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DNA Sequences at the Ends of the Genome of Bacteriophage Mu Essential for Transposition (Mini-Mus/Nudease BAL-31/DNA-Protein Interactions) MARTIEN A

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DNA Sequences at the Ends of the Genome of Bacteriophage Mu Essential for Transposition (Mini-Mus/Nudease BAL-31/DNA-Protein Interactions) MARTIEN A Proc. Natl. Acad. Sci. USA Vol. 82, pp. 2087-2091, April 1985 Genetics DNA sequences at the ends of the genome of bacteriophage 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 plasmid 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 Plasmids. 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.
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