Human Color Vision

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Editors
Jan Kremers Rigmor C. Baraas
University of Erlangen-Nürnberg Department of Optometry and Visual
Erlangen, Germany Science
University College of Southeast Norway
N. Justin Marshall Kongsberg, Norway
Queensland Brain Institute
The University of Queensland
Brisbane, Australia

Springer Series in Vision Research

ISBN 978-3-319-44976-0    ISBN 978-3-319-44978-4 (eBook)
DOI 10.1007/978-3-319-44978-4

Library of Congress Control Number: 2016955085

© Springer International Publishing Switzerland 2016

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Preface

Color vision is considered to be a visual sensation that is intimately related to emo-

tions. If the world is “colorful,” then it is positively full of wonder and surprises.
When it is “bleak” or “gray,” then the prospects are sad and pessimistic. Red is
warm but can mean danger. Blue is cold. Yellow is a color of warning.
Color vision is a marvelous subdiscipline in vision research, embraced by those
who study it and sometimes carefully avoided by those for whom it is only of indi-
rect interest.
In the recent years, our understanding of human color vision has been tremen-
dously advanced by many new developments, ideas, and achievements. It has been
and still is an exciting time for color vision scientists. We therefore think that it is
timely that these new developments are brought together in a book, particularly if it
is one in a series on Vision Research. In this book, many new developments have
been assembled, covering many different levels from genetics to perception, and
studied with state-of-the-art methods such as genetics, morphology, imaging tech-
niques, electrophysiology, psychophysics, and computational neuroscience. The
genetics of cone photopigments is discussed in Chap. 1. Further new exiting devel-
opments have been obtained in the study of cone mosaics (Chap. 3), in the physiol-
ogy of color vision in retinal (Chaps. 2 and 4) and cortical circuitries (Chap. 7), and
in color psychophysics and perception (Chaps. 5, 6 and 8). Going beyond the ques-
tions about the processes leading to visual perception within an individual, the book
also considers the latest computational models (Chap. 9), clinical implications and
the question how retinal disorders can compromise color vision (Chap. 10), and
finally the evolution of color vision (Chap. 11). We hope that the reader will find the
chapters inspiring and helpful in defining scientific topics that will be of interest in
the future. We think that there will be many interesting challenges. To name but a
few, the following topics may emerge: the molecular basis of color vision; the study
of single cells and pathways and their visual responses in the living retina; the
responses of cells in their intact circuitries; the mathematical description of color
processing; the improved use of color vision in diagnosing and monitoring inherited
and acquired disorders of the retina; a better understanding of the many perceptual
aspects of color vision.

v
vi Preface

We were supported by world experts who contributed to the book and wrote
chapters on the new developments in their field of interest. We encouraged them to
seek contact and collaborate with other experts. The result often was an interesting
discussion amongst the authors and with the editors. We are extremely glad and
proud that all authors have put so much effort in writing their chapters. We asked the
authors to keep the text as simple and understandable as they could (without com-
promising on the scientific content), so that it also would entice and interest nonex-
pert scientists and students. The result is a book of which we think highly of and we
are confident that it brings the latest developments in color vision research for a
broader scientific audience. We hope you, as a reader, will agree.
We would like to thank the authors for their brilliant efforts. We appreciate it
enormously. The collaboration between the series editors and with Springer was
also extremely positive and inspiring.
It remains to thank those who continuously supported us. More particularly:
Rigmor C. Baraas: Finn Erik, Rasmus, the rest of the family, and all present and
former members of the lab.
Jan Kremers: Andrea, Leon, Finy, the rest of the family, and all present and for-
mer members of the lab.
Justin Marshall: Sue for endless support and patience

Erlangen, Germany Jan Kremers

Kongsberg, Norway Rigmor C. Baraas
Brisbane, Australia N. Justin Marshall
Contents

1 The Genetics of Color Vision and Congenital Color Deficiencies....... 1

David M. Hunt and Livia S. Carvalho
2 The Retinal Processing of Photoreceptor Signals................................. 33
Jan Kremers, Luiz Carlos L. Silveira, Neil R.A. Parry,
and Declan J. McKeefry
3 Functional Imaging of Cone Photoreceptors......................................... 71
Lawrence C. Sincich, Ramkumar Sabesan, William S. Tuten,
Austin Roorda, and Wolf M. Harmening
4 Cone Opponency: An Efficient Way of Transmitting
Chromatic Information........................................................................... 105
Barry B. Lee and Luiz Carlos L. Silveira
5 Psychophysical Correlates of Retinal Processing.................................. 133
Rigmor C. Baraas and Andrew J. Zele
6 Color Constancy and Contextual Effects on Color Appearance......... 159
Maria Olkkonen and Vebjørn Ekroll
7 Color in the Cortex.................................................................................. 189
Elizabeth N. Johnson and Kathy T. Mullen
8 Interactions of Color Vision with Other Visual Modalities.................. 219
Frederick A.A. Kingdom
9 Computational Modeling of Color Vision.............................................. 243
Thomas Wachtler and Christian Wehrhahn

vii
viii Contents

10 Color Vision in Clinical Practice............................................................ 269

Cord Huchzermeyer, Jan Kremers, and John Barbur
11 Evolution of Color Vision........................................................................ 317
Almut Kelber and Gerald H. Jacobs

Index.................................................................................................................. 355
Contributors

Rigmor C. Baraas National Centre for Optics, Vision and Eye Care, Department
of Optometry and Visual Science, Faculty of Health Sciences, University College of
Southeast Norway (USN), Kongsberg, Norway
John Barbur Applied Vision Research Centre, School of Health Sciences, City
University London, London, UK
Livia S. Carvalho Lions Eye Institute, University of Western Australia, Perth,
WA, Australia
Vebjørn Ekroll Laboratory of Experimental Psychology, University of Leuven
(KU Leuven), Leuven, Belgium
Wolf M. Harmening Department of Ophthalmology, University of Bonn, Bonn,
Germany
Cord Huchzermeyer Department of Ophthalmology, University Hospital
Erlangen, Erlangen, Germany
David M. Hunt, Ph.D., F.R.S.B. Lions Eye Institute, University of Western
Australia, WA, Australia
School of Animal Biology, University of Western Australia, Stirling Highway,
Perth, WA, Australia
Gerald H. Jacobs Department of Psychological and Brain Sciences, University of
California, Santa Barbara, CA, USA
Elizabeth N. Johnson Department of Neurobiology and the Duke Institute for
Brain Sciences, Duke University School of Medicine and Duke University, Durham,
NC, USA
Almut Kelber Department of Biology, Lund University, Lund, Sweden

ix
x Contributors

Frederick A.A. Kingdom Department of Ophthalmology, McGill Vision

Research, McGill University, Montreal, QC, Canada
Jan Kremers Department of Ophthalmology, University Hospital Erlangen,
Erlangen, Germany
School of Optometry and Vision Science, University of Bradford, Bradford, West
Yorkshire, UK
Barry B. Lee School of Optometry, State University of New York, New York, NY,
USA
Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany
Declan J. McKeefry School of Optometry and Vision Science, University of
Bradford, Bradford, West Yorkshire, UK
Kathy T. Mullen Department of Ophthalmology, McGill Vision Research,
Montreal, QC, Canada
Maria Olkkonen Department of Psychology, Science Laboratories, Durham
University, Durham, UK
Institute of Behavioural Sciences, University of Helsinki, Helsinki, Finland
Neil R.A. Parry School of Optometry and Vision Science, University of Bradford,
Bradford, West Yorkshire, UK
Centre for Hearing and Vision Research, Institute of Human Development,
University of Manchester, Manchester, UK
Vision Science Centre, Manchester Royal Eye Hospital, Central Manchester
University Hospitals NHS Foundation Trust, Manchester Academic Health Science
Centre, Manchester, UK
Austin Roorda School of Optometry, University of California, Berkeley, CA,
USA
Ramkumar Sabesan School of Optometry, University of California, Berkeley,
CA, USA
Lawrence C. Sincich Department of Optometry and Vision Science, University of
Alabama at Birmingham, Birmingham, AL, USA
Luiz Carlos L. Silveira Tropical Medicine Nucleus and Biological Sciences
Institute, Federal University of Pará, Belém, Pará, Brazil
Ceuma University, São Luís, Maranhão, Brazil
William S. Tuten School of Optometry, University of California, Berkeley, CA,
USA
Contributors xi

Thomas Wachtler Computational Neuroscience, Department Biologie II, Ludwig-­

Maximilians-­Universität München, Planegg-Martinsried, Germany
Christian Wehrhahn Vision Center Laboratory, The Salk Institute, La Jolla, CA,
USA
Andrew J. Zele School of Optometry and Vision Science and Institute of Health
and Biomedical Innovation, Queensland University of Technology (QUT), Brisbane,
QLD, Australia
Chapter 1
The Genetics of Color Vision and Congenital
Color Deficiencies

David M. Hunt and Livia S. Carvalho

Abstract Primates are unique among mammals in possessing trichromacy. In Old

World primates, it is based on three cone classes in the retina, each expressing a
different class of visual pigment. These pigment classes are each orthologues of
pigments present throughout the vertebrate kingdom, the short wavelength-sensitive
(SWS1, SWS2, LWS and MWS) pigment and two representatives of the long wave-
length-sensitive (LWS) pigment, L cone opsin and M cone opsin. The latter two
pigments arose from a duplication of the LWS gene that occurred at the base of the
Old World primate lineage to give an array of two closely adjacent opsin genes on
the X chromosome. This close proximity and the extensive sequence identity of the
L and M genes promotes mispairing of the genes and thereby underlies the high
frequency of red-green color blindness seen in humans. The consequences of this
mispairing are the loss of either the L or M gene to give full dichromacy, or the
generation of hybrid genes to give anomalous trichromacy. Generally, red-green
color blindness is not associated with loss of acuity, although this is present in a rare
form of dichromacy called Bornholm eye disease where cone dysfunction and myo-
pia is also present. Other forms of color blindness include the X-linked disorder of
blue cone monochromatism where L and M cones are absent, the dominant disorder
of tritanopia where S cone are severely reduced or absent, and the recessive disorder
of achromatopsia where all cone classes may be absent.

Keywords Color vision • Evolution • Color blindness • Trichromacy • Dichromacy

• Achromatopsia • Visual pigments • Visual opsins

D.M. Hunt, B.Sc., Ph.D., FRSB (*)

Lions Eye Institute, University of Western Australia,
Verdun Street, Perth, WA 6009, Australia
School of Animal Biology, University of Western Australia,
Stirling Highway, Perth, WA 6009, Australia
e-mail: david.hunt@uwa.edu.au
L.S. Carvalho, B.Sc., M.Sc., PhD
Lions Eye Institute, University of Western Australia,
Verdun Street, Perth, WA 6009, Australia
e-mail: liviacarvalho@lei.org.au

© Springer International Publishing Switzerland 2016 1

J. Kremers et al. (eds.), Human Color Vision, Springer Series in Vision
Research, DOI 10.1007/978-3-319-44978-4_1
2 D.M. Hunt and L.S. Carvalho

1.1 Introduction

The vertebrate retina contains photoreceptors cells that are specialized for the
capture of light. These cells are subdivided into two classes, the rods and cones.
Rods are responsible for monochromatic vision in dim light and cones for
vision at normal light levels and for color vision. In rod and cone photorecep-
tors, the outer segments are composed of a stack of membranous disks in which
the key molecules for photon capture, the photosensitive visual pigments, are
embedded.
The mechanism of color vision depends critically on a comparison of the amount
of light (photon capture) falling on spectrally different types of cone photoreceptors
that are maximally sensitive to different wavelengths (For further details see Chap. 4).
This is the process of cone opponency whereby each photoreceptor type is stimu-
lated to a different extent by light of differing spectral content. Comparison of these
signals by the brain provides the sensation of color. From this it follows that color
vision requires a minimum of two spectrally different types of cone photoreceptors
to be present.
In primates, trichromatic color vision is provided by the presence of three
classes of cone photoreceptors with wavelengths of maximal sensitivity (λmax) in
the yellow-­green (around 560 nm, longwave-sensitive, L), green (around 530 nm,
middlewave-­sensitive, M), and blue (around 430 nm, shortwave-sensitive, S)
regions of the spectrum [1–3]. The light-sensitive components of photoreceptors,
the visual pigments, are members of the GPCR family of proteins. They comprise
a seven transmembrane (TM) opsin protein that is covalently linked to a Lys resi-
due via a Schiff base (SB) to the chromophore. In mammals, this is invariably
11-cis-retinal derived from vitamin A1, so the peak spectral absorption (λmax) of a
visual pigment is determined not by the chromophore, but by the amino acid
sequence of the opsin protein, with certain residues tuning the pigment to particular
spectral locations.

1.2 Phototransduction Cascade

The ability to see in dim and bright light arises in vertebrates from differences in the
light sensitivity of rods and cones. In general, rod photoreceptors are more sensitive
than cones, have less “dark noise’, adapt over a much narrower range of light intensities,
but have slower response kinetics [4–7]. In contrast, cones have a higher frequency of
spontaneous thermal isomerizations of the chromophore [8], and a substantial propor-
tion of cone opsin exists in an apo form lacking bound chromophore. This apo-pig-
ment is able to activate phototransduction and may account, in part, for the faster and
larger response of cones [9, 10]. The presence of either a rod or cone pigment however
makes only a minor contribution to the kinetics of the photoresponse, as demonstrated
by the photoreceptors present in the retina of the nocturnal Tokay gecko, Gecko gecko,
which have a rod-like morphology [11] and rod-like photokinetics [12], but contain
1 The Genetics of Color Vision and Congenital Color Deficiencies 3

only cone visual pigments [13]. The fundamental difference between rods and cones
with respect to sensitivity to light is not therefore simply determined by the presence
of either a rod or cone visual pigment.
The visual process involves the conversion of the signal from the light-activated
visual pigment to an electrical impulse and this is achieved by the process of photo-
transduction within the photoreceptor; this process also results in a substantial
amplification of the original signal. A striking feature of vertebrate photoreceptors
is the number of rod- and cone-specific isoforms that form the phototransduction
cascade (Fig. 1.1). Absorption of light causes the isomerisation of the chromophore,
11-cis-retinal, to the all-trans form in a photobleaching sequence with consequent
conformational changes in the opsin protein, leading to the activation of the G pro-
tein transducin by the activated form of the visual pigment, metarhodopsin II (meta
II) (Reviewed in Ref. [14]). Meta II activates the heterotrimeric GTP-binding pro-
tein transducin, which is composed of α, β, and γ subunits. GDP bound to transducin
is replaced by GTP and the GTP-α-subunit conjugate dissociates from the βγ com-
ponent. Different isoforms for all three of these subunits are present in rods and
cones. Phosphodiesterase (PDE) in rods is composed of catalytic α and β-subunits
and two inhibitory γ subunits, whereas the cone form is composed of two identical
α′ subunits and two inhibitory γ subunits. Activation of PDE involves the interac-
tion with GTP-α-transducin and the dissociation of the inhibitory γ subunits. This
activation results in the breakdown of cGMP and the closing of the cGMP-gated
(CNG) channels, which also differ between rod and cones, leading to reduced levels
of intracellular Ca2+. Cone channels are generally more permeable to Ca2+ than rod
channels, and this may underlie, in part, the more rapid and larger light dependent
changes in Ca2+ concentration in cone cells [15].
The restoration of the cGMP and Ca2+ resting states is achieved by the activation
of retinal-specific guanylate cyclases (GCs) by the Ca2+-binding guanylate cyclase
activating proteins (GCAPs) [16]. At the low levels of Ca2+ that exist after the clo-
sure of the CNG channels, GCAPs activate the cyclase function of GC, resulting in
the production of cGMP, the reopening of the CNG channels and an increase in Ca2+
concentration to the resting state level. Two isoforms of GC, GC1 and GC2, have
been identified, but GC1 is clearly the more important enzyme for phototransduc-
tion since null mutations in the corresponding GUCY2D gene cause the severe
blinding disease of Leber Congenital Amaurosis [17], and altered photoreceptor
survival in gene knockout mutant mice [18]. Missense mutations are also a major
cause of dominant cone dystrophy [19, 20] (see Chap. 10). GUCY2D is expressed
in both rods and cones, although the level of expression is higher in the latter [21].
Multiple isoforms of retinal GCAPs have also been identified [22–25], but GCAP1
encoded by GUCA1A appears to play the more important role. Mutations in GCAP1
are known to cause dominant cone dystrophy [26–30].
The inactivation of the cascade is necessary for recovery from a photoresponse.
This occurs as a two-step process, involving phosphorylation of the activated pig-
ment (meta II) by rhodopsin kinase targeted to serine residues in the carboxy termi-
nus of the opsin protein, followed by binding of the inhibitory protein arrestin [31].
Two retinal-specific kinases, GRK1 [32, 33] and GRK7 [34], are present in the
Fig. 1.1 Phototransduction cascade. (a) Gene isoforms of component processes that are expressed
in either rods or cones. (b) Schematic diagram showing role of component processes in the activa-
tion of the cascade. Dark state shows the component processes with the cGMP-gated CNG chan-
nels open through the production of cGMP by activated retGC. Cascade activation results from the
conversion of GDP to GTP and the release of the α-transducin subunit. This in turn activates PDE
by removal of the inhibitory γ-subunit, leading to the breakdown of cGMP, the closure of the CNG
channels and a fall in Ca2+ levels. Ca2+-free GCAPs bind to retGC, enabling the activation of the
cyclase activity and the production of cGMP. The restored levels of cGMP lead to a reopening of
the CNG channels and the restoration of Ca2+ levels. Disease-associated subunits identify the
component processes that are involved in defect in color vision
1 The Genetics of Color Vision and Congenital Color Deficiencies 5

human retina; GRK7 is preferentially expressed in human cones [35]. Inactivation is

then completed by the binding of the inhibitory protein arrestin [31] to the phos-
phorylated terminus of the pigment; two isoforms of arrestin are present, S-antigen
(SAG) in rods, and C- or X-arrestin (ARR3) in cones [36].

1.3 Evolution of Visual Pigments in Vertebrates

1.3.1 Ancestral Vertebrate Complement

The vertebrate blueprint for color vision consists of four spectrally distinct visual
opsins, each encoded by a different gene, that first arose in the agnathans, as dem-
onstrated by their retention by the southern hemisphere lamprey, Geotria australis
[37, 38]. Orthologues of these four classes survived the split between the agnathans
and gnathostomes which occurred over 540 MYA [million years ago] [39, 40] and
have been retained by most vertebrate classes. They consist of a longwave-sensi-
tive (LWS) pigment with λmax 500–570 nm, a middlewave-sensitive (MWS or RH2)
pigment with λmax 480–530 nm, and two shortwave-sensitive pigment classes,
SWS2 with λmax 400–470 nm and SWS1 with λmax 355–445 nm. In vertebrate visual
pigments with λmax values >385 nm, the SB is protonated, with a negatively charged
residue at site 113 (usually Glu113) acting as a counterion to stabilize the proton
of the SB [41]. In a subset of SWS1 pigments that show ultraviolet-sensitivity with
λmax values around 360 nm, the SB is unprotonated in the resting state [1, 42] (For
an extended account of evolution of color vision see Chap. 11).

1.3.2  oss of Cone Pigment Classes in the Early Evolution

L
of Mammals

Not all of these cone pigment classes are found in mammals. Cone pigment loss is
thought to have arisen during a nocturnal phase that marks the early evolution of the
mammals around 150–200 million years ago (MYA) [43]. In marsupials and euthe-
rian mammals, the LWS gene is paired with the SWS1 gene, with the loss of the
SWS2 and RH2 genes. The egg-laying protherian mammals, the platypus,
Ornithorhynchus anatinus, and echidna, Tachyglossus aculeatus, belonging to the
Order Monotremata that diverged from the marsupial/placental mammal lineage
around 200 MYA, have also retained the LWS gene but this is paired the SWS2 gene
[44, 45]. In contrast therefore to marsupial and eutherian mammals, protherians
have lost the SWS1 and RH2. The presence of the SWS2 gene in monotremes also
means that ancestral mammals prior to the protherian–therian split must have
retained both SWS genes which, in combination with the LWS gene, would have
provided the basis for trichromacy.
6 D.M. Hunt and L.S. Carvalho

1.4 Evolution of Trichromacy in Primates

1.4.1  ene Duplication and Gene Conversion in Old

G
World Primates

Amongst the mammals, only primates show true trichromatic color vision. The evolu-
tionary drive behind the acquisition of trichromacy is thought to be improved color
discrimination in the red/green region of the spectrum for the detection and evaluation
of ripe fruits [46–49] and young nutritious leaves [50] against the green foliage of the
rainforest. This has been achieved in Old World primates by a ~40 kb duplication of
the X-linked LWS gene which occurred at the base of the Old World primate lineage
[3, 51, 52] to give rise to an array of two adjacent genes (Fig. 1.2). The duplication
generated a copy of the entire coding regions of the LWS opsin gene plus an almost
complete copy of the TEX28 gene [52, 53], a gene that is expressed in testis and not
thought to be involved in vision. The duplicated LWS genes have subsequently
diverged to give an upstream copy (OPN1LW) encoding a long wavelength-­sensitive
(L) pigment with λmax around 560 nm and a downstream copy (OPN1MW) encoding
a middle wavelength-sensitive (M) pigment with λmax around 535 nm. In humans,
intron 1 of the L opsin gene is generally longer by ~2.9 kb than intron 1 in the M opsin
gene. This long intron is rarely present (1 %) in the M opsin gene of Caucasians but is
polymorphic in African-Americans at a frequency of 35 % [54]. Individual cones
express just one copy to give L and M cones, which together with S cones expressing
the autosomal SWS1 gene (OPN1SW), give rise to full trichromacy.
In females with two X chromosomes, the process of X chromosome inactivation
will ensure that only one opsin gene array is active in any given photoreceptor, but
another mechanism must exist that ensures that only one gene, either L or M, is
switched on. This may be the role of the locus control region (LCR), a highly con-
served segment of DNA located in humans between 3.1 and 3.7 kb upstream of the
opsin gene array [55]. The LCR acts as an enhancer and is present in species where
there is only a single X-linked opsin gene [52]; activation of this gene is n­ evertheless
thought to require interaction between the LCR and the promoter region immediately

Fig. 1.2 Origin of opsin gene duplication on the X chromosome of Old World primates. The
exons for the L and M opsin genes and for the TEX28 gene are shown as black bars. Arrows indi-
cate the direction of transcription. The positions of the upstream LCR and the minimal promoter
for the M opsin gene are shown
1 The Genetics of Color Vision and Congenital Color Deficiencies 7

upstream of the coding region [56]. Since the cone-specific phototransduction genes
described above are all expressed in both L and M cones, the selection of either an
L or M opsin gene for expression [57] would appear to be the single determinant for
the production of either an L or M cone. This process may depend on the stochastic
selection of one of two stable and mutually exclusive states. The L and M genes each
possess a minimal promoter immediately upstream of exon 1, so this region could
interact in a gene-specific manner with the LCR to activate only the adjacent gene.
This was tested with transgenic mice that carried the human LCR and the L and M
promoters driving different reporter genes within a transgene [58]. As shown in
Fig. 1.3, the activity of the gene driven by the more proximal promoter to the LCR

Fig. 1.3 The role of the LCR in directing reporter gene expression from L and M opsin gene
promoters. (a) The relative activity of the L and M promoters in four different transgene arrays
assessed in a number of transgenic mouse lines by scoring individual cone cells for the expression
of the reporter genes. PL human L opsin gene promoter, PM human M opsin gene promoter,
AP human placental alkaline phosphatase, lacZ E. coli β-galactosidase. Redrawn from Ref. [58].
(b) Potential mechanism for the stochastic expression of L or M opsin genes via LCR-opsin gene
promoter interaction
8 D.M. Hunt and L.S. Carvalho

was higher, while a 9 kb spacer interposed between the L and M reporter genes
reduced the expression of the downstream gene even further, indicating that physical
distance from the LCR may reduce the frequency of promoter activation. In this way
therefore, only a single opsin gene within the array is activated, with the probability
of interaction with either the L or M promoter dependent on the distance from the
LCR and on certain key regions of the promoter, with different promoters showing
stronger or weaker affinities [56]. This may also in part explain the differences in the
relative number of L and M cones in different individuals (For more on L:M ratios
see Chaps. 2, 3 and 5). Most subjects show a fourfold range [59] but this can extend
in rare cases to a 30-fold range. It should be emphasized however that this variation
in L and M cone ratios is not associated with color vision defects.

1.4.2 Spectral Tuning of Primate Visual Pigments

The spectral shifts between primate L and M pigments are largely due to substitutions
at three sites, 180 encoded by exon 3 and 277 and 285 encoded by exon 5 [60],
although smaller changes arise from substitution at 116 in exon 2, and 230 and 233 in
exon 4 [61]. The residues present at the three former sites are polar Ser, Tyr, and Thr,
respectively, in the L pigment and nonpolar Ala, Phe, and Ala, respectively, in the M
pigment [51, 62]. As shown by site-directed mutagenesis and in vitro expression
studies, the spectral shifts achieved by substitution at these sites are approximately
additive [63, 64]. Site 180 is polymorphic in humans; Ser180 is the more common
residue in the L pigment, but Ala180 is present at a significant frequency to give a
short wavelength-shifted L pigment in some individuals [65].
UV-sensitive (UVS) SWS1 pigments are found throughout the vertebrate kingdom
and almost certainly represent the ancestral form of the pigment [2, 66]. Within the
mammalia, UVS pigments are relatively common in marsupials but are restricted in
eutherians to just a subset of species from the Orders Rodentia, Chiroptera, and
Insectivora [1]. All species of primates possess violet-sensitive (VS) pigments [67,
68] but the tuning method to change from UV to violet sensitivity remains uncertain. It
has been proposed from a comparison with the mouse UVS pigment that tuning from
UV to violet requires the simultaneous replacement of residues at three sites, Phe86Leu,
Thr93Pro, and Ser118Thr [69, 70]. However, other residue changes at these sites are
found in members of the Haplorrhini (New World and Old World monkeys and tarsi-
ers), and the Strepsirrhini (prosimians excluding tarsiers), including Phe86 in one spe-
cies, the aye-aye, a lemur endemic to Madagascar [71]. Phe86 is generally associated
with a UVS pigment [66] and it is the replacement of this residue by either Tyr or Ser
or Val [72–74] that is responsible for the loss of UV-sensitivity in a number of non-
primate species [75]. In vitro expression of the aye-aye pigment however gave a peak
sensitivity at 409 nm [71], so Phe86 does not in this case result in a UVS pigment. The
only residue that is consistently present in primate VS pigments is Pro93. Pro93 is also
found in the VS pigment of the clawed frog [76] so a Thr93Pro substitution may under-
lie primate VS pigments. Consistent with this is the observation that a Pro93Thr substi-