Mclemore_immune Genes In Whales

Identification and Isolation of Stress Related Genes in Grey (Eschrichtius robustus) and Fin Whales (Balaenoptera physalus)

FISH 495 Research Paper Christin McLemore June 04, 2009 School of Aquatic and Fishery Science University of Washington Box 355020 Seattle, WA 98195

Abstract Marine mammals such as cetaceans have been a symbol of traditional and modern day Pacific Northwest for centuries, from Native American aspects of divinity to current day elementary school mascots. Most of these majestic symbols include Beluga whales (Delpinapterus leucas), Killer whales (Orcinus Orca), Gray whales (Eschrichtius robustus), Fin whale (Balaenoptera physalus), and all of which fall under two suborders of the Order Cetacea, Odontoceti (tooth whales) and Mysticeti (baleen whales). Odontoceti and Mysticeti have diverse ecosystems from as far south as the coast of California to the most northern latitudes in the Arctic Ocean. For over a decade, concerns over pollutants, climate changes and diseases in the ecosystems have played an important role in causing a decrease in populations of cetaceans. Currently, a majority of efforts to identify environmental stress in cetaceans are the development of functional assays and molecular techniques. In this study, we will be identifying and isolating stress related genes from Grey and Fin whale tissue.

Introduction The North Pacific is home to over 80 species of cetaceans which are composed of whales, dolphins, and porpoises (Stoneg et al. 1990). Some of the most well known cetaceans of the North Pacific are Beluga whales (Delpinapterus leucas), Killer whales (Orcinus Orca), Gray whales (Eschrichtius robustus), Fin whale (Balaenoptera physalus), and all of which fall under two suborders of the Order Cetacea, Odontoceti (tooth whales) and Mysticeti (baleen whales). Odontoceti and Mysticeti species are wide-ranging and respond to the variability in marine ecosystems by changes in distribution patterns (Forney 2000). Odontoceti, such as Beluga whales and Killer whales, have many types of distribution and migratory patterns. Beluga whales migrate very small distances from the coastal shores of Northern America to the open waters of the Arctic Ocean. During the summer Belugas take advantage of the coastal waters to feed and rear their calves and later are seen in groups in the open Arctic during winter. Killer whales distribution patterns can be seen throughout the world’s

oceans, though they are more abundant in cooler waters (Baird 2002). Killer whales seem to migrate in a pattern that allows them to follow their prey, but not to find suitable breeding and rearing sites like other odontoceti's and mysticeti. Unlike Odontoceti, the distribution and migratory patterns of Mysticeti include the longest known annual movements of any mammal (Figure 1) (Stone et al. 1990). Fin whales use the northern and southern hemisphere to feed and subtropical areas near California to breed and rear their young. In comparison Gray whales have a very distinct migration that follows the same pattern year after year. Gray whales migrate annually between summering areas extending from the Pacific Northwest to Alaska, feeding on epibenthic amphipods, planktonic mysid shrimp and crab larvae (Reeves. 1987), and then migrate to wintering areas that extend from southern California to Mexico (Moore, 2008) to breed and rear their calves.

Figure 1: Map of the North Pacific showing the distribution and migration route of the eastern North Pacific population of grey whales. (Reeves 1987)

For over a decade, pollutants, climate changes and disease have played an important role in causing a decrease in populations of cetaceans. When Mysticeti migrate, they are exposed to different levels and compositions of contaminants at different life stages and different times of the year (Brunstrom et al. 2000). Pollutants such as organochlorine compounds like DDTs, hexachlorocyclohexanes (HCHs), chlordane compounds (CHLs) and polychlorinated biphenyls (PCBs) can disrupt normal endocrine physiology in marine mammals (Tanabe, 2002). Substantial evidence points to organochlorine compounds having, at environmental levels, a number of adverse effects on marine mammal populations. These include depression of the immune system, subsequent triggering of infectious diseases, reproductive impairment, lesions of the adrenal glands and other organs, cancers, alterations in skeletal growth and ontogenic development as well as the induction of bone lesions (Aguilar et al. 2002). Currently, a majority of efforts to identify environmental stress in cetaceans are the development of functional assays, such as the lymphocyte transformation assay enables the investigation of T and B cell immune response. Furthermore, natural killer cell activity, respiratory burst as well as the phagocytic and cytotoxic activity of leukocytes can be measured by immunological methods in different cetacean species (Beineke et al. 2006). Humoral immune responses can be investigated by measuring different immunoglobulins in the serum of marine mammals, using radial immunodiffusion. Additionally, molecular-techniques, such as reverse transcription-polymerase chain reaction (RT-PCR) enable the detection and quantification of gene expression of pro- and anti-inflammatory cytokines in activated lymphoid cells during inflammation or immune responses (Beineke et al. 2006). One approach that would complement these efforts is using transcriptomic analysis to characterize a more global response in immune responses and other stressors.

In this study, we will be identifying and isolating stress related genes from Grey and Fin whale tissue.

Materials and Method DNA Extraction Grey whale tissue was obtained from Jina Ylitalo at the National Oceanic and Atmospheric Association (NOAA) in Seattle, Washington. Fin whale tissue was obtained from Greg Jensen at the School of Aquatic and Fishery Science in Seattle, Washington.Q Genomic DNA was then extracted from both the Grey and Fin whale tissue by following the procedures of a DNeasy Tissue Kit. Tissue stayed overnight in a 55o C warming plate to breakdown thick tissue further. Then the DNA of Grey and Fin whale was quantified by using a spectrophotometer, with grey whale at 142.85 ng/ul and fin whale at 49.51 ng/ul. Primers Degenerate PCR primers were designed in Geneious from the genes of interest of bottlenosed dolphins, Tursiops truncates (see Table 1). These genes of interest include glutathione S-transferase A4 (GStra), Superoxide dismutase [Cu-Zn] (SD), Interleukin-2 precursor (IL-2), heat shock 70kDa protein 6 (HSP6), interleukin 17 (IL17), interleukin 13 precursor (IL13), Toll-like receptor 5 (TLR5) obtained from a BLAST done on the National Center of Biotechnology Information (NCBI), and cyclooxygenase (COX-2) obtained from a study by Sitt et al. 2008.

Table 1 Degenerate Primers from Bottlenose dolphin, Tursiops truncatus Gene

Species

Primer

Sequence: 5’ to 3’

GStra

Dolphin

HSP6

Dolphin

SD

Dolphin

IL17

Dolphin

IL13

Dolphin

TLR5

Dolphin

IL2

Dolphin

COX2

Dolphin

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

GCAAGGCTCAGCTGATTACC TCAGCAAAAGGAAGTGGTGA CCAAGCAGACCCAGACTTTT ACCCTCATACACCTGGATCG CGGGATCCATTACAGGATTG CATTCTGGTCAGCCTTCACA CCACCTCACCTTGGACTCTC TCCCTTCAGCATTGACACAG CACAGAGGTGCCTTCTGGTT CTCCCTCTACAGCCCTCAAG CACCACCCGTTTTCTAAGGA GGAGCAGCAGAAAGGTTGTC CCTCTGGAGGATGTGCTAAA ATCGAGTTCTGGGTTTTGCTT GAGCTCTTCCTCCTGTGSCTGA CTTTGRCTGTSGGMGGATACAYCT

Amplicon Length (bp) 469 523 548 324 279 365 287 353

PCR and Gel Electrophoresis PCR proceeded as follows: Master mix included 50.0 ul of 2x GO TAQ Mm, 2.0 ul of each of the forward and reverse primers, and 42.0 ul of distilled water. A master mix was made up for each individual forward and reverse primers of GStra, HSP6, SD, IL17, IL13, IL2, TLR5, and COX-2. 24 ul of the master mix was then added to a microcentrifuge tube containing 1 ul of either the genomic DNA of the grey whale or fin whale and a control (water). In all, each microcentrifuge tube contained 25 ul of genomic DNA and the master mix. Reaction conditions were as follows: 95o C for 10 min., 40 cycles of 95o C for 45 sec., 55o C for 1 min., 72o C for 1 min., and then 72o C for 10 min. A gel was made from 1.5 grams of Agarose with 100 mL of 1% TAE and then infused with 6 ul of ethidium bromide. The lanes were loaded with 25 ul of the samples and let run for 1

hour at 115 volts. Once completed, bands were then cut out of the gel, placed in a microcentrifuge tube and put in a -20o C freezer. The extraction of the genomic DNA from the gel previously stored was completed by using a Millipore Ultrafree-DA kit. The centrifuge was set at 5,000 rpm for 10 minutes allowing DNA to move out of the agarose and through the Ultrafree-MC and gel nebulizer to the bottom of the microcentrifuge tube. The purified genomic DNA was then sent off to be sequenced.

Sequence Alignments Each sequence was BLAST in NCBI and the top blast was then aligned with the sequences in Geneious. Annotations were then made on the sequences of where they laid in the alignments for introns and exons.

Results DNA Extraction Once DNA was extracted from the provided tissue of Grey and Fin whale, quantification of the DNA was necessary. The Grey and Fin whale DNA was then quantified by using a spectrophotometer, showing Grey whale at 142.85 ng/uL and fin whale at 49.51 ng/uL. PCR and Gel Electrophoresis Bands were identified by electrophoresis for the genes of interest. In Figure 1, Row 1: lanes three (bp~850) and four (bp~850) containing Grey and Fin whale Gstra, lane six (bp~350) containing Grey whale HSP6, lane nine (bp~1000 and bp~300) containing Grey whale SD (for the 1st and 2nd band), fin whale SD in lane ten (bp~1000) and lastly in Row 2: lane three (bp~800)

and four (bp~800) for Grey and Fin whale COX2, bands where cut out of the gel and turned in for sequencing. In Figure 2, lane three (bp~1200) and four (bp~1200) of Grey and Fin whale IL17 and lanes six (bp~600) of Grey whale IL13 and lane seven (bp~450 and bp~250) fin whale IL13 (for 1st and 2nd bands), bands were also cut out for sequencing.

Figure 1: Row one: Lane (1)100 bp ladder, (2) Negative control Gstra, (3) Grey whale Gstra, (4) Fin whale Gstra, (5) Negative control HSP6, (6) Grey whale HSP6, (7) Fin whale HSP6, (8) Negative control SD, (9) Grey whale SD, (10) Fin whale SD. Row 2: Lane (1) 100 bp ladder, (2) Negative control COX2, (3) Grey whale COX2, (4) Fin whale COX2.

Figure 2: Lane (1) 100 bp ladder, (2) Negative control IL17, (3) Grey whale IL17, (4) Fin whale IL17, (5) Negative control IL13, (6) Grey whale IL13, (7) Fin whale IL13, (8) Negative control IL2, (9) Grey whale IL2, (10) Fin whale IL2, (11) Negative control TLR5, (12) Grey whale TLR5, (13) Fin whale TLR5, (14) 100 bp ladder.

Sequences and Alignments After sending the samples of Grey and Fin whale genomic DNA off to be sequenced only IL17 Grey whale forward and reverse (1267 bp), IL17 Fin whale forward and reverse (1287 bp), SD Grey whale reverse (377 bp), COX2 Grey whale forward and reverse (1525 bp), GStra Grey whale forward and reverse (512 bp), and GStra Fin whale reverse (537 bp), were able to be sequenced. Each sequence was BLAST in NCBI and the top blast hits were then aligned with the sequence in Geneious. The top blast hits, for each gene sequence, accession numbers are; IL17:

AY460616, SD: BC122671, COX2: AY229989, GStra: AY878121. Annotations were then made on the sequences to show non-coding (introns) and coding (exon) regions. As seen in Table 2, The Grey whale IL17 consensus sequence made from the forward and reverse sequences were aligned with a Homo sapien interleukin 17 (AY460616) between the base pairs of 1,729 - 3,093. Within this alignment, a coding region (exon) was annotated onto the Grey whale gDNA sequence between base pairs of 194 - 214. Fin whale IL17 forward and reverse sequences were also aligned with the Homo sapien interleukin 17 (AY460616) between the base pairs of 2,385 – 2,756. The sequence of the Fin whale IL17 was annotated to have a coding region between the base pairs of 1245 – 1287. The Grey whale SD consensus sequence for the reverse sequences was aligned with the Bos taurus alanyl-tRNA synthetase (BC122671) and was annotated to be a non-coding sequence. The Grey whale COX2 consensus sequence for both forward and reverse sequence was aligned with the Homo sapien prostaglandinendoperoxide synthetase-2 (AY229989) between the base pairs of 4,041 – 5,563. Within this alignment, a coding region was annotated on the sequence between base pairs of 1 -8 and 13491432. Lastly, the Grey whale GStra was aligned with the Homo sapien glutathione S-transferase A4 (AY878121) between base pairs of 12,394 – 12,942. Within this alignment, a coding region was annotated on the sequence between the base pairs of 1 - 29. The Fin whale GStra sequence was also aligned with the Homo sapien qlutathione S-transferase A4 (AY878121) between the base pairs of 12,790 - 13,423 and was annotated to have a coding region between the base pairs of 287-425.

Table 2: Align regions of Grey and Fin whale IL17, COX2, SD, GStra with the top blast hits found in NCBI.

Reference Reference Taxa Size

Grey Whale Coverage

Fin Whale Coverage 2,385 – 2,756

Interleukin 17

Homo sapien (AY460616)

7,422

1,729 - 3,093

Prostaglandinendoperoxide synthetase-2

Homo sapien (AY229989)

11,449

4,041 – 5,563

tRNA synthetase

Bos taurus (BC122671)

1,538

1 - 360

Glutathione Stransferase A4

Homo sapien (AY878121)

21,305

12,394 – 12,942

12,790 - 13,423

Sequences that were extracted from Grey (Eschrichtius robustus) and Fin whale (Balaenoptera physalus) tissues are as follows: > Eschrichtius robustus Interleukin17 (exon # range 194 - 214) CTCCAGATCACAGAGGGGTACCTCTCAGGGTCCTCATTGCGGCTATAAGG GAGACAAAAGCCAGGAAGAGAGTGAAATCCTCGTAGAGCAGGAAGGAAAT CAAACAGGAATAAACAAAGGGAGACAAATTCACAAGGGTGCAAATGATAG GCATTGTGAATTCTCTGAGTGCTGTTAGTCTGAAAAAGAAGTAGTTTGGG GAAAACTTGTGTTTGCAAGCCCATGAAGTGTTATAGAAAAAAAGACAATG ATTAGTTTTTCCCAATTTCTTTCCAACATACACAAAAGAAGATAGACTGA AACTACAGAATGGATTTAAGTTTGACACAAAGGTTAATTTGTTTAGGGTA CATTTGATGGATGAAAAAAGACTAAAGGAATAGTATAGAAACAAGATAAC TGTTAATCTGACTTAATGAAGTCTGTCTTTGAAACAGAGAATGCACCCAA ATTTTATCTGACAGCACGCTGATGGAACAGAGCCCACGGCTGATATGAAG GGCTGCAAACAAAATCTTAAGCCCCTTTATAACCTTCTCTTCTCACTCCC CTCCAGCTGAGTGAATAATTTGCTTCGACTTCTGAATTAATTTATTTGAA CTATTCTTAGTAAGTAGAAGTAAGAAGACAAGAAACCCTACAGTCAGGTG AATCTGTAGGCTCCATATAACTGAGGTTTATTTGCCTAAAATCAAATTAT CAAAATACATTTTGTACTTAATCCATCTAAATACTAGATGTACTAAGGAC ACAGCAAANTATTGKTCTGTTGGGGATGTTGTCTAAGTCAAAAAAGGGTG GAAATACCCAGCATGATACTTCTTCCACTGGTATTCAAGCTATATTTTCC CAGTGTTAATACAATCAAAAAAGCACAAAAATCCCAGTTAATTAATAGTT ATTTTCAACAAATCTTCTCTGGGAGCTCTGGTAAAGAGAATTCAAGCACT AATCTCCTACGATAAATGTCTATTTCTTTTAAAAATAAATAAACAACAGA CTTGGGGTGTCATAAATATGAGAATGATGACCACCGTTCATTTGGATGGA TTTCTGGGACAATGAGAACTAGTAGAATAGGTGTTATGGGGGGTGGGGTG AGCTGTGCTCTATCAAAGAAATCTGTAGAATGAAAGTTTGAGAATCATTG

GTTTACACAATGGCCCTTCCAGTTTTGACGGATGATTCTGAGTGAGTAAC ACCATTCTCAAGGTGATTTAACTGGCAGTCTGATACATACAGGGTAGAAG AATACTGAGATTATCCT >Balaenoptera physalus Interleukin 17 (exon # range 1245 – 1287) AGGATAAATCTCTGTATTCTTCTACCCTGTATGTATCAGACTGCCAATTA AATCACCTTGAGAATGGTATTACTCACTCAGAATCATCCGTCAAAACTGG AAGAGCCATTGTGTAAACCAATGATTCTCAAACTTTCATTCTACAGATTT CTTTGATAGAGCACAGCTCACCCCACCCCCCATAACACCTATTCTACTAG TTCTGATTGTCCCAGAAATCCATCCAAACGAACGGTGGTCATCATTCTCA TATTTATGACACCCCAAGTCTGTTGTTTATTTATTTTAAAAAGAAATAGA CATTTTTCATAGGAGATTAGTGCTTGAATTCTCTTCACCAGAGCTCCCAA AGAAGATTTGTTGAAAATAACTATTAATTAACTGGGATTTTTGTGCTTTT TTGGTTGTATTAACACTGGGAAAATATAGCTTGAATACCAGTGGAAGAAG TATCATGCTGGGTATTTCCACCCTTTTTTGACTTAGACAACATCCCCAAC AGACCAATATTTGCTGTGTCCTTAGTACATCTAGTATTTAGABNNATNAA GGGTTAAATACNAAATGTATTTTGATAATTTGATTTTAGGCAAATAANNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNTCCATCAAATGTACCCTAAACAA ATTAACCTTTGTGTCAAATTTAAATCCATTCTGTAGTTTCAGTCTATCTT CTTTTGTGTATGTTGGAAAGAAATCGGGAAAAACTAATCATTGTCTTTTT TTCTATAACACTTCATGGGCTTTGAAACACAAGTTTTCCCCAAACTACTT CTTTTTCAGACTAACAGCACTTAGAGAATTCACAATGCCTATCATTTGCG CCCCTGTGAATTTGTCTCCCTTTGTTTATTCCTGTTTGATTTCCTTCCTG CTCTACGAGGATTTCACTCTCTTCCTGGCTTTTGTCTCCCTTGTAGACGC TAATGAGGACCCTGAGAGGTACCCCTCTGTGATCTGG > Eschrichtius robustus Prostaglandin-endoperoxide synthetase-2 (exon # range 1 -8 and 1349-1432) GTGTGAAGGTGAGTGAGAGGAAGCAATTAGACATGTATTAACTGTAACTG AGATTGGATTGCTACCTAGAAAATTTGACCATAAACCATGATTTATTTGT TCATAAAGCCATTTTTTCCTTATGGATATCTCTGGAGATTTTTTGAAAGT GAGTCCATGTAAACTACTCCATGAAAGTGAATGGGCATTTTGGTATAATG GCATGATGTTTTCAAATTCTATCATTAAAACTGAATTAATGCTGTAAACA AAACANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAGGT GGACTTAAATCATGTTTATGGTGAATCTTTGGAGAGACAGCATAAACTGC GCCTTTTCAAGGATGGAAAAATGAAATATCAGGTTTGTCCCATTTAAATA TTAAGAATTAGTTATGACTCACCACCCACCTATGTTCAAAATCTCTCATG ATTAAAATTTGTTCCTTACCTTTTT > Eschrichtius robustus tRNA syntetase CTGTGGGAATTAATTGGTGCTAAGGTCTAACATTGTTCTAATCTGAGTGC CAGGGAATGTTTTCTGTCCCTTCTCACCCCTACATGCTTGTGGTATTTTT TTTTTATCTTAGGGAATTCCCTGGCASGACCGTGGCTAAGGCTCTGCGCT TTCACTGCCAAGGGTGCAAGTTAAATCCCTGGTCAGGGAACTAAAATCCC ACAAGCTGTGCTGTGTGTCCCCCGCCCCCCCCCCCCAAAAAAAAAAAAAA AACCYCAGGCATTTTTAATTTTTTGGGGCCCAACCCCTTAGGGGACCCCG AATCCATCTCAGGACCTTTGCACTTACTAGTTCTGCTTGAAACCCTCTTT CCTCAATCCTGTAATGGATCCCGATTT > Eschrichtius robustus Gstra (exon # range 287-425) TGACCCCGTAANACCTGCAGGGTATAAACATTTTACATTGGTTATAATAC TTGAGATGGAGAAAATGATGGAGCTGTGGAATCAGTGCAGTATCTGAGAA GGGCTGAAACGACGACCTTGTCCTTTTATCCTTTTACGAATAACATCAAC TAAGAAGAAGTTCCCTATATGAGAGGGAGAGAGCGAAAAAGGTGTAGAAA TGAAAGCATCCTTAAGTATCTGCTTTACATCAGAATGAGACCCTAAAAAG GGGAAGGAAATATTAATTCTAGATTTAGCATCCAGGGCTTTAAAAGGATA CCTCTATTCCATAAATGTATCTACAGAAAAGAATTAGCCCTGATGCATTC AGGGGAAGTGATGTAAGACGTAAAGCCACTGGTCCTGTCCAAGGAGTCTA ATCTCATTTGCCAGTTCTGTGTTTCTGTGTGTCAGGACTCACAGTCATAA ACTAGACATGACAGTAAAAAAGCAGGCTTCCTTTCACGTCTTCTAAGACA GGGAAAATTAAA

>Balaenoptera physalus Gstra (exon # range 1-29) AATWAGATACTTTCCTGTGTTCGANAAAGGTAGGTGGCAGTGAAGCCTGT GAACGCGGCATCACCAGACCACAAACGCCCCTTTCTGCTCAGGCAGAGTA AAGCTGAGCCCTGCTAGACCAACCTCCTGAGGGCTCACAGGTGCCATCAG CACAAAGCCAGGATGTCTTGGAGCCGCCCAGACTCTTCCAGAGTCTTCCA TGTTTTCTGGGAAAAATATGGGCTCTCCCACGTGTGCATTGTCATTGCTT ACATTCGCCAGGTGCCACCTTGTGCAGGGTACAGAAGAGAAGAAGCCACC TACTTCATTCATTCGTTCATCATCCTACCTGATAGTTTGGTATAAAGAGA AGAACCATTGCTGTTTTATTTCTCTTCCCTGTGCTCTTTTTGTTTTCTCT TTTGTTACTTCTTGTCTCATTGCCGTATCTGTTTTTCTGAGCTTCAAACT CCTCCTCTCTTTTCCTCAACTGAGAGGGACTATTTTAAATGCTTCAATTA AAAAACAAGAGAACGTGAAAATCTGGAATTGGCAGAC

Discussion Four genes that are associated with an immune response were identified in Grey and Fin whale tissue. These genes are Interluekin 17, Prostaglandin-endoperoxide synthetase-2, tRNA synthetase, Glutatione S-transferase A4, and a description of them are as follow: Interluekin 17 Interluekin (IL) -17 is expressed by activated memory T-lymphocytes which helps stimulate the innate immunity and host defense (Kolls and Linden, 2004). IL-17 receptor protein also can be found in peripheral blood T lymphocytes and in vascular endothelial cells from humans (Moseley et al. 2003), as well as, the mRNA for IL-17R in humans, can be found in epithelial cells, fibrobalsts, B and T lymphocytes (Silva et al. 2003). As in this study, we found that IL-17 was being produced in the epithelial cells and underlying blubber of both Grey and Fin whales showing homology for this gene in human and cetaceans. Increasing scientific evidence is proving that "IL-17 family members are playing an active role in inflammatory diseases, autoimmune diseases, and cancer" (Kolls and Linden, 2004). Prostaglandin-endoperoxide synthetase-2 Prostaglandin endoperoxide sythetase-2 (PGES), also known as Cyclooxygenase-2 (COX-2), is classified as a pro-inflammatory enzyme and is known for its role in treating inflammatory diseases (Gilroy et al. 1999). PGES enzymes belong to the superfamily of glutathione S

transferase (GTS) and “requires GSH as an essential co-factor for activity” (Helliwell et al. 2004). Glutathione S-transferase in this study was found in both Grey and Fin whale tissue while PGES was only found in Grey whale tissue. tRNA synthetase Aminoacyl-tRNA synthetase is a group of enzymes that are structurally diverse. Functions of aminoacyl-tRNA enzymes are promoting protien synthesis (Chang and Dignam, 1990) and cell viability (Park et al. 2008). Bacteria and fungi both regulate aminoacyl-tRNA in high numbers in response to several physiological conditions including amino acid restriction, growth rate, heat shock, and nutrient limitation (Chang and Dignam, 1990). Glutatione S-transferase A4 A significant feature of glutatione S-transferase A4 is its high catalytic efficiency for cytotoxic and mutagenic products of radical reactions and lipid peroxidation (Hubatsch et al. 1998). Studies done by Xu et al. 2007, Yang et al. 2004, and Misra et al. 1995, have shown that GSTA4-4 plays a significant role in protecting endothelial cells and vascular smooth muscle cells from damage by toxins and oxidants. Furthermore, GST families can “detoxify chemical carcinogens, environmental pollutants, and antitumor agents", as well as, "inactivate endogenous α,β-unsaturated aldehydes, quinones, epoxides, and hydroper-oxides formed as secondary metabolites during oxidative stress" (Xu et al. 2007). Phylogenetic trees can further show the divergence of these immune related genes of interest. A neighbor-joining tree were created for each gene of intersest, Interleukin 17 (Figure 3), Prostaglandin-endoperoxide sythetase 2 (Figure 4), tRNA sythetase (Figure 5), and Glutatione S-transferase A4 (Figure 6).

Figure 3: Homology of the gene of interest Interleukin 17 between Bos taurus, Cervus elaphus, Equus caballus, Mus musculus, Rattus norvegicus, Balaenoptera physalus, Homo sapiens, Eschrichtius robustus.

Figure 4: Homology of gene of interest Prostaglandin-endoperoxide synthetase-2 between Bos Taurus, Eschrichtius robustus, Equus caballus, Homo sapiens.

Figure 5: Homology of the gene of interest tRNA synthetase between Ovis aries, Eschrichtius robustus, Bos taurus, Bos taurus1 (aligned sequence).

Figure 6: Homology of the gene of interest Glutathione S-transferase A4 between Balaenoptera physalus, Homo sapiens, Bos taurus, Sus scrofa, Eschrichtius robustus.

Acknowledgements First, I would like to thank Jina Ylitalo and Greg Jensen for supplying my study with the Grey and Fin whale tissue. Secondly, thank you to Sam White for all of his patience and guidance on experimental protocols and sequencing and thank you to Dr. Steven Roberts for being my faculty mentor throughout my research project.

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