■ . . .
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■. ■... X Chromosome Three alleles ■ CL.'; ;.';.';' ; o AutosomeI
____________________ SW Lqcus_________________ Males 3 :C Z Z Z - n i
Female Dichromats $ 3E S : 3):a;
a ... - f l ... :...J $ ..amrzzzmzD
cm
e - z - :..z& : : ) c : . z : ... :.... ... m ...) Female TrichromatsL :...- .... .S.:..:.:..:..:,
c : = 3 = 1CZZZZZZZEZZZZZai^aZD
A a . . . : . :...C L 3 :...: . - M l u _ — _ — - j M iU — i nFigure 1.18 Diagramatic representation of chromosomes
inherited resulting in dichromacy and trichrom acy in NWM. The circles to the right represent cone types expressed in the retina of those animals.
obtained from the MW and LW human opsin clones by Nathans et al. (1986a, b) included approximately 450 bp of upstream sequence from each gene. The TATA
boxes for both genes were identified.
Wang et a l (1993) extended the sequence information to include 6 kb upstream
of the LW opsin gene of human, as well as 5 kb upstream of mouse LM W cone opsin,
and two regions upstream of the bovine L/MW cone opsin. By simple sequence
alignment they identified a conserved 39 bp stretch, present approximately 2.9 kb
upstream of exon 1 of the human LW gene and a comparable distance in the other two
species. This they labelled as the LCR core sequence.
In experiments where the LCR core sequence was investigated by placing it
immediately upstream of a reporter gene, no transcripts were produced (S. Deeb,
personnel communication). This suggested that the relative position of this region to
the gene array is important in regulating transcription of the genes, acting as a master
switch, due to its influence on gross chromatin structure.
Distal promoter elements are known for a large number of genes (Li and Rosen,
1994). Two functions have been attributed to these sites: tissue specificity and
transcription efficiency. Experiments in which these regions are mutated or deleted
cause a reduction in the efficiency of transcription (Jones et a l, 1988). Chiu and
Nathans (1994b) demonstrated that sequences 5.4 kb upstream of the human SW opsin
gene, when placed in an appropriate reporter vector, direct expression of the reporter
gene only in the SW cones (and a subset of cone bipolar cells) of transgenic mice. A
similar set of findings has been reported for the mouse SW cone opsin (Chiu and
Nathans, 1994a), suggesting that all the sequence elements necessary for the control of
SW cone-specific expression are encoded within the 6.4 kb flanking region. It should
be remembered that whereas the human LCR region resides upstream of a opsin gene
array that may contain as many as 10 genes, both the mouse and bovine LCR is thought
to “overlook” just a single gene. The question then arises as to what function the LCR
Introduction
1 . 1 4 . 1 Globin gene cluster as a model
The genes of the human a-globin and p-globin loci are expressed in a specific
temporal pattern, and in specific haemopoietic tissues during development. The
embryonic globins and e) are expressed in the yolk sac blood islands until about the
fifth week of gestation. At that time, adult a globin (a) and foetal p globin and ^ y )
genes begin to be expressed in the liver. The liver is gradually replaced as the major
site of haemopoiesis by the spleen and bone marrow, which express the adult globin
genes (a, 6 and p; Kulozik et al, 1988). The switches which ensue are governed by a
region upstream of the array, which is analogous to the LCR region identified in the
opsin gene array (Tuan et al, 1985). The two types of gene array may therefore be
regulated in an analogous manner. These regions are discussed fully in chapter 4.
1 . 1 4 . 2 The mouse model
Cone development and topography have been studied in the mouse. Two cone
types, as well as rods, have been defined by immunohistochemistry in the mouse retina:
one type (hereafter referred to as the M-LW cones) contain visual pigments that react
with an antibody, mAb COS-1, and with antibodies raised against human LW and MW
visual pigments, and the second type (hereafter referred to as the SW cones) contain a
visual pigment that reacts with another antibody, mAb OS-2, and with antibodies raised
against the human SW visual pigment (Szél et a l, 1992; Wang et a l, 1992). The M-
LW cones and the SW cones most likely give rise, respectively, to the 510-nm and 360-
nm response maxima observed in the mouse electroretinogram under photopic
conditions (Jacobs et a l, 1991). The mouse retina shows that immunohistochemical
staining and morphologic analysis both show that the two cone types are not uniformly
distributed throughout the retina (Szél et a l, 1992; Wang et a l, 1992). Instead, a high
concentration of M/LW cones is found in the upper retina and low concentration in the
lower retina. The SW cones show a reciprocal pattern, with the result that the overall
cone density is approximately uniform. The transition between the upper and lower
zones occurs over a narrow equatorial band which is populated by cones which are
labelled by both antibodies (Rohlich et a i, 1994). It is apparent that these transitional cones express both classes of opsin, in contract to the generally accepted view of one
visual pigment per cone cell (Rohlich et a i, 1994). The retina of the rabbit and guinea
pig show the same pattern. As yet unidentified factors must be responsible for
determining the pattern of cell expression in the retina of these animals. The
coexpression of pigments in transitional cones is probably the result of overlapping
regulatory factors.
1 . 1 4 . 3 The rat anomaly
Work performed by Szél et al. (1994) on the retinae of rodents (rats and
gerbils) suggests that, in these species at least, SW photopigments are expressed before
MW photopigments. Further, MW cones developed from SW cones, that is, a
proportion of SW cones differentiate into MW cones in a temporal manner. This
suggests that SW cone development must be the default pathway, with the switch to the
MW cones occurring as a result of the action of an unknown factor. Such a temporal
Introduction
1 . 1 5 Aims of the project
• Determine the sequence and evolutionary relationships of the X-linked opsin
genes of apes and OWM.
Is spectral tuning of other OW primates dependent upon the same amino
acid sites as those of man? Do the opsin genes of other primates also exhibit
polymorphism? Is it possible to correlate the evolution of the LW and LW opsin
genes with the accepted evolutionary emergence of extant simians?
• Determine the regulatory important regions 5’ flanking the opsin genes of apes,
OWM and NWM.
In order to elicit the mechanisms responsible for determining the class of
opsin gene expressed in any one cone cell, it is necessary to determine which
differences exist in the regulatory regions of these genes. Do the proximal
promoter regions of the LW and MW opsin gene show differences in sequence,
and in the presence of binding sites for known transcription factors? Which 5’
flanking region does the corresponding region from the NWM opsin L/MW
resemble?
• Investigate the genetic basis for two classes of human red-green colour vision
anomalies.
Is it possible to correlate differences in amino acids at particular sites
with the differences in colour perception exhibited by these subjects?
• Examine the genetic basis of the colour vision defect of John Dalton, who died
over one hundred and fifty years ago, utilising tissue from his preserved
eyes.
Is it possible to isolate and amphfy DNA from tissue 200 years old?
Was Dalton an protanope?