Rod-like Properties of Small Single Cones: Transmutated Photoreceptors of Garter Snakes (Thamnophis proximus)

by

Clement Guang Yu Yang

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Cell and System Biology University of Toronto

© Copyright by Clement Guang Yu Yang, 2010

ii

Rod-like Properties of Small Single Cones: Transmutated

Photoreceptors of Garter Snakes (Thamnophis proximus)

Clement Guang Yu Yang

Master of Science

Graduate Department of Cell and System Biology

University of Toronto

2010

Abstract

While nocturnal basal snakes have rod-dominant retinae, diurnal garter snakes have all-cone

retinae. Previous work from the Chang lab identified three visual pigments expressed in the

photoreceptors of Thamnophis proximus: SWS1, LWS and RH1. I further characterized T.

proximus photoreceptors using electron microscopy, immunohistochemistry, and in vitro protein

expression. T. proximus have four types of morphological cones: double cones, large single

cones, small single cones, and very small single cones. Some small single cones have rod-like

features, such as rod-like outer-segment membranes and a lack of micro-droplets.

Immunohistochemistry showed that rod-specific transducin is expressed in some T. proximus

photoreceptors. In vitro expression of T. proximus RH1 produced a functional rhodopsin with

λmax at 485nm, which corresponds to microspectrophotometry measurement from some small

single cones. Current results suggest that small single cones of T. proximus may have evolved

from ancestral rods, and secondarily acquired a cone-like morphology as adaptation to diurnality.

iii

Acknowledgments

At the end of this journey, I feel blessed in many ways. I have my supervisor to thank, Dr.

Belinda Chang. She let me work on this project, studying the visual system of a beautiful animal.

Belinda’s encouragement, guidance, and support kept me and this project going. The idea for

this project was conceived 6 years ago, as a brainchild of Belinda and Dr. Johannes Müller, a

former post-doc and current collaborator of the Chang lab. Since Johannes returned to Germany,

this project fell on the hands of Mengshu Xu and Natalie Chan. Thanks to their efforts, snake

retinal cDNA library was built and three opsin genes were sequenced. This thesis would not be

the same without the graduate students of the Chang lab; I can not thank them enough. Thank

you James Morrow for constructing the expression vector and trouble-shooting the large scale

protein expression. Thank you Ilke van Hazel, for always being patient with me and helpful with

my questions. Thank you Jingjing Du, for sharing your knowledge on bioinformatics and

phylogenetics. Thank you Cameron Weadick and David Yu, for providing many healthy

discussions on (mostly) scientific topics. Thank you Ekaterina Hult, Constanze Bickelmann, and

all Chang lab members for reading and commenting on this thesis. Thanks to Norman White for

taking good care of the snakes in the animal facility. Thank you Henry Hong and Audrey

Darabie from Cell and Systems Imaging Facility, for your expert opinion and guidance on

electron microcopy. Thank you Dr. Ellis Loew for carrying out the microspectrophotometry.

Thank you Dr. Vince Tropepe for guidance on immunohistochemistry. Thanks Dr. Sonke

Johnson for your advice on future behavioral studies. Thank you Kevin Van Doorn for

measuring the absorbance of snake lens and corneas. Thank you Dr. Steve Tobe and Dr. Les

Buck, for being on my thesis committee, and assessing my progress periodically with insightful

comments.

Thank you to my parents, and Jielin. Your love and support made me who I am.

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Table of Contents

Abstract.......................................................................................................................................... ii

Acknowledgments ........................................................................................................................ iii

Table of Contents.......................................................................................................................... iv

List of Figures............................................................................................................................... vi

List of Tables ............................................................................................................................... vii

List of Appendices...................................................................................................................... viii

List of Abbreviations .................................................................................................................... ix

1 Introduction................................................................................................................................1

1.1 Retina and Photoreceptors ..................................................................................................1

1.2 Visual Pigments and Photo-transduction............................................................................5

1.3 Garter Snakes....................................................................................................................10

1.4 Evolution of Snake Visual System ...................................................................................12

1.5 Visual System of Thamnophis proximus ..........................................................................15

1.6 Objectives of Present Study..............................................................................................22

2 Materials and Methods.............................................................................................................23

2.1 Animal Subjects................................................................................................................23

2.2 Electron Microscopy.........................................................................................................23

2.3 Immunohistochemistry .....................................................................................................24

2.4 Cloning into p1D4 ............................................................................................................25

2.5 in vitro Expression and Purification .................................................................................26

3 Results......................................................................................................................................27

3.1 Scanning Electron Microscopy.........................................................................................27

3.2 Transmission Electron Microscopy ..................................................................................33

3.3 Immunohistochemistry .....................................................................................................38

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3.4 RH1 in vitro Expression ...................................................................................................40

4 Discussion................................................................................................................................45

4.1 Morphology of Photoreceptors .........................................................................................45

4.2 In vitro Expression of T. proximus RH1...........................................................................49

4.3 Transmutation of Small Single Cones ..............................................................................55

4.4 Future Directions ..............................................................................................................59

Appendix.......................................................................................................................................64

References.....................................................................................................................................68

vi

List of Figures

Figure 1. Schematic diagram of a rod and a cone photoreceptor Page 2

Figure 2. Simplified representation of the visual cycle of bovine rhodopsin. Page 6

Figure 3. Opsin families and the ranges of spectral absorbance peaks Page 7

Figure 4. Summary of MSP measurements of T. proximus photoreceptors Page 16

Figure 5. Phylogenetic analysis of reptilian opsin genes Page 18

Figure 6. Established phylogenetic relationship of reptiles Page 19

Figure 7. Amino acid sequence alignment of RH1 genes Page 21

Figure 8. A cross-sectional view of T. proximus retina Page 29

Figure 9. SEM overview of T. proximus retina Page 30

Figure 10. SEM of T. proximus photoreceptors Page 31

Figure 11. Percentages of different photoreceptors of T. proximus Page 32

Figure 12. TEM of T. proximus double cone Page 35

Figure 13. TEM of T. proximus large and small single cone Page 36

Figure 14. TEM of T. proximus small and very small single cone Page 37

Figure 15. K-20 antibody staining of T. proximus retina Page 39

Figure 16. Absorbance spectra of bovine RH1 in vitro Page 42

Figure 17. Absorbance spectra of T. proximus RH1 in vitro Page 43

Figure 18. Western blot of bovine and T. proximus RH1 Page 44

Figure 19. Dark spectra of large scale T. proximus RH1 Page 53

Figure 20. Curve-fit of T. proximus RH1 difference spectrum Page 54

Figure 21. Amino acid sequences of T. proximus RH1 and mutants Page 60

Figure 22. TEM of T. proximus very small single cones Page 61

Figure 23. SEM of T. proximus lens Page 63

vii

List of Tables

Table 1. Summary of published data on visual system of snakes. Page 14

viii

List of Appendices

Appendix 1. Amino acid sequence alignment of reptile SWS1 Page 64

Appendix 2. Amino acid sequence alignment of reptile LWS Page 65

Appendix 3. Amino acid sequences of T. proximus SWS1 Page 66

Appendix 4. Amino acid sequences of T. proximus LWS Page 67

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List of Abbreviations

Gαt: G-protein α-subunit

GPCR: G-protein coupled receptors

LWS: Long-wavelength sensitive

λmax: Spectral absorbance peak

MWS: Middle-wavelength sensitive

MSP: Microspectrophotometry

RH1: Rhodopsin

SEM: Scanning electron microscopy

SWS: Short-wavelength sensitive

TEM: Transmission electron microscopy

UV: Ultra-violet

1

1 Introduction

1.1 Retina and Photoreceptors

The vertebrate retina is a light sensitive tissue lining the inner surface of the eye. The vertebrate

retina contains these main classes of cells: photoreceptors, horizontal cells, bipolar cells,

amacrine cells, and ganglion cells (Masland, 2001; Wassle, 2004). Light travels through the

ganglion cell layer, inner nuclear layers, outer nuclear layers, and finally reaches the

photoreceptors. Each photoreceptor has an outer segment, an inner segment, and synaptic

terminals (Figure 1). The outer segment is the membranous site specialized for photon detection,

and the inner segment contains the nucleus and organelles such as mitochondria and Golgi

apparatus for cellular metabolism (Kawamura and Tachibanaki, 2008). The inner segment of a

typical vertebrate photoreceptor can be further divided into three regions: ellipsoid, paraboloid

and myoid (Fein and Szuts, 1982). The ellipsoid is packed with mitochondria to supply the cell

with metabolic energy such as ATP; the paraboloid contains intracellular vacuoles and glycogen

granules; the myoid region houses organelles such as Golgi apparatus for protein synthesis (Fein

and Szuts, 1982).

Two basic classes of photoreceptors can be found in most vertebrate retinae: rods and cones.

Rods are more light-sensitive receptors for the dim-light (scotopic) visual pathway, while cones

are optimized for day-light (photopic) visual pathway (Schultze, 1866). The different

sensitivities of rods and cones originate in their distinct morphological, biochemical properties,

and neuronal synapses. In vertebrate retina, rods and cones are often distinguishable based on

morphology: rods are tall, slender cells with elongated outer segments; cones are shorter and

stouter cells with tapering outer segments. The large outer segments of rods have increased

surface area for photon capture, boosting visual sensitivity in low light environment. Inside the

outer segments of rods are individualized membranous discs unattached to the plasma membrane.

In contrast, outer segment membranes of cones are formed by invaginations of the plasma

membrane. This distinguishing feature of the outer segment membranes has been extensively

applied when characterizing rod versus cone photoreceptors. However, there are substantial

differences between rods and cones beyond their morphology. For example, rods have a high

convergence onto rod bipolar cells, which improves the signal-to-noise ratio of the rod-pathway

(Kefalov, 2003). Although this thesis focuses on the difference between rods and cones based on

2

morphology and biochemistry, it is important to remember that post-receptor neurons play an

important role in the visual pathways as well.

Figure 1. Schematic diagram of a rod and a cone photoreceptor. Note the outer segment

membrane disks of rods, and the presence of oil-droplets in some cones.

3

Vertebrates live in environments that vary in intensity and spectral range of light. Duplex retinae

with rods and cones enable animals to see during day and night. Vertebrate visual systems have

an extensive array of adaptations to different lifestyle and visual environment. One such

adaptation is through varying the proportion of rods and cones. In mammals, for example, rod-

cone ratios roughly correlate with the daily activity pattern (Peichl, 2005). Nocturnal species

have between 0.5% and 3% cones among their photoreceptors, crepuscular and arrhythmic

species have between 2% and 10% cones, and diurnal mammals show a larger range of cone

proportions from 8% to 95% cones (Ahnelt and Kolb, 2000; Peichl et al., 2000). The current

understanding is that all mammals have a duplex retina containing rods and cones (Peichl, 2005).

Although in some cases, vertebrates may appear to have all-rod or all-cone retinae as extreme

adaptations to a strongly nocturnal or diurnal lifestyle, respectively (Kolmer, 1936; Walls, 1942;

Rochon-Duvigneaud, 1943). For example, diurnal mammals such as tree shrews (Tupaia

belangeri) appear to have “pure-cone” retina (Samorajski et al., 1966; Tigges et al., 1967). Based

on EM, rod-like cells were later found to make up about 4% of the photoreceptors (Foelix et al.,

1987). These photoreceptors do not have typical rod-like morphology, since they are relatively

short and narrow cells. They are distinguished from cones based on staining pattern with

toluidine blue (Foelix et al., 1987). The cone-dominant retina of T. belangeri is attributed to its

great reliance on photopic vision, and its visually guided behavior (Samorajski, 1966).

Cone photoreceptors can be further classified into subtypes according to cellular features or

protein expression. Homo sapiens, for example, have rods and three spectral classes of single

cones. These different cone types are responsible for our color vision. However, human retina is

rather simple when compared to that of some birds. Bird retina contains rods, four spectral

classes of single cones, and a class of double cones (Hart, 2007). The bird cones also possess an

array of color filters in the form of oil droplets. Found in the inner segment of cones, an oil

droplet is a large spherical organelle with high lipid content (Johnston and Hudson, 1976). The

oil droplets can be clear and non-pigmented, like those in chondrostean fishes (Walls and Judd,

1933), aunuran amphibians (Hailman, 1976), geckos (Ellignson et al., 1995), monotremes (Walls,

1942) and marsupials (O’day, 1935; Arrese et al., 2002; Arrese et al., 2005). In birds, turtles, and

lizards, however, the oil droplets can have a pale green, greenish yellow, golden yellow or ruby

red coloration (Walls and Judd, 1933; Robinson, 1994; Bailes et al., 2006). The colored oil

4

droplets can enhance color discrimination by filtering out specific wavelength of light (Vorobyev,

2003).

Oil droplets are found in many reptiles but not in snakes, since ancestral snakes lost oil-droplets

as an adaptation to fossorial lifestyle (Walls, 1942). Instead, the photoreceptors of common

garter snakes (Thamnophis sirtalis) are packed with microdroplets of high refractive index

(Wong, 1989). The microdroplets of a snake photoreceptor may function collectively to replace a

single oil droplet as light filters (Wong, 1989). Since the oil droplets are cone-specific organelles,

it will be interesting to examine whether snake cones differentially express microdroplets.

Different subtypes of photoreceptors can form regular retinal mosaic patterns, commonly found

in fish species. In the velvet cichlids (Astronotus ocellatus), for example, the cone photoreceptors

are arranged in a repeating square mosaic pattern with one single cone surrounded by four

double cones (Braekevelt, 1992). These regularly spaced arrangements of cells have predictable

pattern relative to neighboring cell types. There are two primary hypotheses for photoreceptor

mosaic patterning in vertebrates: one postulates that cell fate is determined by a series of

inductive and sequential events, based on preexisting mosaic position (Raymond et al., 1995;

Stenkamp and Cameron, 2002; Raymond and Barthel, 2004); and another postulates that

differential adhesion between cell subtypes establishes mosaic position (Galli-Resta, 2001;

Mochizuki, 2002; Reese and Galli-Resta, 2002; Tohya et al., 2003). Migratory fish such as

rainbow trout (Oncorhynchus mykiss) depend on well-defined square cone mosaic pattern for

polarized light detection (Hawryshyn, 2000). Salmonid and trout species can alter their retinal

mosaic during different lifestages, through ontogenetic loss of UV sensitivity (Allison et al.,

2003). Rapid retinal development of most model systems makes photoreceptor developmental

events difficult to study (Hoke et al., 2006). Mosaic pattern of photoreceptors have not been

reported from any snake species.

Microscopy has been employed extensively in comparative studies of surface morphology,

internal organelles, and mosaic pattern of photoreceptors. The present study used electron

microscopy to characterize the cellular features of Thamnophis proximus photoreceptors. The

reasons for studying this particular species of snake are discussed in later sections. When

combined with molecular data of phototransduction proteins, the photoreceptors can be better

characterized to provide a more accurate reflection on their evolution and functional capability.

5

1.2 Visual Pigments and Photo-transduction

In vertebrate photoreceptors, visual pigments capture light energy and activate the photo-

transduction cascade. A visual pigment is made up of an opsin protein bound to an 11-cis retinal.

Opsin proteins belong to the super family of G protein-coupled receptors (GPCR), characterized

by the seven-transmembrane domain, and the ability to activate a G-protein. The best studied

opsin protein is the rhodopsin (RH1) of bovine, readily available in large quantity from the rod

photoreceptors of cows. When regenerated with 11-cis retinal, bovine RH1 pigments in their

dark state absorb at around 500nm (Figure 2). After photons strike the outer segment membrane,

light energy induces a series of conformational changes to the retinal, eventually breaking its

bond with the opsin. This is also known as light bleaching the visual pigment. When the visual

pigment is in its biologically active conformation, known as the meta-II stage, it binds and

activates the G-protein transducin. Transducin then activates cGMP phosphodiesterase (PDE)

which hydrolyzes cGMP. The drop in cGMP concentration causes closure of cation channels in

photoreceptor plasma membrane, leading to hyperpolarziation of the plasma membrane and

neuronal signaling (Hargrave et al., 1993; Ebrey and Koutalos, 2001). The shut-off mechanism

of opsins are regulated by phosphorylation and arrestin binding (Kefalov, 2003). The only action

of light in vision is to isomerize the retinal from 11-cis to all-trans configuration (Hubbard and

Kropf, 1958), all the subsequent changes in chemistry and physiology are “dark” consequences.

Therefore the dark state absorbance is usually referred to as the absorbance of a visual pigment,

even though each intermediate stage has its own characteristic absorbance.

The absorbance range of a photoreceptor is dependant on what opsin is expressed in their outer

segment membranes. Specialized in dim-light detection, RH1 pigments of most vertebrates

absorb at around 500nm (Figure 3). For their high-sensitivity to photon, RH1 pigments are often

expressed in rod photoreceptors for dim-light detection. In cone photoreceptors, four spectrally

distinct classes of cone opsins can be found. Light from 355-440nm is absorbed by short-wave

sensitive class SWS1, 410-490nm is absorbed by another short-wave sensitive class SWS2, 480-

535nm is absorbed by middle-wave sensitive class RH2, 490-570nm is absorbed by middle- to

long-wave sensitive class LWS (Yokoyama, 2000; Bowmaker, 2008). So far, three classes of

opsins have been sequenced from sunbeam snakes (Xenopeltis unicolor) and python (Python

regius): RH1, SWS1, and LWS (Davies et al., 2009).

6

Figure 2. Simplified representation of the visual cycle of bovine rhodopsin.

Visual pigments play an important role in the functional difference between rod and cone

photoreceptors. Rhodopsin pigments are typically 100 times more photosensitive than cone

pigments, but rod response kinetics are several times slower (Baylor, 1987). Different kinetics

between rod and cone pigments translate into high-sensitivity of rod photoreceptor, but a slower

recovery from light bleaching compared to cones. Complete recovery of mammalian

photoreceptors takes around 20 min for a rod (Thomas & Lamb 1999), but only 20 ms for a cone

(Kenkre et al. 2005). A part of this difference is due to a 10-fold longer lifetime of meta-II state

of rhodopsin, and its lowered rate of spontaneous isomerization. By one account, red cone

pigment isomerizes spontaneously 10000 times more frequently than rod pigments (Kefalov,

2003). Cone pigments are more prone to spontaneous isomerization than rod pigments,

contributing to the dark-noise of cone photoreceptors.

7

Vertebrates evolved visual pigments about 500 million years ago, before the appearance of jaws

(Bowmaker, 2008). Four classes of cone opsin evolved from gene duplication, followed by the

rod opsin class that arose from the duplication of RH2 opsin class. The primary selective

pressure driving the opsin evolution is the spectral range and intensity of light of the visual

environment (Bowmaker, 2008). Vertebrate opsins are tuned to specific spectral regions based

on a number of spectral tuning sites. A spectral tuning site is an amino acid substitution which

results in a shifted λmax of opsins. A single nucleotide substitution can lead to a different amino

acid at the tuning site, resulting in altered interaction between the opsin and chromophore, thus a

spectral shift. This shift, even based on one amino acid change, can range from a few nanometers

to greater than 60nm (Wilkie et al., 2000). Rhodopsin can be spectrally tuned by several amino

acid residues, such as site 83 and 292. With D83 and A292, wild-type bovine RH1 pigments

absorb at 500nm, but after substituting with N83 and S292, the λmax was shifted to 485nm

(Fasick and Robinson, 1998). Amino acid N83 is found in rhodopsin of bottle-nosed dolphin and

deep-water teleost fish as an adaptation to monochromatic deep-water environment (Fasick and

Robinson, 1998; Suguwara et. al., 2009). However, these amino acid substitutions can not be

explained exclusively by deep-water adaptation, since it is also found in terrestrial vertebrates

like elephants (Yokoyama et. al., 2005). In later sections, spectral tuning sites of snake opsins

will be further discussed.

Figure 3. Phylogenetic relationship between opsin families, and the respective range of λmax

(spectral absorbance peak) according to recent reviews (Yokoyama, 2000; Bowmaker, 2008).

8

Another way of visual adaptation based on the four ancestral cone classes is the loss of one or

more cone classes, or the gain of new opsin genes by further gene duplication. There are many

examples of visual pigment loss during species evolution. Mammals lost SWS2 and RH2

pigments, while retaining three functional opsin genes: SWS1, M/LWS, and RH1 (Yokoyama

and Yokoyama 1996; Ebrey and Koutalos, 2001). The SWS2 and RH2 pigments were lost some

time before the divergence of modern mammals (Levenson and Dizon, 2003). In addition,

modern cetaceans and several amphibious mammals lack functional SWS visual pigments

(Fasick et al. 1998; Peichl & Moutairou 1998; Peichl et al. 2001). In these marine mammals the

loss of SWS cones is adaptive for vision under water in photopic conditions (Peichl et al., 2001).

While some vertebrates have lost certain visual pigments, others have gained novel visual

pigments through gene duplication. It has been suggested that genome wide duplication occurred

at the base of teleost radiation (Amores et al., 1998; Meyer and Malaga-Trillo, 1999;

Postlethwait et al., 2000). Further genome duplications have occurred in salmonids, goldfish, and

carp (Larhammar and Risinger, 1994). Zebrafish (Danio rerio), for example, have two red

(LWS-1 and LWS-2), four green (RH2-1, RH2-2, RH2-3, and RH2-4), and single blue (SWS2)

and ultraviolet (SWS1) opsin genes in the genome (Chinen et al., 2003). Mutations in the

duplicated copy can lead to divergent evolution resulting in two or more spectrally distinct

pigments within single opsin classes, exemplified by the two short-wave SWS1 and SWS2 opsin

classes (Bowmakers, 2008). In snakes, three types of opsins are sequenced from two basal

snakes, SWS1, LWS, and Rh1 (Davies et al., 2009). It is unclear whether other snakes, especially

derived species, have gained or lost any opsin beyond the three.

Visual pigments have varying absorbance range and kinetics, but with respect to downstream

signaling of phototransduction cascade, opsins are biochemically equivalent (Kefalov, 2003).

Besides opsins, rods and cones express different sets of proteins for the subsequent photo-

transduction cascade, such as rod-specific and cone-specific transducin (Strathmann, et al., 1990).

Identifying rod- and cone-specific downstream proteins has been used previously to characterize

photoreceptors of Tokay geckos (Zhang et al., 2006). Even though the gecko photoreceptors are

rods based on morphology and light response, only cone-specific versions of photo-transduction

proteins were found, including transducin (Zhang et al., 2006). Transducin is a heterotrimeric G

protein with α, β and γ subunits. Most interest in G proteins has been focused on the α-subunit

9

(Gαt), since it binds and hydrolyzes GTP into GDP. Antibodies that label rod- and cone-specific

Gtα are commercially available.

K-20 antibody, for example, is an affinity-purified rabbit polyclonal antibody raised against rod

α-transducin. K-20 antibody has been used as a rod marker in mice (Kerov et al., 2007), hamsters

(Boughter et. al., 1997), and pigeons (Wada et. al., 2000). In two species of diurnal rodents

(Arvicanthis ansorgei and Lemniscomys barbarus), only the cell body of rods but not cones is

labeled with K-20 (Bobu, 2008). K-20 antibody also distinguished the rod-like photoreceptor

from the cone-like cells in the primitive retina of lamprey (Muradov et al., 2008). In addition, K-

20 antibody is used as rod-specific transducin marker for western blots of retinal extract on

model organisms such as chicken and mice (Cai et al., 2001; Bell et al., 2000; Kasahara et al.,

2001; Sokolov et al., 2004). K-20 antibody has not been used in snake species before this study,

and there is no molecular data on phototransduction cascade of T. proximus.

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1.3 Garter Snakes

Garter snakes (Thamnophis) are a genus of snakes from the family Colubridae. They are perhaps

the most common and widespread snakes in North America (Rossman et al., 1966). Garter

snakes are found in every state in the mainland United States, in Canada from the Maritime

Provinces west to British Columbia, and through much of Mexico and Central America. They are

often chosen as study subjects by biologists and ecologists, because they are abundant, easy to

capture, and usually harmless to human (Rossman et al., 1966).

Garter snakes display a wide range of scale variations, pattern polymorphism, and sexual

dimorphism. Ruthven (1908) thought that attempts to classify Thamnophis “has long stood in the

minds of herpetologists as a synonym for chaos.” At least 30 species of Thamnophis have been

recognized and the number is most likely to increase in future taxonomic studies. The common

garter snakes, Thamnophis sirtalis, were once considered to be the base stock from which the

other taxa arose (Ruthven, 1908), but recent phylogenetic studies based on combined DNA

sequences and allozyme data put T. sirtalis as sister species with western ribbon snakes, T.

proximus (de Queiroz and Lawson, 1994).

Thamnophis proximus is a diurnal garter snake species known as the western ribbon snakes

(Rossman et al., 1966). Western ribbon snakes mainly feed on amphibians and their larvae, but

they are also hunters for small fish and reptiles (Tinkle, 1957; Clark, 1974). Foraging behavior

has been described for T. proximus, for example, using rapid and repeated striking motion to

probe for frogs hiding in vegetation (Wendelken, 1978). Feeding behavior of garter snakes have

been described qualitatively (Ford 1995) and quantitatively (King and Turmo, 1997).

Vision is an important sense for garter snakes. Prey movement was shown to be important in the

feeding behaviour of colubrid snakes (Herzog and Burghardt, 1974). A similar preference for

visually detected movement was demonstrated in adult T. sirtatis (Burghardt and Denny, 1983).

Experienced water snakes orient to and capture fish that contrast with their background, instead

of those that are color-matched (Czaplicki & Porter, 1974). Several Thamnophis species were

observed making air-to-water attacks on aquatic prey when the distance or angle of the prey

precluded any tongue contact, and the snakes repeatedly made underwater attacks on fish well

outside tongue-flicking range (Drummond, 1983). While chemical cues can facilitate the attack

11

in experienced or ingestively naive snakes, garter snakes rely on vision for both initial detection

of and orientation to prey (Drummond, 1985; Teather, 1991).

Besides hunting, garter snakes also use vision for mate recognition and chase sequence during

reproduction (Perry-Richardson et al., 1990). Female red-sided garter snakes (Thamnophis

sirtalis parietalis) exhibit a complex array of behavioral patterns for reproduction (Whittier and

Crew, 1986). Females may mate once, more than once, or not at all from spring to late summer

before returning to winter hibernation. Large year-to-year variation in reproductive rates of

female garter snakes suggest that other factors may play an important role in mating frequencies,

such as nutrition and stored energy reserves (Whittier and Crew, 1986). The reproductive

pressure for male Thamnophis species can be intense. In Manitoba, male T. sirtalis emerge from

hibernation ready to engage in courtship with females as they emerge a few days later (Rossman

et al., 1966). The severely biased operational sex ratio leads to the formation of mating balls, due

to clusters of males courting a single female. Under intense sexual selection pressure, visual cues

are used by garter snakes for recognizing and chasing mates (Perry-Richardson et al., 1990).

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1.4 Evolution of Snake Visual System

While there are many behavioral studies on garter snakes, snake visual systems deserve special

attention (Walls, 1942). As a comparative biologist, Walls (1942) reported that the eyes of

snakes are drastically different from other reptilian species. Instead of mobile eye-lids, snakes

have a modified layer of scale covering the cornea, known as spectacle. Corneas of lizards are

bared and are connected to ciliary muscle and scleral ossicles, both of which are lacking in

snakes. Snakes also have achieved an entirely different mechanism for focusing light on the

retina, by moving the lens in- or outwards (Caprette et al., 2004). Photoreceptors of snakes also

lacked paraboloid and myoid region found in other vertebrate species (Walls, 1942).

After examining the eyes of an extensive collection of snake species, Walls (1942) proposed that

snake ancestors must have undergone a period of visual degeneration, followed by regeneration

of the visual system. After primitive snakes regained functional duplex retinae, their

photoreceptors further “transmutated” to produce the enormous diversity of the ophidian retina

(Walls, 1942). In higher snakes, the duplex retina with both rods and cones is also primitive

while all subsequent modifications are based on a “viperine” pattern with four types of

photoreceptors: large single cones, double cones, small single cones, and rods (Underwood, 1951;

1966). While duplicity is the ancestral state of snake visual system, it has been lost several times

and never regained in some species (Underwood, 1966).

Transmutation of snake photoreceptors can be better understood by comparing the visual system

of basal snakes (henophidians) to derived snakes (caeonophidians). Three species of henophidian

snakes have been characterized for their visual system: ball python (Python regius), boa (Boa

constrictor), and sunbeam snakes (Xenopeltis unicolor). A member of primitive Boidae family,

ball python is partly nocturnal and found primarily in West and Central African grasslands (De

Vosjoli, 1990). Another Boidae snake, boas are found in Central and South America, also known

to have nocturnal lifestyle (McGinnis and Moore, 1969). Common in south Asian countries,

sunbeam snakes are nocturnal species with semi-fossorial lifestyle, distinguished by the

iridescent sheen of their scales (O’Shea, 2007).

Henophidian snakes studied so far have duplex retinae with a large number of rods expressing

RH1, and a small number of cones expressing LWS and SWS1 (Table 1). The retina of ball

13

python has been characterized using scanning electron-microscopy (SEM) and

microspectrophotometry (MSP). P. regius have a duplex retina dominated by rods (90%) with

long, narrow outer segments, and two types of single cones (Sillman et al., 1999). The rods of P.

regius express a rhodopsin with λmax of 494nm. Their large single cones express LWS pigments

with λmax of 551nm, and their rare small single cones express SWS1 pigments with λmax of

360nm. The retina of Boa constrictor imperator, another henophidian snake, was also examined

using SEM and MSP (Sillman et al., 2001). The retina of boa is highly dominated by rods (89%)

over cones (11%). The rods express RH1 with λmax of 495 ± 2 nm. Boas have two types of single

cones distinguishable by size. The more common large single cones expressed LWS with λmax of

549 ± 1 nm, and the rare small single cones express SWS1 with λmax of 357 ± 2 nm (Sillman et

al., 2001). Rods also dominate the retina of sunbeam snakes (X. unicolor), expressing RH1

pigments with λmax around 499nm, while a smaller number of cones expressing LWS with λmax

between 558 and 562nm (Davies, et al., 2009). The SWS1 gene is expressed in the ocular cDNA

of X. unicolor, but this cone type was so rare that MSP was unable to pick up the short-

wavelength signal (Davies, et al., 2009).

Although some henophidian snakes have rod-dominant retinae, caeonophidian snakes such as

diurnal garter snakes have been found to have an all-cone retina. For example, the common

garter snakes (Thamnophis sirtalis) have four major morphological types of cones: double cones,

large single cones, small single cones, and very small single cones (Wong, 1989; Sillman et al.,

1997). Using SEM, the photoreceptor population was determined to be 45% double cones, 40%

large single cones, and 15% small single cones (Sillman et al., 1997). The small single cones

were further divided into two subtypes. Using MSP, it was found that the large single cones and

double cones have λmax of 554nm, and the small single cones have λmax of either 482nm or

360nm (Sillman et al., 1997). In the same study, Sillman and colleagues (1997) found that some

small single cones are labeled with at least two rhodopsin-specific antibodies (AO and B6).

Despite this evidence, it was concluded that T. sirtalis retinae have only cones (Wong, 1989;

Sillman et al., 1997). This conclusion is confirmed by Electroretinogram (ERG) on garter snakes,

as the retinal gross potential peaked from 550nm to 570nm under varying experimental settings

(Jacobs et al, 1992).

14

Henophidian snakes have rod-dominant retina because nocturnal vision is important for their

survival. Diurnal caeonophidian snakes such as common garter snakes have only cones, because

they are mostly active during the day. Visual systems of snakes are adapted to their particular

visual ecology and life-style. Vertebrates in general adapt their visual systems to their

environments, one of many ways they can achieve this is through photoreceptor transmutation.

Photoreceptor transmutation in snakes can be better understood by drawing a comparison

between the visual systems of basal snakes and derived snakes. Photoreceptor morphology and

visual pigments have been characterized in basal snakes (Sillman et al., 1999; Davies et al.,

2009), yet no opsin data from derived species such as garter snakes has been published. Only one

garter snake species, T. sirtalis, has been studied for photoreceptor physiology and morphology

so far (Jacob et al., 1992; Wong, 1989; Sillman et al., 1997). To understand the evolution of

snake visual systems, the retina of garter snakes and other caeonophidian snakes should be

further characterized.

Table 1. Summary of published data from recent papers on visual system of snakes.

15

1.5 Visual System of Thamnophis proximus

The Chang lab and its collaborators have been studying the visual system of a species of garter

snakes, Thamonophis proximus, commonly known as the western ribbon snake. From the retinal

cDNA library of T. proximus, postdoctoral fellow Johannes Muller sequenced the full-length

SWS1 opsin gene, undergraduate student Mengshu Xu sequenced the full-length LWS opsin

gene, and undergraduate student Natalie Chan sequenced the full-length RH1 opsin gene.

Phylogenetic analysis was carried out by fellow graduate student Jingjing Du based on reptile

sequence alignments generated by me. Microspectrophotometry (MSP) was carried out on the T.

proximus by Dr. Ellis Loew from Cornell University.

Microspectrophotometry (MSP) is a technique for measuring the visual pigment absorbance

from individual photoreceptors. A MSP machine works by generating a precise beam of light of

known wavelength, passing it through the outer segments of individual photoreceptors mounted

on a microscope slide, and measuring the absorbance due to visual pigments using a detector on

the output end. Since each visual pigment family has its own characteristic absorbance range,

repeatedly measuring the same type of photoreceptors gave us a good idea about what visual

pigments are expressed in a particular type of photoreceptors.

Previous MSP results published on T. sirtalis were experimentally carried out by Dr. Ellis Loew

at Cornell University (Sillman et al., 1997). Six T. proximus were sent him, and Dr. Ellis Loew

performed MSP on their photoreceptors. At least three types of visual pigments were identified

by MSP from T. proximus photoreceptors (Figure 4). Based on MSP, T. proximus have the same

three types of visual pigments as T. sirtalis (Table 1). Based on measurements from two large

single cones, five principal double cones, and eight accessory double cones of T. proximus, one

type of visual pigments have λmax around 544 ± 1.2 nm, within range of a LWS type pigment.

Small single cones can be distinguished into two subtypes based on MSP. Out of twelve small

single cones, four have λmax around 365 ± 1.0 nm, most likely due to a SWS1 type pigment.

The other eight small single cones absorb around 483 ± 1.5 nm, which is within the range of

RH2 pigments or blue-shifted RH1 pigments. Finally, the very small single cones do not produce

any absorption according to MSP.

16

Figure 4. Summary of MSP measurements of T. proximus photoreceptors, showing λmax (nm)

that met the selection criteria in terms of noise and bandwidth, and sample absorbance from each

type of pigments (Ellis Loew, email communication). Median, standard deviation (S.D.) and

sample size are also shown. Experimental methods of MSP have been described previously

(Loew, 1994).

17

Based on opsin sequences obtained by Johannes Muller, Mengshu Xu, and Natalie Chan from

the retinal cDNA library of T. proximus, a phylogenetic analysis was carried out by lab member

Jingjing Du (unpublished), to determine the phylogenetic relationship between T. proximus

opsins to other reptilian opsins (Figure 5). Opsin sequences from sunbeam snake, python, iguana,

gecko, true lizard, anole, and alligator species were included in the phylogenetic analysis. Beside

garter snake opsin genes sequenced by the Chang lab, other sequences used for generating the

tree are from NCBI database: (1) RH1 opsins of sunbeam snake (Xenopeltis unicolor), FJ497233;

python (Python regius) FJ497236; iguana, (Uta stansburiana), DQ100323; alligator (Alligator

mississippiensis), U23802; (2) RH2 opsins of lacerta (Podarcis sicula), AY941829; iguana (Uta

stansburiana), DQ100324; gecko (Gekko gecko), M92035; (3) SWS2 opsins of: iguana (Uta

stansburiana), DQ100326; anole (Anolis carolinensis); (4) SWS1 opsins of sunbeam snake

(Xenopeltis unicolor), FJ497234; python (Python regius), FJ497237; iguana (Uta stansburiana),

DQ100325; gecko (Phelsuma madagascariensis), AF074045; (5) LWS opsins of python (Python

regius), FJ497238; sunbeam snake (Xenopeltis unicolor), FJ497235; and gecko (Gekko gecko),

M92036.

Vertebrate visual opsins can be classified into five families. The three opsin genes from T.

proximus clustered within SWS1, LWS, and RH1 families. For SWS1 and LWS opsin families,

the garter snake genes always cluster with opsins of python and sunbeam snake. While the

general phylogenetic relationship between snakes and reptiles are well established, the

phylogenetic relationship within snake species is debatable, depending on whether molecular

(Figure 6A) or osteological data (Figure 6B) is used to resolve phylogeny (Lee and Scanlon,

2002; Vidal and Hedges, 2004) .

For the LWS and SWS1 families, the opsin phylogeny agrees with molecular tree (Figure 6A)

from Vidal and Hedges (2004). For the RH1 opsin family, the garter snake forms a monophyletic

group with iguana (Uta stansbriana), instead of grouping with python and sunbeam snakes. This

phylogenetic position is not supported by the molecular tree or the osteological tree.

18

Figure 5. Tree topologies reconstructed by Chang lab graduate student, Jingjing Du. Amino acid

sequences of opsin genes were analyzed with Bayesian method in MrBayes 3.12. MrBayes is a

free software program which performs Bayesian inference of phylogeny. Blosum model was

chosen as the substitution model from a distribution of mixed amino acid models in MrBayes

3.12. The tree is drawn proportional to the branch length. Numbers associated with major nodes

indicate Bayesian posterior probabilities. T. proximus sequences are boxed.

19

Figure 6. Phylogenetic relationship of reptilian species used in our analysis. Tree A is supported

by molecular data (Vidal and Hedges, 2004), while Tree B is supported by osteology and soft

anatomy (Lee and Scanlon, 2002).

20

Since T. proximus photoreceptors are expected to have cone-like morphology like those of T.

sirtalis, the expression of RH1 in their retina is unusual. To find out whether T. proximus RH1

have interesting amino acid substitutions, its amino acid sequence was aligned with published

RH1 sequences from sunbeam snake, python, iguana, and bovine (Figure 7).

Several key amino acid residues are conserved in T. proximus rhodopsin. The site for

chromophore attachment and its counterion, K296 and E113, are conserved in T. proximus RH1.

Palmitylation sites conserved in rhodopsins but not in cone opsins, C322 and C323, are found in

T. proximus RH1. The disulfide bond between C110 and C187 is likely conserved in T. proximus

RH1. Another characteristic residue, E122, is conserved in T. proximus RH1, which is typical of

RH1 and RH2 classes, but not found in other cone opsins (Imai et al., 1997).

The RH1 of T. proximus also have amino acid substitutions that are typically associated with

cone opsins. RH1 class opsins exhibit a kink around the double glycine residues, G89 and G90,

which brings G90 closer to E113, and gives rhodopsin a tighter retinal binding pocket (Nickle

and Robinson, 2007). In RH2 and SWS2 classes, only G90 is conserved, while in SWS1 and

M/LWS classes, neither glycine residue is conserved. These two residues are V89 and G90 in T.

proximus RH1, suggesting a looser conformation of the retinal-binding pocket compared to

bovine rhodopsin, perhaps more comparable to RH2 and SWS2 opsins.

S185 is a unique amino acid substitution for T. proximus RH1. With its close proximity to the

disulfide bond, C185 is conserved in most vertebrate species whose RH1 sequence is available.

Cysteine is a hydrophobic amino acid with a nonpolar thiol side chain, while serine is classified

as a polar amino acid due to its hydroxyl group. C185S substitution is rarely found in vertebrate

RH1, and its functional implications are unknown.

At least two amino acid residues from T. proximus RH1 have known spectral tunings properties:

N83 and S292. Besides RH1 of T. proximus, N83 is found in python, sunbeam snakes, side

blotched lizards, while D83 is found in bovine. N83 is also found in diving mammals such as

bottle-nosed dolphin (Fasick and Robinson, 1998), deep-sea fish (Hunt, 2001), and deep-water

lake cichlids (Sugawara et. al., 2009), which lead to the suggestion that it is an adaptation to

monochromatic deep-water environment. However, N83 can not be explained exclusively by

21

deep-water adaptation, since it is also found in terrestrial vertebrates like elephants (Yokoyama

et. al., 2005) and now in garter snakes.

Residue 292 is another known spectral tuning site for rhodopsin. It is conserved as A292 in

python, sunbeam snake, lizards and bovine rhodopsin genes. However, S292 is found in T.

proximus, dolphins (Fasick and Robinson, 1998), and deep-water lake cichlids (Sugawara et. al.,

2005). A292S evolved several times independently in deep-water cichlid species, suggesting

parallel evolution at this particular site (Sugawara et. al., 2005). The particular combination of

N83 and S292 has been found to blue-shift RH1 by at least 10nm in dolphin, making them

important substitutions for adaptation to deep-water marine environment (Fasick and Robinson,

2000). The T. proximus RH1 could be blue-shifted, according to MSP peak at 483 ± 1.5 nm, due

to the combined effect of N83 and S292.

Figure 7. Amino acid sequence alignment of RH1 expressed in T. proximus, sunbeam (X.

unicolor), python (P. regius), iguana (U. stansbriana), and cow (B. tauras), translated from

nucleotide sequences. The transmembrane domains are denoted by the black boxes below the

alignment. The three highlighted amino acid residues are N83, S185, and S292. A dot represents

the same amino acid as T. proximus RH1, while a line represent a gap.

22

1.6 Objectives of Present Study

Based on published data on snake visual system and previous research in the Chang lab prior to

my arrival, T. proximus may express RH1 pigments in cone-like photoreceptors. To better

characterize the T. proximus photoreceptors and look for evidence of transmutation, three

hypotheses were tested in the present study.

Hypothesis 1: Based on photoreceptor morphology, T. proximus have four types of cones

and no rods.

T. proximus is a sister species to the common garter snakes T. sirtalis, therefore I expect them to

have comparable retinal morphology. Since T. sirtalis is known to have all-cone retina (Sillman

et al., 1997), I expect to find the same types of photoreceptors in T. proximus. To establish

photoreceptor morphology, T. proximus retinae can be examined with electron-microscopy.

Scanning electron microscopy (SEM) can be used to determine surface morphology, while

transmission electron microscopy (TEM) can be carried out to examine internal anatomy of the

photoreceptors.

Hypothesis 2: Rod-specific transducin can be found in some T. proximus photoreceptors.

Research conducted by previous members of the Chang lab isolated both cone opsin genes

(SWS1 and LWS), as well as rod opsin gene (RH1) from T. proximus retinal cDNA library. I

expect to find more rod-specific downstream proteins, such as the transducin (Gαt). To determine

whether rod-specific Gαt maybe expressed in the retina, K-20 antibody was used in the present

study to probe the cryosections of T. proximus photoreceptors for rod-specific Gαt.

Hypothesis 3: RH1 pigments are expressed in some small single cones.

According to MSP results from Dr. Ellis Loew, some small single cones have λmax at 483 ± 1.5

nm. To determine if RH1 might be expressed in these small single cones, the λmax of rhodopsin

pigments can be measured in vitro by protein expression using mammalian cell culture. T.

proximus rhodopsin gene was cloned into an over-expression vector, expressed in vitro,

regenerated with 11-cis retinal, and measured for spectral absorbance using a spectrophotometer.

23

2 Materials and Methods

2.1 Animal Subjects

This study was approved by the U of T animal Care Committee and conforms to the Guide to the

Care and Use of Experimental Animals Vol. 2, as determined by the Canadian Council of

Animal Care regarding relevant guidelines for the care of experimental animals. Adult

Thamnophis proximus were bought from licensed reptile dealers in Ontario, namely Boreal

Scientific and Ward’s Natural Science. The animals were ordered through the departmental

animal facility and handled according to animal care protocol. The snakes were sacrificed by

decapitation before enucleating the eyes. T. proximus can be distinguished based on a pair of

yellow dots on their heads, which are absent from T. sirtalis, the common garter snakes.

2.2 Electron Microscopy

Electron microscopy was carried out in the Cell and Systems Biology Imaging Facility. In total,

ten retinae from five T. proximus were processed for EM: four retinae for SEM, two retinae for

TEM, and four retinae for SEM again in a repeated trial. After hemisection of the T. proximus

eye, the eyecup was placed in 3% glutaraldehyde (SPI Supplies) 0.1 M phosphate buffer, pH 7.8,

where the retina was separated from its pigmented epithelium. After separation, the retina was

kept in fixative at room temperature overnight. The tissue was then washed in 0.1 M phosphate

buffer to remove primary fixative. The retina was incubated in secondary fixative, 1.0% osmium

tetroxide in 0.1 M phosphate buffer, for one hour at room temperature. The tissue was

dehydrated by immersion in increasing concentrations of ethanol (two 5 min immersion in each

of 30, 50, 70, 80, 90% ethanol). From this point on the preparations for SEM and TEM become

different.

Tissues for SEM were soaked four times in 100% ethanol for 3 minutes each. They were then

infiltrated with Hexamethyldisilizane (HMDS) series for 30 minutes at 3:1, then 1:1, then 1:3

ethanol to HMDS ratio respectively. To displace as much ethanol in tissue sample with HMDS,

the final infiltration was three 30 min immersion in 100% HMDS. The HMDS in tissue was

allowed to volatilize overnight. The “dried” retina was taped onto a specimen holder, with

photoreceptors facing outwards, then Sputter coated (50sec) with gold-palladium using the Bal-

24

Tec SCD050. The sample was examined with the Hitachi S-2500 at 20 kV and acquired images

using Quartz PCI.

Tissues for TEM were soaked four times in 100% ethanol for 10 minutes each. The sample is

then infiltrated with modified Spurr’s epoxy resin (16.4g ERL4221, 23.6g NSA, 5.72g DER736,

and 0.4g DMAE) for 30 min. The sample was then embedded in flat molds, and all blocks

polymerized overnight in 65°C oven. Semithin sections (0.5-1 micron) were cut from the blocks

and stained with Toluidine blue (Fisher BioReagents, BP107-10) and methylene blue (British

Drug House Ltd., England, #201383) mixture for 1 minute. Ultrathin sections (60 – 90 nm) were

also cut from the blocks and picked up using high transmission grids. Grids were stained with

3% uranyl acetate in 50% methanol for 45 min and post-stained with Reynold’s lead citrate for

15 min. These ultra-thin sections were examined with the Hitachi H-7000 at 75 kV and images

were acquired using AMT 11 megapixel digital camera.

Overall, more than one hundred SEM images were taken from six pieces of retinal tissues. These

tissues are well preserved, whereas other pieces of T. proximus retinal tissues were not used

because they became damaged during SEM preparation. One piece of retinal tissue with

particularly well-preserved cellular integrity was used for calculating photoreceptor ratios across

nine zones. In total, more than one hundred and fifty TEM images were taken from over twenty

ultra-thin sections, made from two retinae from two different T. proximus individuals.

2.3 Immunohistochemistry

Four retinae from two T. proximus were processed for immunohistochemistry. After enucleation

of eyes from T. proximus, the eyecups were placed in 1% glutaraldehyde prepared in 0.1 mol·l-1

phosphate buffer, where they remained for 1h. The eyes were then washed thoroughly, first in

0.1 mol·l-1 phosphate buffer and then in 0.1 mol·l

-1 TRIS-HCl buffer. Two of the eyes were

processed for retinal whole-mount, by surgically separating the retinae from eye cups and

removing the pigment epithelium. Each retinal tissue as a whole was then placed on a glass

microscope slide, under a cover slide that was slightly elevated to avoid crushing the tissue. For

the cryosections, two T. proximus eyes were dehydrated with ethanol and embedded in resin. A

Leica CM3050 cryostat was used to cut serial semithin (0.2um) tangential sections from the

central part of each retina. The sections were treated with K-20 antibody (Santa-Cruz) diluted

1:1000 for 30 min. K-20 is an affinity purified rabbit polyclonal antibody raised against a peptide

25

mapping within a highly divergent domain of Gαt1. K-20 was used on retinal sections from

pigeons (Wada et. al., 2000) and rodents (Boughter et. al., 1997) to selectively label rod

photoreceptors. The secondary antibody used for both whole-mount and cryosection was Alexa

Fluor 488 diluted 1:1000, a goat anti-rabbit IgG antibody. The sections were placed in 1%

solution of bovine serum albumin in phosphate-buffered saline to block non-specific binding site.

The sections were exposed to the primary antibody K-20 for 2 h, then Alexa Fluor 488 for 1 h.

The sections were mounted with Prolong Gold and photographed in a Zeiss Axioscope. The

whole mount was mounted with Prolong Gold as well. Due to the scarcity of T. proximus retinal

tissue samples and the technical challenges of removing the RPE from smaller snakes, no

negative control was available for the present immunohistochemistry study.

2.4 Cloning into p1D4

Working with T. proximus SWS1, LWS, or RH1 opsin genes in cloning vector pJET, primers

were designed to add EcoRI restriction site to the 5’ and BamHI site to the 3’ end of the opsin

genes. The amplified bands were ligated into the pJet blunt end cloning vector. The genes were

isolated from the pJet vector with EcoRI and BamHI restriction endonucleases, and ligated into

the p1D4 vector treated with the same restriction enzymes. The p1D4 vector consists of the

monoclonal antibody 1D4 epitope, which were the last 9 amino acids from bovine rhodopsin,

fused to the pIRES vector at the multiple cloning site. The expression construct containing the

desired opsin inserts were sequenced to rule out mutations due to polymerase errors.

Mutants of T. proximus RH1 were generated by using QuickChange site-directed mutagenesis kit

(Stratagene). To make a point mutation on a gene, a pair of mutagenesis primers was designed to

anneal to the desired region of the gene, with one mismatch between the primer sequences and

the gene sequences, hence the mutation. All three point-mutants were generated by altering one

nucleotide in the annealing regions through PCR. The PCR product was treated with Dpn I

endonuclease, which specifically digest the methylated and hemimethylated DNA, therefore

destroying the parental DNA template and selecting for mutation-containing synthesized DNA.

All DNA products that were subjected to mutagenesis were sequenced to rule out spurious

mutation.

26

2.5 in vitro Expression and Purification

The expression constructs containing T. proximus RH1 genes in p1D4 vector were transfected

into mammalian cells. This experiment was repeated ten times in total. The expressions were

carried out according to previously described methods (Han et al. 1996), essentially by transient

transfection into HEK293T cells using Lipofectamine 2000 (Life Technologies), harvested after

47 h, regenerated in 5 mM 11-cis retinal, solubilized in 1% n-dodecyl-b-D-maltoside detergent,

and immunoaffinity purified using batch methods with the 1D4 monoclonal antibody. Bovine

RH1 opsin was expressed using the same methods each time as a positive control. For each in

vitro protein sample, absorbance spectroscopy was performed at 25°C using a Varian Cary4000

spectrophotometer, using quartz cuvettes with a 1-cm pathlength. The solublization buffer was

first measured as the blank. For each regenerated opsin sample, dark spectrum was recorded first,

and then light spectrum was recorded after bleaching the sample with full-spectrum white light

for 30 seconds. The difference spectrum can be produced by subtracting the light spectrum from

the dark spectrum. To determine λmax, the difference spectrum was then fitted to a standard

Govardovskii rhodopsin A1 template (Govardovskii, 2000).

27

3 Results

3.1 Scanning Electron Microscopy

All the data presented in the results section, including electron microscopy,

immunohistochemistry, in vitro protein expression were generated by me.

To characterize the photoreceptors based on morphology, retina of T. proximus was examined

with scanning electron microscopy (SEM). The gross morphology of the T. proximus retina

shows both similarities to basal snakes and adaptations to diurnal lifestyle. The basic

stratification pattern of retinal cells (Figure 8A) appears to be the same as Boidae snakes (Walls,

1942). Underneath the pigment epithelium layer are the outer segments of photoreceptors. Below

the outer segments are the ellipsoid regions of photoreceptors, which are darkly stained with

toluidine blue due to high mitochondrial content (Figure 8B). The nucleus and synaptic terminals

of the photoreceptors form the outer nuclear layer. In a duplex retina, the nucleus of rods and

cones tend to occupy different zones of the outer nuclear layer, such as found in Boidae snakes

(Walls, 1942). The outer nuclear layer of T. proximus retina is made up of a relatively thin layer

of cone nucleus, compared to the inner nuclear layer. A nocturnal animal will tend to have a

retina with a thicker outer nuclear layer to accommodate a high number of rod nuclei (Wong,

1989).

Based on cellular morphology, vertebrate photoreceptors can be cone-like, which are shorter and

stouter cells with tapering outer segments, or rod-like, which are taller and slender cells with

enlarged outer segments. Under SEM, all photoreceptors found in T. proximus retina are cone-

like (Figure 9), resembling those from its sister species, T. sirtalis (Wong, 1989; Sillman et al.,

1997). None of the photoreceptor of T. proximus resembles the rods found in henophidian snakes

(Sillman et al., 1999; 2001). Some photoreceptors of T. proximus appear to be arranged in a

pattern of concentric rings (Figure 9).

One type of double cone and three types of single cones can be distinguished under SEM (Figure

10). A T. proximus double cone is composed of a large principal member and a small accessory

member. The two members are joined together by their outer segments, but each member has a

separate stack of outer segment membranes. The T. proximus single cones can be classified,

28

based on their relative size, as large single cones, small single cones, or very small single cones.

The large single cones of T. proximus are comparable in size to the principal members of its

double cones.

It becomes apparent from SEM, that the retina of T. proximus has an abundance of double cones

and large single cones. More scarce are the small single cones, and the very small single cones

are rarely found. To assess the relative abundance of each type of photoreceptors, nine zones

across the surface of a T. proximus retina were surveyed under SEM (Figure 11). Large single

cones and double cones were very common, making up about 45% and 44% of the total

photoreceptor population respectively. The small single cones and very small single cones make

up about 9% and 2% of total photoreceptor population. The overall relative abundance of each

type of photoreceptors is comparable between T. proximus and T. sirtalis (Sillman et al., 1997;

Table 1).

29

Figure 8. A cross-sectional view of T. proximus retina, several layers of retinal tissue can be

distinguished under SEM (A) and light microscopy (B). From the posterior end of the eye, the

layers are R.P.E. - retinal pigment epithelium; P.C. – photoreceptor cells; O.N.L - outer nuclear

layer; I.N.L - inner nuclear layer; G.C - ganglion cells. Scale bar = 5um. The outer nuclear layer

of T. proximus retina is relatively thin compared to the inner nuclear layer.

A B

30

Figure 9. Scanning electron micrographs of the retina of T. proximus. All photoreceptors are

cone-like based on morphology. The white dotted line marks the concentric-ring pattern of T.

proximus photoreceptors. Calibration bar: 5 µm.

31

Figure 10. Scanning electron micrographs of the retina of T. proximus. There are four types of

cones based on morphology: p- principal double cone; a- accessory double cone; ls- large single

cone; ss- small single cone; v- very small single cone. Calibration bar: 5 µm.

32

Figure 11. Nine zones were surveyed from a T. proximus retina under SEM (A). The four types

of photoreceptors were visually scored from SEM from each zone, which were then converted

into percentages (B). An average percentage for each type of photoreceptor was calculated by

taking the means from all nice zones (C). The overall ratio of different photoreceptors types were

calculated to be 44% double cones, 45% large single cones, 9% small single cones, and 2% very

small single cones.

33

3.2 Transmission Electron Microscopy

To look for distinguishable cellular features of rods and cones, ultra-thin sections of T. proximus

retina were examined with transmission electron microscopy (TEM). The ellipsoids of snake

photoreceptors are usually darkly stained due to high mitochondrial content (Walls, 1942; Wong,

1989). Two types of ellipsoids can be found in T. proximus cones. TEM shows that the principal

members of double cones (Figure 12B) and large single cones (Figure 13C) have similar

ellipsoids, but small single cones (Figure 13B) and accessory member of double cones (Figure

12B) have another type of ellipsoids.

This difference is partly due to clusters of brightly stained micro-droplets in the ellipsoids,

Micro-droplets were found in the mitochondrial cristae of T. sirtalis photoreceptors (Wong,

1989). Upon closer examination, micro-droplets are differentially expressed in T. proximus

photoreceptors. Micro-droplets are found in the principal member of double cones (Figure 12B)

and in large single cones (Figure 13C). The accessory member of double cones (Figure 12B) and

some small single cones (Figure 13B) do not have micro-droplets in their outer segments. Instead,

electron-dense pigments are found in the small single cones and accessory double cones. These

dark pigments are even smaller in size than micro-droplets, and they can be occasionally found

scattered amongst micro-droplets (Figure 13C).

The micro-droplets have only been found in snake species, and they are thought to function

collectively to replace oil-droplets, which were lost in snakes but found in lizard and bird cones

(Walls, 1942; Wong, 1989). When cone lattice were directly observed through the pupil of live

garter snakes, dark holes in the cone lattice were also observed (Land and Snyder, 1985; Wong,

1989). These holes could contain structures slightly smaller than the visible cones themselves

(Land and Snyder, 1985). It is possible that these dark holes are the small single cones. They

may appear dark due to the accumulation of dark pigments in their inner segment. Since TEM

can only be photographed in black-and-white, the color of the droplets remains unknown.

The outer segment of a photoreceptor is the membranous site specialized for photon detection

(Kawamura and Tachibanaki, 2008). Rods and cones are known to differ in their outer segment

membranes (Cohen 1972). Rods have outer segment membranes that are discontinuous from

plasma membranes, while cones have outer segment membranes that are open to extracellular

media. Under TEM, some small single cones of T. proximus were found to have rod-like outer-

34

segment membranes, which are disk-like and discontinuous from the plasma membrane (Figure

14B).

The very small single cones were found to lack any outer-segment membrane (Figure 14A). For

a functional visual pigment to participate in phototransduction cascade, it must be properly

packed in the outer segment membranes. This is perhaps why MSP can not detect any spectral

absorbance from these very small single cones.

35

Figure 12. Transmission electron microscopy of a tangential section of T. proximus retina,

showing (A) the outer segment of principal (pc) and accessory (ac) member of a double cone; (B)

close-up of the boxed area in (A), micro-droplets are found in principal member but not

accessory member. Scale bars = 0.5 µm. Dark arrows: electron-dense pigments; white arrows:

micro-droplets.

pc

ac

BBBB

AAAA

pc ac

36

Figure 13. Transmission electron microscopy of a tangential section of T. proximus retina,

showing (A) the outer segment of a small single (ss) cone and a large single (ls) cone; (B)

electron-dense pigments in the small single cone; (C) micro-droplets and electron-dense

pigments in the large single cone. Scale bars = 1 µm. Dark arrows: electron-dense pigments;

white arrows: micro-droplets.

ls ss

ls

ss

AAAA

BBBB CCCC

37

Figure 14. Transmission electron microscopy of a tangential section of T. proximus retina,

showing (A) the outer segments of a very small single (vs) cone and a small single (ss) cone; (B)

outer segment membranes of the small single cone from (A). Note the absence of micro-droplets

from both cones, and the lack of outer segment membranes from the very small single cone. The

outer segment membranes of this small single cone are mostly discontinuous from the plasma

membrane. Scale bar = 1 µm.

BBBB

AAAA

ss

vs

38

3.3 Immunohistochemistry

Besides visual pigments, rods and cones also differ in expression of downstream photo-

transduction proteins, such as transducin. Since RH1 may be expressed in the retina of T.

proximus, it is interesting to find out whether rod-specific transducin is also expressed. K-20

targets rod-specific transducin alpha subunit, and selectively stained only rods in rodents

(Boughter et. al., 1997), pigeons (Wada et. al., 2000), and lampreys (Petromyzon marinus)

(Muradov et. al., 2008). In two species of diurnal rodents (Arvicanthis ansorgei and

Lemniscomys barbarus), only the cell body of rods but not cones were labeled with K-20 (Bobu,

2008).

The retina of T. proximus was isolated and processed for retinal whole-mount and cryosection,

before incubating with K-20 antibody. The retinal whole-mount shows the photoreceptor layer

from a top-down view (like the SEM), while the cryosection shows the cross-section of the retina

(like the TEM). In both methods of preparation, a subpopulation of photoreceptors was stained

by the antibody K-20 (Figure 15). Positive staining with K-20 antibody suggests that some T.

proximus cones are expressing rod-specific transducin, an important upstream component of the

photo-transduction cascade. It is unclear at this point whether these positively stained cones are

small single cones. A negative control was not carried out due to sample limitations. This

preliminary result warrants further investigation into the downstream photo-transduction

cascades in T. proximus.

39

Figure 15. Fluorescence micrographs of retinal wholemount (A) and cryosection (B) of T.

proximus. Photoreceptors appear green in retinal wholemount to indicate rod-specific transducin

antibody (K-20) reactivity. The cryosection is shown with a light-microscopy image as reference

for retinal cell layers, and the arrows indicate positively stained photoreceptors. Calibration bar:

30 µm.

40

3.4 RH1 in vitro Expression

Based on MSP data and opsin gene sequences from T. proximus, the RH1 may be responsible for

the 483 ± 1.5 nm MSP peak. To test whether T. proximus RH1 absorbs at 483 ± 1.5 nm, wild-

type T. proximus RH1 gene was expressed in vitro using previously described methods (Chang et

al., 2002). The full length opsin cDNA sequence was cloned into over-expression vector p1D4,

which is a vector constructed by fellow graduate student James Morrow. In vitro expressed T.

proximus RH1 appears to be a functional pigment with blue-shifted λmax compared to bovine

RH1 control.

Bovine rhodopsin has been established as a model system for opsins. Bovine RH1 in p1D4 was

used to transfect HEK293T cells as a positive control, because the transfection protocol was

established and optimized for bovine RH1. After harvesting and regeneration with 11-cis retinal,

the in vitro expressed bovine rhodopsin pigments were measured for spectral absorbance in the

dark, and then after bleaching with light. Dark-state of bovine RH1 pigments absorb at 500nm,

as measured by the dark spectrum (Figure 16A). After bleaching the sample with light, the light

spectrum shows a decrease of the 500nm peak, and an increase of the 380nm peak (Figure 16A).

The 380nm peak was also noticeable from the dark spectrum. This is most likely due to light leak

during sample preparation, which prematurely activates the in vitro RH1 pigments. Between the

dark and light spectra, the shift in absorbance is mostly due to the activation of dark-state RH1

by light, and the accumulation of Meta-II intermediates.

To reduce artifacts and noise in absorbance spectra, a difference spectrum of bovine RH1 can be

produced by mathematically subtracting the light spectrum from the dark spectrum (Figure 16B).

The resulting 500nm peak corresponds to the λmax of bovine RH1, and the dip in absorbance at

380nm is due to an accumulation of Meta-II intermediates after bleaching with light. The

formation of Meta-II intermediates is an important feature of RH1 pigments, since these

intermediates active the G-protein, transducin. A peak around 500nm and a dip at 380 nm from

the difference spectrum indicates that the in vitro bovine RH1 pigments absorb at 500nm and can

form Meta-II intermediates once light activated.

Compared to bovine RH1, T. proximus RH1 was more challenging to express in vitro. Small

scale transfections of T. proximus RH1 produced dark and light spectra without noticeable

41

absorbance peaks (Figure 17A). The difference spectrum of T. proximus RH1 produced a noisy

peak that seems to be blue-shifted from 500nm, while the dip in absorbance at 380nm was also

not as pronounced as bovine RH1 (Figure 17B).

On a western blot, the in vitro expressed opsins are detected with 1D4 monoclonal antibody

(Figure 18). Both the bovine and T. proximus RH1 pigments are 34kDa according to their amino

acid sequences. While bovine RH1 was visualized on western blot, T. proximus was only

detectable after the in vitro sample was concentrated four times.

42

Figure 16. Dark and light absorbance spectra (top), and the difference spectra (bottom) of in vitro

expressed RH1 visual pigments of bovine, transfected with one plate of cells.

43

Figure 17. Dark and light absorbance spectrum (A), and the difference spectrum (B) of in vitro

expressed RH1 visual pigments of T. proximus, transfected with six plates of cells.

44

Figure 18. Western blot of in vitro expressed RH1 pigments of bovine and T. proximus using

antibody against 1D4 epitope. On the right lane, the T. proximus RH1 sample was four times as

concentrated as the sample in the middle lane. Based on amino acid composition, RH1 proteins

have molecular weight of approximately 34kDa.

45

4 Discussion

4.1 Morphology of Photoreceptors

All photoreceptors of T. proximus are short and stout cells with tapering outer-segments. Based

on cellular morphology, four types of cones can be identified in T. proximus retinae: double

cones, large single cones, small single cones, and very small single cones. Based on SEM from T.

proximus, their photoreceptors resemble the cones identified in the sister species T. sirtalis

(Wong, 1989; Sillman et al., 1997). TEM images show that some T. proximus small single cones

have morphological features that distinguish them from other cone types.

T. proximus photoreceptors are similar in size compared to their counter-parts in T. sirtalis. In T.

proximus, the inner segments of double cones and large single cones are about 9-11um in length,

and the inner segments of small single cones are about 4-6 µm in length. The very small single

cones are difficult to identify and measure; due to the small size and rareness. In T. sirtalis, the

inner segments are 7-10um for double and large single cones, 3-5 µm in length for small single

cones (Wong, 1989). In both T. sirtalis and T. proximus, inner segments of small single cones are

about half the size of large single cones. The inner segment lengths of T. proximus

photoreceptors were estimated from both SEM and TEM images, since there are limitations to

both techniques. SEM images are two-dimensional representations of three-dimensional surface

structures, so structures in greater depth of view appear smaller in size. SEM images are also

usually taken from an angle, which makes accurate measurement of photoreceptors even more

difficult. TEM sections can show the vertical cross-section of individual photoreceptors, but only

a few of the photoreceptors in section are through the central vertical plane of the photoreceptors.

To get a more accurate measurement of photoreceptor size, more ultra-thin sections for TEM

would be needed.

Based on SEM, the cone photoreceptors of T. proximus are morphologically distinct from the rod

photoreceptors of henophidian snakes. Rods of boas are tightly packed, with elongated outer

segments that can exceed 20 µm in length (Sillman et al., 2001). The tapering outer segments of

T. proximus photoreceptors are 3-5 µm in length. Due to the conical shape of inner segments, the

T. proximus photoreceptors appear to be more loosely packed than rods from boas. After

hundreds of SEM images were taken from eight pieces of T. proximus retinae, not a single

46

photoreceptor was found to resemble rods of henophidian snakes. Therefore based on cellular

surface morphology, T. proximus do not appear to have typical rod shaped photoreceptors.

Based on photoreceptors scored from nine zones of the retinal surface from a T. proximus retina

(Figure 11), large single cones and double cones were very common, making up about 45% and

44% of the total photoreceptor population respectively. The small single cones and very small

single cones make up about 9% and 2% of total photoreceptor population. In T. sirtalis, similar

photoreceptor ratios were found (Sillman et al., 1997). Any minor differences in photoreceptor

ratios between the two species could be due to individual variation, but it could also be due to

ecological differences. The differences in photoreceptor ratios are more striking when the all-

cone retina of T. proximus is compared with the rod-dominant retinae of boas and pythons,

where more than 90% of photoreceptors are rods (Sillman et al., 1999; 2001). The rod dominant

retina of henophidian snakes could be an adaptation to nocturnal environment, while the all-cone

retina of T. proximus may be an adaptation to diurnal environment.

Previous studies on garter snake photoreceptors reported irregular mosaic pattern (Wong, 1989;

Sillman et al., 1997), as opposed to the regular square mosaic found in fish (Braekevelt, 1992).

Although photoreceptors of T. proximus also appear be arranged in irregular mosaic pattern,

some cones appear to form concentric-ring pattern under SEM (Figure 9). This type of

photoreceptor mosaic has not been reported by previous studies on snakes. Although some

photoreceptors of T. proximus appear to be in a concentric ring pattern, this observation needs to

be repeated in other snakes, and the functional implications of this cone mosaic pattern require

further investigations.

Even though all the T. proximus photoreceptors have cone-like surface morphology, some small

single cones were found to have rod-like outer segment membranes under TEM (Figure 14B).

Disk-like outer segment membranes are one of the hallmarks of rod photoreceptors, and they

may be crucial to the rod’s function under dim light (Lamb, 2009). While some small single

cones have rod-like outer segment membranes, the current TEM result can not conclude whether

all small single cones have rod-like disks, or maybe some small single cones have cone-like

invaginations. This is due to technical limitations of TEM. Ultra-thin sections of TEM are often

not cut through the central plane of the photoreceptors; therefore a large single cone could appear

to look like a small single cone in size. The principal member of a double cone can also look like

47

a large single cone under TEM, if the section did not capture the accessory member. However,

when rod-like disks of outer segments are found using TEM, they are always associated with

small single cones.

Besides their rod-like disks, these small single cones can also be distinguished from other cone

types by their ellipsoids. Under TEM, two types of ellipsoids can be found in the photoreceptors

of T. proximus. Principal double cones and large single cones have ellipsoids packed with

microdroplets (Figure 12B; Figure 13C). Some small single cones (Figure 13B and 14A) and

accessory double cones (Figure 12B) do not have microdroplets in their ellipsoids.

Microdroplets have been previously found in T. sirtalis (Wong, 1989), although it was not clear

whether they were ubiquitously expressed in all cones, or differentially expressed in certain types

of cones. In the one hundred and fifty TEM images taken from over twenty ultra-thin sections of

T. proximus retinae, the microdroplets are only found in large single cones and principal double

cones.

There have been speculations on the function of microdroplets, one of which proposes that these

microdroplets may function collectively as an oil droplet (Wong, 1989). Found in the ellipsoids

of cones, single oil droplets are continuous spherical structures with diameters of 2-3 µm (Hart et

al., 2000). Smaller in size, microdroplets of T. proximus photoreceptors are around 0.1-0.15 µm

in diameter, and found scattered throughout the ellipsoids. Like microdroplets of T. sirtalis

(Wong, 1989), microdroplets of T. proximus are spheroids because in every plane of section they

remained circular in shape.

Instead of microdroplets, some small single cones have a large amount of electron-dense

pigments in their ellipsoids (Figure 13B and 14A). These electron-dense pigments have not been

previously reported in snakes, since they are much smaller in size compared to microdroplets.

These electron-dense pigments appear to be tiny spheroids, and their small size makes accurate

measurement of diameter a difficult task. These electron dense pigments are also found

occasionally in other T. proximus cones (Figure 13C), but they are found most abundant in the

small single cones, which have rod-like disks and lack micro-droplets. These electron dense

pigments are unlikely to be TEM artefacts, since they are not found outside ellipsoids, and they

appear to be most abundant in the ellipsoids of small single cones, and to a lesser degree in

accessory double cones. The accumulation of these dark pigments could make a photoreceptor

48

appear darker in the snake retinal mosaic in vivo. Photoreceptors with darker ellipsoids and

smaller in size have been noted by previous studies on garter snake retinae (Land and Snyder,

1985; Wong, 1989). Large single cones and principal double cones appear to have brighter

ellipsoids in the retinal mosaic (Land and Snyder, 1985; Wong, 1989), possibly due to funnelling

of light by the microdroplets (Wong, 1989).

Even though snake microdroplets are not single oil droplets, there appears to be a conserved

pattern between localization of microdroplets of snakes and single oil-droplets of birds. Oil

droplets are not found in rods or the accessory member of double cones of birds (Hart et al., 2000)

and turtles (Kolb and Jones, 1982). Since oil droplets may act as light filters, the lack of oil

droplets in rods could be an adaptation for enhanced photo-sensitivity. TEM on T. proximus

photoreceptors shows that microdroplets are not expressed in some small single cones (Figure

13B; Figure 14A), or the accessory member of double cones (Figure 12B). Since rods don’t have

oil droplets, a lack of microdroplets in some small single cones could be a rod-like feature. These

small single cones also have rod-like outer segment disks (Figure 14B). The rod-like cellular

features suggest that some small single cones could be the candidates for expressing RH1

pigments. The functional roles of the small single cones can be better understood by considering

data from RH1 in vitro expression.

49

4.2 In vitro Expression of T. proximus RH1

In total, ten rounds of in vitro expression of T. proximus RH1 pigments were carried out, and for

each round at least one plate of HEK293T cells were transfected with bovine RH1 as positive

control. Bovine RH1 pigments can be reliably expressed as positive control, whereas T. proximus

RH1 pigments were challenging to express and measure in vitro.

Bovine RH1 is a model system for studying GPCR protein (Pierce et al., 2002) and its crystal

structure was the first GPCR to be elucidated (Palczewsk, 2000). Bovine RH1 is therefore

commonly expressed as a positive control of in vitro studies. My sample data of bovine RH1

pigments show in vitro λmax of 500nm for dark-state RH1, and 380nm peak from accumulation

of Meta-II intermediates after bleaching (Figure 16). This corresponds to the established λmax of

bovine RH1 in vitro (Yokoyama 2000). This result is typical of bovine RH1 pigments from every

in vitro expression experiment carried out by me. Bovine RH1 pigments expressed in vitro can

also be visualized on Western blot using 1D4 antibody (Figure 18).

In contrast, in vitro expressed T. proximus RH1 pigments as solubilized sample do not show any

noticeable absorbance peak before or after bleaching (Figure 17A). The sample data shows

spectral absorbance of T. proximus RH1 pigments harvested from six plates of HEK293T cells.

There appears to be a trend of increasing absorbance in shorter wavelength, but it is most likely

an artifact due to the Tris-based buffer used for solubilization, which tends to absorb in the UV.

The resulting difference spectrum of T. proximus RH1 does eliminate the trend (Figure 17B), but

the absorbance peak is still too low and too noisy for calculating λmax. Western blot results also

indicate that T. proximus RH1 pigments are expressed in lower quantities compared to bovine

RH1 pigments (Figure 18). T. proximus RH1 pigments only become visible on Western blots

after the sample is further concentrated. T. proximus RH1 pigments appear to be similar in size

as bovine RH1, as expected from their molecular weights of about 34 kDa. The T. proximus RH1

pigments may be difficult to express and measure in vitro for several reasons.

First of all, the transfection protocol was optimized for bovine RH1, but it may not be ideal for

expressing RH1 pigments of cold-blooded reptiles such as snakes. Vertebrate RH1 pigments are

sensitive to temperatures, and there are considerable variations between species (Hubbard, 1958).

The HEK293T cells transfected with over-expression construct were incubated at 37°C for

50

47hours. Garter snakes live in environments that can fluctuate between below freezing to around

50°C (Lysenko and Gillis, 1980; Peterson, 1987). Garter snakes prefer to maintain their body

temperatures between 25-30°C through behavioral changes, such as basking or under cover

(Peterson, 1987). When environmental temperatures reach more than 30°C, garter snakes

maintained their preferred body temperature over 90% of the time (Peterson, 1987). It’s unlikely

for a garter snake to have body temperature of 37°C for any extended period of time. The

stability of T. proximus RH1 pigments may be hampered by the incubation steps of in vitro

expression protocol. RH1 pigments of X. unicolor were expressed in vitro at 37°C from twelve

plates of cells, and show λmax of 497nm only according to difference spectrum (Davies et al.,

2009). At 37°C, snake RH1 pigments may be more difficult to express in vitro than bovine RH1

pigments.

Secondly, T. proximus RH1 have multiple amino acid differences compared to bovine RH1, and

these amino acid differences could destabilize the protein structure of T. proximus RH1, or lead

to a lowered amplitude of spectral absorbance. There are a number of amino acid substitutions

found in T. proximus RH1, some of which are highly conserved in other vertebrate RH1 genes.

The lowered amplitude in spectral absorbance could be due to the unique amino acid changes in

T. proximus RH1, such as S185, I11, I130, Q150, L159, and V169. Some of these T. proximus

residues, such as Q150 are not found in rod-dominant species such as bovine, as well as sunbeam

snakes and pythons. RH1 pigments of sunbeam snakes and pythons have been expressed in vitro

by previous study (Davies et al., 2009). These unique substitutions could potentially alter the

protein structure of T. proximus RH1, leading to the lowered absorbance level (or sensitivity)

compared to RH1 pigments of other vertebrates, such as the bovine and henophidian snake. To

confirm this, reverse mutants of T. proximus RH1 with single amino acid substitution can be

expressed in the future for comparison of absorption level.

In addition, in vitro expressed T. proximus RH1 pigments were tagged with 1D4 epitope, which

is useful for affinity purification or antibody detection. Many in vitro studies have used 1D4

epitope for expressing vertebrate pigments (e.g. Pointer et al., 2007; Cowing et al., 2008;

Yokoyama, 2000). In our lab, zebrafish opsins have been expressed successfully with 1D4

epitope using the same protocol (James Morrow, unpublished). However, this epitope could also

alter the protein structure of T. proximus RH1. Other studies have found increased stability of

GPCRs by eliminating the C-terminal tail (Rosenbaum et al., 2007). Extending the tail by fusing

51

the RH1 opsin with a 1D4 epitope could further decrease the stability of T. proximus RH1. To

maintain opsin stability while tagging it with 1D4 epitope, T. proximus RH1 could have the C-

terminal tail truncated before tagging with the epitope.

The low absorption level of T. proximus RH1 in vitro could also suggest that these pigments do

not have a functional role in vision. Only 9% of T. proximus photoreceptors are small single

cones, and only some of them absorb around 483nm according to MSP (Figure 4). ERG of garter

snakes found that the retinal gross potential peaked from 550nm to 570nm under varying

experimental settings (Jacobs et al, 1992). Therefore the absorbance from LWS pigments are

likely the dominant contributor to visual signals sent to the brain. However, MSP result from

some small single cones indicates that their outer segments do absorb at 483 ± 1.5 nm (Figure 4).

If T. proximus RH1 pigments are not responsible for this absorption peak, may be there is

another unidentified RH2 type pigment. Previous members of the lab have looked for RH2

sequence from T. proximus cDNA library but could not find any (Mengshu Xu, Natalie Chan,

personal communication). RH2 pigments have been found in other reptile species. In Italian wall

lizards (Podarcis sicula), RH2 opsin have been isolated and shown to participate in photic

entrainment of behavioural rhythms (Pasqualetti et al., 2003). In side-blotched lizard (Uta

stansburiana), RH2 opsin was isolated from the parietal-eye, which mediate global detection of

dawn and dusk instead of image-forming vision (Su et al., 2006). Besides SWS1, LWS, and RH1,

other opsins have not been reported in snake species so far. Previous study on henophidian

snakes tried to isolated SWS2 and RH2 from retinal cDNA library but failed to find them

(Davies et al., 2009). It is possible that the other opsins, such as SWS2 and RH2, are expressed

in the retina, escaping both degenerate PCR and MSP detection. Since SWS2 and RH2 opsins

have not been sequenced from any snakes, degenerate primers could be designed from lizard

species to look for snake SWS2 and RH2 opsins. The genomic sequences of T. proximus could

also be useful for identifying other opsins such as SWS2 and RH2. Nucleic acid probes can be

designed from genomic sequences for Southern-blot to look for other opsins.

To confirm that the poor absorbance of T. proximus RH1 is not due to my experimental errors, a

large scale expression using 36 plates of HEK293T cells was carried out by fellow graduate

student James Morrow. The RH1 gene was re-cloned into expression vector before transfection.

Based on the large scale expression result, the absorption level from purified T. proximus RH1

pigments is about 100 times lower than bovine RH1 (Figure 19). The resulting difference

52

spectrum (Figure 20) suggests that the T. proximus RH1 pigments can form Meta-II

intermediates, indicated by the dip in absorbance around 380nm. There also appears to be an

absorbance peak that may match the MSP signal from some small single cones. To determine the

λmax of T. proximus RH1 in vitro, template-fitting was carried out on the difference spectrum.

Absorbance spectra of visual pigments can be represented by a common template (Dartnall, 1953;

Lamb 1995; Govardovskii, 2000). While the λmax of visual pigments can be tuned to different

wavelengths, the shape of the absorbance curve remains the same. Although this assumption is

built upon empirical data, many studies have searched for the universal template of visual

pigments and the underlying physical factors (Ebrey and Honig, 1977; MacNichol, 1986;

Partridge and De Grip, 1991; Lamb 1995; Gorvadovski, 2000). Although the most recent

template by Gorvadovski (2000) is based on MSP data, this template has been used extensively

to fit difference spectra of in vitro expressed visual pigments of rodents (Parry et al., 2004), birds

(Carvalho, et al., 2007; Pointer et al., 2007), sharks (Davies et al., 2009), lampreys (Davies et al.,

2007), marsupials (Cowing et al., 2008; Hunt et al., 2009), and snakes (Davies et al., 2009).

According to template fitting of the large-scale difference spectrum, T. proximus RH1 pigments

have in vitro λmax of 485nm (Figure 20B). This value may correspond to the MSP λmax of 483

± 1.5 nm from some T. proximus small single cones. MSP λmax can be slightly different from in

vitro λmax, because other proteins or structures of the outer-segments could skew the absroption.

In X. unicolor, rod outer segments have MSP λmax of 499nm, while the RH1 pigments have in

vitro λmax of 497nm (Davies et al., 2009). Compared to X. unicolor, T. proximus RH1 pigments

have a blue-shifted λmax of 485nm, which could be due to residues N83 and S292 (Figure 7).

Double-mutants of bovine RH1 with N83/S292 substitutions have λmax blue-shifted to 485nm

(Fasick and Robinson, 1998). Based on amino acid sequences, RH1 of sunbeams and pythons

both have residues N83/A292, instead of N83/S292 in T. proximus. In RH1 pigments of lake

cichlids, those with N83/S292 have the most blue-shifted λmax, while those with one of N83 or

S292 residues are still blue-shifted but to a lesser degree (Sugawara et. al., 2005). These

substitutions could explain why rhodopsin pigments of sunbeams (X. unicolor) and pythons (P.

regius) have less blue-shifted λmax compared to T. proximus. Further expression of N83D and

S292A mutants of T. proximus are required to determine whether these spectral tuning sites are

responsible for the blue-shifted λmax of snakes RH1 pigments.

53

Dark Absorbance of Expressed Visual Pigments

0

0.05

0.1

0.15

0.2

0.25

0.3

250 300 350 400 450 500 550 600 650

Wavelength (nm)

Abs

Bov Rh1 Tpx Rh1

Figure19. Dark absorbance of in vitro expressed rhodopsins, from 36 plates of cells transfected

with T. proximus RH1, and 4 plates of cells transfected with bovine RH1 as control. These are

the same samples expressed and purified by James Morrow as shown in Figure 20. Note the

difference in absorbance between bovine and T. proximus RH1 pigments at 500nm.

54

Figure 20. Dark and light absorbance spectrum (A) of large-scale in vitro expressed RH1 visual

pigments of T. proximus, transfected and purified by James Morrow using thirty-six plates of

cells. Curve-fitting using the difference spectrum (B) of T. proximus RH1 from large scale

expression, using previously described templates (Govardovskii, 2000): the dots represent data

points from difference spectrum, and the line represents the absorbance predicted by curve-

fitting. The λmax predicted by curve-fitting is 485nm.

55

4.3 Transmutation of Small Single Cones

Based on the current EM, in vitro expression, and MSP data from T. proximus, RH1 pigments

may be expressed in some small single cones, with a blue-shifted λmax of 485 nm. The

expression of RH1 pigments in the all-cone retina of T. proximus is an unusual combination,

since rhodopsin pigments are typically found in morphological rods of most vertebrates,

including henophidian snakes such as X. unicolor and P. regius (Davies et al., 2009). Based on

cellular and molecular evidence, these small single cones may have evolved from rods of

ancestral snakes.

The evolutionary transition between rods and cones was coined “transmutation” by Walls (1942).

According to Walls’ transmutation theory, the first rods were transmutated from cones, and this

transition has occurred in the reverse direction in some snakes (Walls, 1942). There is growing

evidence in support of photoreceptor transmutation. Rod photoreceptors are specialized dim-light

detectors evolved from cones: the elongated outer segments of rods are a derived feature after

diverging from cones, and rhodopsin pigments evolved high sensitivity after diverging from cone

pigments (Bowmaker, 2008). In mammals, the entire rod pathway is built upon existing cone

pathways (Strettoi et al., 1992). Molecular data and phylogenetic studies found that LWS

pigments are the most basal family while the RH1 pigments are the most derived (Okano et al.,

1992). In most vertebrates the cones and rods have become so specialized (i.e. typical rod-like

morphology matched with expression of RH1), that any evidence of past transmutation has been

obscured. On the other hand, evidence of photoreceptor transmutation has been found in a few

vertebrate species, such as in nocturnal geckos.

The nocturnal Tokay gecko (Gekko gecko) is well studied for photoreceptor transmutation. Their

photoreceptors are rod-like in terms of morphology and electrophysiology (Pedler and Tilly,

1964; Tansley, 1964; Rispoli et al., 1993; Kleinschmidt and Dowling, 1975). There are three

types of photoreceptor cells in nocturnal geckos – type A single rods, type B double rods, and

type C double rods (Underwood, 1951). Gecko photoreceptor cells have three cone-type visual

pigments. The first visual pigment, P521, is similar to chicken red-sensitive cone visual pigment

(iodopsin) belonging to M/LWS subfamily (Kojima et al., 1992). The second visual pigment

P467 has the highest sequence similarity to chicken green-sensitive cone visual pigment and is

also highly related to a subfamily of rhodopsin, RH2, which are found in both rods and cones

56

(Ebrey and Koutalos, 2001; Kojima et al., 1992). The third visual pigment P364 is a short-

wavelength-sensitive cone-like pigment (Yokoyama and Blow, 2001). Besides cone-type

pigments, only cone-specific sequences were obtained for phototransduction proteins, including

transducin α-subunit, phosphodiesterase catalytic and inhibitory subunits, cyclic nucleotide-gated

channel and arrestin (Zhang et al., 2006). Phylogenetically, nocturnal rod-only gecko and

modern diurnal cone-only geckos both evolved from a common ancestor with only cones

(Northcut and Butler, 1974; Roll, 2000). Rod-shaped photoreceptors of geckos were derived

from ancestral cone-like photoreceptors, since cone-specific phototransduction proteins were still

expressed (Walls, 1934; Zhang et al., 2006). The retina of Tokay gecko is a good example of

how the morphology of photoreceptor cells can evolve independently of visual pigments (Kojima

et al., 1992).

Results from the present study suggest that morphology and visual pigments may have also

evolved independently in T. proximus. While all T. proximus photoreceptors have cone-like

ultra-structure, some small single cones have rod-like cellular features. These small single cones

have rod-like outer segment membrane disks, which are usually only found in rods. RH1

pigments may be expressed in these rod-like disks, since MSP peak from some small single

cones may correspond to in vitro λmax of T. proximus RH1 pigments. Unlike large single cones

and double cones, some small single cones were found to lack microdroplets. Instead they have

an accumulation of tiny electron-dense pigments. Considering that T. proximus is a diurnal snake,

these dark pigments may function as neutral light filters, since T. proximus RH1 pigments may

be used for day-light vision.

There is good reason to speculate that RH1 should participate in day-light vision of T. proximus.

One possible function is to provide signal contrast with the other cones. Cone photoreceptors and

cone pigments are usually responsible for providing signal contrast and color vision. For T.

proximus, the λmax for its cone pigments, SWS1 and LWS, are perhaps too far apart (360nm

and 550nm) for signal contrast. There is no record of any vertebrate using only SWS1 and LWS

for color vision, but studies now suggest that rods can participate in color vision in human. Blue-

cone monochromats have rods and only one type of cones, but these patients have color vision

within a limited spectral and intensity range (Reitner et al., 1991). At this point, we can only

speculate on the possibility that T. proximus can use rhodopsin for wavelength discrimination

with short-wavelength and long-wavelength signals. This hypothesis can be tested through

57

behavioral studies. Food rewards have been used to study visual capability of vertebrates such as

goldfish, but it may be more complicated to elicit a response from snakes, as they have high

chemosensitivity as well. Instead, behavioral experiments may be carried out to take advantage

of the optomotor response, well studied in invertebrates (e.g. McCann and MacGinitie, 1965)

and vertebrates. Behavioral studies measuring optomotor response are carried out in a variety of

vertebrate species, such as newts (Manteuffel and Himstedt, 1978), cichlids (Kroger et al., 2003),

salamanders (Joseph et al., 1973), and the tuatara (Ireland and Gans, 1977). Optomotor (or

optokinetic) responses have adaptive significance for orientation, visual acuity, and

distinguishing moving objects (Walls, 1962). The experimental setup will involve putting a

snake inside an optomotor-drum, a revolving chamber lined with contrasting vertical stripes on

the inside. The drum is spun, and if snakes can distinguish the two colors represented on the

vertical stripes, then behaviors typically associated with the optomotor response can be scored.

Our current evidence suggests that while one population of small single cones may be expressing

RH1 pigments, another population may be expressing SWS1 pigments according to MSP. It is

unknown what proportion of small single cones expresses each pigment, and it is unclear

whether cones expressing SWS1 pigments have different morphological features from cones

expressing RH1. Without knowing these answers, we can not rule out another possibility, that

instead of expressing one type of visual pigments, small single cones could be co-expressing

both SWS1 and RH1 pigments. Co-expression of LWS and SWS pigments has been discovered

in mammalian photoreceptors (Szél et al., 2000; Lukáts et al., 2005). The co-expressed pigments

are both linked to the phototransduction cascade (Lyubarsky et al., 1999; Ekesten et al., 2002),

which turns the cone into a spectral broadband detector (Peichl, 2005). Snakes like T. proximus

could be co-expressing SWS1 and RH1 pigments in their small single cones, perhaps using them

as broadband detectors. MSP data can not rule out this possibility due to limitation of techniques,

since only absorbance data that fit the standard absorbance curve was selected. Co-expression of

RH1 and SWS1 pigments could broaden the absorbance curve detected by MSP, making these

photoreceptors appear to be anomalies. Co-expression of visual pigments has not been reported

in reptilian species, so perhaps the small single cones of T. proximus could be candidates for

future studies on co-expression of visual pigments in reptiles.

In diurnal colubrids such as T. proximus, the LWS pigments are likely the major contributor to

visual signals. Large single cones and double cones could be expressing LWS pigments

58

according to MSP on T. proximus. Combined, these two types of cones make up about 85-90%

of the total photoreceptor population. ERG result from T. sirtalis also indicates that LWS

pigments are the major contributor to retinal gross potential (Jacobs et al., 1992). The other two

pigments, SWS1 and RH1, are likely to be expressed in small single cones of T. proximus. In

basal snakes such as boas and pythons, LWS pigments are expressed in large single cones,

SWS1 pigments in small single cones, but RH1 pigments are expressed in rods. One possible

evolutionary scenario is that these rods secondarily evolved cone-like morphology, as some

caeonophidian snakes took on more diurnal lifestyle. It’s also possible that the rods degenerated,

but the rod-pathways were maintained in a small number of cones. To know the answer, the

visual systems of more nocturnal colubrids should be characterized in the future. For example, a

nocturnal colubrid species called Texas Night Snake (Hypsiglena torquata) is known to have all-

rod retina (Walls, 1942; Stovall, 1976). Studying nocturnal colubrids will provide contrast to the

current results from diurnal T. proximus.

The retinae of T. proximus lack typical rods, but rod pigments could be expressed in small single

cones. The RH1 pigments may still be functional and could provide signal contrast with LWS

and SWS1 pigments. Cellular features such as rod-like outer segment membranes of some small

single cones may be crucial for the proper function of RH1 pigments. Other features such as

microdroplets may be used for correcting Stiles-Crawford effect, while electron-dense pigments

perhaps functional as neutral filters for RH1 pigments. Evidence of transmutation has not been

found in henophidian snakes (Davies, 2009), but our evidences suggest that some T. proximus

small single cones could be derived from ancestral rods. Besides geckos, evidence of

photoreceptor transmutation has not been published in other vertebrate species. There are many

other questions left unanswered by the current data, therefore future experiments are outlined in

the last section of the discussion.

59

4.4 Future Directions

The most immediate goal in the near future should be the expression of T. proximus RH1

mutants. Several sites have been identified in the RH1 sequence, some of which have been

known to have spectral tuning properties (e.g. 83 and 292), others are unique to the western

ribbon snake (e.g. 185). Three mutants, N83D, S185C, and S292A, have been generated and

cloned into p1D4 vector (Figure 21). By in vitro expression of these RH1 mutants for site 83 and

292, it is expected that λmax will be shifted closer to 500nm. While mutants for site 185 may or

may not have spectral tuning properties, it is important to measure the change in meta-II decay

rate and protein kinetics, as varying this site may have functional consequence for the visual

pigment beyond spectral tuning.

Figure 21. Amino acid sequence alignment of wild-type T. proximus RH1 (CY527mx), mutants

(CY378mp2, CY528mx, CY534mx) and expected RH1 sequence in p1D4 expression vector

(Tpx_RH1v). CY378mp2 is miniprep DNA that has N83D mutation; CY528mx is maxiprep

DNA that has S185C mutation; and CY534mx is maxiprep DNA that has S292A mutation.

These sequences are derived from contigs using sequencing results. The RH1 gene is denoted by

a black line with boxed area representing the transmembrane domains. The 1D4 epitope is

denoted by a double-line. The restriction sites used for cloning are represented by the black and

grey triangles. The stop codon is denoted by an asterisk. Vector sequences that are not part of the

in vitro expressed protein are not denoted.

60

Further immunohistochemistry study should be carried out on the T. proximus retina, using

antibodies targeting rod-specific transducin (K-20), cone-specific transducin (I-20), as well as N-

terminus of rhodopsin. Even though the preliminary staining result shows that the rod-specific

transducin antibody K-20 labeled some photoreceptors in the T. proximus retina. In future

staining, an effort can be put into distinguishing the cell-type of labeled photoreceptors, perhaps

by measuring the size of outer segments.

The ophidian double cones were thought to be evolved within snakes, although double-unit

photoreceptors have been found extensively in teleosts, amphibians, reptiles, and birds

(Underwood, 1951; Walls, 1967; Crescitelli, 1972). There have been speculations on the function

of double cones, such as increasing packing density (Pedler and Tilly, 1964), analyzing polarized

light (Underwood, 1970), sampling a limited visual field with two instruments (Cohen, 1963), or

detecting intensity differences (Stovall, 1976). In T. proximus, the principal member of double

cones have the same ellipsoid region as large single cones, while the accessory members lack

microdroplets, like small single cones. The two members of the double cones may combine their

absorption signals, one for the intensity of light while the other for the wavelength information.

There is still much to learn about these double cones.

TEM have also captured some very small single cones as they appear as budding photoreceptors

(Figure 22). The very small single cones are another mystery, as they lack outer segment

membranes and do not produce a MSP signal. One possibility is that these very small single

cones are the precursors to the accessory members of double cones. Since large single cones

(Figure 13) and principal double cones (Figure 12) appear to have similar ellipsoid regions, a

double cone is perhaps formed by one large single cone and one very small single cone. To test

this hypothesis would require a better understanding of the developmental stages of these

ophidian photoreceptors.

61

Figure 22. Transmission electron microscopy of a tangential section of T. proximus retina,

Arrows indicate the very small single cone. Note the absence of outer segment membranes. Scale

bar = 5um.

62

Developmental studies of photoreceptors have been carried out in visual system of migratory fish,

such as rainbow trout. Rainbow trout (O. mykiss) is known to degenerate their SWS1 cones and

lose UV sensitivity before migration from rivers to ocean (Hawryshyn, 2000). This

transformation is called Smoltification, and it is triggered by elevated thyroid levels.

Smoltification and the loss of UVS cones can also be induced by treating juveniles with thyroid

hormone (Browman and Hawryshyn, 1994). Thyroid hormone levels control the shedding

behavior in snakes (Chiu and Lynn, 1971). Garter snakes are known to get blue-eyed 4 days

before skin shedding (King and Turmo, 1997). Clear-eyed garter snakes have different visual

capabilities when compared to blue-eyed individuals, such as shorter latency to move, but blue-

eyed snakes have significantly greater response distance (King and Turmo, 1997). It is possible

that cycling thyroid hormone levels play an important role in the visual system of snakes, which

may be measured by the relative expression level of visual pigments with respect to

developmental events. Developmental study of the photoreceptors may be difficult to carry out,

but it will provide useful information about the cell-fate of T. proximus photoreceptors.

One more observation on T. proximus visual system should be noted: the shape of the T.

proximus lens. During dissection of the retina, the lens appears to be a spherical structure, which

is confirmed under SEM (Figure 23). Cracking of the lens is likely an artifact of SEM

preparation, but it reveals the layered structure of the T. proximus lens. Spherical lenses are not

common in vertebrates, except in fish. Spherical lenses have different refractive properties from

the thinner, more elastic ones, like those found in mammals. Attached to ciliary muscles, the thin

lens can be stretched to accommodate focusing on different depths of view. Garter snakes like T.

proximus do not have ciliary muscles; instead they have a spherical lens which can be moved in-

or outward of the focal axis (Walls, 1942). The spherical lens of fish, such as those found in

carps, have been recently shown to have multifocal properties (Malkki and Kröger, 2005). The

spherical lens of garter snakes is unique amongst reptiles (Walls, 1942). Like many other unique

features of the snake visual system, they have evolved as snakes have to adapt to new habits and

different lifestyles. Studying their visual system will shed light on the evolutionary history of

snakes, and will contribute evidence to solving the phylogenetic relationships between snake

species.

63

Figure 23. Scanning electron micrographs of the lens of T. proximus. It has a spherical shape,

with cracking induced from SEM preparation. Calibration bar: 0.1mm.

64

Appendix

Appendix 1. Amino acid sequence alignment of SWS1 expressed in T. proximus, X. unicolor, P.

regius, U. stabriana, and G. gecko. The transmembrane domains are denoted by the black boxes

below the alignment, which are predicted for T. proximus SWS1 gene online using TMHMM

Server Version 2.0 (http://www.cbs.dtu.dk/ services/TMHMM/). In the alignment sequences, a

dot represents the same amino acid as T. proximus SWS1, while a line represent a gap. Residue

F86 of TM2 is boxed with solid line.

65

Appendix 2. Amino acid sequence alignment of LWS expressed in T. proximus, X. unicolor, P.

regius, O.anatinus, and T. guttata. The transmembrane domains are denoted by the black boxes

below the alignment, which are predicted for T. proximus LWS gene online using TMHMM

Server Version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). In the alignment sequences, a

dot represents the same amino acid as T. proximus LWS, while a line represent a gap.

66

Appendix 3. Amino acid sequence alignment of T. proximus SWS1 clone (CY541mx) and

expected SWS1 sequence in p1D4 expression vector (Tpx_SWS1v). CY541mx is maxiprep

DNA, with one putative polymorphism I205V. The sequence for CY541mx is derived from

contig using sequencing results. The SWS1 gene is denoted by a black line with boxed area

representing the transmembrane domains, which are predicted online for T. proximus SWS1 gene

using TMHMM Server Version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). The 1D4

epitope is denoted by a double-line. The restriction sites used for cloning are represented by the

black and grey triangles. The stop codon is denoted by an asterisk. Vector sequences that are not

part of the in vitro expressed protein are not denoted.

67

Appendix 4. Amino acid sequence alignment of T. proximus LWS clone (CY344mp1) and

expected LWS cDNA sequence (Tpx_LWS). CY344mp1 is miniprep DNA in pJET cloning

vector, with three putative mutations: D18E, I146T, and S244T. The sequence for CY344mp1 is

derived from contig using sequencing results. The LWS gene is denoted by a black line with

boxed area representing the transmembrane domains, which are predicted online using TMHMM

Server Version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). There are three amino acid

differences between the two sequences, none of the three residues from CY344mp1 were found

in other snake species (Appendix 2). Therefore these should be mutated back to wild-type

residues before protein expression.

68

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