5 - (O p 4 + /b a se 6 50 ) (O p 4 -/b a s e 7 6 5 ) 3’ Exon 5 119
5’-GAA TCC ACC CAG AAG GCA GAG-3’ 5’-CGG TGA TAG ATG TTG GGG CA-3’
(O p 5 + /b a se 830) (O p 5 -/b a se 981)
PCR parameters were: initial dénaturation at 94°C for 4 minutes. Denature
94 °C for 45 seconds, anneal at 64°C for 60 seconds, extend at 72°C for 45 seconds,
for 35 cycles. 10 minutes at 72°C final extension. Total volume per tube = 50 |il,
containing 1.5 mM M gCh, 2.5 U Taq polymerase, and approximately 200 ng of
template DNA.
Following PCR a 20 \i\ aliquot of each reaction was loaded on to a 1.8 % low
melting point agarose gel to check the success, specificity, and yield of the amplification
(section 2.4.4).
Target bands were excised and eluted (section 2.5.3.1) and then sub-cloned into
pCRlOOO™ or pCRII™ vectors (section 2.5.4). Following transformation into
competent cells (either commercially obtained or produced in-house), plating on
appropriate plates, and amplification in culture media (sections 2.4.5.3, 2.4.5.4, and
2.4.5.5), the recombinant plasmids were harvested by utilising the GeneClean™
protocol (section 2.4.6.3). Positive clones (those containing an insert of the correct
size) were identified by digesting a small (5 p,l) aliquot of the plasmid prep with
appropriate restriction enzymes (for vector pCRlOOO used Notl, and for pCRII used
jEcoRI). Clones containing inserts of the right size were subjected to sequencing (see
section 2.6 and 2.7).
To overcome an inherent problem with the PCR amplification process, whereby
incorporation of incorrect bases occurs at a low frequency in an enzyme dependent
manner (for Taq it is calculated at 10*^; Eckert and Kunkel, 1990), at least three PCR
Figure 3.4 Regions (dark cicles) of the LW and MW opsin that were sequenced in the present study
clones from a number of independent PCRs it was possible to identify those sequences
which result from incorporation errors and thus eliminate them from the analysis.
Sequencing by the manual dideoxy method using T7 polymerase and 35§_
labelled dATP (section 2.6.2), or automated sequencing using the ABI sequencer and
Taq Dyedeoxy terminator kits (section 2.7.4), was carried out. For manual
sequencing, products of the sequencing reactions were loaded on to 6 . 0 %
polyacrylamide gels, separated at 1500V for about 4.5 hours, and autoradiographed
overnight. Sequence data was read directly off the autoradiograph. Automated
sequencing generated electropherograms, from which sequence data was directly
imported into DNA alignment programmes (GeneWorks, MacVector, or the UW-GCG
package - see section 2.8). All clones were sequenced in both directions.
3 . 2 . 4 Phylogenetic tree construction
Using the neighbour-joining algorithm of Saitou and Nei (1987) incorporated
into the computer programme MEGA (Kumar and Simons, 1993) phylogenetic trees
were constructed for the LW and MW opsin genes using the nucleotide sequence and
deduced amino acid sequence data generated. The equivalent gene sequences from the
marmoset 563 nm allele (Hunt et a i, 1993a) was included in this analysis, and the
chicken iodopsin (Kuwata et a l, 1990) was used as an outgroup to root the tree. The
average number of base pair substitutions per nucleotide site (silent and coding) was
calculated. To make maximal use of the data, for each species the sequences obtained
for exons 3,4, and 5 were combined, and all substitutions were included.
Individual pair-wise comparisons between all the OW primates were made for
each gene, as well as a comparison of each species with the corresponding human,
marmoset 563 nm allele, and the chicken iodopsin sequences. The topology and branch
lengths of the tree were based on p values (where, the p value is the frequency of amino
acid replacements per site). Support for branches of the tree were estimated by
Prim ate Opsin Evolution The relatively low frequency of nucleotide differences prompted the calculation
of the average divergence of the group from several other closely related genes of other
species (Table 2). The sequence from an equivalent region of a single MW/LW
sensitive photoreceptor gene (560 nm) of the marmoset (Williams et aL, 1992), was
included to test the accuracy of the tree. The sequence of the LW photoreceptor gene of
the chicken (Kuwata et al., 1990) was used as an outgroup.
3 .3 R esu lts
3 . 3 . 1 Sex of the primates
As seen in Figure 3.5, a band of the target size, in the corresponding lane
indicates that the animal from which the sample of gDNA was obtained was a male;
absence of a band of the correct size suggests that that animal lacked a Y chromosome,
and thus by inference was a female. DNAs of known sex were included as positive
controls. The human female (lane 3), and male (lane 6) did provide the appropriate
result. These primers were designed based on the human sequence, therefore they were
expected to work only with the male. The results of lanes 1, 2, 7, and 9 show that
these primers were able to differentiate correctly between OWM of known sex. Finally,
lanes 4, 8, 10, and 11 show the sex of the unknown individuals; orang-utan was a
male, as was the chimpanzee, whereas, the gorilla and baboon were females. The
experiment was repeated a second time at a later period and gave identical results. The
findings are summarised in Table 3
3 . 3 . 2 Opsin gene sequences of the primates
For all eight primate samples (includes human control) an intensely staining
bands of the expected size (164 bp for exon 3, 156 bp for exon 4, and 191 bp for exon
5) was obtained (data not shown). Sequencing of the clones confirmed the presence of
both MW and LW opsin products in the amplified fragments from each PCR. Some
clones (a minority) gave sequences that were neither MW or LW opsin fragments, nor
did they correspond to any other opsin-like gene. These were considered to be