molecular characterization of hobemediated inversions in ......n-7547 4- h-2750 direction of hobo...

Copyright 0 1996 by the Genetics Society of America

Molecular Characterization of hobeMediated Inversions in Drosophila rnelanogaster

William B. Eggleston,” Nac R. Rimt.’ and Johng K. Limt

*Department of Biology, Virginia Commonwealth University, Richmond, Virgznia 23284 and tDepartment of Biology, University of Wisconsin, Eau Claire, Wisconsin 54702

Manuscript received April 10, 1996 Accepted for publication June 22, 1996

ABSTRACT The structure of chromosomal inversions mediated by hobo transposable elements in the Uc-1 X

chromosome was investigated using cytogenetic and molecular methods. Uc-1 contains a phenotypically silent hobo element inserted in an intron of the Notch locus. Cytological screening identified six independent Notch mutations resulting from chromosomal inversions with one breakpoint at cytological position 3C7, the location of Notch. In situ hybridization to salivary gland polytene chromosomes determined that both ends of each inversion contained hobo and Notch sequences. Southern blot analyses showed that both breakpoints in each inversion had hobo-l\iotch junction fragments indistinguishable in structure from those present in the Uc-1 Xchromosome prior to the rearrangements. Polymerase chain reaction amplification of the 12 hobeNotch junction fragments in the six inversions, followed by DNA sequence analysis, determined that each was identical to one of the two hobo-Notchjunctions present in Uc-1. These results are consistent with a model in which hobvmediated inversions result from homologous pairing and recombination between a pair of hobo elements in reverse orientation.

M OBILE or transposable genetic elements (TEs) are ubiquitous components of the genomes of

all organisms tested to date (see BERG and HOW 1989 for review). Although TEs occur in many shapes and sizes, all share the common ability to insert into new sites in genomes. Some also have the ability to excise from preexisting sites. TEs first were discovered by MCCLINTOCK (1946, 1947) based on their ability to induce chromosome breakage and rearrangement. How- ever, this feature of their biology has received little at- tention relative to investigations of their ability to cause insertion mutations, their transposition mechanisms and their utility as tools in molecular biology.

In terms of evolutionary importance, the long-term impact of TE-mediated chromosome restructuring is likely on par with their ability to induce “point” mutations following insertions in and near genes. Many families of TEs in numerous organisms have been shown to mediate chromosomal rearrangements such as deletions, duplications, inversions and translocations (see BERG and HOW 1989 and LIM and SIMMONS 1994 for reviews). Yet little is known about the mechanism(s) by which eukaryotic TEs mediate such events.

In Drosophila, hobo elements are one of several families of TEs known to mediate frequently chromosome rearrangements (reviewed in LIM and SIMMONS 1994). The largest hobo elements are 3-kbp long and contain a 1.9-kbp major open reading frame (OW) (STRECK et

Cmrsponding author; William Eggleston, Department of Biology, Virginia Commonwealth University, P.O. Box 842012, 816 Park Ave., Richmond, VA 23284. E-mail: [email protected]

’ Prrsent address: Department of Biology Education, Chonbuk Na- tional University, Chonju 560-756, Republic of Korea.

Genetics 144 647-656 (October, 1996)

al. 1986; BLACKMAN et al. 1989). At least one such 3- kbp element, denoted HFL1, expresses a trawacting product capable of inducing hobo movement, and therefore is defined as an autonomous element (BLACKMAN et al. 1989). CALW and GELBART (1994) have further shown that restriction of hobo movement primarily to the germline is due to tissue-specific regulation at the level of transcription. Many strains also contain shorter, internally detected hobo elements (STRECK et al. 1986; BOUSSY and DANIELS 1991). Both complete and incom- plete hobo elements are bounded by 12-bp terminal inverted repeats and during insertion they generate 8-bp target site duplications typically ending in the dinucleo- tide AC (STRECK et al. 1986). A truncated version of the terminal inverted repeat is located -240 bp from the 3‘ end of the full-size hobo, as defined by the direction of transcription of the 1.9-kbp OW. Two additional types of repeats also are found in larger hobo elements: three to 10 tandem copies of a 9-bp sequence called the short or “S” repeats and two tandem copies of a 20-bp sequence called the long or “L” repeats (STRECK

Based on sequence similarities in their protein cod- ing region and production of an &bp target site duplication during insertion, hobo elements are thought to be- long to a superfamily of evolutionarily related TEs that includes the following: Tam3 in Antirrhinum, Ac/Ds and Bg in maize, Tag1 in Arabidopsis and H m e s in housefly (POHLMAN et al. 1984; CALVI et al. 1991; FELD- MAR and KUNZE 1991; HARTINGS et al. 1991; ATKINSON et al. 1993; TSAY et al. 1993; WARREN et al. 1994; O’BROCHTA et al. 1996).

et d . 1986; LIM 1988).

648 W. B. Eggleston, M. R.

An important hallmark of several instances of hobo- induced phenotypic instability in Drosophila is the frequent association with cytologically visible chromosome rearrangements (BLACKMAN et al. 1987; YANNOPOULOS et al. 1987; LIM 1988; HO et al. 1993; SHEEN et al. 1993). To date most hobomediated rearrangements observed involve intrachromosomal events such as deletions, tandem duplications and inversions. Interchromosomal events such as translocations have been observed relatively infrequently.

The focus of the present report is the unstable X chromosome known as Uc-1. This chromosome and its progenitor Xchromosomes have been undergoing hobomediated rearrangements in the homozygous and hemizygous conditions for almost 200 generations (LIM 1979, 1981, 1988; LAVERTY and LIM 1982; JOHNSON-

SCHLITZ and LIM 1987; MORRISON et al. 1988; HO et al. 1993; SHEEN et al. 1993). Molecular analyses of Uc-1 have shown that the Notch locus of this chromosome contains the insertion of a 2.95-kbp hobo element that does not affect Notch expression (LIM 1988; HO et al. 1993). Typical sublines of Uc-1 produce Notch mutations at frequencies between 4 and 1096, all of which are rearrangements involving cytological position 3C7, the location of Notch (Ho et al. 1993; J. LIM, unpublished observations), The vast majority of such lesions are deletions. The cloning and determination of relative orientations of hobo elements on the Uc-1 chromosome in the above reports indicated that the elements interacted in an orientation-dependent manner to induce chromosomal rearrangements. LIM (1988) observed that preexisting hobo elements in direct orientation mediate chromosome deletions while those in reverse orientation mediate inversions. These observations suggested the possibility that hobomediated chromosome rearrangements occur via homologous pairing and recombination between hobo elements at ectopic sites on the Uc-1 chromosome (Ho et al. 1993; SHEEN et al. 1993; LIM and SIMMONS 1994), as has been suggested for several other families of mobile elements in Drosophila and in other species (reviewed in LIM and SIMMONS 1994).

The objective of the current study is to use cytogenetic and molecular methods to characterize hobo-mediated chromosomal inversions of the Uc-I X chromosome further to deduce a molecular mechanism for these events. Notch mutations of independent origin associated with chromosomal inversions were recovered, and the inversion breakpoints examined by in situ hybridization, Southern blot, PCR amplification and DNA sequence analysis. The purpose of this analysis was to determine whether inversions were likely to have arisen via homologous recombination between hobo elements in reverse orientation or with alternative mechanisms. The results are discussed in relationship to models for rearrangements and transposition involving other TE families in Drosophila and in other eukaryotes.

Rim and J. K. Lim

MATERIALS AND METHODS

Genetic stocks: All stocks and experimental cultures were maintained at 22-25' as described previously (Ho et al. 1993). See LINDSLEY and ZIMM (1992) for genetic symbols and termi- nology.

Uc-Z: This strain contains numerous complete hobo elements as determined by Southern blot analysis (Ho et al. 1993). The results of in situ hybridization during recent years consistently has detected - 100 total sites of hobo homology in this genome, of which 10-35 are on the X chromosome (J. LIM, unpublished observations). The X chromosome in this stock, called Uc-I, carries the three recessive markers y5"', z and u'. The Uc-Z chromosome was derived from the rearranged unstable X chromosome Df(1)cm-In as described in JOHNSON-%HI.ITZ and LIM (1987) and LIM (1988). Uc-1 has a hobo element at cytological position 3C7, location of the Notch locus. This insertion does not interfere with Notch function. Homozygous stocks containing the Uc-I chromosome produce Notch mutations at a frequency of up to 10% per generation, all involving chromosome deletions or inversions with a breakpoint at 3C7 (LIM 1988; HO et al. 1993; J. LIM, unpublished observations). Uc-1 sublines producing high frequencies of Notch mutations were maintained as homozygotes by sib or single pair matings between non-Notch individuals.

Canton S (CS): Wild-type strain lacking hobo elements. This strain is routinely used in matings to Uc-1 to detect newly arisen Notch mutations.

y' z' spl sn3: This strain, like Canton S, lacks hobo elements (Ho et al. 1993). This strain routinely is mated to FM7/ Uc-1, Notch females to perform cytological and in situ hybridization analyses of Uc-1 X chromosomes with Notch mutations (see below).

FM7/C(I)DX, y f/Y (abbreviated FM7): a balancer Xchromo- some stock. The free-X chromosome carries the recessive markers y"", sc', u" and B. Newly arisen phenotypically Notch females were mated to FA47 males to maintain Notchbearing Uc-1 X chromosomes since Notch mutations have a dominant wing phenotype and also are recessive lethals.

Cytological and in situ hybridization analysis: y' z' spl sn3 males were mated to FM7/Uc-Z, Notch females and the prog- eny female larvae with light brown mouth parts (y' larval phenotype of y59/' from Uc-1 and y' from y' z' spl sn') were selected, polytene chromosomes were prepared, and analyzed as described in LIM and SNYDER (1968). For each inversion, at least 20 nuclei clearly showing inversion breakpoints were examined by cytological analysis, and at least 30 nuclei were analyzed by in situ hybridization analysis. Biotinylated DNA probes were hybridized in situ to salivary gland polytene chromosomes as described in LIM (1993).

Isolation of genomic DNA and Southern blot analysis: Ge- nomic DNA was isolated from FM7 males, Uc-l homozygotes and females heterozygous for FM7 and each of six X chromosomes containing inversions with breakpoints in 3C7 using the Puregene Kit (Gentra). Purified DNA was digested with restriction endonucleases, separated on 1% agarose slab gels, transferred to Durulon-UV and crosslinked as described in EGGLESTON et al. (1995). Filters were hybridized with radiola- beled DNA fragments, posthybridized, subjected to autoradi- ography for 1-5 days and stripped as described in ECCLESTON et al. (1995) using standard methods (SAMBROOK et al. 1989).

PCR amplification, DNA cloning and DNA sequence analysis: The approximate location and relative orientation of the hobo element inserted at cytological location 3C7 initially was deduced by comparison of the restriction map of hEC245 (LIM 1988) to the restriction map and sequence of the-wild type Notch locus (KIDD et al. 1983; KIDD et al. 1986). As shown in Figure 1, two pairs of oligomers were designed to amplify each hobo-Notchjunction for the hobo element inserted in 3C7 in the Uc-I Xchromosome. At the hobo 5' end were H-69 5'-

hobplllediated Inversions

N-7063 4

H-118 * H - 6 9

649

N-7 1 04 -4- -+ - + Direction of Notch H-2890 transcription

hobo !3= -3’ 4- N-7547

4- H-2750

Direction of hobo N-7569 transcription +

& 7326 7333

EiGiEl 7326 7333

FIGURE 1.-Schematic showing relative orientation and insertion site of hobo element in intron 5 of Notch locus and oligomers used in PCR amplification of hobo-Notch junction fragments from Uc-I and inversion X chromosomes. Open box represents hobo element and thick lines Notch sequences. Small arrows show relative positions of oligomers in 5’ + 3’ orientation. Notch coordinates are from KIDD et al. (1986). Boxed sequences represent 8-bp target site duplication of Notch DNA flanking hobo element and their coordinates. See MATERIALS AND METHODS for sequences of hobo and Notch oligomers.

AAGCTTGATGTGCGTGGCGAGT-3‘ and H-118 5”AAGCTT- GGTGTCTTGTCTGCATGT-3’. At the hobo 3’ end were H- 2750 5’-AAGCTTGTTGACTGTGCGTCCACT-3’ and H-2890 5’-AAGClTTGCCTAGAGTACCGAGTGT-3’. In the region of Notch 5’ to the hobo insertion site were N-7063 5”GCACTC- TTATTGCAACGT-3‘ and N-7104 5”CGTGTCGGTTTC- GTTGT-3’. In the region of Notch 3’ to the hobo insertion site were N-7547 5’-AGCCGGAGATCGTGTGAA-3’ and N-7569 5’- GCGCATATGAGAAGTGCA-3’. Oligomers were synthesized by the MCV-VCU Nucleic Acid Synthesis and Analysis Core Facility and by Integrated DNA Technologies. Typical PCRs were carried out based on protocols in INNIS et al. (1990) using one Notch and one hobo oligomer in a total volume of 50 p1 overlayed with mineral oil. Reaction conditions included -10 ng of genomic DNA, 1 pg of each oligomer, 2-4 mM MgCle, 10 mM Tris (pH 7.4), 250 mki dNTPs and 1 unit Taq DNA polymerase (Fisher Scientific). Reactions were per- formed in a Hybaid thermal reactor (Gibbco-National Lab- net). Depending on the oligomer combination, typical cycling conditions were 30-33 rounds of 25-30 sec at 92”, 60 sec at 60-65” and 60 sec at 72”. To test for amplified fragments, 5- 10 pl of each sample was subjected to electrophoresis in a 2% agarose, 0.5X TBE slab gel, stained with ethidium bromide (SAMBROOK et al. 1989) and photographed with an Eagle Eye Photo Imaging System (Stratagene) under W transillumina- tion. Amplified fragments were ligated to ddT-tailed, EcoRV- digested pBluescript I1 (pBSII) (Stratagene) prepared essen- tially as described by (HOLTON and GRAHAM 1991) with minor modifications recommended by H. ROBERTSON (personal communication). Standard methods (SAMBROOK et al. 1989) were used to subclone EcoRI fragments from XEC245 (see below) into the EcoRI site of pBSII. Sequences of inserts in plasmids derived from PCR amplification or from subcloning phagederived fragments were determined using Sequenase v2.0 (United States Biochemical). The Ks or SK oligomers flanking the multiple cloning site in pBSII (Stratagene) or the hobo and/or hobo oligomers listed above were used to direct DNA synthesis under conditions recommended by the manufacturer for double-stranded plasmid DNA. Following termination of sequencing reactions, DNAs were separated on 6% Long Ranger (Calbiochem) vertical gels under conditions recommended by the manufacturer, dried under vacuum and autoradiographed for 1-3 days at room temperature.

DNA probes: Probes used in in situ and Southern blot hybridizations include the EcoRI Notch fragment in p2.2R-N2, 1.1- and 1.8-kbp HindIII/BamHI fragments from the plasmid pSB4 and the entire phage AEC294. p2.2R-N2 is a 2.3-kbp EcoRI fragment inserted into the EcoRI site of pBSII (Figure 2). The 2.3-kbp fragment corresponds to wild-type Notch sequences flanking the hobo insertion site in 3C7 in the Uc-I X chromosome. This fragment encompasses coordinates 6662- 8983 on the Notch sequence map of KIDD et al. (1986). For

simplicity, this EcoRI fragment will be referred to as p2.2R- N2. pSB4 contains a PCR-amplified fragment spanning the entire 2.95-kbp hobo element from XEC245 inserted into the BamHI site of pBSII. XEC245 is a phage clone containing the hobo element and flanking Notch sequences from the Uc-I X chromosome ( LIM 1988). For simplicity, probes made from the two HindIII/BamHI fragments from pSB4 will be collec- tively referred to as pSB4. hEC294 contains a 2.95-kbp hobo element and flanking DNA from cytological position 9A of the Uc-1 Xchromosome (LIM 1988).

RESULTS

Characterization of the hobo element insertion site in the Notch locus in the Uc-I X chromosome: Restriction map analysis (LIM 1988) established that the hobo element integrated in the Notch locus in the Uc-1 Xchromo- some had inserted in reverse orientation, relative to Notch transcription, in a 2.3-kbp EcoRI fragment encompassing a portion of the wild-type locus. This hobocon- taining EcoRI fragment was contained in the phage clone XEC245 (LIM 1988). As summarized in Figure 2, EcoRI digestion releases fragments of 1.7 and 2.9 kbp containing the junctions between hobo and Notch sequences. The 1.7-kbp fragment contains the hobo 3’ end and Notch sequences 5’ to the hobo insertion site, and the 2.9-kbp fragment contains the hobo 5’ end and Notch sequences 3’ to the insertion site in XEC245 (LIM 1988). These two fragments were inserted separately into the EcoRI site of pBSII, and the sequence of both strands of each hobo-Notch junction was determined by DNA sequencing using the hobo and Notch oligomers shown in Figure 1 and listed in METHODS AND MATERIALS to prime synthesis. As summarized in Figure 1, the hobo element in XEC245 is inserted in Notch intron 5 at coordinates 7326-7333 from the sequence map of KIDD et al. (1986). The hobo element is flanked by a direct duplication of the 8-bp target site AAGCAAAC. This duplication is consistent with previous observations that hobo elements generate 8-bp direct target site duplications upon insertion, frequently ending in the dinuclec- tide AC (STRECK et al. 1986). No differences were detected in the 50-bp of hobo sequence and 50-bp of adjacent Notch sequence determined at each end of the hobo insertion, relative to published hobo and Notch sequences (KIDD et al. 1986; STRECK et al, 1986).

650 M’. B. Eggleston, M. R. Rim andJ. K. L.im

R 0.65-kbp 1.65-kbp R r 1

I~ 2.3-kbp I

t R

1.7-kbp R

I R

2.9-kbp I R

FIGLIRE 2.-Diagram showing the 2.Skhp k,hRI fragment in the wild-type ,\b/ch locus and in the Uc-1 X chromosome. The 2.Skhp fragment in the wild-type No/ch locus is shown at the top with the approximate site of the IroOo element insertion in the Uc-1 Xchromosome and distances from the insertion site to each end of the fragment. The LcoRI fragments released from the corresponding region i n the Uc-1 Xchromo- some are shown at the bottom along with the origin of the 1.7- and 2.9-kbp I h R I hobn”Vo/ch junction fragments with homology to p2.2R-N2 and holm probes. The .5’ end of the “Wrh locus is to the left.

Identification and cytological analysis of inversions with breakpoints in Notch: In a previous study (Ho pt

nl. 1993) of 80 independent No/ch mutations of the Ur-I chromosome screened cytologically, 73 were associated with visible chromosome rearrangements involving 3C7, and only three of these were simple inversions. Upon further examination, each of the remaining seven mutations was shown to be associated with a small deletion with one breakpoint at 3C7 ( J . LIk1, unpuk lished observations). In the current study, more than 120 new, independent Notrh mutations of the Ur-I X were screened cytologically to recover additional simple inversions with one breakpoint in 3C7. Nearly all Notch mutations involved deletions with a breakpoint at 3C7. However, three additional, simple inversions involving 3C7 were identified, as well as one translocation between 3C7 and chromosome 2 [ T ( 1 ; 2 ) 3 C ; 3 8 B j . Table 1 lists the six Nofch mutations having simple inversions, and their breakpoints, recovered in this and in the previous study by H o ut nl. (1993).

All six inversions analyzed by in sitzc hybridization

TABLE 1

Description of inversions derived from the Uc-1 Xchromosome

Name Cytological hreakpoints Source

5 In(1)3C7;2R This work 6 In(1)3C7:3111-2 This work 5j 5 In(l)3C7;4E H o d nl. (1993) 22j4 In(1)3C7;6A Ho d nl. (1993) 298 In(l)3C7;14R This work 8d.5 In(1)3C7;19E H o c/ ( I / . (1993)

B - FIGLRE S.--S;lli\.al-\. glm~tl poly~cnc Xrhromosomes hctertr

zygous for a normal X chromosome (J’ :’ si)/ sn’) and a holm mediated inversion chromosome hyhridizctl with hobo prohes. A was hybridized with XE(294 and panel R with pSR4. (A) The normal X chromosome heterozygous with In(l)3C7; 191.: (inversion 8d.5). (B) The normal X chromosome hetcrozy- gous with ln(1)3C7;14R (inversion 298). The inversion hreakpoints are indicated hy small arrows in each panel. A short horizontal har in each panc,l reprcsents “10 p m .

analysis had homology to hobo and No/ch probes at both inversion breakpoints. Examples of in .s i /?! hybridization results with hoho probes are shown in Figure 3 for h ( l ) 3 C 7 ; 19I:’and In(1)3C7; 14N. This probe labels both ends of each inversion, showing that each breakpoint contains hobo sequences. In A, i n addition to label at the breakpoints at positions X 7 and 19E, -25 additional sites of hobo homology were observed. Included among the labeled sites are cytological positions 2R, 4E, 6A and 19E. These four sites coincide with the second breakpoint in four o f the five remaining No/clr inversions studied. Although the ln(1)3C7; 191:‘ chromosome failed to show Iloho homology at position 14R, the second breakpoint in the sixth inversion chromosome examined, In( l )3C7;14B, analysis of most other Uc-1 X chromosomes did detect label at this position.

The ectopic recornhination model of inversion formation predicts that each breakpoint will have a portion of the No/rh locus adjacent to the respective Itobo element. As shown in Figure 2, the I w A o insertion site relative to p2.2R-N2 is “6.50 bp from the 5’ end. Therefore, if inversions involve sequences within two ectopic llobo elementq in reverse orientation, one breakpoint should label more intensely than the other i n in .si/?( Ilybridiza- tions with p2.2R-N2 due to different Icngths of homol-

o<q. to this probe. In Fact, all six inversions had different intensities of label at each breakpoint. Weaker labeling at the distal breakpoint than at 3 C f was ohsetTed for the sole inversion in which the second breakpoint w a s distal to 3 C i , In(I)j'C7;2N (Figure 4D). Conversely, a s shown for three such inversions in Figures 4, A-C, stronger labeling was ohsewed at the proximal breakpoint than at 3C7 i n the five remaining inversions i n which the second breakpoint was proximal t o Nolrh.

Southern blot analysis of Notch mutations with inversions involving 3C7: Cytological and in s i / u hybridization data were consistent with each of the six 1 W r . h

mutations arising 7 1 i o recombination in or near the hoOo elemcmt in >\i)/ch and a second hoBo element elsewhere in the C'c-1 Xchromosome. To characterize these events further. genomic DNA from FM7 males, Uc-1 flies and females lwteroz!y)rls for fi\liand each o f the inversion chromosomes was digested with f M V I plus fh/XI, South- ern blotted and h~~hridizccl with p2.2R-N'L. Neither o f these two enzymes cleave inside canonical hoboelements (S7'RI.:(:li r/ (I / . 1986). Results are shown in Figure 5. Rased on sequence analysis ( K m ) r / ai. 1986), a single 3.3f-kbp I3s/NI/fJs/XI fragment with homology to p2.2R-X2 was expected to be released from the wild- type .\'o/ch locus. As seen in lane 1, a fragment of 3.3 khp is released from F M i , which is wild type for Notch. I n lane 2, a 6.2-kbp fragment is released from Uc-1, as expected for the insertion of a 2.95-kbp hobo element into the 3.3-kbp fragment. The presence o f the faint 3.3-kbp fragment in LJc-1 DNA indicates a precise or nearly precise excision of the hobo element in some cells or flies.

I n lanes 4-8, the 3.3-kbp fragment released from I347 is retained, consistent with all individuals being heterozygous for this chromosome. However, in each of these lanes the 6.2-kbp fragment released from Uc-I is replaced by two novel size fragments. These results indicate that each inversion involved a breakage in or near the hoOo element in ,Vo/rh and that physical separa- tion o f the No/ch locus into two fragments had occurred. These results are consistent with in s i / u hybridization results showing that portions of the Notch locus with homology t o p2.2R-N2 arc present at each end of each inversion. Also noteworthy is that the two new fragments in each lane have differential laheling intensities, as obsened for pairs of breakpoint in in s i / u hybridizations with this probe. That each inversion has a unique pair of new fragments is consistent with an interaction between the l~obo element in ,Vo/ch and second hoAo elements at various sites in the genome with distinct nearby patterns of Il.s/sc\II and fWXI recognition sites.

The above results did not address whether a hobo element w a s present at both breakpoints in each inversion or the relative orientations o f the hobo elements. To examine these questions, each of the above DNA samples w a s digested with EcoRI and hybridized with p2.2R-N2. The results are shown in Figure 6. EcoRI cleaves at two sites in canonical ho00 element5 (STRECK

FKX'KE 4.--Salivav gland polytene Xchromosomes heterozygous for a normal X chromosome (J' z' spI sn') and a hob^ mediated in\wsion chromosome hyhridized with the Notch locus prohe p2.2R-N2. In each panel, the No/rh locus in the normal X is indicated by a small arrow. Two additional labels in each panel mark the two hreakpoints in the inversion chromosome. (A) The normal X chromosome heterozygous with ln(l)?C7;19E (inversion 8d.5). (R) The tip of the Xchromo- somes heterozygous for the normal X chromosome and In(1)3C7;3111-2 (inversion ti). ( C ) The normal Xchromosome heterozygouswith In(l)?C7; 14B (inversion 298). (D) The normal Xchromosome heterozygous with In(I)?C7;2B (inversion 5 ) . A short horizontal bar in each panel represents "10 pm.

P/ nl. 1986; C A I . \ ~ I P/ nl. 1991). As expected, a single fragment of 2.3 kbp is released from FM7 (lane l ) , equal i n size to the 2.3-kbp fragment expected based on the previous sequence analysis of Nolch (UDD rl nl. 1986). Since EcoRI cleaves within the hoho element in-

652 W. B. Eggleston, M. R. Rim and J. K. Lim

. 1 " . 2 .." 3 .cb 4 ..D3 5 - 6--7 8 1 2 3 4 5 6 7 8

6.2- mIr --e

3.3- = " FIGURE 5.-Drosophila genomic DNA digested with BstNI

and BstXI and hybridized with p2.2R-N2. Lanes are as follows: (1) FM7, (2) Uc-1, (3) FM7/In(1)3C7;2B, (4) FM7/ FIGURE 7.-Drosophila genomic DNA digested with EcoRI In(1)3C7;3D1-2, (5) FM7/In(1)3C7;4E, (6) FM7/In(1)3C7;6A, and hybridized with pSB4. Rehybridization Of filter PrevioUs1Y (7) I;M7/In(1)3C7;14B, (8) FM7/In(1)3C7;19E. Numbers to hybridized with p2.2R-N2 (fo1lowing strippin@. Lanes are as left indicate sizes in kilobase pairs. Sizes are 20.1 kbp. follows: (1) FM7, (2) Uc-1, (3) FM7/In(1)3C7;2B, (4) FM7/

In(l)3C7;3Dl-2, (5) FM7/In(1)3C7;4E, (6) FM7/In(1)3C7;6A, serted into the EcoFU Notch fragment used as a probe (7) FM7/In(1)3C7;14B, (8) FM7/In(1)3C7;19E. Numbers to

(see Figure 2), two fragments of 1.7 and 2.9 kbp were left indicate sizes in kilobase pairs. Sizes are 20.1 kbp.

expected to be released from Uc-1 flies. As shown in lane Lanes 3-8 show that the wild-type 2.3-kbp fragment 2' a wi1d"We fragment (2'3 kbp) plus two additiona1 released from the FM7chromosome is present in each fragments are present in Uc-1. As discussed above, the wild-type fragment likely results from excision of the lane as expected because all six inversion chromosomes

hobo element from the Notch locus in a fraction of indi- are heterozygous for this chromosome. Also shown are

viduals or in tissue of individuals from which DNA was the fragments indistinguishable in size from the 1.7-

prepared. The 1.7- and 2.9-kbp fragments therefore and 2.9-kbp fragments present in Uc-1 released from

likely are released from the Uc-1 X chromosome re- each inversion chromosome. This indicates that follow-

taining a hobo insertion in the Notch locus. ing each inversion event, both pieces of the Notch locus remain adjacent to at least a portion of a hobo element

1 2 3 4 5 6 7 8

1.7-

FIGURE 6.-Drosophila genomic DNA digested with EcoRI and hybridized with p2.2R-N2. Lanes are as follows: (1) FM7, (2) Uc-1, (3) FM7/In(I)3C7;2B, (4) FM7/In(1)3C7;3Dl-2, (5) FM7/In(1;)3C7;4E, (6) FM7/In(1)3C7;6A, (7) FM7/ In(1)3C7;14B, (8 ) FM7/In(1)3C7;19E. Numbers to the left indicate sizes in kilobase pairs. Sizes are 20.1 kbp.

- at the same relative location and in the same relative orientation as in the Uc-1 Xchromosome.

To confirm that hobo sequences remain adjacent to the Notchsequences present at both breakpoints in each inversion, after hybridization with p2.2R-N2, filters were stripped and rehybridized with pSB4, which contains only hobo sequences from the Drosophila genome. The results are shown in Figure 7. Although partially ob- scured by the many other EcoRI fragments with homology to the hobo sequences, both the 1.7- and 2.9-kbp fragments in Uc-1 and in each inversion line contain hobo homology. In contrast, the 2.3-kbp wild-type fragment in all eight lanes with homology to Notch is not recognized by the hobo sequences in pSB4. These results are consistent with the conclusion that following breakage of the Notch locus, both pieces of the gene remain adjacent to hobo sequences at the same approximate site and in the same relative orientation as present in the Uc-1 X chromosome. Lack of signal from the 2.3-kbp wild-type size fragment in Figure 6 shows that hybridization to the 1.7- and 2.9-kbp fragments is not due to residual signal from hybridization with p2.2R-N2.

PCR and DNA sequence analysis of Notch inversions

hobeMediated Inversions 653

involving 3C7: Neither the precision with which the above inversions occurred nor potential sites of breakage could be determined with the above methods due to relatively low resolution. To characterize further the structure of each inversion, the region encompassing each hobo-Notchjunction in Uc-I, and in each inversion, was PCR amplified using combinations of the oligomers shown in Figure 1. Combinations used successfully included: N-7063 + H-2890 and N-7104 + H-2890 to amplify the junction between the 3' hobo end and adjacent Notch sequence and N-7547 + H-69 and N-7547 + H- 118 to amplify the junction between the 5' hobo end and adjacent Notch sequences.

For each of the two combinations of oligomers designed to amplify each hobo-Notch junction present in the Uc-1 X chromosome, amplifications produced fragments from Uc-1 of the expected size based on the hobo and oligomer positions shown in Figure 1. All four combinations of oligomers produced amplified fragments identical in size to those recovered from Uc-1 when used to amplify DNA from each of the six inversions. in no instance were fragments similar in size to those expected for the hobo-Notch junctions in Uc-I produced in parallel amplifications with FM7. To insure that the FM7 DNA was capable of supporting amplification, Notch primers alone, such as N-7063 + N-7569 and N-7104 + N-7547, were used in amplification reactions. In each instance, fragments of the size expected for a wild-type Notch locus were produced showing that FM7 DNA could support amplification. Since no Uc-I-size fragments were observed in amplifications with FM7 DNA, these results indicate that the amplified fragments produced from the six inversion lines are not the result of inadvertent contamination with Uc-I DNA.

For each of the six inversions, at least one hobo-Notch junction fragment for each hobo end was subcloned into the ddT-tailed EcoRV site of pBSli and subjected to DNA sequence analysis. Thus, more than 14 independent clones were characterized, a minimum of two from Uc- I and from each of the six inversion lines. The sequence of both hobo-Notch junctions derived from Uc-1 were identical to the corresponding hobeNotch junctions present in each inversion (data not shown). Thus, inversions did not result in any observable sequence changes at the Notch-hobo junctions tested.

DISCUSSION

Mechanism of hobemediated inversions in Uc-1 X chromosome: Previous work from several laboratories has shown that hobo elements are capable of mediating frequent chromosome restructuring (BLACKMAN et al. 1987; YANNOPOULOS et al. 1987; LIM 1988; H o et al. 1993; SHEEN et al. 1993). Typically, restructuring was intrachromosomal rather than interchromosomal. Where tested, such rearrangements typically had hobo sequences at each breakpoint before and after restructuring (BLACKMAN 1987; YANNOPOULOS et al. 1987; LIM 1988; HO et al. 1993; SHEEN et al. 1993).

1 hobo 3 2 hobo 4 ---"..................~ FIGURE 8.-Model for the mechanism of hobemediated in-

versions in the Uc-1 X chromosome. The region defined by two elements in reverse orientation to each other (in dotted line with loci 2 and 3) is the region to be inverted. A recombination event within the interacting hobo elements (at center) is envisioned to yield the inversion chromosome shown at the bottom.

LIM (1988) further showed that the outcome of hob@ mediated rearrangements was orientation dependent. When preexisting elements in the same relative orientation in a chromosome interacted, the outcome was a deletion of intervening material and the presence of a hobo at the deletion breakpoint. Conversely, when preexisting elements were in reverse orientation, the outcome was an inversion with hobo elements at both breakpoints.

Taken together, the above observations indicate that most chromosome restructuring involves interactions between preexisting hobo elements, two at a time, and the outcome of the interaction is dependent on the relative orientation of the hobo elements. Based on these observations, LIM (1988) and LIM and SIMMONS (1994) proposed a model in which hobo elements induce chromosome restructuring via homologous pairing and recombination between elements at ectopic sites in the genome.

As predicted based on the ectopic recombination model, following each of the six inversions tested, hobo and Notch sequences were present at each breakpoint, and the orientation, position and sequence of adjacent hobo and Notch sequences were unaltered during the process of chromosome restructuring. Additionally, the hobo elements must be in reverse orientation relative to each other following each inversion. If the hobo elements were in direct orientation following each inversion, one hobo-Notch junction would be identical to a junction present in Uc-1 while the other would be altered in size relative to Uc-l. This would occur because the relative orientation of the Notch sequences on at least one side of the original hobo would be reversed relative to the hobo at the second site in the genome following inversion. Such a change would alter the dis- tance between the EcoRI site in Notch sequences and the EcoRI sites within the adjacent hobo element. Thus, if the hobo elements are in direct orientation following

654 W. B. Eggleston, M. R. Rim and J. K Lim

inversion, one fragment observed on Southern blots would remain in common with Uc-I while the other would be altered in mobility in hybridizations with Notch and hobo sequences. Instead, as seen in Figures 6 and 7, fragments with homology to p2.2K-N2 and pSB4 released from Uc-I were indistinguishable in size to those present in all six inversions. The only way in which this result could be produced is if the hobo elements at each breakpoint are in reverse relative orientation following each inversion.

For technical reasons, it was not possible to determine the relative orientation of the hobo elements before each particular inversion. However, for the hobo elements at each breakpoint to be in reverse orientation following inversions necessitates that they also be in reverse orientation before each inversion event. This conclusion is supported by LIM’S observation (1988) that only preexisting hobo elements in reverse orientation produced inversion events. Figure 8 shows the basic model for inversions based on homologous pairing and recombination between hobo elements at ectopic sites on the same chromosome. While not addressed di- rectly, the results of this report also are consistent with hobomediated deletion events resulting from a similar type of homologous recombination between hobo elements in direct relative orientation.

Relationship of hobemediated chromosomal rearrangements and rearrangements mediated by other families of TEs: Numerous families of TEs in many eukaryotes have been shown to mediate chromosome rearrangements (reviewed in BERG and HOWE 1989; LIM and SIMMONS 1994). To date, all structural classes of TEs have been shown to mediate chromosome restructuring. This includes retrotransposons, with long terminal direct repeats, such as BEL, B104 and roo in Drosophila (GOLDBERG et al. 1983; DAVIS et al. 1987; TSUBOTA et al. 1989; MONTGOMERY et al. 1991) and Ty elements in yeast (KOEDER and FINK 1983; BOEKE 1989), retroposons or LINES, which lack terminal repeats, such as Doc and I in Drosophila (SCHNEUWLY et al. 1987; BUSSEAU et al. 1989) and Alu in humans (LEHRMAN et al. 1987; STOPPA-LYONNET et al. 1990), elements with short terminal inverted repeats, such as P, Ac/Ds, Tam3 and a mariner transposon-like element in Drosophila, Antirrhinum, maize and humans (MCCLINTOCK 1946, 1947; ENGELS and PRESTON 1984; MARTIN et nl. 1988; REITER et al. 1996), and lastly, elements with long terminal inverted repeats such as FB in Drosophila (COLLJNS and KUBIN 1984).

As reviewed in some detail in LIM and SIMMONS (1994), four basic types of mechanisms have been proposed to account for TE-mediated chromosomal rearrangements. These results are based primarily on a combination of cytogenetic and restriction map analyses. In the simplest mechanism, proposed to account for rearrangements mediated by Alu, BEL, B104, Doc, FB, roo, and hobo elements and by some Tam3 and Ty, chromosome restructuring results from pairing and homologous recombina-

tion between preexisting TEs at ectopic sites in the genome. Only in rearrangements explained by this type of mechanism has DNA sequence analysis confirmed a lack of DNA sequence alterations breakpoints. This includes Alzcmediated deletion (STOPPA-LYONNET et al. 1990) and duplication breakpoints (LEHRMAN et ul. 1987), BI04-mediated duplication breakpoints (TsuBOrA et al. 1989) and Doc-mediated inversions (SCHNEUWLY et al. 1987).

As reviewed in LIM and SIMMONS (1994), other types of rearrangements appear to involve more complex mechanisms resulting from aberrant transposition of TEs and/or double-strand breaks generated by TEs during transposition. Where sequenced, the rearrangement breakpoints have had additions, losses or replacement of bases at the TE ends for elements including I (BUSSEAL! et al. 1989), Ac/Ds (DORING et nl. 1990; WEIL and WESSLER 1993) and Tam3 (MARTIN et al. 1988; LISTER et al. 1993). In spite of the putative role of element-encoded transposases in chromosome restructuring, to date, only rearrangements mediated by Ac/Ds and P elements have been shown to be dependent on the presence of an element-encoded transposase (EN- GLES and PRESTON 1984; DORING et al. 1990; DOONER and BELACHEW 1991; WEIL and WESSLER 1993). For most other elements, tools currently are not available to test this question. In the current study, it was not possible to assess the role of the hoboencoded transposase in recombination with the materials used.

The most striking feature of TE-mediated rearrangements is that elements which transpose via distinct mechanisms appear to promote ectopic recombination. This similarity could simply reflect that independent of transposase activity, random breaks made in elements are capable of inducing homology searches and pro- moting homologous recombination by repair mechanisms (e.g., SZOSTAK et al. 1983). Alternatively, host pro- teins involved in mediating transposition and/or each element’s transposase could make single- or doublestrand nicks and breaks at the element ends or internal to element termini, in effect, enhancing recombination and repair mechanisms. In this case, transposase would only act to make breaks acted upon by host enzymes that participate in homologous recombination and repair. This possibility is made more likely by the recent observation by CRAIG (1995) that there appears to be similar and unifying enzymatic reactions involved in transposition of RNA-intermediate and by cut-and-paste type elements leading to single- and double strand breaks at the ends of TEs. Thus, while initiated by transposase, transpositions and rearrangements would not otherwise be mechanistically related.

For hobemediated rearrangements in the Uc-1 Xchro- mosome, current evidence is clearly consistent with models based on ectopic, homologous recombination. However, this evidence does not exclude other models such as the random breakage and rejoining proposed for Pelements (ENGELS and PRESTON 1984), or viaaber-

hobo-Mediated Inversions 655

rant transposition as proposed for Ac/Ds (DORING et al. 1990; WEIL and WESSLER 1993) and Tam3 elements (MARTIN et al. 1988; LISTER et al. 1993). However, in the present report, it is noteworthy that no sequence changes or replacement of 8-bp target site duplication were identified near the hobo ends, as previously observed for rearrangements ascribed to aberrant transposition mechanisms. Additionally, the results do not address the role of hobo transposase in the restructuring process. As more rearrangements are examined at the molecular level, it is possible that events consistent with other rearrangement mechanisms may be identified. Rather than indicating that the homologous recombination model is incorrect, such data may show that hobo elements mediate chromosomal restructuring via a vari- ety of mechanisms.

This work was supported by American Cancer Society institution grant IN-105s to W.B.E. and by National Science Foundation grant MCB-9318934 to J.K.L.

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Communicating editor: 1,. L. SEARI.ES

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