining or deleting sequences within indicates that the Tdd-2 element may be adjacent to another the repeat and generating unique bands of hybridization. In repeat element on the right side. Thus, the rightward BamHI the region of the repeat that hybridizes with the probe, there site of pDdB14 lies outside the element, and a similar is only one EcoRI site; genomic EcoRI fragments hybridiz- analysis indicates that the HindIll site defining the right side ing with the probe have one site within the conserved repeat of probe VI is outside the element (data not shown). In the and the other end at the first EcoRI site outside an individual region beyond this, probes (e.g., probe VII) hybridize repeat family member. Thus, -10 bands of hybridization of strongly to one band and very weakly to one or two other equal intensity are seen on an EcoRI Southern blot, in bands (Fig. 8A). For a number of restriction enzymes, the contrast to the intense band observed on a BglII Southern strong band of hybridization seen is the size expected if the blot, showing that the locations of the repeat in relation to probe was hybridizing to its own single-copy genomic frag- the first EcoRI site outside the repeat are quite different among the strains (Fig. 3A). These properties are those expected of a mobile genetic element with 10 to 15 copies in the genomes of the different strains; we have designated this LL _ i -j B o U -j ` < < element Tdd-2. A 2 0 < z < B 2~: 0 z Extent of the Tdd-2 element. Only the leftward end of the Tdd-2 element is contained within the cloned fragment of pDdDiscl-CD. Using this as a probe, we have isolated a second member of the Tdd-2 family on a cloned 12-kb BamHI fragment (designated clone pDdB14; map, Fig. 4) from strain Ax-3L. The BglII subfragment labeled VIII lines 2.0 up on Southern blots, with the intense genomic band of hybridization seen in Fig. 3B. To characterize the extent of 1.4 the Tdd-2 element in this large fragment, various subfrag- ments of pDdB14 were hybridized to genomic Southern blots .84- X of the wild-type strains (Fig. 5 through 7). As expected, all subfragments from within the 3.6-kb BglII fragment (e.g., probe IV of Fig. 4) hybridize intensely with the conserved .50--

3.6-kb and to other, bands (Fig. SA). - BgIII fragment unique .38 * The rightward 380-bp BglII fragment (probe V) is part of the Tdd-2 element since it behaves similarly, hybridizing with a 380-bp BglII fragment conserved between the strains (Fig. 6A). Thus, the rightward end of Tdd-2 lies somewhere beyond this small BgIII fragment. When the adjacent BglII- HindlIl fragment (probe VI in Fig. 4) is used to probe a Southern blot of DNA from different FIG. 6. Characterization of the small BgIlI fragments of BamHI-BglII-digested pDdB14. DNA from the strains was digested with BglII, separated a is seen, and the pattern is not strains, repeat pattern on a 1.5% agarose gel, transferred to nitrocellulose, and hybridized conserved among the wild-type strains (Fig. SB). As with the with probe III (B) or V (A). Strains are as in the legend to Fig. 1. The EcoRI digestion described above (Fig. 3A), multiple uncon- relatively low intensity of the hybridization to the small fragment is served bands are expected if this fragment overlaps the end likely an artifact of the relatively poor binding of the 0.38-kb of the repeat element, although the number of bands seen fragment to the nitrocellulose. VOL. 4, 1984 MOBILE ELEMENTS AND DISCOIDIN I GENES 675

ment (Fig. 8A and data not shown), confirming that this 0 0 0 0 LL LL S Y ) -J LL LL = Y .) -J region is outside of the Tdd-2 element. A S E 0E < z <4 B .e 12 2 0 4 z 4 pDdB14 contains a second . We used a similar approach to determine the nature of the sequences adjacent to the left end of the Tdd-2 element in pDdB14. 0 Southern blot hybridizations showed that a large region to 12. - 0.4i the left was moderately repeated. When restriction frag- 7.5 4 ~- an=r ments from this region (e.g., probe II) were used to probe a -6 Southern blot of BglII-digested or BglII-BamHI-digested 51- - _ DNA from the various strains, a pattern of the same general nature as that of the Tdd-2 hybridization was seen (Fig. 7). There is intense hybridization to a conserved 4.2-kb BglII 2.8- band and weaker hybridization to other nonconserved bands. However, the 370-bp BglII junction fragment (probe III) is not conserved (Fig. 6B), and other restriction frag- ments crossing this junction are also not conserved (data not shown). These results show that they are different elements, FIG. 8. The extreme ends of pDdB14 hybridize to the same not one large element or two adjacent copies of the same genomic BamHl fragments. DNA from the different strains was element. We designate this second element Tdd-3. digested with BamHI, separated on a 0.8% gel, transferred to All subfragments from within the 4.2-kb BglII fragment nitrocellulose, and hybridized with probe I (B) or VII (A). Strains show intense hybridization with the 4.2-kb genomic band are as in the legend to Fig. 5. and weaker hybridization to other, nonconserved bands, which may represent partial elements or elements lacking one of the BglII sites. Since the Tdd-3 element thus has two conserved BglII sites, it must extend beyond these sites. On background repeat hybridization most likely is due to the left the right side, however, it extends less than 370 bp since it end of the Tdd-3 element. The weak intensity of the back- abuts the Tdd-2 element. Thus, of the two BglII sites that ground hybridization indicates that the Tdd-3 element ex- define the 370-bp junction fragment (fragment III, Fig. 4), tends only a short distance past the BglII site. the left site is one of the conserved sites of the Tdd-3 The Tdd-3 element is mobile. We have shown above that element, whereas the right site is one of the conserved sites the extreme rightmost and leftmost restriction fragments of of the Tdd-2 element. the 12-kb BamHI insert of pDdB14 hybridize predominantly The left end of Tdd-3 most likely does not extend much as single-copy sequences. Indeed, when either of these past the leftmost BglII site of pDdB14, since probe I fragments (probes I and VII) are used to probe a BamHI hybridizes strongly to one major band and weakly to a digest of the different strains, both fragments, which do not number of other bands (Fig. 8B). The strong band represents cross-hybridize, hybridize to the same major single-copy the probe hybridizing to its own genomic fragment, and the genomic BamHI fragments in the various strains (cf. Fig. 8A and B). The size of the hybridizing fragment differs in each strain, ranging from -2.8 kb in strain Dd-Ohio to -12 kb in c0 strain Ax-3L. Thus, these two single-copy regions have LY LL O C) i varying amounts of DNA separating them, and in the case of ~d2 4 z 4 strain Ax-3L, we showed above that this extra DNA consists of the Tdd-2 and Tdd-3 elements. Note that in Fig. 8 this band is -12 kb in strain Ax-3L but is only -7.5 kb in both its parental strain, NC-4, and a sibling axenic strain, Ax-3K. 9. Figure 9 demonstrates that this difference in size reflects the recent insertion of the Tdd-3 element into this region of 4.2 . strain Ax-3L sometime after the separation of the two axenic stocks. The Tdd-2 element has a single PstI site and a single XhoI site. When the approximately single-copy rightward HindIll fragment of pDdB14 (fragment VII, Fig. 4) is used to probe a Southern blot of Ax-3K or Ax-3L DNA digested with BamHI-XhoI or BamHI-PstI, the major band of single- copy hybridization seen is the same size in both strains, 2.0- indicating that the Tdd-2 element is present in both strains (Fig. 9A). However, when the left BamHI-BglII fragment of pDdB14 (fragment I) is used as probe, the size of the major single-copy band of hybridization is smaller in strain Ax-3K than in strain Ax-3L by -4 kb (Fig. 9B). Thus the region to the left of the PstI and XlhoI sites in the Tdd-2 element in strain Ax-3L has recently gained an -4-kb insertion, proba- bly resulting from the transposition of a copy of the Tdd-3 element. FIG. 7. pDdBI4 contains a second mobile element. DNA from Preferred site of insertion of Tdd-3 element. The Tdd-3 the different strains was digested with BamHI plus Bg/II, separated element has many of the typical characteristics of transpos- on a 0.8% agarose gel, transferred to nitrocellulose, and hybridized able elements. It is a large repeat with a conserved structure, with probe II. Strains are as in the legend to Fig. 5. is found in different locations in different strains, and can 676 POOLE AND FIRTEL MOL. CELL. BIOL.

-j Y J i 3 element shares either of these characteristics, we se- < < A< < < <: quenced the regions near the BglIl sites conserved in the Tdd-3 element (Fig. 4, sites B' and B"), including the 370-bp Tdd-3-Tdd-2 junction fragment, to compare the left and right ends of the element (Fig. 10). Note that when the Tdd-3 -9.4 9 9 element inserted into the pDdBl4 region near the Tdd-2 5.04M4 --- 44 3.0- a _ element, it either inserted adjacent to the Tdd-2 element or it actually inserted into the end of the Tdd-2 element, splitting -2.0 off some portion of the Tdd-2 left end. The sequences reveal that the latter event occurred. The sequences around the site of insertion of the Tdd-3 element in pDdB14 are shown in Fig. 10, and the sequence of the left end of the Tdd-2 element upstream from the Discl-y gene is shown in Fig. 11 (top lines). Regions of homology between these two sets of sequences define Tdd-2 DNA and are shown by solid lines underneath the sequences. Compar- ing the two sets sequences, a a b b a a b b of it is clear that in pDdB14 the Tdd-3 element has inserted into the end of the Tdd-2 FIG. 9. The Tdd-3 element is mobile. DNA from strain Ax-3K or element, since the leftmost 45 bp of the Tdd-2 element are Ax-3L was digested with either BamHI plus Pstl (lanes a) or BamHI separated from the rest of the element by 4.2 kb of Tdd-3 plus XhoI (lanes b), separated on a 0.8% agarose gel, transferred to DNA (Fig. 10, wavy lines). nitrocellulose, and hybridized with probe 1 (B) or VII (A). The major When we compare the left and right junctions where the Ax-3L band of hybridization to probe I in lanes a and b is large and Tdd-3 element has inserted, two features of interest are seen. probably transferred less efficiently. The first is that the insertion of the Tdd-3 element into this region apparently caused the direct duplication of 9 to 10 bp insert into new locations. Transposable elements generally of Tdd-2 DNA at the site of insertion (Fig. 10, horizontal have two other characteristics: direct or inverted terminal arrows over junctions between solid and wavy lines). The repeats and production of a short of the target second is that a 22-bp region of Tdd-2 adjacent to the site of DNA at the site of insertion. To determine whether the Tdd- insertion (Fig. 10 and 11, open boxes) is homologous with a

CACCACAATT AAGTGAGTAA CAACTAAAAG CCTACAGT,GA TCAAACGGAT ACTAGATACA,

GAAAAACATA AAAAAACCGC ACCGCGATCA AGAGGATACA AGATACACGT GAAAASTAAT

TCA'rCTTTT CTATCCTTAA TCTCGGTCAA TTTTAACCAA TATTTCAAAA AAAATCACCA B' X AT,CACCACG ATOTACA*GAT CT...... <4.2 Ksof Tdd-3 DNA> ...... TCTAGAAT B" ACATCAAAAA AGCRAAAAGAT4 CTGAAAAXCT TATCAGCAAA AGACCATAAT AACTrCAAAG

AT-CTAAJAT "CITCTCACT TCAACGATCA AGCTAAGAAA AACAACA'JGr AATTACTATT

CTATTCCAGA ATCATCTCTC CCAAATATTA TATCTTITGA TCAATTCATA TGATTAAAAT

AACCCTTCAC CTTAAATGAT AAGCCTTTAA ATAAATATTA AATAAAACTC OTATTAACAC

AGATCJACAT ATATCAATCT TGTTAATCCA ATATTAAAAA AAAAAAAAAA AAAG4AAAAC

ATATCCATCA TCAATCTAAC ATCAACTACA TTCAATCTAT CTACTCTACA CACTAATTCC B"' 4 TTCAATCCTT GGATTAGGAA TAAGAATAGA TCTGAATAAA CGAGAAAGAA AAAAAAAAAA

A4AATAAAAA TAAAAATAAA AAATATTTTT ATATAAACAC ATAATTAAAA AAAAAATAAT

AAAAATAATA TAAAAAAAAA AAAAAAAAAA AAAAAAAAA FIG. 10. Nucleotide sequences of the Tdd-3 insertion site in pDdB14. The orientation of the sequence is the same as that of the map in Fig. 4. The upper region of the figure shows the sequences leftward from the B' Bgll site. The lower portion of the sequence shows the sequence rightward from the XhoI site 4.2 kb away. The restriction sites X, B', B", and B"' refer to sites in the map of Fig. 4. The solid lines underneath the sequences show regions of Tdd-2 DNA. The wavy lines show regions of Tdd-3 DNA. The boxes show the 22-bp region of homology near the left termini of Tdd-2 and Tdd-3. The horizontal arrows over the sequences show the duplication of Tdd-2 DNA presumably caused by insertion of the Tdd-3 element. We have drawn the arrows over the 9-bp duplication. Since we do not know the exact structure of the Tdd-3 left end, it is also possible that the duplication extends to the A residue 1 bp farther leftward. The A residue rightward is not part of the dupli- cation. VOL. 4,1984 MOBILE ELEMENTS AND DISCOIDIN 1 GENES 677

X A ********* ********I* **§******* u*******r** I*****I**** ********** [Ax3L]..CTCGAGGTCCT AGGATCGAAA CCTAGAGAAG CTAAATTTTA AAATTATACA GtATATGTCG [Ax3K]..CTCGAGGTCCT AGGATCGAAA CCTAGAGAAG CTAAATTTTA AAATTATACA GAA1'ATGTCG

***I****u* ****,*§**u* ********** u*****I**** ******.I** W**I*§**** **I****}*** CTAGTTCGAT TCCTATTTTA AAGAATTAAA TTCCATATTG ATTTAATTTT AAAAAAAT,TA AAAGCCTACA CTAGTTCGAT TCCTATTTTA AAGAATTAAA TTCCATATTG ATTTAATTTT AAAAAAATITA AAA.GCCTACA

5TT,GATCAAA CGGATACTAG ATAC4GAAAA ACATATCCAT CAATCTAACA TCAACTACAT TCAATCTATC GTTAAAAAAA CCGCACCGCAG ATCAAGAGGA TACAAGATAC ACGTGAAAAG TAATTCATCC TTTTCTATCC B TACTCTACAC ACTAATTCCT TCAATCCTTG, GATTGGGAAT AAGAATA#GAT CTGAATAAAC GTAAAGAAAA

TTAATCTCGG TCAATTTTAA CCAATCTTTC AAAAAAAATC ACCAATCCAC CACGATCTAC AGATCTAATT

B AAAAAAATkA AAATAAAAAT CAAAAATATT TTTATATAAA CACATAATTA AAAAAAAAAA AATAATAAAA

TAC'.AAATCG CCGATTCAAA 33AATCCCAT CGAGATGAAT TC ...... E ATA.ATTAAA AAAAAAAAAA AAAAAAAAAA A ..... [Ax3L]

...... [Ax3Kl FIG. 11. The Tdd-3 element has a preferred site of insertion. The nucleotide sequences rightward from the far XhoI site upstream from the Discl-y gene in strains Ax-3L and Ax-3K are shown. The orientation of the sequences is the same as that of the map in Fig. 2. Regions of ho- mology between this region of the two strains are designated by asterisks above the sequence. Upper line: sequence from the XhoI site of Ax- 3L. Lower line: sequence from the Xhol site of Ax-3K. Other symbols are as in the legend to Fig. 10.

22-bp region just inside the left end of the Tdd-3 element has inserted into the Tdd-2 element upstream from the Discl- (Fig. 10, hatched box). It may be that the Tdd-3 element -y gene. Again, the site of insertion is next to the 22-bp inserted at this point because of this homology. homology region, but in this case it is to the left of the This interpretation is strengthened by an analysis of the homology, whereas in pDdB14 it was to the right of the region upstream from the Discl--y gene in strain Ax-3K. As homology. We have not isolated the region rightward from mentioned above, the 7.2-kb EcoRI fragment containing the the EcoRI site, so we cannot determine whether this second region 5' to the Discl--y gene of NC-4 is missing in strain Ax- insertional event also caused a short direct duplication of the 3K, and a new band 5.1 kb in length is present (Fig. 1A). We target site DNA. have cloned this EcoRI fragment, and the restriction map of Transposons in other systems exhibit terminal repeats. this clone, pDdDiscK5.1, is shown in Fig. 2C. This fragment We have found no evidence for terminal direct or inverted has the same restriction sites as does pDdDiscl-CD for most repeat structures at the ends of either the Tdd-2 or Tdd-3 of its length, differing only at the far right end, near the site elements. The termini of either Tdd-2 or Tdd-3 do not cross- of the Tdd-2 element in pDdDiscl-CD. Probes made from the hybridize with each other, and the sequences of >300 bp at Tdd-2 element located in pDdB14 do not hybridize with either end of the Tdd-3 element are not homologous (data not pDdDiscK5.1. To better define the nature of the changes in shown). However, both the left and right ends of the Tdd-3 this region between the two strains, we have sequenced the element in pDdB14 terminate in a stretch of homopolymer differing region of pDdDiscK5.1 to compare it with the same deoxyribosyladenine (dA) residues (7 and 18 nucleotides region of pDdDiscl-CD (Fig. 11), starting at the rightward long, respectively). These short runs may be equivalent to XhoI site (map, Fig. 2). Rightward from this site, the two the terminal repeats of other transposons. Alternatively, it is sequences are identical for 131 bp and then diverge. It is possible that this type ofD. discoidelum element does not use interesting that this point of divergence is 15 bp into the Tdd- terminal repeats for movement. However, since we have not 2 element, exactly at the left side of the 22-bp Tdd-2-Tdd-3 yet isolated additional members of these repeat families, we homology (Fig. 11, open box under Ax-3L sequences). Since cannot rule out the possibility of DNA rearrangement or this sequence was closely associated with the insertion of the deletion at the right ends. Tdd-3 element in the pDdB14 region of strain Ax-3L, we compared the divergent sequences in strain Ax-3K with the left end of the Tdd-3 element in pDdB14 (Fig. 10, wavy lines DISCUSSION in upper region). The two sets of sequences are identical Genomic instability near the discoidin I genes. We have from the point of divergence in clone pDdDiscK5.1 to the shown by genomic Southern hybridizations that the regions EcoRI site at the end of the cloned fragment, with the surrounding two linked discoidin I genes can undergo rapid exception of a single base pair within the 22-bp homology changes in structure. A similar assay of restriction sites region. Thus, it appears that in strain Ax-3K a Tdd-3 element surrounding the actin showed a remarkable 678 POOLE AND FIRTEL MOL. CELL. BIOL.

NC-4

-Tdd-2-, -v'---cl---. .-( Dsis-Yupstream region) Disc l-Y + ~-(Tdd-2(pDdBp 14 rregion)

Ax-3K Ax-3L

l_ Tdd-3 - Tdd-3- V --MENU-/ (Discl-Yupstream (pDdB14 region) I/ 1.-Td-2---0 region)

FIG. 12. Transposition of the Tdd-3 element into two different copies of the Tdd-2 element. A model to explain the sequencing results of Fig. 10 and 11. The symbols used are the same as in Fig. 10 and 11. Thick solid lines represent the Tdd-2 element, wavy lines represent the Tdd-3 element, and the open and hatched boxes represent the 22-bp homology regions. The figure is not drawn to scale. Strain NC-4 has one copy of the Tdd-2 element in the pDdB14 region. The Tdd-3 element preferentially inserts at the site of the 22-bp homology. In strain Ax-3L, the Tdd-3 element has inserted in the pDdB14 region at the right side of the homology, splitting the Tdd-2 element at the site shown and causing a short direct duplication of the target site DNA. In strain Ax-3K, a Tdd-3 element has instead inserted into the Discl--y upstream region, this time to the left of the homology. We assume that this results in short, direct duplication at this target site also. The mechanism of insertion is unknown. stability, even for the largest fragments and despite the identified two repeat elements that behave as mobile genetic number offragments resulting from the 17 to 20 actin genes. elements. Both the Tdd-2 and the Tdd-3 elements have The instability around the Discl-1 and DiscI-y genes was conserved structures and are found in different locations in particularly evident among three closely related strains. different strains of D. discoideum. Neither one of these These strains are a wild-type laboratory stock, NC-4, and elements is found in two other species, Dictyostelium pur- two independently maintained isolates, Ax-3L and Ax-3K, pureum or Dictyostelium mucoroides (R. A. Firtel, unpub- of an axenic derivative of NC-4. These axenic strains are lished data). By genomic Southern blot analysis and DNA capable of growing in liquid media in the absence of the sequencing, we have examined these individual family mem- bacterial food source required by wild-type strains. Com- bers in wild-type strain NC-4 and its two sibling derivatives pared with their wild-type parent, both of these axenic Ax-3L and Ax-3K. A model consistent with the DNA derivatives have undergone a rearrangement involving the sequencing data is shown in Fig. 12. The structures of region upstream from the two linked discoidin I genes. individual Tdd-2 elements at two different locations in strain In strain Ax-3L, this rearrangement is a duplication event NC-4 are shown. One of these is located upstream from the starting -1 kb upstream from the Discl-y gene, extending Discl--y gene, and the other is located in the pDdB14 region. through both tandemly linked genes and the proposed pseu- In two separate occurrences in two axenic derivatives of dogene, and continuing for as far as we can detect. Both the strain NC-4, each of these copies of the Tdd-2 element has original DiscI--y gene and its duplicate, Discl-y', have close been interrupted by the insertion of a second transposable XhoI and HaeIII sites -1 kb upstream from the genes, and element, Tdd-3. Upon insertion, the Tdd-3 element gener- both sequences are identical from the XhoI sites toward the ates a 9- to 10-bp duplication of the target site DNA. This genes for over 150 bp. However, the XhoI site upstream behavior is typical of procaryotic and eucaryotic transpos- from the duplicated Discl-y' gene is -25 bp closer to the able elements, which cause short duplications of target site gene than the XhoI site upstream is from the Discl-y gene sequences (7, 9, 13, 28, 29), presumably resulting from a (20). We assume that a small deletion accompanied this staggered cut in the target DNA. duplication. An unusual aspect of this model is the preferential inser- Previously we showed that a moderately repeated se- tion of the Tdd-3 element into the Tdd-2 element. The site of quence that was found 3' to the DiscI-P was also found insertion is immediately to the left or right side of a 22-bp upstream from the duplicated Discl--y' gene (20) but is not region of close homology shared between the two elements, within the duplicated region. When the same restriction suggesting that this homology is the basis for the insertion fragment carrying the repeat from the region 3' to the Discl- site preference of Tdd-3. Procaryotic transposons can insert , gene is hybridized to genomic Southern blots of DNA from at many locations but have preferred sequences at the target the various wild-type strains, different patterns of hybridiza- site (11). Similarly, the D. melanogaster transpo- tion are seen (Fig. 1C). This repeat element may, therefore, son can insert at a variety of locations but does have certain be associated with yet another mobile element. It is possible hotspots of insertion (6, 10), although it is not known that this repeat was involved in the duplication event. The whether a specific nucleotide is the repeat copy downstream from the tandemly linked genes is cause of a hotspot. itself just upstream from the presumed discoidin I pseudo- The two elements do have a common terminal structure, gene, so it is also possible that the repeat was involved in the the 22-bp homology. This small region is located 15 to 16 bp duplication that generated the pseudogene. from the left terminus of both Tdd-2 and Tdd-3. In the Two mobile genetic elements of D. discoideum. We have regions we have sequenced, this is the only homologous VOL. 4. 1984 MOBILE ELEMENTS AND DISCOIDIN I GENES 679 stretch we have located, and the two elements do not cross- regions had no inserted DNA (Fig. 8). The size of the hybridize. The homology may indicate a role for this 22-bp fragment in strains NC-4 and Ax-3K is -7.5 kb, resulting sequence in the movement of the elements. That the Tdd-3 from the insertion of the Tdd-2 element, and in strain Ax-3L element has inserted in two cases exactly adjacent to this it is -12 kb due to the additional insertion of the Tdd-3 region also implicates it in element movement. element. The sizes seen in the other wild-type strains are >5 The Tdd-3 element has many of the characteristics of kb, as if other elements had inserted or inserted and then transposable elements, and we would expect that the Tdd-2 partially deleted. Thus, this small region of the is element will also share these characteristics. However, there extremely fluid in its behavior. It may be that this region has is a major difference between the Tdd-2 and Tdd-3 elements a preferred target sequence for one or more of the D. and known transposons, their lack of terminal repeats. The discoideum transposon-like elements. yeast element Tyl has -0.3-kb terminal direct repeats (1, 7); a nematode transposon has terminal inverted repeats (4); D. ACKNOWLEDGMENTS melanogaster copia-like elements have terminal direct re- We thank Anthony R. Hunter, Alan Kimmel, Wolfgang Nellen, peats ranging in size from -260 to 480 bp (28); D. melano- Mona Mehdy, and Christophe Reymond for critical readings of the gaster FB elements have long, inverted repeats of various manuscript. We thank David Francis, Richard Kessin, Alan Kim- sizes (30); and D. melanogaster P elements have 31-bp mel, and William F. Loomis for providing D. discoideum strains and Kenn for M13 strains. inverted repeats (29). Neither the Tdd-2 nor the Tdd-3 Buckley This work was supported in part by grants from the National element appears to have such a structure. The termini of the Institutes of Health to R.A.F. For part of the period of this work, Tdd-2 element do not cross-hybridize. The Tdd-3 termini R.A.F. was supported by a Faculty Research Award from the also do not cross-hybridize, and the DNA sequences of the American Cancer Society, and S.P. was a trainee of the Public ends do not reveal any terminal repeats other than a short Health Service. stretch (7 to 18 nucleotides) of deoxyribosyladenine resi- dues. The deoxyribosyladenine residues could serve the LITERATURE CITED same purpose as the terminal repeats of other eucaryotic transposons. It is also possible that these mobile elements 1. Cameron, J., E. Loh, and R. Davis. 1979. Evidence for transpo- sition of dispersed repetitive DNA families in yeast. Cell may in fact be unlike transposons in relation to terminal 16:739-751. structures or that the copies of these elements we have 2. Denhardt, D. 1966. A membrane filter technique for the detec- isolated are partial elements lacking the terminal repeats. tion of complementary DNA. Biochem. Biophys. Res. Com- Another class of D. discoideium transposable elements being mun. 23:640-646. studied in our laboratory does have inverted repeats near the 3. Devine, J., A. Tsang, and J. Williams. 1982. Differential expres- termini. However, sequences common to several isolated sion of the members of the discoidin I multigene family during transposons extend beyond the terminal repeat on one side growth and development of Dictyostelium discoideum. Cell of the element (21). Thus, D. discoideum mobile genetic 28:793-800. elements may exhibit unusual terminal structures. It will be 4. Emmons, S. W., L. Yesner, and K. Ruan, D. Katzenberg. 1983. interesting to see whether these other elements Evidence for a transposon in Caenorhabdites elegans. Cell with atypical 32:55-65. termini will insert at regions having extensive homology with 5. Engels, W. 1979. Extrachromosomal control of mutability in the termini. Drosophila melanogaster. Proc. Natl. Acad. Sci. U.S.A. The left termini of the two Tdd-3 elements that we have 76:4011-4015. sequenced are identical except for one difference in the 22- 6. Engels, W., and C. Preston. 1981. Identifying P factors in bp homology region. The left termini of the Tdd-2 elements Drosophila by means of chromosomal breakage hotspots. Cell are homologous but not identical. Just inward from the 22-bp 26:421-428. homology region of Tdd-2, there is an -70-bp stretch that 7. Farabaugh, P., and G. Fink. 1980. Insertion of the eukaryotic consists of degenerate repeats of the trinucleotide ATC, and transposable element Tyl creates a 5 bp duplication. Nature (London) 286:352-356. the Tdd-2 element upstream from the gene has one Discl-y 8. Firtel, R., R. Timm, A. Kimmel, and M. McKeown. 1979. fewer ATC trinucleotide than does the Tdd-2 element in Unusual nucleotide sequences at the 5' end of actin genes in pDdB14. They also differ in the lengths of several homopoly- Dictvostelium discoideum. Proc. Natl. Acad. Sci. U.S.A. mer adenosine tracts and have three other base substitutions 76:6206-6210. in this region. 9. Gafner, J., and P. Phillippsen. 1980. The yeast transposon Tyl We have not detected transcripts from either the Tdd-2 or generates duplications of target DNA on insertion. Nature the Tdd-3 elements. However, fragment V (Fig. 4) has an (London) 286:414-418. open reading frame for its entire 385-nucleotide length, and 10. Green, M. 1977. Genetic instability in Drosophila melanogaster: there is an additional open reading frame in the --150 De novo induction of putative insertion mutations. Proc. Natl. nucleotides sequenced leftward from the EcoRI site of the Acad. Sci. U.S.A. 74:3490-3493. 11. Hailing, S., and N. Kleckner. 1982. A symmetrical sex-base-pair Tdd-2 element upstream from the gene Discl-y (map, Fig. 2). target site sequence determines TnlO insertional specificity. Thus it is likely that the elements encode polypeptides that Cell 28:155-163. we presume are involved with regulating the movement of 12. Kindle, K. L., and R. A. Firtel. 1978. Identification and analysis the elements. of Dictvostelium actin genes, a family of moderately repeated The pDdB14 region of strain Ax-3L consists of two unique genes. Cell 15:763-778. regions separated by two mobile genetic elements. The 13. Kleckner, N. 1981. Transposable elements in prokaryotes. amount of DNA separating these two unique regions can be Annu. Rev. Genet. 15:341-404. assayed by hybridizing genomic Southern blots of BamHI- 14. Liebman, S., P. Shalit, and S. Picoglou. 1981. Ty elements are digested DNA with probes made from these involved in the formation of deletions in DELl strains of S. single-copy cerei'isiae. Cell 26:401-409. regions. Accordingly, almost every strain looked at a has 15. Loomis, W. F. 1971. Sensitivity of D. discoideum to different amount of DNA separating these two regions. The analogues. Exp. Cell Res. 60:285-289. size of the fragment in strain Dd-Ohio is -3 kb, which is 16. Loomis, W. F. 1975. Dictyostelium discoideum, a developmen- consistent with the size expected if the two single-copy tal system. Academic Press, Inc., New York. 680 POOLE AND FIRTEL MOL. CELL. BIOL.

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