The first attempt to isolate the PSII-W gene sequence involved using a cDNA library as the DNA template in a PCR reaction. The library was a gift from Prof. Moroney (Louisiana State University) and was constructed from cells which had been growing photoautotrophically. The cDNA molecules were directionally cloned into a lambda-Zap vector between the E cdRl and X hol sites as shown in Figure 5.3a. The PCR reaction was carried out using the T7 prim er and a gene specific primer. The only information available for the p s b ^ gene in C. reinhardtii
is the partial N-terminal sequence reported by de Vitry et al. (1991). This was used to infer the most likely DNA codons which would represent this protein sequence. The codon usage data, used in this case, came from a review by Rochaix (1987) where the nucleotide sequence o f eight nuclear genes was examined and the frequency o f the different codons was determined. The consensus sequence used is shown below in Figure 5.2.
Figure 5.2 The N-terminal protein sequence of PSII-W and the inferred DNA sequence used to design a gene specific primer.
Protein
L
V
D
E
R
M
N
G
DNA
cug
g u c /g gac
ga g
c g c
aug
a a c
g g% Frequency
94.5
(44 /52)
83.9
100
89
100
98.1
A PCR reaction was carried out as described in Section 2.7.10, using the gene specific primer and T7, with an annealing temperature o f 50°C for thirty cycles. The product amplified by this reaction is shown in Figure 5.3b. The fragment shown is approximately 900bp in length and contains DNA complementary to the gene primer designed against the PSll-W sequence. This was subsequently purified and blunt cloned into the vector pUC19 which had been
Figure 5.3a A diagram of a l ZAP cDNA clone
T3 T7
cDNA
AAAAAA
EcoRI Xhol
Figure 5.3b A gel photo of the PC R pro d u ct amplified from the X-ZAP library
PCR
4000 2213
1074 —
The PCR reaction was carried out using X-ZAP library DNA with T7 and a gene specific primer.
- 900
cut with HincU.. After transformation into D H 5a and preparation o f the plasmid (pPsbw) the insert was sequenced using an automated sequencer.
The 800bp o f sequence data which was produced was GC rich indicating that it is likely to be due to a nuclear sequence but there was no homology to the
p sb W sequences published on the database nor any similarity to a particular gene
type.
5.3 Rapid Amplification of cDNA Ends (RACE)
The technique known as RACE allows the researcher to amplify a desired cDNA from only a small length o f known sequence (reviewed Frohman 1990, Schaefer 1995). The process requires the preparation o f total cellular mRNA from the cell sample which is then used with reverse transcriptase to generate a corresponding population o f cDNA molecules. The primer used in this synthesis is complementary to the poly A tail found on most mRNA molecules but it has additional nucleotides added after the thymines which are used in the next stage o f the protocol. A summary o f the amplification o f sequence 3' to the known area can be seen in Figure 5.4. After synthesis o f the cDNA pool the method involves amplification o f the desired sequence by PCR. The primers used in this are complementary to the region o f known sequence and to the introduced tag positioned after the poly-A tail. Any product amplified in this way can be screened by a second round o f PCR using two internal primers. The purified PCR product is used as the template and the two primers lead to amplification o f a slightly smaller product than the original.
The work to be described in this chapter used the 3' RACE reaction to amplify the genomic sequence o f the C. reinhardtii p sb W gene.
5.3.1 RACE primers
The process o f RACE requires a region o f known sequence in order to design primers. The only data available was the N-terminal protein sequence so this had to be the starting point in the planning o f the experiment. W hen designing a new gene specific primer for this technique I decided to use a more degenerate prim er to ensure that the correct sequence was not missed. In addition, I had
Figure 5.4 Schematic diagram to show the process of 3 ’ RACE used to amplify th ep sh ^ g e n e sequence mRNA __ dt58 primer a a a a a a a a t t t t t t t t Reverse transcriptase cDNA Primers W1 Racel First Round PCR Race product 1 Primers W2 Race2 Second round PCR Race product 2 178
access to a different table o f codon usage for nuclear genes which was compiled by using the data from 176 genes (www.dna.affrc.go.jp/nakamura-bin) whereas the older one, used in the previous section, was compiled from only eight sequences. Figure 5.5 shows the two overlapping gene specific primers designed for use in this protocol.
The first step o f the RACE protocol requires the preparation o f a population o f cDNA molecules which is achieved using a poly T primer with an added tail section (DT-58). The 3' primers for the PCR sections o f this method (racel and race2) are complementary to the tag added at the cDNA synthesis stage. The sequence o f each o f these primers is given in the methods section. Table 2.5.
5.3.2 3 ’ RACE to amplify the p sbW gene
The RACE protocol requires the preparation o f mRNA from C. reinhardtii
cells and then synthesis o f a corresponding cDNA pool. These two manipulations were carried out by Dr. N. Gumpel in our laboratory. I then used the cDNA as the template for the PCR steps o f the p sb W isolation initially with primers race 1 and W l. The details o f the amplification are given in Section 2.8.3. The product o f this reaction was run on an 2% agarose gel with a lOObp ladder and a fragment o f approximately 670 bp was seen. This was subsequently gel purified and used as the template in the second PCR reaction which was designed to ensure that the product amplified corresponded to the p sb W sequence. Internal primers race2 and W2 were used under the same conditions as the original amplification. Figure 5.6 shows a photo o f a 2% agarose gel with both the first and second stage RACE products run against lOObp ladders. The product o f the first reaction is approximately 50bp larger than the second product which is to be expected with the primers used.
Having potentially isolated a copy o f the coding region for p sb W we then needed to sequence the RACE product to ensure that it did correspond to the gene. This was achieved by direct sequencing o f the purified PCR product on an automated sequencer. Details o f the sequence will be given in a later section but it was obvious that the data did relate to the p sb W gene through the levels o f homology to published sequences from other species. The purified RACE product
Figure 5.5 The p sb W specific primers used for RACE
Given below is the known protein sequence o f PSII-W together with the codons which relate to the different amino acids. Where multiple codons are possible all alternatives were included in the primer design
Primer Wl
Protein
L
V
D
E
R
M
N
Codons
cug
gug
g a c
gag
cg c
aug
aac
c
c
u
gu
Primer
t s
g t s
g a ygag
cgb
a t g
aa
Primer W2
Protein