1983; Brookfield et al. 1984), 412 (Martin et al. 1983; Brookfield et al. ... 6525 North Sheridan Road, Chicago, Illinois 60626. Mol. Biol. Evol. 7(6):589-606. 1990.
Distribution of hobo Transposable Elements in the Genus Drosophila’ Stephen B. Daniels, * Arthur Chovnick, * and Ian A. Boussyy *Department of Molecular and Cellular Biology, University of Connecticut, TDepartment of Biology, Loyola University of Chicago
and
This study describes the distribution of hobehybridizing sequences in the genus Drosophila. Southern blot analysis of 134 species revealed that hobo sequences are limited to the melanogaster and montium subgroups of the melanogaster-species group. Of the hobo-bearing species, only D. melanogaster and two of its sibling species, D. simulans and D. mauritiana, were found to contain potentially complete hobo elements. The distribution of hobo sequences is one of the narrowest distributions thus far described for any Drosophila transposable element.
Introduction It is becoming increasingly apparent that transposable elements may play an important role in eukaryotic evolution (Finnegan 1989a), although, at present, very little is known about their origins and modes of transmission. The first step in understanding the evolutionary history of a particular transposable element is to examine its phylogenetic distribution. Within the genus Drosophila, distribution patterns have been described for a number of different mobile element families, e.g., 297 (Martin et al. 1983; Brookfield et al. 1984), 412 (Martin et al. 1983; Brookfield et al. 1984), copia (Martin et al. 1983; Brookfield et al. 1984; Stacey et al. 1986), gypsy (Stacey et al. 1986), F (Stacey et al. 1986), I (Bucheton et al. 1986; Stacey et al. 1986), and P (Brookfield et al. 1984; Stacey et al. 1986; Anxolabehere and Periquet 1987; Daniels et al. 1990). The last two elements are particularly interesting in that they have been shown to be the causative agents of the I-R (Finnegan 19896) and P-M (Engels 1989) systems of hybrid dysgenesis in D. melanogaster. Hybrid dysgenesis is a syndrome of correlated genetic abnormalities that is induced in the germ line of progeny from certain intraspecific crosses (Kidwell et al. 1977). Recently, a third, independent system of hybrid dysgenesis has been documented in D. melanogaster (Blackman et al. 1987; Yannopoulos et al. 1987; Stamatis et al. 1989). This system has been shown to be causally related to the activity of the hobo transposable element, although many of the genetic and environmental factors that modulate hobo activity are still not well understood. On the molecular level, the hobo element was first described by McGinnis et al. ( 1983) and later was characterized in more detail by Streck et al. ( 1986). In some respects, hobo bears a close resemblance to the P element (see Engels 1989). Both elements have short inverted terminal repeats and can be divided into two functionally 1. Key words: Drosophilaspecies, transposable elements, hobo element, phylogenetic distribution, molecular evolution. Address for correspondence and reprints: Dr. Ian A. Boussy, Department of Biology, Loyola University, 6525 North Sheridan Road, Chicago, Illinois 60626. Mol. Biol. Evol. 7(6):589-606.1990. 0 1990by The University of Chicago.All rights reserved. 0737-4038/90/0706-0006.$02.00 589
590
Daniels et al.
distinct size classes, complete and defective. In the hobo family, complete elements are 3.0 kb in length and are capable of producing the trans-acting product necessary for hobo mobilization. Defective elements are smaller and variable in size and are derived from complete elements by internal deletions; they can undergo transposition only in the presence of complete elements. (For a recent review, see Blackman and Gelbart 1989. ) To date, there has been very little information concerning the phylogenetic distribution of hobo sequences, beyond the observation that they are found in D. melanogaster and in two of its sibling species, D. simulans and D. mauritiana (Streck et al. 1986). To gain a better understanding of the phylogenetic distribution of this element, we have undertaken a more thorough survey of the genus, the evolution of which has been described in some detail by Throckmorton ( 1975) and is depicted diagrammatically in figure 1. The genus Drosophila arose from one of several early radiations within the family Drosophilidae and underwent extensive diversification in the form of five major radiations, the earliest of which gave rise to the subgenus Scaptodrosophila. This was followed by the Sophophora radiation, which produced the melanogaster, obscura,
modified-mouth-parts
lmmigrans
and Hirtodrosophila radiations
mesophragmatica
uirilis-repleta
Drosophila radiation
Sophophora Victoria
radiation latifasciaeformis
coracina Scaptodrosophila
radiation
FIG. 1.-Depiction of evolution of genus Drosophila, showing major radiations and present-day species groups that have resulted from each (atIer Throckmorton 1975). Only subgeneraand species groups surveyed in this study are included, several derivative genera are also shown.
hobo Distribution in Drosophila
59 1
willistoni, and saltans lineages. The Sophophora radiation was in turn followed by the complex Drosophila radiation, which is composed of three main branches, represented by the virilis-repleta, immigrans, and Hirtodrosophila radiations. In the present study, we have surveyed all the major phylogenetic lineages within the genus for the presence of hobo-hybridizing sequences and have found that the hobo element has one of the narrowest distributions of any Drosophila transposon thus far examined. Material and Methods Fly stocks
All of the species used in the present study are listed in table 1; each listing includes one of the following designations to denote the laboratory from which the stock was obtained: BG (National Drosophila Species Resource Center, Bowling Green State University), AC (A. Chovnick, University of Connecticut), WH (W. Heed, University of Arizona), HK (H. Krider, University of Connecticut), JC (J. Coyne, University of Chicago), FA (F. Ayala, University of California, Davis), LE (L. Ehrman, State University of New York at Purchase), and MS (M. Seiger, Wright State University ) .
Plasmid DNA pRG2.6X contains the internal 2.6-kb XhoI fragment from a complete hobo element cloned into the Sal1 site of the plasmid vector pUC8 (Blackman et al. 1987). The complete hobo element was derived from the dpp’/CyO strain of Drosophila melanogaster. This plasmid is shown in figure 2 and was provided by R. Blackman. Southern Blotting Genomic DNA samples were routinely prepared from -0.12 g of adult llies by the method described by Daniels and Strausbaugh ( 1986). DNA was quantitated by the fluorometric assay described by Kissane and Robins ( 1958). Procedures for restriction-enzyme digestion, agarose-gel electrophoresis, gel blotting, and preparation of nick-translated probes are described by Rushlow et al. ( 1984). Gels were blotted to either Nytran (Schleicher and Schuell) or nitrocellulose (Schleicher and Schuell), with the same blotting conditions being used for both. The pRG2.6X plasmid was used as probe in all experiments. Hybridizations were carried out overnight in a mixture containing 50% formamide (Fluka), 5 X SSPE (0.75 M NaCl, 0.05 M NaHP04, 0.01 M ethylenediaminetetraacetate, pH 7.0), 2 X Denhardt’s solution [0.04% (w/v) ficoll (type 400), 0.04% (w/ v) polyvinylpyrrolidone, 0.04% (w/v) bovine serum albumin], 1% sodium dodecyl sulfate (SDS), 100 pg salmon sperm DNA/ml, and 0.1 pg 32P-labeled probe DNA/ ml. Filters were washed for 3 h in a series of solutions of increasing stringency, the final one of which contained 0.1 X SSPE and 0.5% SDS. Hybridizations and washes were done at 37°C. Stringency is directly related to the melting temperature (T,,,) of DNA/DNA hybrids. The T, depends primarily on temperature, ionic strength, percentage of formamide, G/C content, and number of mismatched pairs and can be calculated by an empirical relationship described by Beltz et al. ( 1983 ) . In this way, we have estimated that our hybridization and final wash conditions will promote and maintain DNA hybrids between probe and target only when the two have a sequence similarity equal to or greater than - 74%, if it is assumed that G/C content is 38%, which is the content of the canonical D. melanogaster hobo element (Streck et al. 1986).
Table 1 Distribution of k&o-hybridizing
Source”
g I.J
BG 11010-0011.0 BG 11010-0021.0 BG 11010-0031.0 BG 11010-0041.0 BG 11020-0051.0 BG 11020-0061.0 BG 14030-0741.1 FA .._,_......_.,.., LE (C-2) FA BG 14030-0801.0 MS (“willi 7”) BG 14030-0721.1 BG 14030-0751.1 MS (“neb 2”) BG 14030-0791.1 BG 14041-0831.0 BG 14042-0841.0 BG 14042-0851.0 BGl4043-0861.0 BG 14043-0871.0 BGl4045-0881.0 BG 14045-0891.0 BG 14045-0901.0 BG 14045-0911.0 BG 14012-0141.0 BG 14012-0151.0 BG 14012-0161.0 BG 14012-0171.0 BG 14012-0181.0
Sequences within the Genus Drosophila and among Several Related Genera
Genus Drosophila
Subgenus Scaptodrosophila
Species Group
subgroup
Victoria
coracina latifasciaeformis willistoni
bocainensis
_. _. saltans
cordata elliptica
._... saltans
.._._
_. obscura
_.
a#inis
Species lebanonensis casteeli lebanonensis lebanonensis pattersoni stoni dimorpha lattfasciaeformis equinoxialis insularis paulistorum pavlovskiana tropicalis willistoni capricomi fimipennis nebulosa sucinea neocordata emarginata neoelliptica milleri sturtevanti austrosaltans lusaltans prosaltans saltans a#inis algonquin athabasca azteca btjasciata
hobo Hybridizationb _ _ _ _ _ _ _ _ _ _ _ _ _ _ -
...................... ...................... ...................... ...................... ......................
E .3 % E
Table 1 (Continued)
Source”
2 P
BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG BG WH BG BG
14028-060 1.O 14028-06 11 .O 14028-0621.0 14028-063 1.O 14028-064 I .O 14028-0651.0 14028-066 1.O 14028-0671.0 14028-0681.0 14028-069 1.O 14028-070 1.O 14028-07 I 1.O 14023-0331.0 14023-0341.0 14023-035 1.O 14023-0361.0 14022-027 1.O 14022-028 1.O 14022-029 1.O 14022-0301.1 14022-03 11 .O 15120-1911.0 15120-1921.0 15120-1931.2 15120-1941.0 15120-1951.1 15040-1181.0 15040-I 191.0 15100-1711.0 15020-1111.1
Genus
Subgenus
Species Group
Subgroup
Species
orosa parvula pennae punjabiensis quadraria
suzukii
takahashii
Drosophila
.funebris
annulimana melanica polychaeta robusta
4a seguyi serrata triauraria tsacasi vulcana lucipennis mimetica pulchrella rajasekzri lutescens paralutea prostipennis pseudotakahashii takahashii funebris macrospina limpiensis macrospina macrospina multispina subjimebris gibberosa talamancana melanica polychaeta
hobo Hybridizationb
++ +
+ ++
-
-
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
Table 1 (Continued) hobo Sourcea BG BG BG BG BG BG BG BG BG BG BG
15284-1501.0 15285-2521.0 13000-008 1.O 20000-262 1.O 20000-263 1.1 60000-275 1.O 80000-276 1.O 31000-2641.0 31000-2651.0 50000-2746.0 50000-274 1.O
Genus
Subgenus
Species Group
Subgroup
hawaiiensis pilimana Dorsilopha Chymomyza Liodrosophila Samoaia Scaptomyza Zaprionus
Parascaptomyza
Species
gymnobasis aglaia busckii amoena procnemis aerea leonensis adusta elmoi inermis tuberculatus
Hybridizationb
_ _ _ _ _ _ _ -
NOTE.-Taxonomic groupings are those of Throckmorton (1975). Genomic blots were analyzed by scanning densitometry (see Material and Methods). a Letter designations are as defined in Material and Methods and denote the laboratories from which the stocks were obtained. b The pRG2.6X plasmid was used as probe. A plus sign (+) indicates that hybridization was observed, and a minus sign (-) indicates that no hybridization was seen at the stringency employed; the number of pluses indicates the relative strength of the hybridization signal. An F indicates that one or more bands were faintly discernible on the blot but that the strength of the signal was not significantly above the baseline level of negative samples.
hobo Distribution in Drosophila
597
2.6 kb I
I
X
X
1
kb
FIG. 2.-Depiction of complete hobo element (after Streck et al. 1986). The complete element is 3.0 kb, has short inverted repeats (open boxes), and contains one long open reading frame (grey bar). There is an XhoI (X) site near each terminus. The pRG2.6X plasmid contains the internal 2.6-kb XhoI fragment cloned into the Sal1 site of the plasmid vector pUC8 (stripedbars).
Scanning Densitometry Autoradiograms of the hobo-element survey were subjected to scanning densitometry (LKB UltroScan XL) in order to roughly quantitate relative levels of hobo hybridization. Each autoradiogram had a positive control lane (containing DNA from the Harwich-77 strain of D. melunoguster), as well as lanes containing DNA from several different species. Identical amounts of DNA were loaded in each lane, with the exception of the Harwich-77 control, which had one-fifth the amount of DNA of the other samples. The entire length of each lane was scanned, and the density of all bands was measured; a single, composite “raw score” was then calculated by integrating the area under all of the peaks. So that comparisons could be made between different autoradiograms, a final, standardized score was computed for each sample by expressing its raw score as a percentage of the Harwich score. All samples were then placed on an arbitrarily defined, five-point scale to indicate relative strength of hybridization (see table 1). Results The goal of the present study was to determine the phylogenetic distribution of hobo-hybridizing sequences in the genus Drosophila and, to a limited extent, among several of the closely related derivative genera. In all, 142 species were examined, 134 from within the genus ( - 10% of the presently described species) and representing 26 species groups and all the major radiations, and eight from outside the genus and representing five other genera. Information concerning the evolution and taxonomy of the genus Drosophila can be found in the work of Throckmorton ( 1975) and Beverley and Wilson ( 1984). The phylogenetic relationships of the groups examined in the present study are depicted in figure 1; the arrangement of subgenera, species groups, and subgroups is given in table 1. To assay for hobo-hybridizing sequences, genomic DNA samples were digested with PvuII and were subjected to Southern blot analysis as described in Material and Methods, using the pRG2.6X plasmid as probe. The PvuII restriction enzyme does not cut within the canonical hobo element ( Streck et al. 1986)) so each band potentially represents a unique fragment of genomic DNA containing a single hobo sequence. Autoradiograms with positive signals were analyzed by scanning densitometry (see Material and Methods), and samples were placed on an arbitrarily defined, five-point scale to indicate relative strength of hybridization. The results of the survey are shown in table 1. Within the genus Drosophila, hobo sequences were found only in the melano-
Table 2 Distribution of Eight Drosophila Transposable
Elements TRANSPOSABLE ELEMENT’
GENUS Drosophila
SUBGENUS Scaptodrosophila
Sophophora
SPECIESGROUP Victoria , coracina Iatifasciaeforrnis willistoni saltans
obscura melanogaster
Drosophila
funebris melanica polychaeta robusta tumiditarsus virilis canalinea dreyfusi mesophragmatica
SUBGROUP
willistoni bocainensis cordata elliptica sturtevanti saltans a&is obscura ananassae elegans eugracilis jicusphila melanogaster rnontium suzukii takahashii
hobo
_ _ _ _ _ _ + +I_ _ _ _ _ _ _ _
Ib
297c
-
_ _ _
? _ (+)
+ _
-
Pd
412’
(+) _
+ + + +
(+I + + + + + t+;+ +/+/+/(+)/_
(+I
+ + +
+
+
_ _
_ _ +
-
copia f
gypsy’
F8
+ + + + + + + + + + +/+ + _ + + + + +
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + +
-
+
+
_
+
+
_
+
+
+
+
+
+++
+ ++
+
+
I
I
I
I
c.
I
I++
+-+
++
+
I
+
I
II
+++
II++
I
III1
603
I
+
t
I
604
Daniels et al.
these weakly hybridizing elements from different species. Besides the usefulness of sequence data in determining relatedness between sequences found in different species, such data might shed light on the long-term fate of transposable elements within a lineage and on the molecular processes that impinge on these intriguing components of the eukaryotic genome. To fully understand the evolutionary history of a particular transposable element within a lineage, one must determine its initial point of entry, its subsequent distribution, and its mode of transmission between species (see Stacey et al. 1986). If transmission has been strictly vertical (i.e., mating dependent), then descendants of an ancestral species bearing the element should also possess homologues of the element. If during evolution the element has been lost from a species, then all of its descendants should be element free. Thus, mating-dependent transmission should result in distribution patterns that are virtually continuous. Alternatively, if transmission has occurred horizontally between reproductively isolated species, then distribution patterns may not follow phylogenetic groupings, i.e., they may be discontinuous. The fact that vertical and horizontal modes of transmission are not mutually exclusive often makes it difficult to decipher the exact sequence of events leading to present-day distribution patterns, since very complex patterns can result from the combination of occasional horizontal transfer and subsequent vertical transmission. Moreover, the picture can be even further complicated by the possibility that a lineage may have been invaded at more than one point during its evolution or by the possibility that the complete loss of a sequence family from a species and its descendants may occur more often than supposed. There are at least two hypothetical scenarios that can be proposed to account for the current distribution of hobo homologues in Drosophila The most parsimonious interpretation of the data posits a single introduction of hobo elements into the melunogaster-species-group lineage at some point prior to the divergence of the melanogaster and montium subgroups, with homologues being transmitted vertically between species. However, such a scenario does not address the large quantitative differences in hobo hybridization between the melanogaster complex and the rest of the melanogasterand montium-subgroup species. Alternatively, it can be proposed that the present-day distribution of hobo homologues results from two independent introductions: an initial introduction into the melanoguster-species-group lineage prior to the separation of the melanogaster and montium subgroups and a more recent reintroduction into the progenitor of the melunoguster complex, perhaps by horizontal transmission. This second introduction would account for the apparent difference between the hobo elements in the melunogaster complex and those in the rest of the hobo-bearing species of the melunogaster-species group. The notion of horizontal transmission between reproductively isolated species has been invoked to account for the current distribution patterns of several Drosophila transposable elements (e.g., see Bucheton et al. 1986; Stacey et al. 1986; Anxolabehere and Periquet 1987; Daniels et al. 1990). To account for the patchy distribution of hobo-hybridizing sequences within the montium subgroup, both models must necessarily propose that elements have been lost in some species during the course of their evolution. A theoretical treatment of the evolution of a hypothetical transposable element has suggested that under certain conditions such sequences can be eliminated from the genome (Kaplan et al. 1985). It is quite possible, then, that the predominantly weak hobo homologues in the montium subgroup represent sequences that are gradually drifting to extinction.
hobo Distribution in Drosophila 605
Acknowledgments We thank Ron Blackman for the gift of the pRG2.6X plasmid. Much of this work was supported by U.S. Public Health Service grants GM-3724 1 and GM-09886 to A.C. A portion of the study was conducted in the laboratory of Margaret Kidwell (U.S. Public Health Service grant GM-367 15 ) . LITERATURE
CITED
ANXOLAB~H~RE,D., and G. PBRIQUET. 1987. P-homologous sequences in Diptera are not restricted to the Drosophilidae family. Genet. Iber. 39:21 l-222. BELTZ, G. A,, K. A. JACOBS, T. H. EICKBUSH, P. T. CHERBAS,and F. C. KAFATOS. 1983. Isolation of multigene families and determination of homologies by filter hybridization methods. Methods Enzymol. 100:266-285. BEVERLEY,S. M., and A. C. WILSON. 1984. Molecular evolution in Drosophila and the higher Diptera. II. A time scale for fly evolution. J. Mol. Evol. 21:1-13. BLACKMAN,R. K., and W. M. GELBART. 1989. The transposable element hobo of Drosophila melanogaster. Pp. 523-529 in D. BERG and M. HOWE, eds. Mobile DNA. American Society for Microbiology, Washington, DC. BLACKMAN,R. K., R. GRIMAILA, M. MACY, D. KOEHLER, and W. M. GELBART. 1987. Mobilization of hobo elements residing within the decapentaplegic gene complex: suggestion of a new hybrid dysgenesis system in Drosophila melanogaster. Cell 49:497-505. BROOKFIELD,J. F. Y., E. MONTGOMERY,and C. H. LANGLEY. 1984. Apparent absence of transposable elements related to the P elements of D. melanogaster in other species of Drosophila. Nature 310:330-332. BUCHETON,A., M. SIMONELIG,C. VAURY, and M. CROZATIER. 1986. Sequences similar to the I transposable element involved in I-R hybrid dysgenesis in D. melanogaster occur in other Drosophila species. Nature 322:650-652. DANIEL& S. B., K. R. PETERSON,L. D. STRAUSBAUGH,M. G. KIDWELL, and A. CHOVNICK. 1990. Evidence for horizontal transmission of the P transposable element between Drosophila species. Genetics 124:339-355. DANIEL& S. B., and L. D. STRAUSBAUGH.1986. The distribution of P-element sequences in Drosophila: the willistoni and saltans species groups. J. Mol. Evol. 23~138-148. ENGELS,W. R. 1989. P elements in Drosophila melanogaster. Pp. 437-484 in D. BERG and M. HOWE, eds. Mobile DNA. American Society for Microbiology, Washington, D.C. FINNEGAN, D. J. 1989a. Eukaryotic transposable elements and genome evolution. Trends Genet. \ 5:103-107. -. 19896. The I factor and I-R hybrid dysgenesis in Drosophila melanogaster. Pp. 5035 17 in D. BERG and M. HOWE, eds. Mobile DNA. American Society for Microbiology, Washington, D.C. KAPLAN, N., T. DARDEN, and C. H. LANGLEY. 1985. Evolution and extinction of transposable elements in Mendelian populations. Genetics 109:459-480. KIDWELL, M. G., J. F. KIDWELL, and J. A. SVED. 1977. Hybrid dysgenesis in Drosophila melanogaster: a syndrome of aberrant traits including mutation, sterility, and male recombination. Genetics 36:8 13-833. KISSANE, H. M., and E. ROBINS. 1958. The fluorometric measurement of DNA in animal tissues with special reference to CNS. J. Biol. Chem. 233: 184-l 88. LEMEUNIER,F., L. TSACAS,J. R. DAVID, and M. ASHBURNER. 1986. The melanogaster species group. Pp. 147-256 in M. ASHBURNER,H. L. CARSON,and J. N. THOMPSON,JR., eds. The genetics and biology of Drosophila. Vol. 3e. Academic Press, London. MCGINNIS, W., A. W. SHERMOEN,and S. K. BECKENDORF. 1983. A transposable element inserted just 5’ to a Drosophila glue protein gene alters gene expression and chromatin structure. Cell 34:75-84.
606
Daniels et al.
MARTIN, G., D. WIERNASZ,and P. SCHEDL. 1983. Evolution of Drosophila repetitive-dispersed DNA. J. Mol. Evol. 19:203-213. RUSHLOW,C. A., W. BENDER,and A. CHOVNICK. 1984. Studies on the mechanism of heterochromatic position effect at the rosy locus of Drosophila melanogaster. Genetics 108:603615. STACEY,S. N., R. A. LANSMAN,H. W. BROCK, and T. A. GRIGLIATTI. 1986. Distribution and conservation of mobile elements in the genus Drosophila. Mol. Biol. Evol. 3:522-534. STAMATIS,N., M. MONASTIRIOTI,G. YANNOPOULOS,and C. LOUIS. 1989. The P-M and the 23.5 MRF (hobo) systems of hybrid dysgenesis in Drosophila melanogaster are independent of each other. Genetics 123:379-387. STRECK, R. D., J. E. MACGAFFEY, and S. K. BECKENDORF.1986. The structure of hobo transposable elements and their insertion sites. EMBO J. 5:36 15-3623. THROCKMORTON,L. H. 1975. The phylogeny, ecology and geography of Drosophila. Pp. 42 l469 in R. C. King, ed. Handbook of genetics, vol. 3: Invertabrates of genetic interest. Plenum, New York. YANNOPOULOS,G., N. STAMATIS,M. MONASTIRIOTI,P. HATZOPOULOS,and C. LOUIS. 1987. hobo is responsible for the induction of hybrid dysgenesis by strains of Drosophila melanogaster bearing the male recombination factor 23SMRF. Cell 49:487-495. BRIAN CHARLESWORTH, reviewing
editor
Received
January
received
Accepted
May 23, 1990
4, 1990; revision
May 10, 1990
