Figure 1.
Schematic representation of plasmids.
A) The vector pAAVRepURA contains wild type Rep gene and the yeast URA3 marker replacing the Cap gene flanked by the two ITRs. B) The vector pAAVpokURA contains a stuffer sequence, pok, instead of Rep gene. The ITRs are the only AAV sequence present in the vector. C) The vector pRepURA has the same sequences as pAAVRepURA without ITRs.
Figure 2.
The frequency of colonies carrying the AAV genome increased when Rep68 is expressed.
The plasmid pAAVRepURA3, pAAVpokURA linearized with PvuII and the control plasmid, pRepURA3, digested with XbaI, were co-transformed with the plasmid pGAD424 or pG.Rep68 containing the LEU2 marker gene (A, B). (A) Representative plates comparing colonies obtained from the transformed yeast RSY12 strain with plasmid pRepURA and pGAD424 (i), pRepURA and pG.Rep68 (ii), pAAVRepURA and pGAD424 (iii); pAAVRepURA and pG.Rep68 (iv). (B) Transformed yeast cells were scored for their ability to form colonies on selective medium lacking leucine and uracil. The frequency was calculated as described in material and methods. Results are the mean of 4 independent experiments±standard deviation.
Figure 3.
Rep protein expression in yeast.
Western blotting of total yeast cell lysate from cells transformed with pAAVRepURA (A) and pG.Rep68 (B). (A) The two lanes were loaded with the protein obtained with first extraction (lane 1) and second extraction (lane 2). The second extraction contains Rep78, not present in the first extraction. (B) The two lanes were loaded with proteins from the second extraction of a yeast clone obtained from transformation with control plasmid pGAD (lane 1) and pG.Rep68 (lane 2). In (A) and (B), the amount of proteins loaded was detected with 3-phoshoglycerate kinase antibody (3-PGK).
Figure 4.
(A) Southern blot analysis of low Mr DNA of two different yeast clones URA3+ (lane 1 and 2) derived from transformation with pAAVRepURA3 using the URA3 marker gene as probe. (B, C) Southern blot analysis of genomic DNA of the same two clones as in B undigested (lane 1 and 3), and digested with AseI (lane 2 and 4) probed with URA3 (B) or ITR probe (C). (D) Low Mr DNA of the two yeast clones URA3+LEU2+ derived from transformation with pAAVRepURA3 and successively transformed with pG.Rep68 (lane 1 and 2) analyzed on Southern Blot using the URA3 probe. (E) Low Mr DNA of yeast clones URA3+LEU2+ derived from co-transformation of pAAVRepURA with control plasmid pGAD424. Lanes 1 and 3 show the undigested DNA and lanes 2 and 4, DNA digested with DpnI and subjected to Southern blot analysis using URA3 marker gene as probe. Lane 5 shows the result of DpnI digestion of the pAAVRepURA plasmid. DpnI was performed in order to demonstrate that AAV DNA replicated in yeast. (F) Low Mr DNA of yeast clones URA3+LEU2+ derived from co-transformation of pAAVRepURA with plasmid pG.Rep68. Lanes 1, 3, 5, 7 show the undigested DNA and lanes 2, 4, 6, 8 DNA digested with DpnI and subjected to Southern blot analysis using URA3 marker gene as probe. (G) Schematic representation of DpnI/MboI (indicated with D) and AseI (indicated with A) restriction map of pAAVRepURA plasmid. The DpnI/MboI restriction endonucleases do not cut in the URA3 gene.
Figure 5.
S1 and Mung Bean nuclease sensitivity.
(A, B) Low Mr DNA from a URA3+LEU2+ clone derived from co-transformation of pAAVRepURA with plasmid pG.Rep68, was digested with DpnI (loaded in the lane 2), MboI (loaded in the lane 3) , S1 nuclease (in the lane 4) and was analyzed on Southern Blot using URA3 probe (A) or ITR probe (B). (C) Southern blot analysis of low Mr of two URA3+LEU2+ clone derived from co-transformation of pAAVRepURA with plasmid pG.Rep68 was digested with Mung Bean nuclease (in the lane lane 2 and 4) and compared with the not digested DNA (lane 1, 3). DNA was detected using the URA3 probe. The arrows indicate the ssDNA which disappeared after digestion with S1 and Mung Bean nuclease.
Figure 6.
Characterization of newly replicated DNA.
(A) Low Mr DNA of four clones derived from yeast co-transformed with pAAVRepURA and pG.Rep68 digested with DpnI and BamHI (lane 2, 4, 6, 8) and not digested (lane 1, 3, 5, 7) was analyzed on Southern Blot and probe with the URA3 gene. BamHI cuts once in AAV construct. (B, C) Low Mr DNA of one clone obtained from the co-transformation of pAAVRepURA with pGAD not digested (lane 1) and digested with DpnI and BamHI (lane 2); the low Mr DNA of one out four clones analyzed in panel A undigested (lane 3) and digested with DpnI and BamHI (lane 4) was analyzed on Southern Blot and detected with URA3 probe (B) and F1 probe (C). (D, E) Equal amount of low Mr DNA of one out of four clones analyzed in panel A was loaded in two gel slots and transferred on nylon membrane. The membrane was cut in two pieces corresponding to gel slots and further cut to leave only ssDNA. The ssDNA was detected using as probe a 100-mer oligonucleotide complementary to the filament (+) of the URA gene URA(+) (D), or a oligonucleotide probe complementary to filament (-) of the URA gene URA(-) (E), to determine the polarity of ssDNA. (F) Restriction maps of predicted replicative intermediates: i) linear monomer, ii) tail-to-tail dimer, iii) head-to-head dimer, iv) linear vector. Position of BamHI is indicated with B. The sizes of the fragments liberated following BamHI cleavage and recognized by the URA probe are shown next to corresponding structures. ITRs are indicated as black boxes.
Figure 7.
ssDNA formation is dependent on ITRs and Rep68.
Southern blot of low Mr DNA obtained from a clone expressing Rep68 transformed with pAAVpokURA (A) or pRepURA (B) and probed with URA3. (A) Lane 1 shows low Mr DNA undigested and lane 2, low Mr DNA subjected to S1 nuclease. (B) Low Mr DNA of two different clones (lane 1 and 2) expressing Rep68 and transformed with pRepURA.
Figure 8.
Diagram model showing ssDNA formation from a plasmid containing the AAV genome in yeast.
(A) For the sake of clarity, the plasmid carrying ITRs is depicted as linear molecule. Rep nicks at the trs (white star) (B). A replication complex is assembled and replication commences through the ITR towards the vector (C). The new synthesized ITR fold into a hairpin conformation displacing the replication strand. Such displacement determines a template switch so that the originally nicked strand is copied during replication. The replication passes through the other ITR and proceeds into the vector sequence (D). After replication fork has completed a full circle, Rep produces a second nick and the newly synthesized DNA is displaced as ssDNA (E). The new ssDNA is nicked again and two ssDNA containing only one complete ITR are formed (F). The missing ITR is repaired by gene correction mechanism (G).