Proc. Natl. Acad. Sci. USA Vol. 82, pp. 2087-2091, April 1985

DNA sequences at the ends of the of Mu essential for transposition (mini-Mus/nudease BAL-31/DNA-protein interactions) MARTIEN A. M. GROENEN, ERIK TIMMERS, AND PIETER VAN DE PUTTE Laboratory of Molecular Genetics, Leiden State University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands Communicated by Franklin W. Stahl, November 19, 1984 ABSTRACT We have determined the minimal DNA se- att L at quences the ends of the genome of bacteriophage Mu that 13 21 23 20 15 14 5 29 are required for its transposition. A mini-Mu was constructed Ill JI Il506 on a multicopy that enabled the manipulation of the 10 20 30 40 S0 do DNA sequences at its ends without affecting the genes essential 5 TGTATTGATT CACTTGAAGT ACGAAAAAAA CCGGGAGGAC ATTGGATTAT TCGGGATCTG for transposition. The genes A and B, which were cloned out- side the ends of the mini-Mu on the same plasmid, were both 70 80 90 100 110 120 needed for optimal transposition. In our experimental system ATGGGATTAG ATTTGGTG$ GCTTGCAAGC CTGTAGTGCA AATTTTAGTC CTTAATCAAT the predominant end products of the transposition are cointe- 28 4 27 24 26 11 1618 1 III 11 grates both in the presence and in the absence of B. Two re- 130 140 150 160 170 180 gions ending approximately 25 and 160 bp from the left end GAACGCGAA AGATAGTAM AAATTGCTTT TGTTTCATTG AAAATACGAA AAACAAAAAC and one ending approximately 50 bp from the right end appear to be essential for optimal transposition. Overlapping with these regions, a 22-base-pair sequence was recognized with the 190 200 210 220 consensus Y-G-T-T-T-C-A-Y-T-N-N-A-A-R-Y-R-C-G-A-A-A- ACTGCAAATC ATTTCAATAA CAGCTTCAAA AAACGTTCAA A, where Y and R represent any pyrimidine and purine, re- spectively. At the left end these sequences occur as direct re- 220 210 200 190 peats; at the right end this sequence is inverted with respect to GTGGTACACA AATTTAATCA GTATCGCTAC ATCAGATTCC those at the left end. 180 170 160 150 140 130 Bacteriophage Mu is a very efficient transposon as it repli- TGAACAAACG AGCMGGAAG CGGCTAAATA CCAAACTATT CAAGGTTCAG CCATACCCTA cates an by active process of transposition (for a recent re- 52 view, see ref. 1). Two phage-coded proteins A and B (2, 3) 1I11l1 II are essential for this process. A is absolutely required, 120 110 100 90 80 70 whereas in the absence of B there is a decrease of the trans- AGTGATCCCC ATGTAATGAA TAAAAAGCAG TAATTAATAC ATCTGTTTCA LT[GAAGCGC position frequency by a factor of 100 (4, 5). Other phage- 60 53 58 61 64 66 43 coded functions-e.g., ner (6), arm (7), and kil, gam, lig, or 60 60 40 30 20 10 sot (8)-are involved in modulating the efficiency of trans- GAAAGCTAAA GTITTCGCAT TTATCGTGAA ACGCTTTCGC GTTTTTCGTG CGC4GCTTCA 3 position. Typically the nucleotide sequences at the ends of most transposons are more or less perfect inverted repeats att R varying between approximately 18 and 40 base pairs (bp) (9, 10). The terminal inverted repeats of bacteriophage Mu, FIG. 1. Nucleotide sequences at the ends of bacteriophage Mu. Comparison of the sequences at the left (12, 13) and right (14, 15) however, are only 2 bp in length. It was shown (11) that there end of bacteriophage Mu reveals considerable stretches of homolo- is an essential Mu sequence required for transposition be- gy. Sequences homologous to the consensus Y-R-C-G-A-A-A-A tween nucleotides 27 and 116 from the right end. Examina- ( -.), Y-G-T-T-T-C-A-Y-T (- --), and T-G-A-A-G-C-G (-* *--) tion of the sequences near the left end (12, 13) and right end are underlined (Y and R represent any pyrimidine and purine, re- (14, 15) reveals considerable stretches of homology in both spectively). The direction of the arrow represents the relative orien- inverted and direct orientation (Fig. 1). To investigate the tation of the sequence. A 12-bp palindrome found at positions 80-91 importance of these sequences we have determined the se- ofthe left end is boxed. The positions ofthe BAL-31-generated dele- quences at the ends of the genome of Mu that are minimally tions are indicated with vertical bars. The positions of those that are for To this a mini-Mu was numbered are determined by sequencing using the dideoxy method required transposition. purpose (16). The numbers correspond to the isolation numbers shown in constructed that allowed the digestion ofDNA sequences by Table 2. exonuclease BAL-31 from within the mini-Mu towards the ends without affecting the genes essential for transposition. These genes were cloned under the control of a strong induc- MATERIALS AND METHODS ible promoter on the same plasmid but separated from the Recombinant DNA Techniques and DNA Sequencing. Re- mini-Mu. By using this system A and B are still provided in striction endonucleases, T4 DNA ligase, Klenow fragment cis, which might be important for optimal transposition. The of DNA polymerase 1, exonuclease BAL-31, BamHI linkers results obtained with this mini-Mu indicate that two regions d(C-G-G-G-A-T-C-C-C-G), and Sal I linkers d(C-G-T-C-G- at the left end and one at the right end are essential for opti- A-C-G) were purchased from Boehringer Mannheim, Miles mal transposition. Laboratories, and Biolabs Bethesda. Incubation conditions

The publication costs of this article were defrayed in part by page charge Abbreviations: R, resistant; s, sensitive; Amp, ampicillin; Strep, payment. This article must therefore be hereby marked "advertisement" streptomycin; CAM, chloramphenicol; bp, base pair(s); IS, inser- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tion sequence.

2087 Downloaded by guest on September 28, 2021 2088 Genetics: Groenen et aL Proc. NatL Acad Sci USA 82 (1985) were as recommended by the suppliers. Sequencing reac- tions were done according to Sanger et al. (16). DNA restric- tion fragments were cloned in M13 vectors mp8 or mp9 (17) and clones were checked for the insert by isolation of repli- cative form DNA 4 hr after infection (18). Bacterial Strains and . Bacteria were grown as de- scribed (19). Plasmid POX38 is an F factor lacking the inser- tion sequence (IS) elements (20). The other plasmids were constructed by cloning Mu sequences on pPLc2833 (21). The Eco RI bacterial strains used were JM101 (17), KMBL1164 (Alac- proXIII, thi2O9, supE; our laboratory), PP135 [KMBL1164 (X)], PP455 (KMBL1164 containing an F pro+ lac+ epi- PstI some), and PP1573 [a streptomycin-resistant (StrepR) deriva- EcoRI tive of PP135]. Brm HI BalI HpaI SailI Assay of Transposition by Conjugation. Transposition of -v--I,,," v---I ~~~~~~~~~I, mini-Mu sequences to an F pro' lac+ episome was moni- pGP 618 tored by a conjugation assay as described (22), except that ner A B the transposition was induced by shifting the culture to 380C Sol I 4 for 1 hr. The donor strain PP455 contained, in addition to the pGP 619 A F pro' lac+ episome, the mini-Mu on a pBR322 derivative and the compatible kanamycin-resistant plasmid pCI857 (20) HpaI SalI containing the temperature-sensitive repressor gene C1857 of 1 I 620 Wb bacteriophage X. The recipient strain PP1573 is a StrepR X pGP ca AA BB lysogen. After the mating, the cells were pelleted by low- BamHIBaII Sall speed centrifugation. The bacteria were resuspended in 1 ml of 0.9% (wt/vol) NaCl and 100-1.l aliquots were plated on pGP 621 e4 _ the appropriate minimal glucose plates provided with the ap- ner A propriate antibiotics to select for StrepR pro' and StrepR BalI HpaISaII chloramphenicol-resistant (CAMR) pro' exconjugants to p GP 634 ' measure the total transfer of the F pro' lac+ episome and ner A B the transfer of the transposed mini-Mu on the F pro' lac+ Bat I HpaI Sal I BamHI EcoRI episome. In some experiments the CAMR exconjugants were 4 pGP 630 checked for the presence of ampicillin-resistance (AmpR) ner A B (cointegrates) or absence of AmpR (simple insertions). FIG. 2. Construction of plasmid pGP618 and deletion deriva- RESULTS tives. Mu sequences are shown with thick lines, whereas the non- Mu DNA is shown with thin lines. Positions of the left (L) and right Construction of Mini-Mus. The mini-Mu that was used for (R) end of Mu are shown with arrows pointing from within the Mu the isolation of the different deletion derivatives is shown in part. The genes ner, A, and B and the ends of Mu were cloned sepa- Fig. 2. First the left 850 bp and right 792 bp of Mu were rately on pPLc2833 (21). For cloning of the left end of Mu, pGP20 was used, which is a deletion derivative of pGP2 (23). pGP20 con- cloned in their proper relative orientation on pPLc2833. For tains the left 850 bp of Mu fused to a partially deleted tetracycline an easy monitoring of the transposition of the mini-Mu, the resistance gene containing the Sal I but not the BamHI site. The Pst I fragment of Tn9 containing the CAMR gene was cloned EcoRI-Sal I fragment of pGP20, containing the left end of Mu, was between the ends of the mini-Mu, resulting in plasmid cloned between the EcoRI and Sal I site on pPLc2833, resulting in pGP614. Subsequently a DNA fragment containing the ner, pGP604. To clone the right end, the Bcl I-HindIII fragment of A, and B genes of Mu was also cloned on this plasmid just pGP204 (24), containing the right 792 bp of Mu, was cloned between outside the left end of the mini-Mu under control of the PL the BamHI and HindIII site on pGP604. The Pst I fragment of Tn9 promoter of bacteriophage X, resulting in plasmid pGP618. containing the gene conferring CAMR was cloned in the Pst I site When induced, functional A and B proteins are made, as was present between the ends of the mini-Mu, resulting in pGP614. Fi- shown by complementation with Mu Aam and Bam phages. nally, the EcoRI-HindIII fragment of pLP103-6 (25), containing the genes ner, A, and B, was cloned downstream of the PL promoter in Several deletion derivatives of pGP618, which were used in the EcoRI site of pGP614. The HindIII site of the fragment was first the experiments, are also shown in Fig. 2. converted to an EcoRI site. Plasmid pLP103-6 has a deletion of Gene Products Essential for Transposition of the Mini-Mu. about 100 bp in the lIpa I-EcoRI region rendering it Kil-. The We first measured the influence of the expression of differ- EcoRI, Pst I, and HindIII sites present on pGP618 are indicated in ent combinations of the ner, A, and B genes on the transposi- the circular representation. These sites are also present in ail other tion frequency of the mini-Mu. When all three genes are ex- plasmids shown. Additional restriction sites, which are not present pressed the frequency of transposition is 4.9% (Table 1). in all plasmids, are indicated in the linear representation. In plas- This transposition frequency is somewhat lower than might mids pGP619 and pGP620 the part of the ner gene left of the Bal I be expected for this system, in which the mini-Mu is carried site has been removed. This was done by using the EcoRI-HindIII on a multicopy plasmid and in which ner, A, and B are pro- fragment of pGP90 (6) containing the A and B genes instead of the EcoRI-HindIII fragment of pLP103-6. BAL-31-generated deletion vided in cis. It is possible that transcription of the PL tran- derivatives pGP619 and pGP621 have a deletion of approximately script across the left end reduces the transposition frequen- 1000 bp over the Hpa I site, rendering it B-. These deletions re- cy, as was also found for the IS] element (26, 27). On the moved about 500-600 bp of the B gene. BAL-31-generated deletion average, 90% of the transposition events are due to cointe- derivatives pGP630 and pGP634 contain 216 bp of the right end, re- grates (CAMR AmpR exconjugants) and 10% are due to di- spectively, and 170 bp of the left end of Mu. Kb, kilobases. rect transposition [CAMR Amp-sensitive (Amps) exconju- gants]. When A and B are expressed without ner the same factor of 100. This result is in agreement with earlier obser- results are obtained, indicating that Ner is not directly in- vations with a complete Mu phage (4, 5) or mini-Mus (28) volved in transposition. The absence of a functional B prod- and with in vitro experiments (29). Under B- conditions the uct leads to a decrease in the transposition frequency by a frequency of transposition is only slightly above the values Downloaded by guest on September 28, 2021 Genetics: Groenen et aL Proc. NatL Acad Sci USA 82 (1985) 2089

Table 1. Effect of the expression of different combinations of the Table 2. Transposition frequency of mini-Mu deletion derivatives ner, A, and B genes on the transposition frequency of T s i the mini-Mu PBase pairs present Transposetqon Plasmid Transposition At left end At right end frequency Mu genes frequency pGP618 850 792 4.9 Plasmid expressed 0 min 60 min 1, 16 170, 164 792 3.8-4.5 18 163 792 5.8 pGP618 ner, A, B 0.03 4.9 11 156 792 2.6 pGP620 A, B 0.02 4.6 26 147 792 0.50 pGP619 A 0.03 0.04 5, 24, 27, 4, 28, 29, 14 135-33 792 0.30-0.50 pGP621 ner, A 0.01 0.04 15 25 792 0.35 pGP614 - 0.02 0.03 20 20 792 0.01 Several deletion derivatives of pGP618 were made (Fig. 2) and 13, 21, 23 2-9 792 0.01-0.03 tested for their ability to mediate transposition of the mini-Mu. The 52, 60 850 65, 54 4.0-4.5 transposition frequency, determined 0 min and 60 min after induc- 53 850 52 4.1 tion, is expressed as the percentage ofthe exconjugants (StrepR) that 56 850 45 2.0 is CAMR. The number of exconjugants was between 101 and 106 per 61 850 38 0.7 conjugation experiment. 64 850 32 0.15 66 850 27 0.03 found for the control pGP614, which is due to the presence 43 850 23 0.03 of IS elements on the F pro' lac+ episome (30). Therefore, 153 170 52 3.5 to determine the percentage ofcointegrates and simple inser- 553 40 52 0.20 tions under B- conditions, the experiments were repeated pGP614 (control) with the F factor POX38 (19), which lacks the IS elements. 0.03 When using this F factor, 99%o of the transposition events Deletions were made within the mini-Mu. To obtain deletions under B- conditions were found to be cointegrates. (The towards the right end, plasmid pGP630 was linearized with BamHI number of exconjugants after 0 min and 60 min of induction and to obtain deletions towards the left end plasmids pGP618 and pGP634 were linearized with Sal I (Fig. 2). After digesting the was 3 x 102 and 9.5 x 103, respectively.) linearized plasmids with BAL-31, the plasmids were ligated with In all experiments described the transposition was induced BamHI or Sal I linkers. Where necessary, the CAMR gene was at 38TC and not at 42TC. Induction for 1 hr at 42TC resulted in restored by subsequent recloning of the original fragment containing a decrease of the transfer of the F pro' lac+ episome by a this gene. Nucleotide sequences of Mu are immediately followed by factor of 200. This interference with F transfer was not de- a BamHI linker, except for plasmids 1, 4, and 5, where it is followed pendent on the presence of the mini-Mu and was only ob- by a Sal I linker. The numbers of the plasmids are isolation numbers served if both the A and B genes were expressed at a high and correspond to those shown in Fig. 1. The exact positions of the level (at 420C). deletions were determined by dideoxy sequencing (16). The trans- Sequences at the Ends ofBacteriophage Mu That Are Essen- position frec ency is expressed as the. percentage of the exconju- tial for Transposition. To determine which sequences at the gants (Strep ) that is CAMR. The number of exconjugants was ends of Mu are required for transposition, deletions were between 105 and 106 per conjugation experiment. made with exonuclease BAL-31 from inside the mini-Mus contribute to the same functional effect as the innermost se- towards one of its ends and the transposition frequencies of quence, deletion of the latter would abolish function and fur- the different deletion derivatives were determined. For dele- ther deletion would have no effect. tions towards the left end, pGP618 and pGP634 were used as We also constructed a mini-Mu that contains only the left starting plasmids, whereas for deletions towards the right 170 bp and the right 52 bp and one that contains the left 40 bp end, pGP630 was used (Fig. 2). Deletions, which end in criti- and the right 52 bp of Mu (plasmids 153 and 553 in Table 2). cal regions where significant changes in the transposition The transposition frequencies of these mini-Mus show that frequency were found, were all sequenced (Fig. 1). Results no additional regions are present between nucleotides 170 obtained with deletions towards the right att site of Mu (Ta- and 850 of the left end and 52 and 792 ofthe right end that are ble 2) indicate that sequences can be deleted without affect- involved in the transposition. ing the frequency of transposition until 52 bp are still pres- Partially overlapping with the three regions, which are es- ent. Deleting nucleotides in the region from 52 to 32 bp from sential for transposition, a 22-bp sequence is present, which the right end results in a gradual decrease of the transposi- shows considerable sequence homology (Fig. 3). The one tion frequency by a factor of 30. If <28 bp are present, trans- found at the right end is in the inverted orientation with re- position of the mini-Mu is no longer observed. spect to the two sequences at the left end. The same se- At the left end quite different results were obtained (Table quence is repeated a second time near the right end but as a 2). Here two regions were found that are essential for trans- direct repeat with respect to those at the left end. This se- position of which the right borders are situated 125 bp apart. quence does not seem to affect transposition. With 163 bp present at the left end, the transposition fre- quency is still normal. A decrease by a factor of 2 might DISCUSSION occur after deleting nucleotides 163-157. When another 9 bp are deleted a reduction in the transposition frequency by a To obtain maximal transposition with the mini-Mu described factor of 10-20 is observed. Then the transposition frequen- above, only the expression of the A and B genes is essential. cy remains constant for a long distance, as removal of nucle- No additional effect was found when also the ner gene was otides 147-26 does not result in a further decrease in transpo- expressed. In bacteriophage Mu itself a ner mutation leads to sition. After deleting nucleotides 25-21 the transposition fre- a strong reduction of Mu transposition (6). This is probably quency of the mini-Mu is reduced below a measurable level. due to the regulatory role of Ner in repressor synthesis. In Apparently two regions at the left end and one at the right this experimental system the normal regulation sites of Mu end of Mu are essential for optimal transposition. We cannot are not present, which explains the absence of any effect of exclude the possibility that regions within the segment be- Ner on the transposition of the mini-Mu. tween nucleotides 25 and 160 of the left end are essential for If both the A and B genes are expressed the predominant transposition that are not detected. If these regions would end products of the transposition are found to be cointe- Downloaded by guest on September 28, 2021 2090 Genetics: Groenen et aL Proc. NatL Acad Sci USA 82 (1985)

ott L sential for optimal transposition (Fig. 3). These 22-bp se- quences are in fact a composition of the two consensus I 100 150 200 50 so iv I I I 1I sequences Y-R-C-G-A-A-A-A and Y-G-T-T-T-C-A-Y-T found several times more in both phage ends both as invert- ed and as direct repeats (Fig. 1). The sequence R-C-G-A-A- ,=* V III A-A is also found at the ends of Tn3 and related transposons II (33). However, part of the stretch of A residues can be de- leted without affecting the transposition frequency. From se- quence II (Fig. 3), the sequence R-C-G-A-A-A-A can even 5 28 be removed completely without affecting the transposition TT ]AC C TT G A A G T A C G A A AAA frequency. If the sequence Y-G-T-T-T-C-A-Y-T is absent, 150 173 transposition (or stimulation of transposition in the case of II TT G IT T T C A T T G A A A A T A C G A A AAA sequence II, Fig. 3) is completely abolished. We suggest that 27 50 this sequence is a very essential part of the recognition site III G C G IT T T C A C G AT A AIA T G C G A A AAC of the transposase. A fourth sequence (sequence IV, Fig. 3), which shares the 78 55 of is found between nucleotides 78 IV C T G T T T TG A AIG C G C GAAAG C same amount homology, L and 55 of the right end as a direct repeat with respect to those found at the left end. This sequence does not affect transpo- consensus N Y G T T T C A YT NNAA R Y R C G A AAAN sition, which may be due to its wrong (not inverted) orienta- tion with respect to those at the left end. The sequence at the FIG. 3. Organization of the att sites of bacteriophage Mu. The right end that is essential for transposition (sequence III, scale at the top represents base pairs. Numbering of nucleotides cor- Fig. 3) is found in the inverted orientation with respect to responds with that in Fig. 1. Derivatives with deletions ending in the those at the left end. Apparently, as in other transposable part indicated with open boxes show normal levels of transposition. elements, inverted sequences seem to play also an important Those that end in the part indicated with a hatched box show a low role in the transposition of Mu. The difference, however, but constant level of transposition, and those that end in the parts with most other transposons is that the (imperfect) inverted indicated with dotted boxes show no transposition. Filled boxes in- repeats in the case of Mu are not at the very ends of the dicate the regions where, upon deletion from inside the mini-Mu to- transposon (apart from a 2-bp inverted repeat) but are locat- wards the end, a decrease in the transposition frequency is ob- ed at some distance from the ends and moreover at a differ- served. The 22-bp consensus sequence found twice in both ends is the right end. Although indicated with an open arrow. This consensus sequence is composed ent distance from the left and from of two sequences, Y-R-C-G-A-A-A-A(--) and Y-G-T-T-T-C-A- the rationale for this type of organization is not yet known, Y-T(--_), that are repeated several times more in both ends (Fig. we want to suggest that it is related to transposition being 1). essential for the development of the phage. The replication of the phage genome proceeds mainly from left to right on grates. In the absence of B, transposition is reduced by a the genome (31, 32) and also phage-packaging is a process factor of 100 but the predominant end products are still coin- that shows polarity, as it is always initiated near the left end tegrates. This differs from the results reported by Harshey (34). An asymmetric organization of the inverted repeats at (28), who found predominantly direct transposition under B- the ends might be required for these processes to occur in conditions. An explanation for this difference could be that the correct direction. simple insertions are produced at a higher frequency but that Finally, as can be seen in Fig. 1, certain parts of the se- with pBR-derived plasmids there is a preference for intramo- quences shown in Fig. 3 are found as both inverted and di- lecular direct transposition under B- conditions, as suggest- rect repeats several times more in both ends. Several of ed by Harshey (R. M. Harshey, personal communication). these sequences can be removed without affecting the trans- The recovery of these products would then be skewed by the position frequency. These sequences could also have other conjugation assay. However, it is not clear why this would functions, such as termination signals for transcription of the not occur when the low copy-number plasmid pSC101 is repressor gene at the left end and the mom gene at the right used (28). end. In the transcripts of these genes some of these se- The B product is not absolutely required for Mu transposi- quences could form stem-loop structures followed by a tion but acts as an enhancer of transposition. This may be stretch of U residues, characteristic for transcription termi- required because Mu replicates by transposition. Probably nation (35). related to the same demand for efficiency is the finding that two regions at the left end are required for optimal transposi- Dr. M. S. Guyer and Dr. D. E. Berg kindly provided POX38. We tion (Fig. 3). These two regions probably contain A binding thank Dr. M. Giphart-Gassler for critical reading of the manuscript sites, as such sites are found in vitro in approximately the and Mrs. N. van Hoek for typing it. These investigations have been same position (K. Mizuuchi, personal communication). Af- carried out under the auspices of the Netherlands Foundation of ter the second region has been removed, transposition is still Chemical Research (SON) and with the financial aid from the Neth- B dependent. This second A binding site, which is not abso- erlands Organization for the Advancement of Pure Scientific Re- lutely required for transposition, could promote the proba- search (ZWO). bility of binding of the transposase to the left end and thus increase the frequency at which the initiation complex is 1. Toussaint, A. & Resibois, A. (1983) in Mobile Genetic Ele- formed. At the right end of Mu only one region is found that ments, ed. Shapiro, I. (Academic, New York), pp. 105-158. is essential for transposition. The presence of two regions at 2. Wijffelman, C., Gassler, M., Stevens, W. F. & van de Putte, P. (1974) Mol. Genet. 131, the left end and only one at the right end that are essential for Gen. 85-96. 3. Wijffelman, C. & Lotterman, B. (1977) Mol. Gen. Genet. 151, optimal transposition might also be related to the observed 169-174. direction of replication of Mu, which is predominantly from 4. O'Day, K. J., Schultz, D. W. & Howe, M. M. (1978) in Micro- left to right (31, 32). biology 1978, ed. Schlessinger, D. (Am. Soc. Microbiol., A common sequence of 22 bp corresponding to the con- Washington, DC), pp. 48-51. sensus Y-G-T-T-T-C-A-Y-T-N-N-A-A-R-Y-R-C-G-A-A-A- 5. Faelen, M., Huisman, 0. & Toussaint, A. (1978) Nature (Lon- A is found partially overlapping with the regions that are es- don) 271, 580-582. Downloaded by guest on September 28, 2021 Genetics: Groenen et aL Proc. NatL Acad Scd USA 82 (1985) 2091

6. Goosen, N. (1984) Dissertation (University of Leiden, Leiden, 22. Chaconas, G., Harshey, R. M., Sarvetnick, N. & Bukhari, A. The Netherlands). (1981) J. Mol. Biol. 150, 341-359. 7. Waggoner, P. T., Pato, M., Toussaint, A. & Faelen, M. (1981) 23. Giphart-Gassler, M., Reeve, J. & van de Putte, P. (1981) J. Virology 113, 379-387. Mol. Biol. 145, 165-191. 8. Goosen, T., Giphart-Gassler, M. & van de Putte, P. (1982) 24. Plasterk, R. H. A., Ilmer, T. A. M. & van de Putte, P. (1983) Mol. Gen. Genet. 186, 135-139. Virology 127, 24-36. 9. Shapiro, I., ed. (1983) Mobile Genetic Elements (Academic, 25. Van Leerdam, E., Karreman, C. & van de Putte, P. (1982) Vi- New York). rology 123, 19-28. 10. Calos, M. P. & Miller, J. H. (1980) Cell 20, 579-595. 26. Chandler, M. & Galas, D. J. (1983) J. Mol. Biol. 170, 61-91. 11. Casadaban, M. J. & Chou, J. (1984) Proc. Natl. Acad. Sci. 27. Biel, S. W., Adelt, G. & Berg, D. E. (1984) J. Mol. Biol. 174, USA 81, 535-539. 251-264. 12. Kahmann, R. & Kamp, D. (1979) Nature (London) 280, 247- 28. Harshey, R. M. (1983) Proc. Natl. Acad. Sci. USA 80, 2012- 250. 2016. 13. Priess, H., Kamp, D., Kahmann, R., Brauer, B. & Delius, H. 29. Mizuuchi, K. (1983) Cell 35, 785-794. (1982) Mol. Gen. Genet. 186, 315-321. 30. Guyer, M. S. (1978) J. Mol. Biol. 126, 347-365. 14. Plasterk, R. H. A., Vollering, M., Brinkman, A. & van de 31. Wijffelman, C. & van de Putte, P. (1977) in DNA Insertion Putte, P. (1984) Cell 36, 189-196. Elements, Plasmids and Episomes, eds. Bukhari, A. I., Sha- 15. Kahmann, R. (1983) Cold Spring Harbor Symp. Quant. Biol. piro, I. A. & Adhya, S. L. (Cold Spring Harbor Laboratory, 47, 631-638. Cold Spring Harbor, NY), pp. 329-333. 16. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. 32. Goosen, T. (1978) in DNA Synthesis Present and Future, eds. Acad. Sci. USA 74, 5463-5467. M. (Plenum, New York), pp. 121- 17. Messing, J. & Vieira, J. (1982) Gene 19, 269-276. Moulineux, I. & Kohiyama, 18. Birnboim, H. C. & Doly, J. (1979) Nucleic Acids Res. 7, 1513- 126. 1523. 33. Reed, R., Young, R., Steitz, J. A., Grindley, N. & Guyer, M. 19. Wijffelman, C. A., Westmaas, G. C. & van de Putte, P. (1972) (1979) Proc. NatI. Acad. Sci. USA 76, 4882-4886. Mol. Gen. Genet. 116, 40-46. 34. Bukhari, A. I. & Taylor, A. L. (1975) Proc. NatI. Acad. Sci. 20. Guyer, M. S., Reed, R. R., Steitz, J. A. & Low, K. B. (1979) USA 72, 4399-4403. Cold Spring Harbor Symp. Quant. Biol. 43, 135-140. 35. Holmes, W. M., Platt, T. & Rosenberg, M. (1983) Cell 32, 21. Remaut, E., Stanssens, P. & Fiers, W. (1981) Gene 15, 81-93. 1029-1032. Downloaded by guest on September 28, 2021