Nucleic Acids Research

Construction of expression plasmids

A plasmid for expression of full-length transposase was constructed as follows. Primers M0804 and M0805 are complementary and contain the sequence at the junction of ORF1 and ORF2 of pogo as indicated by the sequence of pogo cDNAs (8). Primers S5046 and S5049 comprise the sequences at the beginning of ORF1 and end of ORF2, respectively. In S5046 the ORF1 sequence is preceded by a BamHI site and in S5049 the ORF2 sequence is followed by an XhoI site. The sequences of ORF1 and ORF2 were amplified separately using primers S5046 and M0804 and primers M0805 and S5049 and a template from pogoR11XC (8). The products of these reactions were mixed in equimolar amounts and used as templates for a second PCR using primer S5046 and S5049 as primers. The final PCR product was purified by GENECLEAN (BIO101) and cloned into a pGEX4T-2 vector (Pharmacia) via BamHI and XhoI sites in a ligation reaction catalysed by T4 ligase (New England Biolabs). This joins the C-terminus of GST to the second codon of transposase omitting the methionine that is presumed to initiate translation. Competent E.coli NM522 cells were then transformed and the transformants were selected on L-Amp plates (100 µg/ml ampicillin). The positive constructs were examined by DNA sequencing before being used for protein expression.

DNA coding for various fragments of pogo transposase were amplified by PCR using different pairs of primers: S5046 and A05 for N158, S5046 and A06 for N138, S5046 and T6395 for N74, S5046 and T5774 for N59, Z7187 and S5049 for C430, A09 and S5049 for C380, A10 and S5049 for C341, and S5048 and S5049 for C193; these PCR products were cloned into pGEX4T-2 using the same restriction sites as the whole transposase as described above.

Pairs of complementary primers were designed to generate single amino acid substitutions within GST:N74 as follows: V7017 and V7018 for the R39A mutation, V7013 and V7014 for the R44A mutation, V7015 and V7016 for the mutation K48A, W0914 and W0915 for K34A, W0916 and W0917 for R63A, V7912 and V7913 for C34P, and V7914 and V7915 for V42P. The minus strand primers were used together with S5046 to amplify the left hand part of the mutated N74 proteins, while the plus strand primers were used together with T6395 to amplify the right hand part. S5046 and T6395 were then used to join the left and right hand segments of the DNA coding for the proteins using equimolar amounts of the corresponding left and right hand PCR products as templates. The products of these reactions were cloned into pGEX4T-2 as described above for the complete transposase coding sequence.

Protein expression and purification

Each transposase derivative was expressed in E.coli NM522 (28). Expression of the fusion protein was induced at OD₅₅₀ by adding 1 M IPTG to a final concentration of 0.5 mM followed by incubation at 30°C for 2-5 h. Cells from 1.5 ml of culture were resuspended in 300 µl of ice-cold PBS (80 g NaCl, 20 g KCl, 20 g Na₂HPO₄, made up to 1 l with H₂O, pH 7.4) and 1% (v/v) Triton X-100. The cells were lysed by sonication and the insoluble material removed by centrifugation for 5 min at 4°C. The supernatant was then transferred to a fresh tube together with 50 µl of a 50% slurry of glutathione-agarose beads. This was incubated at 4°C for 0.5-2 h to allow binding of the GST fusion proteins. The beads were then collected by centrifugation (5 s at 14 000 g) and washed three times with 1 ml of PBS. If the fusion protein was to be analysed by SDS-PAGE the beads were incubated at 100°C for 3 min in loading buffer with 6% SDS and 15% (v/v) [beta]-mercaptoethanol. If the protein was to be used for DNA binding studies, it was eluted using an equal volume of freshly made 50 mM Tris-HCl (pH 8.0) containing 5 mM reduced glutathione (Sigma) pH 7.5.

Preparation of probes for gel retardation assays

A DNA fragment to be labelled was amplified by PCR and digested with restriction enzymes to create a cohesive end at either one or both ends. Different pairs of primers were used to amplify different DNA fragments as follows: A02 and A01 for the 131 bp probe, A02 and T6396 for the 95 bp probe, A02 and T7175 for the 43 bp probe, A03 and A04 for the 22-160 probe, T7880 and A01 for the 43-131 probe, V5498 and T6396 for the -25-95 probe, V5498 and T7461 for the -25-43 probe, V5498 and V6333 for the -25-21 probe, and V8599 and V8600 for the R12 probe. Primers V5498, V7460, V7459 and T6396 were used to generate the internally deleted probe -25-95([Delta]22-42) using a method equivalent to that described above for joining the two coding regions of the transposase. Oligonucleotides V8601 and V9045 were annealed to make the 14BS probe.

DNA fragments to be used as probes were radioactively labelled with [[alpha]-³²P]dCTP using Klenow polymerase (New England Biolabs) to fill in cohesive ends generated by digestion with restriction enzymes.

Gel retardation assay

An aliquot of 1-3 µg of protein was incubated with 1 µg of non-specific competitor poly dI-dC on ice in binding buffer (25 mM HEPES, pH 7.6; 40 mM KCl; 2 mM MgCl₂; 0.1 mM EDTA; 1 mM DTT; 10% glycerol) for 10 min. DNA probe (2 ng) was added to the mixture and the incubation continued for another 20 min. In competition assays a 10-100-fold molar excess of unlabelled competitor DNA was added to the reaction at the same time as poly dI-dC. Two microlitres of loading buffer (binding buffer with 0.05% bromophenol blue) was added to each sample before it was run on a 5% polyacrylamide gel. The gel was then dried under vacuum and autoradiographed.

Sequence-specific DNA binding activity of pogo transposase

In order to investigate the DNA binding properties of pogo transposase we have expressed the protein in E.coli as a fusion with GST. The two exons of the transposase gene were joined according to the sequence of pogo cDNAs (8) using PCR as described in Materials and Methods. The complete transposase coding sequence, except for the initiating methionine, was then inserted downstream of the GST gene in the vector pGEX4T-2 (Pharmacia) to give the plasmid pGEX-pogo. This plasmid was introduced into the E.coli strain NM522 and GST-transposase expression induced by the addition of IPTG after which the fusion protein was purified on glutathione-agarose beads (Materials and Methods). A protein of about the expected size, 85 kDa, was produced from cells carrying pGEX-pogo but not from those carrying unmodified pGEX4T-2 (Fig. 1A). The fusion protein contains a thrombin cleavage site at the junction between GST and transposase. A polypeptide corresponding in size to GST itself was isolated from cells carrying pGEX-pogo. This is presumably the result of premature termination of translation or proteolytic cleavage at the junction between the GST and transposase sequences.

Figure 1. (A) Purification of GST-transposase fusion protein. Extracts of E.coli strain NM522 with or without an expression plasmid and grown in the presence of IPTG were mixed with glutathione-agarose beads. Proteins released from the beads by boiling in the presence of SDS were separated on a 12% SDS polyacrylamide gel and stained with Coomassie Blue. Lane 1, marker proteins (Sigma SDS-6H) of 29, 45, 66, 97, 116 and 205 kDa; lane 2, protein from cells with no plasmid; lane 3, protein from strain NM522 carrying the vector pGEX4T-2; lane 4, proteins from strain NM522 carrying the fusion plasmid pGEX-pogo. (B) Pogo transposase binds to the left hand end of pogo in a sequence-specific manner. A fragment comprising the first 131 bp of pogo was labelled with ³²P and incubated with purified GST:transposase with or without unlabelled competitor DNA before being separated on a 5% polyacrylamide gel. The position of the probe fragment is indicated by an arrowhead. Lane 1, probe alone; lanes 2-6, probe plus GST:transposase; lanes 3 and 4, incubation with a 10- or 100-fold molar excess of unlabelled probe fragment; lanes 5 and 6, incubation with a 10- or 100-fold molar excess of a 190 bp fragment from the left hand end of the mariner transposable element.

Transposases are believed to recognise the ends of their own elements as the first step in transposition to form synaptic complexes before catalysing endonuclease cleavage and strand transfer reactions. Sequence-specific DNA binding should therefore be a characteristic of every transposase. Since the sequences recognised by transposases studied so far lie within the terminal inverted repeats of an element (7,23,29) or sequences adjacent to them (6,30) we have used a probe containing 131 bp from the left hand end of pogo as a probe with which to investigate the DNA binding properties of its transposase in gel retardation assays.

The purified GST-transposase fusion protein bound the end probe in a sequence-specific manner as binding was competed by a 100-fold molar excess of unlabelled probe fragment but not by a similar amount of a 190 bp fragment from the left hand end of the transposable element mariner when it was used as a non-specific competitor (Fig. 1B). No binding of the pogo probe was seen when it was incubated with either purified GST itself or with whole cell extracts of the host strain NM522 (data not shown).

The DNA binding domain of pogo transposase

We have determined the region of pogo transposase that is responsible for DNA binding by making fusion proteins with GST and various C- or N-terminal deletions of the transposase and testing their ability to bind to the 131 bp probe. The C-terminal deletions resulted in fusion proteins containing the N-terminal 158, 138, 74 and 59 amino acids of the transposase, respectively, and of these all but the last were able to bind the 131 bp probe (Fig. 2, lanes 1-10). This suggests that a DNA binding domain of pogo transposase is located at the N-terminus of the protein with at least one end lying between residues 59 and 74. None of the fusion proteins containing N-terminal deletions of the transposase sequence bound the probe (Fig. 2, lanes 11-13). Since the longest of these, GST:C430, contained all but the N-terminal 70 residues, we conclude that the DNA binding capacity of pogo transposase is due to a sequence, or sequences, within the N-terminal 74 residues of the protein.

Figure 2. The DNA binding domain of pogo transposase is contained within the N-terminal 74 residues. (A) A fragment comprising the left hand 131 bp of pogo was labelled with ³²P and incubated with 1 or 3 µg of GST-transposase fusion proteins and then separated on a polyacrylamide gel. Lanes 1, 10 and 11 show the probe alone; lanes 2-5, 6-9, 12 and 13 show the probe incubated with GST fused to segments of pogo transposase. The length of the transposase sequence is indicated above each lane. Lanes 2, 4, 6, 8 and 12 show the effect of incubating with 1 µg of protein and lanes 3, 5, 7, 9 and 13 of incubating with 3 µg. `N' indicates residues from the N-terminus while `C' indicates residues from the C-terminus. The position of the probe fragment is indicated by an arrowhead. (B) SDS-PAGE gel showing the GST-transposase fusions N59 and C380 used for the gel retardation experiments in (A). The protein concentrations were adjusted appropriately prior to incubation with the probe.

In gel retardation experiments using GST fused to the full-length transposase (Fig. 1), and with GST:N158 and GST:N138 (Fig. 2, lanes 2 and 4), two retarded bands can be seen, the larger being much less prominent than the smaller. These higher molecular weight bands are only seen at higher protein concentrations (Fig. 2, lanes 2-5) and may be due to the binding of transposase dimers. If dimers are formed then the dimerisation domain is probably downstream of the DNA binding domain, since a single retarded band was seen with GST:N74 even at high protein concentrations. The fact that only a single retarded band was seen with GST:N74 suggests that the additional bands seen with other transposase fusions were not due to transposase monomers binding separately to the two binding sites on the probe. It is also unlikely that these minor bands are due to degradation of the transposase fusion proteins, since full-length proteins have always been the major species in our preparations. This can be seen for the full-length protein in Figure 1A, lane 4.

Pietrokovsky and Henikoff have predicted that pogo transposase contains an HTH motif from residues 26 to 47 (25). This lies within the region that we have shown is responsible for DNA binding (Fig. 3A), so we have used site-directed mutagenesis to determine whether or not the residues predicted to form this HTH are required for binding. The residues in an HTH that make contact with the DNA sequence that it recognises are generally in the second, or recognition, helix (31), so we have investigated the effect on DNA binding of changing arginine 35 and arginine 43 to alanine residues (R39A, R44A) (Fig. 3A) in the fusion protein GST:N74. The mutant proteins were expressed in E.coli and purified on glutathione-agarose beads before being tested for DNA binding with the 131 bp pogo probe (Fig. 3B, lanes 4-7). The ability of GST:N74 to bind to the pogo probe was greatly reduced when either of the residues believed to lie in the second helix of the HTH motif was changed, whereas changing the lysine residue just beyond the second helix (K48A) had little effect (Fig. 3B, lanes 8 and 9). We have also made equivalent changes to lysine 34 (K34A) in the putative first helix of the HTH and to arginine 63 (R63A) beyond the recognition helix (Fig. 3A). Neither of these mutations had a significant effect on DNA binding (data not shown).

Figure 3. The N-terminus of pogo transposase may contain an HTH motif. (A) The residues believed to be contained within a helix (H) or turn (t) are indicated above the sequence of the N-terminal 74 residues of pogo transposase. The residues that have been altered by site-directed mutagenesis are underlined. The top line indicates the numbering of residues within the predicted HTH while the bottom line indicates the numbering of the residues within the transposase. (B) The effect on DNA binding of changes to specific residues in the DNA binding domain of pogo transposase. The 131 bp probe from the left hand end of pogo was incubated with wild-type or mutant GST:N74 as indicated, before being separated on a polyacrylamide gel. P indicates the probe alone. The position of the probe fragment is indicated by an arrowhead.

We have investigated the importance of this region of the protein further by introducing proline residues separately into each potential helix of the HTH (C32P and V42P, Fig. 3A). The resulting GST:N74 derivatives also had less affinity for the probe than the wild-type and the retarded complexes that were formed migrated more slowly on the gel (Fig. 3B, lanes 10-13), possibly because of the effect of the proline residues on their conformation.

These results are consistent with the prediction that the ability of pogo transposase to bind ends of the element is due to an HTH motif lying between residues 28 and 47 (25).

Binding site for pogo transposase

In order to identify the sequence recognised by pogo transposase we have tested the ability of GST:N74 to bind to probes comprising different sequences from the left hand end of the element (Fig. 4). Deletion of DNA between nucleotides 43 and 131 did not affect binding (Fig. 5, lanes 1-3) whereas a probe containing nucleotides 43-131 was not bound by transposase (Fig. 5, lanes 10-12) indicating that the sequence recognised by transposase lies within the first 43 bp. This is consistent with our expectation that the transposase binding site is contained within the terminal inverted repeat as is the case for Tc1, Tc3 and Himar1 (7,23). This is not the case, however, as a 46 bp probe containing the 21 bp inverted repeat was not bound by transposase (Fig. 5, lanes 7-9), whereas a probe containing nucleotides 22-161 was bound (Fig. 5, lanes 4-6). The 46 bp probe contained 25 bp of non-pogo sequence to the 5[prime] side of nucleotides 1-21 in case transposase binds less efficiently to short fragments of DNA. We conclude that binding requires nucleotides 22-43 and have confirmed this by showing that a probe containing nucleotides 1-95, but deleted for nucleotides 22-43 (data not shown), is not bound by transposase even though the equivalent 1-95 bp probe is bound (Fig. 4).

Figure 4. The position of the sequences recognised by pogo transposase. The ability of the GST:N74 fusion protein to bind to fragments comprising various sequences from the left hand end of pogo is indicated. The 25 nucleotides to the left of the left hand end of the pogo sequence (-25 to 1) is the chromosomal sequence immediately to the left of the pogo element R11 (8).

Figure 5. Interaction of the DNA binding domain of pogo transposase with sequences from the left hand end of pogo. Increasing amounts (0, 1 and 3 µg) of the GST:N74 fusion protein were incubated with ³²P-labelled probes comprising sequences from the left hand end of pogo as indicated. The positions of the free probe fragments are indicated by arrowheads. The probes correspond to the fragments shown in Figure 4 as follows: lanes 1-3, nucleotides 1-43; lanes 4-6, nucleotides 22-160; lanes 7-9, nucleotides -25 to 21; lanes 10-12, nucleotides 43-131.

Transposase must recognise both ends of pogo during transposition and as the 21 bp terminal inverted repeats at each end are identical we expected that transposase would bind to a subterminal sequence at the right hand end. We have tested this using a probe comprising the 140 nucleotides immediately to the left of the right hand inverted repeat (nucleotides 1960-2100). This was bound by transposase in a gel retardation assay, indicating that it contains at least one transposase binding site (data not shown).

We have determined the nucleotide sequence recognised by transposase by comparing nucleotides 22-43, which contain at least one transposase binding site, with the sequences at each end of pogo. This should identify any sequence that is also present at the right hand end as well as any sequence that occurs more than once at the left hand end. This revealed a 12 bp sequence that is present in inverted orientation and with one mismatch. These lie 14 bp from each end of pogo [nucleotides 14-25 (TTAGCTGCATCG) and 2108-2097 (TTAGCTGCCTCG)]. This extends the terminal inverted repeats, previously thought to be 21 bp long, past a single base mismatch to a length of 26 bp (Fig. 6).

Figure 6. The position of transposase binding sites at the ends of pogo. The upper sequence is from the top strand at the left hand end of pogo and the lower sequence is from the bottom strand at the right hand end. Both sequences are written 5[prime] to 3[prime]. The positions of the 12 bp transposase binding sites are indicated by arrows. The 26 bp terminal inverted repeats are underlined.

We have confirmed that both copies of the 12 bp sequence are recognised by transposase by showing that GST:N74 binds to both a 43 bp synthetic oligonucleotide containing the 12 bp sequence flanked by sequences not present at the ends of pogo (Fig. 7, lanes 1-3) and a 43 bp oligonucleotide comprising nucleotides 2079-2121 from the right hand end of pogo (Fig. 7, lanes 4-6). Copies of the 12 bp sequence are also present at asymmetric sites just inside the inverted repeats, an exact copy at nucleotides 30-41 and a copy with one mismatch (TTACCTGCATCG) at nucleotides 2066-2077 (Fig. 6). The first of these can account for the binding of GST:N74 to nucleotides 22-43 (Fig. 4). DNA containing the 12 bp sequence from nucleotides 2066-2077 is also recognised by this region of the transposase (data not shown). The innermost copies of the 12 bp sequence can be extended by 3 bp to TAGTTAGCTGCATCG, raising the possibility that transposase may bind more strongly to this extended sequence. We have tried to determine the transposase binding site directly in footprinting experiments but for reasons that are unclear have been unable to do so.

Figure 7. A 12 bp sequence is the transposase binding site. Two ³²P-labelled probes each containing a single copy of the 12 bp transposase binding site were incubated with 0, 1 or 3 µg of purified GST:N74 before being separated on a polyacrylamide gel. The probes were as follows: lanes 1-3, a fragment with the sequence indicated below the autoradiogram (upper case indicates the binding site); lanes 4-6, a fragment comprising nucleotides 2079-2121 of pogo; this contains one copy of the binding sequence TTAGCTGCCTCG as indicated in Figure 6. The positions of the free probe fragments are indicated by arrowheads.

DISCUSSION

All transposases investigated so far recognise sequences at the ends of the corresponding transposable element, and this is probably essential for transposition. These binding sites are often in the terminal inverted repeats of the element (7,23,29) although this is not always the case. The transposase of the Ac element of maize binds to sequences both within and adjacent to the terminal inverted repeats (32), while the binding site for the transposase of the P element of Drosophila is adjacent to the terminal repeats (33).

The DNA binding domains of all eukaryotic transposases that have been studied so far lie in the N-terminal part of the protein (22,23,32,34). The transposases of Tc1 and Tc3 have been shown to have bipartite DNA binding domains with the N-terminal part being responsible for recognising sequences within the terminal inverted repeats of these elements. Computer analysis of these regions indicates that HTH motifs may be responsible for sequence-specific binding and this has been confirmed by analysis of crystals of the 65 N-terminal residues of Tc3 transposase and an oligonucleotide containing its binding site (24). This fragment of the protein forms three [alpha]-helices, the second and third of which form an HTH that binds a 20 nucleotide sequence that had previously been identified as the transposase binding site (23). This starts 12 bp from the ends of the terminal inverted repeats. Three residues in the recognition helix contact bases in the binding site. These are arginines at the positions 1 and 5 in this helix and a histidine at position 2.

We have demonstrated that pogo transposase, which is distantly related to the transposases of the Tc1/mariner family of elements, binds specifically to the ends of a pogo element and that its DNA binding domain is contained within the N-terminal 74 residues of the protein. This region is predicted to contain an HTH motif. We have investigated whether residues within the putative HTH are required for DNA binding by testing the effect on binding of changing residues that might be expected to be required for recognition of the target site as well as residues that might not. Changing arginine residues in positions 1 and 6 of the second helix to alanine (R39A and R44A) severely reduced the ability of the GST:N74 fusion protein to bind to the left hand end of pogo, whereas changing the lysine at position 7 in the first helix (K34A) had little if any effect. In contrast, introduction of a proline residue at position 5 of the first helix (C32P) greatly reduced binding as might be expected for a change that would be expected to significantly distort an HTH. Changing the lysine immediately beyond the predicted end of the recognition helix (K48A) or the arginine 15 residues further downstream (R63A) also had no detectable effect on binding.

The protein formed by fusing GST to the N-terminal 59 residues of pogo transposase, GST:N59, did not bind to the left hand end of the element. This may be because the structure of the DNA binding domain that we have identified is unstable in GST:N59 rather than because there are residues essential for binding between positions 59 and 74. A similar result has been found for Tc3 transposase. A fragment comprising the N-terminal 54 residues of this protein does not bind DNA, even though the sequence-specific binding domain of the transposase is contained entirely within this sequence (23,24).

The first of the three [alpha]-helices in the N-terminal 65 residues of Tc3 transposase is involved in dimerisation of these fragments within the crystal (24), although it is not known if this is the case for the complete protein in solution. We have no evidence to indicate whether or not the equivalent region of pogo transposase is involved in binding. No stable [alpha]-helix has been predicted for this region, but neither was the first helix of the Tc3 sequence (25).

We have identified a 12 bp binding sequence for pogo transposase. Two copies of this sequence are present at each end of the element, the outermost lying within the terminal inverted repeats (Fig. 6). Each copy is recognised by transposase. We expect that the binding sites nearest the ends of the element will be essential for transposition. Transposase monomers could bind at these sites by their N-termini leaving their C-terminal catalytic domains oriented towards the junction between pogo and flanking DNA that they must cleave to allow transposition to proceed. The inner copies of the transposase binding site may also be required for transposition. This might be similar to bacteriophage Mu for which there are three transposase (MuA) binding sites at each end. During transposition, a synaptic complex is formed comprising a tetramer of transposase monomers that interact with two of the binding sites at the right hand end and one at the left. Tc3 also has two copies of its transposase binding site within each of its 462 bp terminal inverted repeats, but only the outer sites are required for transposition (van Luenen and Plasterk cited in 24). This is perhaps not surprising as the inner sites are ~200 bp from each end (35).

Pogo transposase is a member of a family of proteins with related amino acid sequences. Other members of the family include the putative transposases of transposable elements including Pot2, Fot1 and Fcc1 (12,36,37) and the products of chromosomal genes including PDC2, RAG3 and jerky (17-19). Each of these putative transposases contains a potential HTH corresponding to that of pogo and these are likely to be involved in recognising the ends of the elements concerned. The proteins that are not transposases do not contain this motif, and the regions of pogo transposase with which they show sequence similarity lie elsewhere in the protein.

ACKNOWLEDGEMENTS

We are grateful to I. Clark and A. Dawson for careful reading of the manuscript. H.W. held a Postgraduate Studentship from The Darwin Trust and this work was supported by a project grant from the Wellcome Trust (042192/Z/94/PMG/JC/YJ3).

REFERENCES

1. Finnegan,D.J. (1989) Trends Genet., 5, 103-107. MEDLINE Abstract

2. Mizuuchi,K. (1997) Genes Cells, 2, 1-12. MEDLINE Abstract

3. Grindley,N.D.F. and Leschziner,A.E. (1995) Cell, 83, 1063-1066. MEDLINE Abstract

4. Vos,J.C., De Baere,I. and Plasterk,R.H.A. (1996) Genes Dev., 10, 755-761. MEDLINE Abstract

5. van Luenen,H.G.A.M., Colloms,S.D. and Plasterk,R.H.A. (1994) Cell, 79, 293-301. MEDLINE Abstract

6. Kaufman,P.D. and Rio,D.C. (1992) Cell, 69, 27-39. MEDLINE Abstract

7. Lampe,D.J., Churchill,M.E.A. and Robertson,H.M. (1996) EMBO J., 15, 5470-5479. MEDLINE Abstract

8. Tudor,M., Lobocka,M., Goodell,M., Petit,J. and O'Hare,K. (1992) Mol. Gen. Genet., 232, 126-134. MEDLINE Abstract

9. Daboussi,M.-J., Langin,T. and Y.,B. (1992) Mol. Gen. Genet., 232, 12-16. MEDLINE Abstract

10. Levis,C., Fortini,D. and Brygoo,Y. (1997) Mol. Gen. Genet., 254, 674-680. MEDLINE Abstract

11. Smit,A.F.A. and Riggs,A.D. (1996) Proc. Natl Acad. Sci. USA, 93, 1443-1448.

12. Robertson,H.M. (1996) Mol. Gen. Genet., 252, 761-766. MEDLINE Abstract

13. Ruvolo,V., Hill,J.E. and Levitt,A. (1992) DNA Cell Biol., 11, 111-122. MEDLINE Abstract

14. Yuan,J., Finney,M., Tsung,N. and Horvitz,H.R. (1991) Proc. Natl Acad. Sci. USA, 88, 3334-3338. MEDLINE Abstract

15. Collins,J.J. and Anderson,P. (1994) Genetics, 137, 771-781. MEDLINE Abstract

16. Earnshaw,W.C., Sullivan,K.T., Machlin,P.S., Cooke,C.A., Kaiser,D.A., Pollard,T.D., Rothfield,N.F. and Cleveland,D.W. (1987) J. Cell Biol., 104, 817-829. MEDLINE Abstract

17. Toth,M., Grimsby,J., Buzaki,G. and Donovan,G.P. (1995) Nature Genet., 11, 71-75. MEDLINE Abstract

18. Hohmann,S. (1993) Mol. Gen. Genet., 241, 657-666. MEDLINE Abstract

19. Prior,C., Tizzani,L., Fukuhara,H. and Wesolowski-Louvel,M. (1996) Mol. Microbiol., 20, 765-772. MEDLINE Abstract

20. Doak,T.G., Doerder,F.P., Jahn,C.L. and Herrick,G. (1994) Proc. Natl Acad. Sci. USA, 91, 942-946. MEDLINE Abstract

21. Robertson,H.M. (1995) J. Insect Physiol., 41, 99-105.

22. Vos,J.C. and Plasterk,R.H. (1994) EMBO J., 13, 6125-6132. MEDLINE Abstract

23. Colloms,S.D., van Luenen,H.G.A.M. and Plasterk,R.H.A. (1994) Nucleic Acids Res., 25, 5548-5554. MEDLINE Abstract

24. van Pouderoyen,G., Ketting,R.F., Perrakis,A., Plasterk,R.H.A. and Sixma,T.K. (1997) EMBO J., 16, 6044-6054. MEDLINE Abstract

25. Pietrokovski,S. and Henikoff,S. (1997) Mol. Gen. Genet., 254, 689-695. MEDLINE Abstract

26. Clubb,R.T., Schumacher,S., Mizuuchi,K., Gronenborn,A.M. and Clore,G.M. (1997) J. Mol. Biol., 273, 19-25. MEDLINE Abstract

27. Schumacher,S., Clubb,T.R., Cai,M., Mizuuchi,K., Clore,G.M. and Gronenborn,A.M. (1997) EMBO J., 16, 7532-7541. MEDLINE Abstract

28. Gough,J.A. and Murray,N.E. (1983) J. Mol. Biol., 166, 1-19. MEDLINE Abstract

29. Vos,J.C., van Luenen,H.G.A.M. and Plasterk,R.H.A. (1993) Genes Dev., 7, 1244-1253.

30. Kunze,R. and Starlinger,P. (1989) EMBO J., 8, 3177-3185. MEDLINE Abstract

31. Brennan,R.G. and Matthews,B.W. (1989) J. Biol. Chem., 264, 1903-1906. MEDLINE Abstract

32. Becker,H.-A. and Kunze,R. (1997) Mol. Gen. Genet., 254, 219-230. MEDLINE Abstract

33. Kaufman,P.D., Doll,R.F. and Rio,D.C. (1989) Cell, 59, 359-371. MEDLINE Abstract

34. Lee,C.C., Mul,Y.M. and Rio,D.C. (1996) Mol. Cell. Biol., 16, 5616-5622. MEDLINE Abstract

35. Collins,J., Forbes,E. and Anderson,P. (1989) Genetics, 121, 47-55. MEDLINE Abstract

36. Kachroo,P., Leong,S.A. and Chattoo,B.B. (1994) Mol. Gen. Genet., 245, 339-348. MEDLINE Abstract

37. Panaccione,D.G., Pitkin,J.W., Walton,J.D. and Annis,S.L. (1996) Gene, 176, 103-109. MEDLINE Abstract

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*To whom correspondence should be addressed. Tel: +44 131 650 5361; Fax: +44 131 650 8650; Email: d_finnegan@ed.ac.uk
⁺Present address: Department of Microbiology, Eastman Research Institute, 256 Gray's Inn Road, London WC1X 8LD, UK

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