Transcript Marine Mammal Vision Poster

THE EFFECTS OF PHOTIC ENVIRONMENT ON MARINE MAMMAL MELANOPSIN
Vanessa Ortiz 1*, April Triano 2* and Jeffry I. Fasick, Ph.D
1
B.S Biology (Biotechnology Option), Kean University, Union, NJ 07083, USA
2 B.S Biology (General Option), Kean University, Union, NJ 07083, USA
* These students contributed equally to this research.
Studies of the retina from cetaceans (dolphins and whales) have revealed that it is quite different from its terrestrial counterparts in that its visual pigments are strongly blue-shifted (Fasick et al., 1998; Fasick & Robinson, 2000). The blue-shifted rod and cone pigments, as well as the lack of color vision found in the
cetacean eye, are believed to be associated with the relatively monochromatic blue light available to the animals at foraging depth. A recently discovered class of retinal photopigment, melanopsin, has been shown to be closely associated with photoentrainment as well as directing the pupil response to light (Provencio et
al., 1998). Our study is designed to determine which photic environment, surface or depth, is responsible for the spectral tuning properties of cetacean melanopsins. To date we have sequenced approximately 900 bp of the dolphin melanopsin cDNA. Alignments with mammalian melanopsin nucleotide sequences show that
dolphin melanopsin, like the dolphin retinal visual pigments, is most closely related (90 % identity) to melanopsin from even-toed ungulates, such as the domestic cow (Bos taurus). Once the full length dolphin melanopsin cDNA is cloned, we will express, reconstitute and purify the resulting pigment to determine its
absorption spectrum and compare it to the absorption spectrum determined from B. taurus melanopsin.
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• Melanopsin is a novel photopigment
found in specialized photosensitive
ganglion cells of the retina.
• Although melanopsin is not involved
with image formations, it is
responsible for the light activation of
the G-protein Gq typically found in
invertebrates photoreceptors.
GENE ID: 515152 OPN4 | opsin 4 [Bos taurus]
• Align subsets of known vertebrate melanopsin amino
acid and nucleotide sequences
Bovine Melanopsin Amino Acid Positions
61 74
91 195 218
203 411
•Melanopsin is composed of an opsin
protein covalently attached to the
light absorbing chromophore 11-cis
retinal.
• Melanopsin, when activated by
light, sends signals through the axons
of ganglion cells, to specific parts of
the brain including the olivary
pretectal nucleus (a center responsible
for controlling the pupil of the eye)
and the suprachiasmatic nucleus of
the hypothalamus (the master
pacemaker of circadian rhythms).
• Studies of the retina from cetaceans
(dolphins and whales) have revealed
that it is quite different from its
terrestrial counterparts in that its
visual pigments are strongly blueshifted.
•The blue-shifted rod and cone
pigments and lack of color vision
found in the cetacean eye are believed
to be associated with the relatively
monochromatic blue light available to
the animals at foraging depth.
PREDICTED: Bos taurus similar to opsin 4 (OPN4), mRNA
• From these alignments, design degenerate
oligonucleotide primers for use in PCR amplification of
marine mammal melanopsin polynucleotide sequences
Score = 1291 bits (699), Expect = 0.0
Identities = 830/894 (92%), Gaps = 7/894 (0%)
Strand=Plus/Plus
Query
2
Sbjct
486
Query
62
Sbjct
546
Query
122
Sbjct
606
Query
182
Sbjct
666
Query
242
Sbjct
726
Query
302
Sbjct
786
Query
360
Sbjct
844
Query
420
Sbjct
903
Query
480
Sbjct
963
Query
540
Sbjct
1023
Query
600
Sbjct
1083
Query
660
Sbjct
1143
Query
720
Sbjct
1203
Query
780
Sbjct
1263
Query
840
Sbjct
1323
GGCCATCGCCCTGGACCGCTACCTGGTGATCACACGCCCACTGGCCACCGTCGGGATGGT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GGCCATCGCCCTGGACCGCTACCTGGTGATCACACGCCCACTGGCCACCGTCGGGATGGT
61
GTCCAAGAGGCGGGCGGCGCTTGTCCTGCTGGGCGTCTGGCTCTATGCCCTGGCTTGGAG
||||||||||||||| ||||||||||||||||||||||||||||||||||| ||||||||
GTCCAAGAGGCGGGCCGCGCTTGTCCTGCTGGGCGTCTGGCTCTATGCCCTAGCTTGGAG
121
TCTGCCGCCCTTCTTTGGCTGGAGTGCCTATGTGCCCGAGGGGCTGCTGACCTCTTGCTC
|||||||||||||||||||||||||||||||||||||| || |||||||||||||||||
CCTGCCGCCCTTCTTTGGCTGGAGTGCCTATGTGCCCGAAGGCCTGCTGACCTCTTGCTC
181
CTGGGACTACGTGAGCTTCACGCCATCGGTCCGCGCCTACACCATGCTGCTCTTCTGCTT
|||||||||||||||||||||||| || ||||||||||||||||||||||||||||| ||
CTGGGACTACGTGAGCTTCACGCCGTCCGTCCGCGCCTACACCATGCTGCTCTTCTGTTT
241
TGTGTTCTTCCTCCCCCTGGTCGTCATCATCTACTGATACATCTTCATCTTCAAGGCCAT
|||||||||||||||| || || ||||||| ||||| |||||||||||||||||||||||
TGTGTTCTTCCTCCCCTTGCTCATCATCATTTACTGCTACATCTTCATCTTCAAGGCCAT
CCGGGAGACGGGCCAAGCTCTCCAGACTTTCNGGG-CCAGCGAGGGGTGGTGGTGAGTG||| ||||||||||||||||||||||| ||| ||| || |||| |||||| |||||||
CCGAGAGACGGGCCAAGCTCTCCAGACCTTC-GGGACCTGCGA-GGGTGGCAGTGAGTGT
605
665
Provencio et al. 2000
725
301
785
359
419
CATCCTTCTCTTCGTGCTCTCCTGGGCCCCCTACTCCACTGTAGCCCTGATGGGCTTTGC
|||||| ||||| ||||||||||||||||||||||||||||| |||||||||||||||||
CATCCTCCTCTTTGTGCTCTCCTGGGCCCCCTACTCCACTGTCGCCCTGATGGGCTTTGC
479
TGGGTACGCACATGTCCTGACGCCCTACATGAACTCGGTGCCAGCTGTCATCGCCAAGGC
||||||||||||| ||||||||||||||||||||||||||||||||||||| ||||||||
TGGGTACGCACATATCCTGACGCCCTACATGAACTCGGTGCCAGCTGTCATTGCCAAGGC
539
CTCTGCCATCTACAACCCCATCATTTATGCCATCACCCACCCCAAGTACAGAATGGCCAT
||||||||||||||||||||||||||| |||||||||||||||||||||||| |||||||
CTCTGCCATCTACAACCCCATCATTTACGCCATCACCCACCCCAAGTACAGATTGGCCAT
599
CGCCCAGCACCTGCCCTGCCTCGGGGTGCTGCTGGGCGTGTCAGGCCAGCGCACTGGCCT
||||||||||||||||||||| |||||||||||||||||||| |||||||||||||||||
CGCCCAGCACCTGCCCTGCCTGGGGGTGCTGCTGGGCGTGTCGGGCCAGCGCACTGGCCT
659
GTACACCAGCTACCGCTTCACCCACCGCTCCACACTGAGCAGCCAGGCCTCAGACCTCAG
||||||||||||||||| ||| || |||||||| ||||| ||||||||||| ||||||||
GTACACCAGCTACCGCTCCACTCATCGCTCCACGCTGAGTAGCCAGGCCTCGGACCTCAG
719
CTGGATCACTGGACGGAGGCGCCAGGTGTCCCTGGGCTCTGAGAGTGAGGTGGGCTGGAC
||||||| |||||||||||||||||| ||||||||||||||||| |||||||||||||
CTGGATCTCTGGACGGAGGCGCCAGGCATCCCTGGGCTCTGAGAGCGAGGTGGGCTGGAT
779
AGACACGGAGGTAACAGCTGCTTGGGGGACTGCCCAGCAAATGAGCGGGTGGTCCCCCTG
||||| || | |||||||||||||||| ||| |||||| ||||||| |||||||||||
GGACACAGAAGCAACAGCTGCTTGGGGGGCTGGCCAGCAGGTGAGCGGATGGTCCCCCTG
839
3’
5’
843
CCCCTGGCAACGGCAGCGTCTGCAGAACGAGTGGAAAATGGCCAAGATCGAGCTGTTGGT
|||| |||| |||||||| || ||||| ||||||||||||||||||||||||||| ||||
CCCC-GGCAGCGGCAGCGGCTACAGAATGAGTGGAAAATGGCCAAGATCGAGCTGCTGGT
CAGTCAGGGCCTGGAGGATGTGGAAGCCAAGGCCCCT-CCCAAGTCCCANGGAC
||||||| ||||||| ||||||||||||||||||| | |||| | |||| ||||
CAGTCAGCGCCTGGATGATGTGGAAGCCAAGGCCC-TGCCCAGGCCCCAGGGAC
Structure of Human Melanopsin Gene
545
Sense Primer
902
N.T. Position: 423
Anti Primer
1400
962
Squid Rhodopsin (0.3811)
1022
Mouse Melanopsin (0.1360)
Cat Melanopsin (0.0924)
Human Melanopsin (0.1105)
Pig Melanopsin (0.0285)
Dolphin Melanopsin (Tufts_3329_seq.2) (0.0967)
Cow Melanopsin (0.0058)
Horse Melanopsin (0.0269)
Dog Melanopsin (0.0990)
1082
1142
1202
1262
1322
892
1375
• PCR amplify from a dolphin retinal cDNA library and
dolphin gDNA to determine if a) the dolphin possess a
melanopsin-like gene and b) is so, is the gene transcribed
into a functional mRNA transcript
• Clone, sequence & express full-length & truncated carboxyl-tail dolphin melanopsin cDNAs
• Sequence positive PCR products
• Perform sequence analyses to compare the evolutionary rates between the dolphin melanopsin and visual pigment
opsin nucleotide and amino acid sequences
• Incorporate sequence data into blast searches for
identification based on homology
• Perform sequence alignments and construct
phylogenetic trees to determine the evolutionary
relationships to other vertebrate melanopsins
• Perform phylogenetic analyses of full-length dolphin melanopsin nucleotide and amino acid sequences with those of
other vertebrate melanopsins to determine relatedness and evolutionary distances
• Clone, sequence and express full-length & truncated melanopsin cDNA from cetaceans occupying different photic
environments to understand which selection pressure, photoentrainment or pupillary light response, most influences the
melanopsin protein
• Compare marine mammal melanopsin sequences and absorption maxima to each other, as well as with bovine
melanopsin, to identify candidate sites for site-directed mutagenesis in order to determine the amino acids involved with
spectral tuning of the melanopsin pigments
References
Fasick JI, Cronin TW, Hunt DM, Robinson PR. 1998. The visual pigments of the bottlenose dolphin (Tursiops truncatus). Vis Neurosci. 15(4):643-51.
Fasick JI, Robinson PR. 2000. Spectral-tuning mechanisms of marine mammal rhodopsins and correlations with foraging depth. Vis Neurosci. 17(5):781-8.
Provencio I, Jiang G, De Grip WJ, Hayes WP, Rollag MD. 1998. Melanopsin: An opsin in melanophores, brain, and eye. Proc Natl Acad Sci U S A. 95(1):340-5.
Provencio I, Rodriguez IR, Jiang G, Hayes WP, Moreira EF, Rollag MD. 2000. A novel human opsin in the inner retina. J Neurosci. 20(2):600-5