Transposon removal

As the Kmr _{gene was part of a bacterial Tn7-derived transposon sequence, it could be} excised from pAd2-BAC mutant DNA in vitro by use of a Tn7 transposition system, TnsABC*, and subsequent religation of the gap (Ruzsics et al in preparation).

TnsABC* (New England Biolabs, Frankfurt, Germany) consists of wt bacterial proteins TnsA, TnsB, which act interdependently to execute the catalytic steps of the transposition reaction (Biery et al., 2000a), and a mutant variant of TnsC. Wt TnsC is an ATP-dependent DNA-binding protein (Gamas and Craig, 1992). TnsC binds DNA without any obvious sequence specificity, but

it depends on Tn7-encoded TnsD or TnsE target selection proteins to enable transposition. Gain of function mutant TnsC* (TnsCA225V_{) can activate TnsA+B in the absence of TnsD or TnsE to} give very robust levels of recombination with low target site selectivity (Biery et al., 2000b; Stellwagen and Craig, 2001). The TnsABC*-mediated transposon excision reaction is initiated following specific recognition of the inverted repeats at the Tn7 transposon ends by TnsB. TnsC* binds to target DNA and interacts with TnsB. TnsA associates with TnsB:DNA. Thus, a three protein, two DNA complex is assembled and allows TnsA and TnsB to carry out the strand transfer reaction in the presence of cofactors ATP and Mg2+_{. The donor DNA is cleaved three} bases 5’ to the transposon in one strand and precisely at the transposon 3’ end in the other strand. This occurs on both sides of the transposon, creating three base single-stranded 5’ overhangs in the donor DNA. In the target DNA a five-base staggered cut is made. Transposon insertion results in a five-base duplication of target sequences (Craig, 1996).

For transposon excision from Km-resistant mutant pAd2-BAC DNA to occur, TnsABC* was applied to an in vitro reaction mix containing both pAd2-BAC donor DNA and plasmid pST76Tet as transposon acceptor (as described in Materials and Methods). Plasmid pST76Tet carries a temperature-sensitive mutation in the pSC101 replicon and cannot replicate at 37-42°C (Posfai et al., 1997; Posfai et al., 1999). Thus, the transposon will be received by a suicide plasmid and during bacterial growth cells are easily cured of that plasmid.

TnsABC*-mediated transposon excision from pAd2-H7-derived mutant BAC vectors created 3’-overhangs which could anneal as they contained complementary bases (Ruszics et al, manuscript in preparation). These cohesive ends were religated by addition of T4 DNA ligase. Correct transposon removal from pAd2-H7-derived mutant BAC vectors and religation of the gap was characterized by creation of a new NheI restriction site (Ruszics et al., manuscript in preparation, and data not shown).

To efficiently eliminate Km-resistant BAC vectors that persist due to incomplete transposition a strong counterselection tool was applied. The reaction mix was electroporated into

E. coli strain RP-12, which constitutively expressed a meganuclease I-Sce I from high copy plasmid

pUC19RP12 (Posfai et al., 1999). Meganuclease I-Sce I (Intron encoded meganuclease from

Saccharomyces cerevisiae) recognizes a specific sequence of 18 nucleotides in the transposon

sequence. Because of the length of the recognition sequence such a meganuclease target site is extremely rare and thus not present in the BAC vector backbone, in the E.coli genome, nor in the Ad2 genomic sequence (Posfai et al., 1999), Ruszics et al, manuscript in preparation). Meganuclease cleavage induces a double-stranded break in the mTn containing DNA, generating free DNA ends that trigger degradation of these DNA species.

Fig. 28 Analysis of mutant pAd2-BACs by restriction cut

Analytical restriction digests of mutant pAd2-BAC DNA were separated on a 0.8% agarose gel.

Enzymes used for the restriction cut are listed above the lanes. Numbers on top of the lanes denote the type of BAC DNA analysed, as listed below the gel pictures. M, 1kb DNA ladder.

To eliminate contamination by high copy plasmid pUC19RP12, the BAC-DNA was isolated from RP-12 cells, retransformed into DH10B and Cmr_/Aps _{clones were selected. The} integrity of the newly generated mutant pAd2-BAC vectors was analyzed by restriction cuts (Fig. 28). For a detailed overview of restriction fragments, see annexe.

XhoI cleavage of the mutant BACs pAd2/14.5ko (no FLAG-sequence), pAd2/10.4LL-F14.5, pAd2/F14.5Y74 (Fig. 28A) yielded the expected band pattern which was similar to that of pAd2/F14.5 (shown in Fig. 28B, XhoI cut of construct 1, and annexe). The top band that was present in all XhoI restriction cuts seemed to result from incomplete digestion. The 10.4ko mutation abolished one XhoI site, thus the 5864 bp and 7918 bp fragments were not generated, but migrated as one band of 13778 bp in size (Fig. 28A, XhoI, and annexe). The mutant BAC clone pAd2/10.4ko-F14.5 was additionally cut by PacI to confirm that the 14.5* ORF in the acceptor BAC DNA had been replaced by the FLAG-14.5K sequence. The mutated 14.5K start codon had been successfully replaced, as no 1.5kb fragment was obtained (Fig. 28B, PacI, annexe). EcoRV and

HindIII digestion of pAd2/10.4ko-F14.5 yielded the correct pattern, identical to the wt situation (Fig. 28B, annexe).

SnaB1 digestion liberated the viral DNA framed by intact flanking ITRs from the BAC vector backbone and a purified preparation of 2-6 µg of linear Ad2 mutant genomes was

transfected into 293 cells (~2*106 _{cells) for reconstitution of viral particles (described in Materials}

and Methods).

5.3. Recombinant Ads expressing 10.4-14.5 mutants are defective in receptor down-