CONCLUSIONES Y RECOMENDACIONES

Mini-preps of cosmid DNA (section 2.6.7.2) were digested with Hin^/III along with genomic DNA and fractionated by agarose gel electrophoresis (figure 5.7). This enzyme cuts twice in the pWB15 vector to generate a 4kb band which is common to all of the digests. For each secondary screen filter, all the colonies give the same restriction pattern, with the exception of A2c. The negative colony from filter B1 shows a different restriction pattern from the positives. The presence of differing clones (A2c and B1-)

The p d e b p ro m o ter region and m appin g o f tran scription al sta rt sites

F i^ re 5.5 Dot blot control filter with serial dilurtions of plasmid DNA containing the target insert on the left ('+' W109/R3) and containing a fragment of the Talapoin monkey opsin gene on

the right Probe = W109/R3 PCR

product. I , ' lo

0

lot

Figure 5.6 (right and below) Secondary screen of the primary picks from master filters A and B by colony blot Each filter is designated according to the area or colony lifted from the p r im ^ screen masters. Only the filters giving clear positive signals on both replicas after overnight exposure are shown. Probe = W109/R3 DNA. The indicated discrete colonies were isolated from the secondary screen master filters.

V I ' B1 A5.2 E ▼ •

The p d eb prom oter region and mapping o f transcriptional sta rt sites

indicates that there was more than one clone growing on the secondary filters. This gel was run out further and transferred to a Hybond-N membrane (section 2.6.9.8).

The secondary screen was repeated for A l, A5 and B l. Several clones were selected from each filter, mini-prepped and digested. At least one clone from each filter gave the same pattern as those isolated in the original secondary screen.

DNA from the original preparations (of better quality) was used as a template for PCR amplification of pdeb exon 1. Figure 5.8 shows the result of this screen, with none of the clones giving a positive signal. The positive control from a 1(P dilution of genomic DNA gives a band of the expected size. As an additional control, an aliquot of each prep was spiked with genomic DNA and subjected to the same amplification as above. All the preparations gave a positive result (data not shown). The results suggest that none of the clones which gave positive signals in the primary and secondary screens contain this exon and are therefore excluded from further characterisation.

Figure 5.9 shows that a negative result was also obtained in a PCR from the 3' end of pdeb in representatives of the groups of A and B positives. Additionally, a primer pair from the mid-portion of the pdeb gene also gave a negative result (data not shown).

A base composition plot of the pdeb cDNA was constructed using the Mac Vector software (section 2.9.1). This reveals (figure 5.10) that the first 5G0bp has a particularly high GC content relative to the remaining 900bp or so of the W109/R3 PCR fragment. Therefore, the alternative probe F8/R3 was used to probe the Southern blot of the A and B secondary screen positives (section 2.6.9 8).

Figure 5.11 shows the result of the tertiary Southern blot hybridisation with probe F8/R3. At a high stringency wash there is hybridisation to specific bands of the digest, suggesting that it was not hybridisation of the GC rich portion of exon 1 in the library screens which gave rise to the positive signals. A single band is hybridised in most cases, except A2c, where 2 bands are seen. There is a faint band in the B l- lane, the negative control from the secondary screen. This may represent some residual binding of the probe, but, since the amount of DNA in this lane is much less than that in the other lanes, this is not a clear negative hybridisation result. For the positive clone groups there is no band sharing.

Th£ pdeh prom oter region and mofjping o f transcriptional start sites

Figure 5.7 Hindlll digest of cosmid DNA preps from the colonies picked from the secondary screen of the genomic library. M = X/Hindlll markers. Each clone is identified by its letter from the secondary screen filters. Lancs 1-3 A2a-c, lanes 4-8 A5.2a-e, lanes 9-14 B la-f. lane 15 B1-. G = genomic DNA.

Figure 5.8 Tertiary screen by PCR. DNA from each of the positive clones was tested for the presence of pdeb exon

1 using primers W 109/W 118. Lanes 1-15, positive clones, lane 16 DNA prepared from an aliquot of the neat library which had been grown up in broth overnight, lane 17 no DNA control, lane 18 genomic DNA control. M = lOObp ladder.

A 5 2

M 1

Figure 5.9 Tertiary screen by PCR.

Representatives from each group of clones as defined by their Hindlll restriction pattern were examined for the presence of 3' pdeb sequences using primers W100/X22R. M = PhiX 174/HaeIll Lane 1 clone A2a, lane 2 clone A2c, lane 3 clone A5.2a, lane 4 clone B la, lanes 5 & 6 genomic DNA, lane 7 no DNA control.

m #

Figure 5.10 Dinucleotide composition plot showing the G+C content (%, y-axis) throughout the l^ c b cDNA sequence given in number of nucleotides from the 5' end of prim er FI x-axis, see figure 3.19). A window size of 50bp was used by the MacVector 3.5 software. Primer R3 maps to nucleotide 1681 whilst primer F8 maps to position 766.

Tïie p d eb p rom oter region and mofjping o f tra n scription al start sites 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 2 3 — 9 4 — 66 — 4 4 — 2 3 -

Figure 5.11 Tertiary screen of genomic library clones positive for pdeb by hybridisation. The gel shown in figure 5.7 was run out further and blotted onto a nylon membrane. This was then hybridised by a F8/R3 canine pdeb probe. Lanes 1-3 A2a-c, lanes 4-8 A5.2a-e, lanes 9-14 B la-f, lane 15 B1-, lane 16 genomic DNA. The positions of X/Hindlll markers are given in kilobase pairs.

10 11

— 4 5 0

Figure 5.12 PCR secondary screen of the primary screen positive areas lifted from master filter C. Using primers W 109/W 118, the presence of pdeb exon 1 in these clones was assayed. Lanes 1- 6 positive areas 1-6 from filter C (figure 5.4), lane 7 no template control, lane 8 sterile loop only negative control, lane 9 opsin colony negative control, lane 10 plasmid pCRll insert W109/R7- containing colony (positive control), lane 11 genomic DNA. L = PhiX 174/Haelll ladder.

The p d eb prom oter region and mapping o f transcriptional sta rt sites

suggesting that each group represents an unrelated segment of genomic DNA. The inserts may contain DNA sequences which contain portions of pdeb outwith the PCR primer binding sites, or represent closely related genes. Since a differential signal was obtained on the primary and secondary screens, this hybridisation is not to the vector sequences. However, the low level of hybridisation seen in the B l- lane is unexpected. Although the lane of genomic DNA shows a major specifc band at the same position as shown in figure 5.2, there is some background smearing which may be enough to give a positive signal on a cosmid clone digest because of the amount of DNA present in each band.

In any event, these clones had been shown not to contain pdeb exon 1 sequences which should have given a product by PCR and were therefore considered to be of no further interest in a genomic walk upstream..