Investigation of the Recloned Vector

The recloned plasmid was analysed using many different molecular biological methods to show that a promoter converted provirus had been isolated and that the conversion had taken place correctly.

3.2.9.1 Southern Blot Analysis

The recloned plasmid was digested with the restriction enzymeKpnI. pWAP-BAGgal was also digested with the same enzyme in parallel. TheKpnI recognition site is present in the R region of both LTRs. After digestion withKpnI two fragments of 7 kb and~4 kb could be seen in the recloned plasmid. In pWAP-BAGgal bands of 7 kb und 0.7 kb were expected. In the recloned plasmid both the 7 kb as well as the 4 kb fragment should contain the WAP promoter if promoter conversion has taken place. In the plasmid pWAP- BAGgal the U3 region of the WAP promoter is only present in the 3’LTR and should only be detected in the 7 kb fragment. The gel was blotted and hybridised to an WAP specific probe. As expected both the 7 kb and the 4 kb band from the recloned plasmid hybridised to the probe gave a hybridisation signal (lane 1), and only the 7 kb product in pWAP- BAGgal was seen to be positive.

Figure 3.55: Southern Blot

1µg plasmid DNA was digested with the restriction enzymeKpnI. This enzyme was chosen as it could be used to show the presence of the WAP promoter in the 5' LTR after hybridisation to WAP specific probe. TheKpnI digest shows a 7 kb fragment and approximately 4 kb fragment in the recloned plasmid. Here it was expected that the WAP promoter would be present in both fragments after reverse transcription. The 4 kb fragment represents the remaining parts of the 5' and 3' LTRs as well as genomic DNA from the integration site. pWAP-BAGgal gave fragments of 7 kb and 0.7 kb as expected. The recloned plasmid is in lane 1 and pWAP-BAGgal in lane 2. After transfer to a nylon membrane, the DNA was hybridised to a radioactively labelled probe. In lane 1 the recloned, promoter converted plasmid shows two hybridised bands corresponding to the 7 and 4 kb fragments from the KpnI digest. In lane 2, pWAP-BAGgal shows only the 7 kb hybridised band as expected.

3.2.9.2 PCR Analysis of the Recloned Vector

PCR was used to further characterise the recloned plasmid. The primers used can be seen in figure 3.56. In the first primer pair the downstream primer binds at the beginning of the WAP promoter region in the 5’ LTR and the other binding to the packaging signal of the vector. In a second pair, the downstream primer binds in the middle of the WAP promoter region in the 5’

LTR, the complementary upstream primer binding in the ß-galactosidase gene. If the WAP promoter region has been duplicated and translocated from the 3' LTR to the 5' LTR 0.9 kb and 2.1 kb bands would be expected from the first and second primer pairs respectively. As expected the 0.9 kb PCR product can be seen in lane 1 and a 2.1 kb PCR product in lane 2 from the recloned plasmid. A 1.6 kb product in lane 5 and a 2.8 kb product in lane 6 arising from the parental plasmid control pWAP-BAGgal as WAP NRE is only present in the U3 of the 3' LTR.

The resulting gel was then blotted to a nylon membrane and hybridised to a 0.32 kb radioactively labelled probe isolated from the WAP promoter in order to check the specificity of the bands present. After hybridisation a strong band could be seen at the expected sizes (Fig.

3.56). This shows the presence of the WAP promoter region in both the 5' and 3' LTR’s of the recloned plasmid.

1 2 3 4 5 6 7 8

pWAPProCon pWAPProCon pWAP-BAGgal pWAP-BAGgal 0.9 kb

1.6 kb 2.8 kb 2.1 kb

PCR

pWAPProCon pWAP-BAGgal

pWAPProCon pWAP-BAGgal

0.9 kb

PCR pair 2 PCR pair 1

1.6 kb

2.1 kb 2.8 kb

PCR Southern

Hybridisation

Southern Hybridisation

Figure 3.56: PCR Analysis of the Recloned Vector

1 ng of plasmid DNA was amplified by PCR using 40 pmol of each primer. In the first primer pair, one primer is specific for the beginning of the WAP promoter region (Wapanf, 5’-AGATGTAGCCACGAACTC-3’) and the other primer specific for the MLV packaging region (125posc2, 5’-GGTCGGCCAGATACAGAGCTAGTTA-3’).

In the second primer pair one primer binds in the middle of the WAP promoter (Wapmit, 5’-GTGTGGCCAAGAAGGAAGTG-3’), the complementary primer being specific for the ß-galactosidase gene (Baggal2, 5'-TTCATCCACCACATACAGGC-3'). The PCR was performed under the following reaction conditions: denaturating for 1 min at 94°C, annealing for 2 mins at 51°C (P1-P2) or 55°C (P3-P4), and elongation for 3 mins at 68°C; 35 cycles were made. The primer pair P1-P2 gave a 0.9 kb PCR product (Lane 1) and P3-P4 gave 2.1 kb (Lane 2) from the recloned plasmid (pWAPProCon) whereas 1.6 and 2.1 kb PCR products (Lane 5 and 6) arose from pWAP-BAGgal. 10 µl of the PCR product was separated on a 0.8% agarose gel and transferred to a nylon membrane. The membrane was then hybridised against anα³²P-labelled 0.32 kb WAP specificXhoI/XbaI fragment.

3.2.9.3 Sequencing of the Recloned Plasmid

Sequencing was used to show that the promoter conversion had taken place correctly at the level of DNA sequence and also to determine the sequence of the integration site. The recloned plasmid was digested withKpnI, which recognises sites present in both LTRs, to separate the 5’

and 3’ LTRs and thus facilitate analysis of both LTRs individually. After promoter conversion it would be expected that the WAP promoter should be present in both LTRs. Two fragments were obtained, a 7 kb fragment and a 4 kb fragment. The 7 kb fragment contained the U3 region of WAP present in the 3’ LTR and a part of the R region and the 4 kb fragment contained the WAP promoter region present in the 5’ LTR and part of the R region. The 4 kb fragment was used for sequencing (see 2.2.5.1.3) as it contains the promoter converted LTR.

Two primers were chosen for the sequencing reactions, one of which was complementary to the U3 region of WAP (S1) and the other which was complementary to the R region of MLV (S2).

Sequencing of the junction between the CrFK flanking sequence and at the 5’end of the provirus revealed that theSacII restriction site and WAP NRE sequences inserted into this site were as expected intact.. The MLV IR was also present, but it had been shortened by 2 bp, as expected, due to processing during the integration event (Goffet al., 1992). Similarly, the sequence at the border between the WAP promoter and MLV R regions in the 5’ LTR revealed that the WAP promoter,MluI restriction site, and MLV R region were also intact (Fig. 3.57).

Figure 3.57: Sequencing of the Junction Regions of a Recloned Provirus

5 µg of plasmid DNA was digested with the restriction enzymeKpnI. A 4 kb fragment, which contains the WAP promoter region present in the 5' LTR, was taken and extracted for sequencing.

Sequencing was carried out with an automatic sequencer (ABI 373a; Applied Biosystems). The reactions were performed with a dye terminator cycle sequencing kit (Applied Biosystems) according to the manufacturer’s instructions (see 2.2.5.1.3). PCR mixtures contained 1µg of plasmid DNA and 4pmol of primer, one of which was complementary to the WAP promoter (S1, 5'-AGTTCGTGGCTACATCTGAG-3') and the other of which was complementary to the R region of MLV (S2, 5'-CCACAAGTCGGATGCAACTG-3'). Shown are the sequences at the U3-R junction of the 5' LTR and the integration site-U3 junction of the 5' LTR. The sequence analysis reveals that (i) the borders between the WAP promoter and MLV R regions are intact after promoter conversion and (ii) the IR has been used for integration. TheSacII restriction enzyme cleavage site and theMluI site used to introduce the WAP promoter region are marked. +1 indicates the position of the classic CAP site. IR represents the position of the inverted repeat. The small letters represent the flanking sequence from the CrFK genome.