Characterization of the Leptin Receptor (Lepr) Isoforms in Bowhead Whales using
RACE and cDNA Sequencing
Rachel Sarah Bright
Department of Biology
Honors Research Project
Submitted to
The Honors College
Approved:
Accepted:
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ABSTRACT
Leptin and its receptor play a key role in adipose tissue regulation. The whale,
Balaena mysticetus (Bowhead whale) has vast amounts of adipose tissue (blubber)
needed to survive the arctic waters and thus represents an extreme organism with respect
to how fat must be regulated. To better understand how this whale may regulate this
large blubber mass further characterization of the leptin receptor gene (Lepr) and its
different isoforms was undertaken. Sequences from GenBank of ungulates were aligned
and used to predict what the sequences of the bowhead whale could be, given that whales
are related to ungulates. To demonstrate the actual presence of these isoforms in whales,
rapid amplification of cDNA ends (RACE) was performed on RNA extracted from
bowhead kidney tissue. Resulting products were cloned, and the nucleotide sequences
determined. Multiple attempts to derive these isoforms from RNA samples proved
problematic. However, a PCR on extracted genomic DNA was successful and produced
a 230 base pair sequence. This sequence was aligned and identified to be most similar to
a portion of the long isoform (ObRb) of ungulates. This sequence confirms the presence
of a receptor gene in bowhead whale similar to that of ungulates as predicted.
2
ACKNOWLEDGMENTS
I would like to thank Dr. Joel Duff and Hope Ball for giving me the opportunity
to participate in this project. Thank you to Dr. Stephen Weeks for organizing the Tiered
Mentoring Program, which sparked my interest in this project and helped me to better my
understanding of genetics and molecular techniques used in a research setting. I thank Dr.
Duff for being supportive on my path to dental school and being both a role model and
mentor. I thank Hope Ball for being so patient and resourceful. Your help with learning
in the lab and making sure everything was going smoothly cannot be thanked enough. I
thank Dr. Hans Thweissen for collecting the Bowhead samples from the Inuit hunts. I
want to thank my project readers: Dr. Rich Londraville and Dr. Francisco Moore. Also,
thank you to the Department of Biology chair, Dr. Monte Turner and my department
Honors Faculty Advisor, Dr. Jim Holda. Thank you to the Honors College for overseeing
this process and encouraging student faculty relations. Lastly, I thank my parents Ellen
and James K. Bright for their support through my undergraduate career and with this
project. I would not be nearly as successful as a student without your support from an
early age and I love you very much. As I continue on to the Ohio State College of
Dentistry this experience will remain as my most valuable and difficult one that I
encountered as an undergraduate.
3
TABLE OF CONTENTS
LIST OF FIGURES ..................................................................................................................5
LIST OF TABLES ...................................................................................................................6
CHAPTER
I. INTRODUCTION ..........................................................................................................7
II. MATERIALS AND METHODS ................................................................................15
III. RESULTS ....................................................................................................................29
IV. DISCUSSION AND CONCLUSIONS .....................................................................33
REFERENCES CITED ..........................................................................................................35
4
LIST OF FIGURES
1. Graph of the structure of the alternately spliced leptin isoforms (Ahima and Osei,
2004).
2. Flow chart of biological responses to varying adipose tissue and leptin levels
(Friedman, 2002).
3. Presence of Lepr isoforms in Bos Taurus tissues Kawachi et al 2007.
4. Map showing location of bowhead whale populations (Braham et al. 1984)
5. Sequencher® alignment of cow mouse sheep and human to make primers.
6. 5’ RACE scan
7. 3’ RACE scan
8. 3’ RACE clone check scan
9. Alignment of Bos taurus Lepr and PCR Clone check of kidney tissue from
Bowhead whale.
5
LIST OF TABLES
1. 5’ RACE primers
2. 3’ RACE primers
3. 5’ RACE sample trial values
4. 5’ RACE PCR reaction specifics
5. 3’ RACE sample trial values
6. Vector primer sequences
7. PCR reaction specifics for clone checks
8. Sequencing reaction specifics
6
CHAPTER I
INTRODUCTION
LEPTIN (GENERAL)
Leptin, a peptide hormone most closely associated with adipose tissue, was first
identified in 1994 (Zhang et al., 1994). With a molecular weight of 16 kd, the amino acid
sequence is 84% similar between human and mouse (Zhang et al., 1994). Produced by
adipose cells, leptin plays a role in appetite control and energy balance through
interactions with the leptin receptor (Lepr) in the hypothalamus (Friedman 2009,
Trayhurn et al., 2006, & Zhang et al., 1994). Individuals with more adipose tissue show
larger amounts of leptin (Villafuerte, 2000). Individuals with lower fat concentrations
exhibit lower levels of leptin, leading to stimulated appetite and decreased energy
expenditure until fat levels increase (Friedman, 2009). Mutations in the genes encoding
the hormone itself and receptor have been linked to profound obesity in both rodents and
humans (Trayhurn et al., 2006, & Zhang et al., 1994). Some leptin deficiency problems
have been found to be treatable with leptin replacement therapies, though obese subjects
tend to be leptin resistant (Freidman 2009). Though highly conserved in mammals, the
leptin gene is poorly conserved in other classes of vertebrates as shown by a study of
leptin in puffer fish (Kurokawa, 2004) and the South African clawed frog (Crespi et al.,
2006).
LEPTIN RECEPTOR
For a peptide hormone to cause a reaction within a cell, a specific receptor for that
hormone must be present on the cell surface. The leptin receptor (Lepr) is a single
7
membrane spanning cytokine-1 family receptor and was identified in 1995 by the
Tartaglia lab group (Friedman 2009, Tartaglia 1995). Lepr, in mammals, is known to
have six isoforms, ObRa-f (Chen, et al., 1996, Lee et al., 1996 and Tartaglia, et al.,
1995). These isoforms are formed from alternate splicing of the same Lepr gene (Lee, et
al. 1996, Figure 1 Ahima 2004, Chen, et al. 1996, & Friedman 2009). The amino acid
sequences of Lepr are highly conserved and are shown to be over 70% similar in
comparing mouse and human isoforms (Chen, et al. 1996, & Kawachi, et al., 2007).
Of the six isoforms three have been studied in detail (ObRb, ObRa and ObRe).
ObRb is the longest of the known isoforms and is expressed predominatly in the
hypothalamus and cerebellum, compared to other tissues. This is thought to help regulate
the weight reducing effects of leptin (Bjørbæk, et al. 1998, Lee, et al. 1996 and Uotani, et
al 1999) . ObRa, a short isoform, is thought to help transport leptin across the blood-brain
barrier (Uotani, et al.1999) (Bjørbæk, et al. 1998) and is expressed on the cell surface much
more often then ObRb possibly due to ObRa being recycled and ObRb being degraded (Uotani,
1999). Lepr isoforms ObRa and ObRb are also thought to contribute to leptin degradation
and transport throughout the body as well (Uotani, 1999). ObRe is soluble, non- membrane
spanning and the shortest of the isoforms and is the major determinant of plasma leptin levels
(Yang, et al. 2004). It is expressed heavily in the hypothalamus, adipose tissue, heart, testes and
produced by the placenta during development (Gavrilova, et al.1997, Lee, et al.1996), though the
effect on circulating ObRe is still unknown (Yang, et al. 2004).
8
Figure 1: Graph of the structure of the alternately spliced leptin isoforms (from
Ahima and Osei, 2004).
LEPTIN SIGNALING
Leptin signaling functions as a negative feedback loop, maintaining homeostasis
of adipose tissue by regulating energy output and food intake through neural control
(Friedman, 2009). Effects of changes of adipose tissue and leptin levels can be seen
below in Figure 2 (Friedman, 2002). To have an effect on a target cell, leptin must bind to
ObRb (the long isoform) that sits on the cells surface causing the activation of a pathway
known as JAK2 (Yang, et al. 2004). When leptin is bound to ObRe, the leptin-receptor
complex is not capable of activating ObRb, and therefore having an effect on a target cell
(Yang, et al. 2004).
9
Figure 2: Flow chart of biological responses to varying adipose tissue and leptin
levels (Friedman, 2002).
OTHER STUDIES
Current studies in ungulates have shown differences in the amounts of each Lepr
isoform found in differing tissues (Kawachi, et al., 2007). Other research groups have
had success at identifying and sequencing the Lepr and its isoforms in bovine (Kawachi,
et al., 2007). They identified and sequenced isoforms Ob-Ra, b and c. In this study, ObRa was found in all tissues examined, Ob-Rb was found in low levels in the lungs and
testes, and Ob-Rc was found in low levels in the lung and spleen. The Kawachi lab group
10
did RACE to determine the cDNA sequence for cows and this approach is similar to what
has been attempted here for the Bowhead whale (2007). Below, figure 3 (Kawachi, et al.,
2007) shows the presence of three Lepr isoforms in tissues throughout the body in cow.
Figure 3: Presence of Lepr isoforms in Bos Taurus tissues Kawachi et al 2007.
BLUBBER:
Cetaceans and other marine mammals have a thick layer of fat, called blubber,
which is useful for insulation in the cool arctic waters and acts as an energy reservoir
(Iverson, 2002). Firm and fibrous in nature, blubber can also affect animal buoyancy and
can help with streamlining (Iverson, 2002). A layer of blubber is almost continuous
11
across the body and can account for over 50% of total body mass (Iverson, 2002).
Blubber is cooler near the skin then it is near the interior (muscles or body core) of the
animal and changes biochemically with depth as well (Iverson, 2002). Bowhead blubber
is thought to have two distinct layers (Budge, 2008). In a study of 64 bowhead whale
samples, blubber fatty acid composition was found to be significantly different for whale
age, season hunted and year hunted, but gender was not noticed to be a factor (Budge,
2008).
WHALE ECOLOGY:
Bowhead whales (Balaena mysticicetus) (Linnaeus 1758) are large, stocky baleen
whales which feed exclusively on one of the smallest organisms in the oceans,
phytoplankton (Mackas et. al. 1985). They are also known as the Greenland whale,
Greenland Right whale or Polar whale (COSEWIC 2005). Their head, ideally shaped for
pushing up through ice to breath, is large in proportion to the rest of their body,
composing around 30% of their total body length (George et al. 1989). They have small
paddle- shaped flippers and no dorsal fin or dorsal bump (Haldiman and Tarpley, 1993).
The lifespan of bowheads may exceed 100 years, with sexual maturity occurring in the
25th year (George et al. 1999). Generally, females give birth to a single calf once every
three years (Miller et. al 1992, & Rugh et al. 1992). Bowheads, along with other arctic
mammals, are equipped with numerous adaptations to survive their cold habitat. They
have their massive energy storage in the form of blubber that also helps to insulate
internal body temperature, a sophisticated acoustic sense for ice navigation and long
range communication (Ellison et al. 1987, & George et al. 1989).
12
Worldwide, all five recognized populations of Bowhead whale (see Figure 4) are
located in the northern hemisphere (Braham et al. 1984). As of May 2005, three
populations of bowhead whales can be found off the coast of Canada, with two
populations considered threatened and one population under special concern (Cosewic
2005). All populations in Canadian waters are designated endangered since 1980, and are
currently listed as endangered in the U.S. under the Endangered Species Act of 1973 and
as depleted under the U.S. Marine Mammal Protection Act of 1972 (Cosewic 2005).
Reasons for population threats include hurt from previous commercial whaling from
1860-1915 (Ross 1974). This whaling left the populations vulnerable and today they are
still threatened by illegal hunting and predation due to reduced ice coverage caused by
human effects (Cosewic 2005). Other threats to population growth include low
fecundity, the small ecological niche where bowheads live and the recent climate change
which is affecting ice coverage (Cosewic 2005). The killer whale, Orcinus orca
(Linnaeus, 1758), is the only known natural predator of the bowhead whale, besides man
(George et al. 1994). Today, bowheads are legally hunted by the Inuit natives in small
quantities bi-annually in the Canadian arctic (NWMB 2000). Samples used in this study
were obtained from whales of the Bering-Chukchi-Beaufort Sea population in hunts of
this nature. (Gavrilova, 1997)
13
Figure 4: Map showing location of bowhead whale populations (Braham et al. 1984)
WHY LEPTIN RECEPTOR IN WHALES
The goal of this project is to provide sequence evidence for bowhead whale Lepr.
By examining sequences of Lepr and its isoforms, we hope to identify known isoforms
and make note of any variation found in the bowhead whale sequence due to alternate
splicing at the 3’ end. Variation at this site is seen when comparing other mammal
sequences. Knowledge of the alternate splicing and sequence at the 3’ end will be useful
to future research on leptin signaling and fat storage of cetaceans. Given the origin of
whales from ungulates it is hypothesized that whales will exhibit similar leptin isoforms
as those that have been identified in other ungulates, specifically Bos taurus.
14
CHAPTER II
MATERIALS AND METHODS
BIOINFORMATICS:
First, using the national data base NCBI, a search was conducted for Lepr
sequences from mammals. Sequences obtained were of Bos taurus, Mus musculus, Homo
sapien, and Ovis aries (bull, mouse, human and sheep). Once obtained, these sequences
were aligned using the program Sequencher® (4.10.1 Gene Codes Corporation). This
alignment was useful when determining location of useful primer sites by finding
portions of the sequence that were highly conserved among all the samples and thus
could be expected to be conserved in the target whale species.
Figure 5: Sequencher® alignment of cow mouse sheep and human to make primers.
Blue=2502, Green= overlap, Yellow=2520, Pink=1469, 410 not shown.
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
505
-AAAAACCAG
GGACCCCCGG
TGACAAACAT
---------------------------605
TTGTTACA-C
ATGTGATAAC
ATGTAACAAC
ATGTAATAAC
ATGTAATAAC
ATGTAATAAC
705
CACAACCGAT
C-C--TT-TT
C-C--TC-TT
C-C--TC-TT
C-C--TC-TT
C-C--TC-TT
805
TTCAAGTGGT
CTCAC-T--T
CT-AC-T--T
CT-AC-T--T
CT-AC-T--T
CT-AC-T--T
905
GAAGGGAAGA
GAAGG-AAGG
GAAGGGAAGG
GAAGGGAAGG
GAAGGGAAGG
GAAGGGAAGG
1005
ATTCATCTGT
ATTCATCTGT
ATTCATCTGT
ATTCATCTGT
ATTCATCTGT
ATTCATCTGT
1105
CCTCTGCCCC
515
ACCCGA-CCG
A-TC-A--AG
G-TC-ATTTG
---------------------------615
TGGGAATTTC
TG--CGTTTA
TG--CATTTA
TG--CATTTA
TG--CATTTA
TG--CATTTA
715
GACTCCT-TT
GCCTGCTGGA
TCCTCCTGGA
TCCTCCTGGA
TCCTCCTGGA
TCCTCCTGGA
815
ATCTACGTTC
TTCTAA-CTATCTAA-TTATCTAA-CTATCTAA-CTATCTAA-CT915
-CACTGGCTT
ACATTTGTTT
-CATTTGTTA
-CATTTGTTA
-CATTTGTTA
-CATTTGTTA
1015
CATATGGAGC
TATGTGGAGT
TATATTGAGT
AATATTGAGT
AATATTGAGT
AATATTGAGT
1115
CACTGAAAGA
525
AGGAATCGTGTGTA-CTTC
AAGAA-CATC
---------------------------625
TTTATGTGAT
ACT-TGTCAT
ACT-TGGCAT
ACT-TGGCAT
ACT-TGGCAT
ACT-TGGCAT
725
CTCTCACCTG
CTCTCA-AAG
ATCTCA-AAG
ATCTCA-AAG
ATCTCA-AAG
ATCTCA-AAG
825
CTGAGTTATC
-----T-ATC
-T-A-TCATC
-T-A-TCATC
-T-A-TCATC
-T-A-TCATC
925
CAGTAGTGAA
CAACAGTAAA
TAACAGTAAA
CAACAGTAAA
CAACAGTAAA
CAACAGTAAA
1025
CATTACCTAA
CATTATTTAA
CATTATTTAA
CATTATTTAA
CATTATTTAA
CATTATTTAA
1125
CAGCTTTCAG
535
TCTGCAA--A
TCTG-AA--G
TGTGTACTTC
---------------------------635
AGCTGCACTT
A--TCCAATT
A--TCCAATT
A--TCCAATT
A--TCCAATT
A--TCCAATT
735
CTGGAGCCCC
-AATA-CTTC
-AACA-CTTC
-AACA-CTTC
-AACA-CTTC
-AACA-CTTC
835
CAAAACAGTC
CAAAGCAACT
AAGAACAACT
CAGAACAACT
CAGAACAACT
CAGAACAACT
935
GGCTTCAGTT
TTCTTTAGTT
TTCTTTAGTT
TTCTTTAGTT
TTCTTTAGTT
TTCTTTAGTT
1035
GAACCCCTTC
GAATCTATTC
GAATCCTTTC
GAATCCTTTC
GAATCCTTTC
GAATCCTTTC
1135
ACTGTCCAAT
545
TC--CAGGTG
T---AAGATG
TCTGAAGATG
-------ATG
-------ATG
-------ATG
645
A-ACC-TGGC
ACTCCTTGGACTCCCTGGACTCCCTGGACTCCCTGGACTCCCTGG745
AAACAATGCC
AAA-TTCGAA
AAA-TTTGAA
AAA-TTTGAA
AAA-TTTGAA
AAA-TTTGAA
845
TTCCACTGTT
TTCCACTGTT
TTCCACTGTT
TTCCACTGTT
TTCCACTGTT
TTCCACTGTT
945
TTTC-GCCAG
TTTCAACAAA
TTTCAACAAA
TTTCAGCAAA
TTTCAGCAAA
TTTCAGCAAA
1045
AAGAATTATG
AGGAATTATA
AAGAATTATG
AAGAATTATG
AAGAATTATG
AAGAATTATG
1145
GCAACTGCAG
555
TACACCTCTG
-ATTTGTC--ATTAGTC--ATTAGTC--ATTAGTC--ATTAGTC-655
ATATCCAA-T
AGATTTAAGT
AAATTCAAGT
AAATTTAAGT
AAATTTAAGT
AAATTTAAGT
755
TCGGC-TT-T
TGGACATTAT
TGGACGTTCT
TGGACGTTAT
TGGACGTTAT
TGGACGTTAT
855
GCTTTGGGAA
GCTTTCGGAG
GCTTTTGGAG
GCTTTTGGAG
GCTTTTGGAG
GCTTTTGGAG
955
CTAGGTGTAA
T-AGATGCAA
C-AGGTGCAC
C-AGGTGCAA
C-AGGTGCAA
C-AGGTGCAA
1055
ACTCTAAGGT
ACTATAAGGT
ACCTTAAGGT
ACCTTAAGGT
ACCTTAAGGT
ACCTTAAGGT
1155
TCTTC--G-G
565
AAGAAAGATG
AAAAATTCTG
AAAAATTCTG
AAAAATTCTG
AAAAATTCTG
AAAAATTCTG
665
-CTCTCCCTG
TGTCTTGCAT
TGTCTTGCAT
TGTCTTGCAT
TGTCTTGCAT
TGTCTTGCAT
765
GAAGGGGGCT
GA-GACAGCT
GA-GGCAGTT
GA-GGCAGTT
GA-GGCAGTT
GA-GGCAGTT
865
TGAGCAAGGT
TGAGCAAGAT
TGAGGAAGAT
CGAGGAAGAT
CGAGGAAGAT
CGAGGAAGAT
965
ACTGGGACAT
ACTGGAACAT
ACTGGAACAT
ACTGGAACAT
ACTGGAACAT
ACTGGAACAT
1065
CCATCTTTTA
CCATCTTTTA
CCATCTTTTA
CCATCTTTTA
CCATCTTTTA
CCATCTTTTA
1165
GGATGTGAAT
575
ATGTG--TCA
-TGTGGTTTT
-TGTGGTTTT
-TGTGGCTTT
-TGTGGCTTT
-TGTGGCTTT
675
GAAATTTAAG
GCCACCAAAT
GCCATCAAAT
GCCATCAAAT
GCCATCAAAT
GCCATCAAAT
775
TCTGAAGCAA
GTTGAACCTA
GTTGAAACTA
GTTGAAACTA
GTTGAAACTA
GTTGAAACTA
875
CAAAACTGCT
AGAAACTGCT
AAAAACTGCT
AAAAACTGCT
AAAAACTGCT
AAAAACTGCT
975
AGAGTGCTGG
ACAGTGCTGG
ACAGTGCTGG
ACAGTGCTGG
ACAGTGCTGG
ACAGTGCTGG
1075
TATGATCTGC
TATGTTCTGC
TATGTTCTGC
TATGTTCTGC
TATGTTCTGC
TATGTTCTGC
1175
GTCATGTGCC
585
G--AAATT-GTTACATTGG
GTTACATTGG
GTTACATTGG
GTTACATTGG
GTTACATTGG
685
TTGTTTTGTG
TCAACCTATG
ACAACATATG
ACAACATATG
ACAACATATG
ACAACATATG
785
TTGTTGAAGC
-AGTTTAATT
-AGCTTAATT
-AGCTTAATT
-AGCTTAATT
-AGCTTAATT
885
CTGCACTCAC
CCTTATGTGC
CTGTATATGC
CTGTATATAC
CTGTATATAC
CTGTATATAC
985
ATGAAAGGGG
CTAAAAGGAG
ATGAAAGAGG
ATGAAAGAGG
ATGAAAGAGG
ATGAAAGAGG
1085
CTGAAGTCAT
CTGAAGTGTT
TTGACCTATT
TTGACGTGTT
TTGACGTGTT
TTGACGTGTT
1185
GGTACCCAGA
595
CTATGTGGTT
GAATTTATTT
GAATTTATTT
GAATTTATTT
GAATTTATTT
GAATTTATTT
695
GACCACCGAA
-ACTAC--TT
-ACT----T-ACT----T-ACT----T-ACT----T795
TAAAT-TTAA
CAAGTGGTACAAGTGGTAA
CAAGTGGTAC
CAAGTGGTAC
CAAGTGGTAC
895
AGACAACACT
AGACAACATT
AGATGACATT
AGATGACATT
AGATGACATT
AGATGACATT
995
ACTTGACATT
ACTTAAAATT
ACTTGAAATT
ACTTGAAATT
ACTTGAAATT
ACTTGAAATT
1095
AGATGATTCG
AGAAGATT CA
AGAAGAGTCA
AGAAGAGT CA
AGAAGAGTCA
AGAAGAGT CA
1195
GCCAAACTCA
15
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
CCTCTGGTTC
CCTCTGCTCC
CCTCTGCTCC
CCTCTGCTCC
CCTCTGCTCC
1205
ACTACGCTCT
ACGACACTCT
ATAACACCCT
ATGACACCCT
ATGACACCCT
ATGACACCCT
1305
CCACCCTTAG
CCACCATTAG
CCACCATTAG
CCACCATTAG
CCACCATTAG
CCACCATTAG
1405
AATATTTAGA
AATATTCAGA
AATATTCAGA
AATATTCAGA
AATATTCAGA
AATATTCAGA
1505
GGTCCAGGTG
GGTTCAGGTG
TGTGCAGGTG
CGCACAGGTG
CGCACAGGTG
CGCACAGGTG
1605
AAAATTCTGA
AAAATTCTGA
AAAATTCTGA
AAAATTCTGA
AAAATTCTGA
AAAATTCTGA
1705
CTAGCTGAGA
TTAGCTGAGA
TTAGCTGAGA
TTAGCTGAGA
TTAGCTGAGA
TTAGCTGAGA
1805
TTACCTATGA
TTACCTATGA
TCACCTATGA
TCACCTATGA
TCACCTATGA
TCACCTATGA
1905
TGACGGGTAC
TGATGGGTAC
TGATGGGTAC
TGATGGGTAC
TGATGGGTAC
TGATGGGTAC
2005
TATTGTCCTG
TACTGTTCTG
TACTGTTCTG
TACTGTTCTG
TACTGTTCTG
TACTGTTCTG
2105
TATTATCTGG
TATTATCTGG
TATTATCTGG
TATTATCTGG
TATTATCTGG
TATTATCTGG
2205
ACCTCCATCT
GCCTCCATCC
GCCTCCATCC
GCCTCCATCC
GCCTCCATCC
GCCTCCATCC
2305
ATTCGATATG
ATTCGCTATG
ATTCGCTATG
ATTCGCTATG
ATTCGCTATG
ATTCGCTATG
2405
CAGTCTATGT
CAGTCTATGC
CAGTCTATAC
CAGTCTATAC
CAGTCTATAC
CAGTCTATAC
2505
TCCTATGAGA
TCCTATGAGA
TCCTATCAGA
TCCTATCAGA
CCCAAAAAGG
CCGAGAAAGG
CCCAGAAAGA
CCCAGAAAGA
CCCAGAAAGA
1215
TCTGATGTAT
CCTTATGTGT
TCTCATGTAT
TCTTATGTAT
TCTTATGTAT
TCTTATGTAT
1315
GTTTGCATAT
GTTTGCATAT
GTTTGCATAT
GTTTGCGTAT
GTTTGCGTAT
GTTTGCGTAT
1415
GAATTCTACA
GAATTCTACA
GAATTCTACA
GAATTCTACA
GAATTCTACA
GAATTCTACA
1515
AGGAGCAAGA
AGGGGCAAGA
AGGAGCAAGA
AGGTGCAAGA
AGGTGCAAGA
AGGTGCAAGA
1615
CTAG-TGTTG
CAAG-TGTTG
C-AGCGGTTG
C-AGCGGTTG
C-AGCGGTTG
C-AGCGGTTG
1715
AAATCCCTGA
AAATTCCTCA
AAATTCCTCA
AAATTCCTCA
AAATTCCTCA
AAATTCCTCA
1815
CGCAGTGTAC
TGCAGTGTAC
CGCAGTGTAC
TGCAGTGTAC
TGCAGTGTAC
TGCAGTGTAC
1915
TTAACTAAAA
TTAACTAAAA
TTAACTAAAA
TTAACTAAAA
TTAACTAAAA
TTAACTAAAA
2015
ATAGTCCATC
ATATTCCATC
ATGTTCCATC
ATGTTCCGTC
ATGTTCCGTC
ATGTTCCGTC
2115
CTATACAATG
CTACACAATG
CTATACAATG
CTATACAATG
CTATACAATG
CTATACAATG
2215
AACGTAAAAG
AGTGTGAAAG
AGTGTGAAAG
AGTGTGAAAG
AGTGTGAAAG
AGTGTGAAAG
2315
GCTTAAGTGG
GTTTAAGTGG
GTTTAAGTGG
GTTTAAGTGG
GTTTAAGTGG
GTTTAAGTGG
2415
GGTCCAGGTT
TGTTCAGGTG
TGTCCAGGTG
TGTCCAGGTG
TGTCCAGGTG
TGTCCAGGTG
2515
GGGCCTGAAT
GGACCTGAAT
GGACCTGAAT
GGACCTGAAT
CAGTTTTCAG
CAGTTTTCAC
CAGTTTTCAG
CAGTTTTCAG
CAGTTTTCAG
1225
TTGGAAATCA
TTGAAAATCA
TTGAAAATCA
TTGAAAATCA
TTGAAAATCA
TTGAAAATCA
1325
GGAAGTCACA
GGAAATCACA
GGAAATCATA
GGAAATCACA
GGAAATCACA
GGAAATCACA
1425
A--T-TGTAA
ACAGTTATCA
AAAAATATGA
AAATATATAA
AAATATATAA
AAATATATAA
1525
GACTGGATGG
GACTGGATGG
GATTGGATGG
GATTGGATGG
GATTGGATGG
GATTGGATGG
1625
GATCGAATGC
GGTCTAATGT
GGTCTAACAT
GGTCTAACAT
GGTCTAACAT
GGTCTAACAT
1725
GATACAGTAC
AAGCCAGTAT
AAGCCAGTAT
AAGCCAGTAT
AAGCCAGTAT
AAGCCAGTAT
1825
TGCTGCAATG
TGCTGCAATG
TGCTGCAATG
TGCTGCAATG
TGCTGCAATG
TGCTGCAATG
1925
TGACTTGCAG
TGACTTGCAG
TGACTTGCAG
TGACTTGCAG
TGACTTGCAG
TGACTTGCAG
2025
TATTCATCCT
TATTCATCCC
TGTTCATCCT
TATTCATCCT
TATTCATCCT
TATTCATCCT
2125
TGGATCAGGA
TGGATTAGGA
TGGATTAGAA
TGGATTAGAA
TGGATTAGAA
TGGATTAGAA
2225
CAGAGATTAC
CAGAAATTAC
CAGAAATTAC
CAGAAATTAC
CAGAAATTAC
CAGAAATTAC
2325
AAAAGAAATA
AAAAGAAGTA
AAAACAAGTA
AAAACAAGTA
AAAACAAGTA
AAAACAAGTA
2425
CGCTGCCGGC
CGCTGTAAGA
CGCTGTAAGA
CGCTGTAAGA
CGCTGTAAGA
CGCTGTAAGA
2525
TTTGGAGAAA
TTTGGAGAAT
TTTGGAGAAT
TTTGGAGACT
ATGGTTCACT
ATTGTTCAGT
GTTGTTCAGT
GTTGTTCAGT
GTTGTTCAGT
1235
CATCTGCCGG
CATCTGGTGG
CATCTGGTGG
CATCTGGTGG
CATCTGGTGG
CATCTGGTGG
1335
GATGATGGTA
GATGATGGTA
GACACCGGTA
GACACTGGTA
GACACTGGTA
GACACTGGTA
1435
GAGAGGCTGC
GAGAAGCTGA
AAAAAGCTGA
GAAAAACTGA
GAAAAACTGA
GAAAAACTGA
1535
TTCAGGAGTC
CCCAGGAATC
CCTAGGAATC
CCTAGGAATC
CCTAGGAATC
CCTAGGAATC
1635
TTCTTTTCAT
TTCTTTTCAC
TTCTTTTCAC
TTCTTTTCAC
TTCTTTTCAC
TTCTTTTCAC
1735
AGCATTGTGA
GATGTTGTGA
GATGTTGTGG
GATGTTGTGG
GATGTTGTGG
GATGTTGTGG
1835
AGCAGGCGTG
AACATGAATG
AGCAAGAGTG
AGCAAGAGTG
AGCAAGAGTG
AGCAAGAGTG
1935
ATGGTCACCC
ATGGTCAACC
ATGGTCACCC
ATGGTCACCC
ATGGTCACCC
ATGGTCACCC
2035
ACGTCTGAGC
ATATCTGAGC
GTATCTGAAC
GTATCTGAAC
GTATCTGAAC
GTATCTGAAC
2135
TCAACCATTC
TCAATCACTC
TTAATCACAC
TTAATCACAC
TTAATCACAC
TTAATCACAC
2235
TGTAAACACT
TATAAACATT
TGTAAAAATT
TGTAAAAATT
TGTAAAAATT
TGTAAAAATT
2335
CAATGGAAGA
CAATGGAAGA
CAATGGAAGA
CAATGGAAGA
CAATGGAAGA
CAATGGAAGA
2435
GGTTGGATGG
GGCTAGATGG
GGCTAGATGG
GCCTAGATGG
GCCTAGATGG
GCCTAGATGG
2535
AATGGATGGG
AATTAATGGA
AATTAGTGAA
AATTAGTGAA
GCAATTGCAG
GCAACTGCAG
GCAACTGCAG
GCAACTGCAG
GCAACTGCAG
1245
TGTGAGTTTT
AGT-AATTTT
AGT-AATTTT
AGC-AGTTTT
AGC-AGTTTT
AGC-AGTTTT
1345
ATTTAAAGAT
ATTTAAAGAT
GTTTAAAGAT
GTTTAAAGAT
GTTTAAAGAT
GTTTAAAGAT
1445
TGAAATTGTC
CAAGATTGTC
TGAGATTGTC
TGAGATTGTC
TGAGATTGTC
TGAGATTGTC
1545
TGGAGTGACT
TGGAGTGACT
TGGAGTGACT
TGGAGTGATT
TGGAGTGATT
TGGAGTGATT
1645
TGCATCTACA
TGCATCTATA
TGTATCTATA
TGTATCTATA
TGTATCTATA
TGTATCTATA
1745
GTGACCGAGT
GTGATCATGT
ATGACCATAT
ATGACCATAT
ATGACCATAT
ATGACCATAT
1845
CCATCACCGC
CCATCATCGC
CCACCATCGC
CCACCATCGC
CCACCATCGC
CCACCATCGC
1945
AGCACAATCC
AGTACAATCC
AATGCAATCC
AATGCAATCC
AATGCAATCC
AATGCAATCC
2045
CCAAAAACTG
CCAAAGATTG
CTAAAGATTG
CCAAAGATTG
CCAAAGATTG
CCAAAGATTG
2145
TTTAGGTTCA
TCTAGGTTCA
GTTGGGCTCA
GTTGGGCTCA
GTTGGGCTCA
GTTGGGCTCA
2245
GGATTATTGA
GGATTATTGA
GGATTATTGA
GGATTATTGA
GGATTATTGA
GGATTATTGA
2345
CACATGAGGT
TGTATGAGGT
TGTTTGAGGT
TGTTTGAGGT
TGTTTGAGGT
TGTTTGAGGT
2445
ACTAGGATAT
ACTGGGATAT
ACTGGGATAT
ACTGGGATAT
ACTGGGATAT
ACTGGGATAT
2545
GACGTTACTA
GATACTATGA
GACACCACTA
GACACCACTA
TGTTCATGAA
TTTTAATGAA
TGTTCATGAA
TGTTCATGAA
TGTTCATGAA
1255
-CAGTCACCT
CCGGTCACCT
TCACTCACCT
TCACTCACCT
TCACTCACCT
TCACTCACCT
1355
TTCTTGGGAC
TTCTTGGTCC
TTCCTGGTCC
TTCGTGGTCC
TTCGTGGTCC
TTCGTGGTCC
1455
TCAGCTACAT
TCAGCTACAT
ACAGCTACGT
TCAGCTACGT
TCAGCTACGT
TCAGCTACGT
1555
GGAGTTCACC
GGAGTACTCC
GGAGCGCCCT
GGAGTGCCCT
GGAGTGCCCT
GGAGTGCCCT
1655
AAAACGAAAA
AGAAGGAAAA
AAAATGAAAA
AAAATGAAAA
AAAATGAAAA
AAAATGAAAA
1755
TAGCAAAGTT
TAGCAAAGTT
TAGCAAAGTT
TAGCAAAGTT
TAGCAAAGTT
TAGCAAAGTT
1855
TATGCTGAAT
TATGCTGAAT
TATGCTGAGT
TATGCTGAGT
TATGCTGAGT
TATGCTGAGT
1955
AATCACTAGT
AGTCACTTGC
AATCACTTGC
AATCACTTGC
AATCACTTGC
AATCACTTGC
2055
CGTCTTACAG
CTATTTGCAG
CCACTTGCAG
CCACTTGCAG
CCACTTGCAG
CCACTTGCAG
2155
CTTGACTCGC
CTTGACTCTC
CTGGACTCTC
CTGGACTCTC
CTGGACTCTC
CTGGACTCTC
2255
AAGTATCTTG
AAATATCTTG
AAATATCTTG
AAATATCTTG
AAATATCTTG
AAATATCTTG
2355
ATTCGATGCA
TTATGATGCA
GTATGATGCA
ATATGATGCA
ATATGATGCA
ATATGATGCA
2455
TGGAGTAATT
TGGAGTAATT
TGGAGTAATT
TGGAGTAATT
TGGAGTAATT
TGGAGTAATT
2555
AAAAGGAGAG
AAAAGGAGAA
AAAAAGAGAG
AAAAAGAGAG
TGTTGTGAAT
TGTTGTGAAT
TGTTGTGAAT
TGTTGTGAAT
TGTTGTGAAT
1265
CTGATGTCACTAATGTCAG
CCAATGTCAG
CCAATGTCAG
CCAATGTCAG
CCAATGTCAG
1365
AGCCAAACAA
AGCCCACCAT
AGCCCAACAT
AGCCCAACAC
AGCCCAACAC
AGCCCAACAC
1465
CTCTGCTGGT
CCCTGCTAGT
CTCTGCTAGT
CTCTGCTAGT
CTCTGCTAGT
CTCTGCTAGT
1565
TCAAGTCTTT
TCGTGTCTTT
TCTTACCTTT
TCTTACCTTT
TCTTACCTTT
TCTTACCTTT
1665
CCAGATTATC
CAAGATTGTT
GAAGATTGTT
GAAGATCGTT
GAAGATCGTT
GAAGATCGTT
1765
ACCTTCTCCA
ACTTTTTTCA
ACTTTTCCCA
ACTTTTCCCA
ACTTTTCCCA
ACTTTTCCCA
1865
TATACGTGAT
TATATGTGAT
TATACGTGAT
TATATGTGAT
TATATGTGAT
TATATGTGAT
1965
GGGAAGCACT
GGAAAGCACT
AGGAAGCAAT
AGGAAGCAAT
AGGAAGCAAT
AGGAAGCAAT
2065
AGAGACGGCT
AGTGATGGTT
AGAGATGGTT
AGAGATGGTT
AGAGATGGTT
AGAGATGGTT
2165
CACCAACGTG
CACCAACATG
CACCAGTTTG
CACCAGCTTG
CACCAGCTTG
CACCAGCTTG
2265
GGAAAAGCCA
GGAAAAGCCA
GGAAAAGCCG
GGAAAAGCCG
GGAAAAGCCG
GGAAAAGCCG
2365
AAGTCAAAGT
AAATCAAAAT
AAATTAAAAT
AAATTAAAAT
AAATTAAAAT
AAATTAAAAT
2465
GGAGCAGTCC
GGAGCAATCC
GGAGTACTCC
GGAGTACTCC
GGAGTACTCC
GGAGTACTCC
2565
AAATGTCACC
AAATGTCACT
AAATGTCACT
AAATGTCACT
GTCTTGTGCC
GTCATGTGCC
GTCATGTGCC
GTCATGTGCC
GTCATGTGCC
1275
CTGCAGCCCA
TT-CAGCCCA
CT-CAGCCCA
CT-CAGCCCA
CT-CAGCCCA
CT-CAGCCCA
1375
TGGCACCATT
TGGTACCATT
TGGTACCATT
TGGTACCATT
TGGTACCATT
TGGTACCATT
1475
AGACAGTGTG
AGACAGTATA
AGACAGTGTG
AGACAGTGTG
AGACAGTGTG
AGACAGTGTG
1575
ACCACACAAG
ACCACACAAG
ACTACACAAG
ACTACACAAG
ACTACACAAG
ACTACACAAG
1675
TCCTCAAAAC
CCCTCAAAAG
TCCTCAAAAA
TCCTCAAAAA
TCCTCAAAAA
TCCTCAAAAA
1775
ACCTGAAAGC
ATCTGAATGA
ACTTGAATGC
ACTTGAATGC
ACTTGAATGC
ACTTGAATGC
1875
CGATGTCAAT
TGATGTCAAT
TGATGTCAAT
TGATGTCAAT
TGATGTCAAT
TGATGTCAAT
1975
GTGCAGCTGA
TTGCAATTGA
TTGCAGCTGA
TTGCAGCTGA
TTGCAGCTGA
TTGCAGCTGA
2075
TTTATGAATG
TTTATGAATG
TTTATGAATG
TTTATGAATG
TTTATGAATG
TTTATGAATG
2175
TGTCCTTCCT
TGTCCTTCCT
TGTCATTCCT
TGTCATTCCT
TGTCATTCCT
TGTCATTCCT
2275
GTCTTTCCGG
GTCTTTCCAG
GTCTTTCCAG
GTCTTCCCAG
GTCTTCCCAG
GTCTTCCCAG
2375
CTGCCAGCCT
CTGTCAGTCT
CTGCCAGTCT
CTGCCAGTCT
CTGCCAGTCT
CTGCCAGTCT
2475
AGCCTATACG
AGCCTACACA
AGCCCACACA
AGCCCACACA
AGCCCACACA
AGCCCACACA
2575
TTGCTTTGGA
TTACTTTGGA
TTGCTTTGGA
TTGCTTTGGA
TGTGCCAACA
TGTGCCAACA
TGTGCCGACA
TGTGCCGACA
TGTGCCGACA
1285
TGCTTGTTGT
TAAATATGGT
TTAATGTTGT
TTAATGTTGT
TTAATGTTGT
TTAATGTTGT
1385
TCCGCTTCAA
TCCACTTCAA
TCAACTTCAA
TCAACTTCAA
TCAACTTCAA
TCAACTTCAA
1485
CTTCCTGGAT
CTTCCTGGGT
CTTCCTGGGT
CTTCCTGGGT
CTTCCTGGGT
CTTCCTGGGT
1585
ATGTTGTGTA
ATGTCATATA
ATGTCATATA
ATGTCATATA
ATGTCATATA
ATGTCATATA
1685
AGATAGTTTG
AGATTGTTTG
AGATTGTTTG
AGATTGTTTG
AGATTGTTTG
AGATTGTTTG
1785
CACCAGACCT
AACCAAACCT
AACCAAACCC
AACCAAACCC
AACCAAACCC
AACCAAACCC
1885
ATCAATATAT
ATCAATATCT
ATCAATATAT
ATCAATATAT
ATCAATATAT
ATCAATATAT
1985
GGTATCACAG
GGTATCATAG
GGTATCATAG
GGTATCATAG
GGTATCATAG
GGTATCATAG
2085
TGTTTTCCAG
CATTTTCCAG
CATATTTCAG
CATATTTCAG
CATATTTCAG
CATATTTCAG
2185
GACTCCGTAG
GATTCTGTGG
GATTCTGTGG
GATTCTGTGG
GATTCTGTGG
GATTCTGTGG
2285
AGAATAACCT
AGAATAACCT
AGAATAATCT
AGAATAATCT
AGAATAATCT
AGAATAATCT
2385
GCTGGTGTCA
CCCAGTTCCA
CCCAGTGCCA
CCCAGTGCCA
CCCAGTGCCA
CCCAGTGCCA
2485
CTTGTCATGG
GTTGTCATGG
GTTGTCGTGG
GTTGTCATGG
GTTGTCATGG
GTTGTCATGG
2585
AGCCCCTGAC
AGCCCCTGAT
AGCCCCTGAT
AGCCCCTGAT
GCCAAACT CA
GCCAAACTCA
GCCAAGCT CA
GCCAAGCTCA
GCCAAGCT CA
1295
GAAACCCGAT
GAAGCCTGAT
GAAGCCTGAT
GAAGCCTGAT
GAAGCCTGAT
GAAGCCTGAT
1395
TATCAGGTGA
TATCAAGT GA
TATCAAGTGA
TATCAAGT GC
TATCAAGTGC
TATCAAGT GC
1495
CTTCATATGA
CTTCGTATGA
CTTCATATGA
CTTCGTATGG
CTTCGTATGG
CTTCGTATGG
1595
TTTTCCAC CC
CT TTCCACCT
TTTTCCAC CT
TTTTCCACCT
TTTTCCAC CT
TTTTCCACCT
1695
GTGGAGGAAT
GTGGATGAAT
GTGGTTGAAT
GTGGTTGAAT
GTGGTTGAAT
GTGGTTGAAT
1795
CGAGGGAA GT
CGAGGAAAGT
CGAGGAAA GT
CGAGGAAAGT
CGAGGAAA GT
CGAGGAAAGT
1895
CATGTGAAAC
CATGTGAA AC
CATGTGAAAC
CATGTGAA AC
CATGTGAAAC
CATGTGAA AC
1995
GCGCAGCCTG
GAGCAGCC TT
GA GCAGCCTG
GAGCAGCC TG
GAGCAGCCTG
GAGCAGCC TG
2095
CCAATCTT TC
CCAATCTTCC
CCAATCTT TC
CCAATCTTTC
CCAATCTT TC
CCAATCTTTC
2195
TAAAACCACT
TGAAGCCA CT
TGAAACCACT
TGAAACCA CT
TGAAACCACT
TGAAACCA CT
2295
TCAATTCC AG
TCAATTCCAG
TCAGTTCC AG
TCAGTTCCAG
TCAGTTCC AG
TCAGTTCCAG
2395
GACCTCTG TG
GACTTGTGTG
GACCTCTG TG
GA CCTCTGTG
GACCTCTG TG
GACCTCTGTG
2495
ATGTAAAAGT
ATATAAAA GT
ATGTGAAA GT
ATGTGAAA GT
ATGTGAAA GT
ATGTGAAA GT
2595
GAAAAATGAC
GAAAAATGAC
GAAAAATGAC
GAAAAATGAC
16
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
mus Mus musculu
Homo Human B219
Ovis Ovis aries
Bos Bos taurus
Bos Bos taurus
Bos Bos taurus
TCCTATCAGA
TCCTATCAGA
2605
TCACTGTGTA
TCATTGTGCA
TCATTGTGCA
TCATTGTGCA
TCATTGTGCA
TCATTGTGCA
2705
TGGACAGAAC
TGGACAGAGC
TGGACAGAGC
TGGACAGAGC
TGGACAGAGC
TGGACAGAGC
2805
TGAGTGCTGT
TAAATATCGT
TAAATATCGT
TAAATATCGT
TAAATATCGT
TAAATATCGT
2905
TATTGAATGG
TATTGAGTGG
TCTTGAGTGG
TCTTGAGTGG
TCTTGAGTGG
TCTTGAGTGG
3005
GAGAAATATC
GAGAAGTACC
GAGAAGTATC
GAGAAATATC
GAGAAATATC
GAGAAATATC
3105
ATGACGCAGG
GTGATGCAGG
ATGATGCAGA
ATGATGCAGA
ATGATGCAGA
ATGATGCAGA
3205
TTGGGACGAT
TTGGGAAGAT
TTGGGAAGAT
TTGGGAAGAT
TTGGGAAGAT
TTGGGAAGAT
GGACCTGAAT
GGACCTGAAT
2615
GTGT-GAGGA
GTGTTCAG-A
GTGT-AAGAA
GTGT-AAGAA
GTGT-AAGAA
GTGT-AAGAA
2715
CAGCGCACAC
AAGCACATAC
AAGCACATTC
AAGCACATTC
AAGCACATTC
AAGCACATTC
2815
GGAGTCACTC
GCAGTCACTC
GCAGTCCCTC
GCAGTCACTC
GCAGTCACTC
GCAGTCACTC
2915
AAGATCCTTA
AAAAATCTTA
AAAATTCTTA
AAAATTCTTA
AAAATTCTTA
AAAATTCTTA
3015
AGTTTAGTCT
AGTTCAGTCT
AGTTCAGTCT
AGTTCAGTCT
AGTTCAGTCT
AGTTCAGTCT
3115
GCTGTATGTC
TTTATATGTA
TCTGTATGTA
TCTATATGTA
TCTATATGTA
TCTATATGTA
3215
GTTCCAAACC
GTTCCGAACC
GTTCCAAACC
GTTCCAAACC
GTTCCAAACC
GTTCCAAACC
TTTGGAGACT
TTTGGAGACT
2625
GGTACGTGGT
GATATGTGAT
GATATGTGGT
GATATGTGGT
GATATGTGGT
GATATGTGGT
2725
TGTTACAGTT
TGTTACGGTT
TGTTATGGTT
TGTTATGGTG
TGTTATGGTG
TGTTATGGTG
2825
AGTGCTTATC
AGTGCTTATC
AGCGCTTACC
AGTGCTTACC
AGTGCTTACC
AGTGCTTACC
2925
ATGAAGATGA
ATGAAGATGG
ATGAAGACAG
ATGAAGACAG
ATGAAGACAG
ATGAAGACAG
3025
TTACCCAGTA
TTACCCAATA
TTACCCAGTA
TTACCCAATA
TTACCCAATA
TTACCCAATA
3125
ATTGTACCCA
ATTGTGCCAG
ATCGTGCCAA
ATTGTGCCAA
ATTGTGCCAA
ATTGTGCCAA
3225
CCAAGAATTG
CCAAGAATTG
CCAAGAACTG
CCAAGAACTG
CCAAGAACTG
CCAAGAACTG
AATTAGTGAA
AATTAGTGAA
2635
GAAGCATCGT
AAACCATCAT
AAAACATCAT
AAAACATCAT
AAAACATCAT
AAAACATCAT
2735
CTGGCTGTCA
CTGGCCATCA
CTGGCCATCA
CTGGCCATCA
CTGGCCATCA
CTGGCCATCA
2835
CCCTGAGCAG
CTTTAAACAG
CTTTAAACAG
CTTTAAACAG
CTTTAAACAG
CTTTAAACAG
2935
TGGAATGAAG
TGAAATAAAA
TGAAATTAAA
TGAAATTAAA
TGAAATTAAA
TGAAATTAAA
3035
TTTATGGAAG
TTTATGGAAG
TTTACGGAAG
TTTACGGAAG
TTTACGGAAG
TTTACGGAAG
3135
TAATTATTTC
TAATTATTTC
TAATAATCTC
TAATAATTTC
TAATAATTTC
TAATAATTTC
3235
TTCCTGGGCA
TTCCTGGGCA
TTCCTGGGCA
TTCCTGGGCA
TTCCTGGGCA
TTCCTGGGCA
GACACCACTA
GACACCACTA
2645
ACTGCCCACA
ACTTCCTGCA
ACTTCCCACA
ACTTCCCACA
ACTTCCCACA
ACTTCCCACA
2745
ATTCCCTCGG
ATTCAATTGG
ATTCAATTGG
ATTCAATTGG
ATTCAATTGG
ATTCAATTGG
2845
CAGCTGTGTC
CAGTTGTGTG
CACTTGTGTA
CAGTTGTGTA
CAGTTGTGTA
CAGTTGTGTA
2945
TGGCTTAGAA
TGGCTTAGAA
TGGCTTAGAA
TGGCTTAGAA
TGGCTTAGAA
TGGCTTAGAA
3045
GAGTTGGAAA
GAGTGGGAAA
GAGTAGGGAA
GAGTAGGGAA
GAGTAGGGAA
GAGTAGGGAA
3145
CTCTTGTGTC
CTCTTCCATC
CTCCTCAATC
CTCCTCAATC
CTCCTCAATC
CTCCTCAATC
3245
CAAGGACTGA
CAAGGACTTA
CAAGGACTTA
CAAGGACTTA
CAAGGACTTA
CAAGGACTTA
AAAAAGAGAG
AAAAAGAGAG
2655
ATGGGACGTG
ATGGAACATG
ATGGAACGTG
ATGGAACGTG
ATGGAACGTG
ATGGAACGTG
2755
CGCTTCCCTT
TGCTTCTGTT
TGCTTCCTCT
TGCTTCTTCT
TGCTTCTTCT
TGCTTCTTCT
2855
ATCCTTTCCT
ATTGTTTCCT
ATTCTTTCCT
ATTCTTTCCT
ATTCTTTCCT
ATTCTTTCCT
2955
TTCCCTCGAA
TCTCTTCATC
TCCCTTCCTC
TCCCTTCCTC
TCCCTTCCTC
TCCCTTCCTC
3055
ACCAAAGATA
ACCAAAGATA
ACCGAAGATA
ACCGAAGATA
ACCGAAGATA
ACCGAAGATA
3155
CTACTGCTCG
TTATTGCTTG
TTATTGCTTG
TTATTGCTTG
TTATTGCTTG
TTATTGCTTG
3255
ATTTCCAAAA
ATTTTCAGAA
ATTTTCAGAA
ATTTTCAGAA
ATTTTCAGAA
ATTTTCAGAA
AAATGTCACT
AAATGTCACT
2665
GTCAGAAGAT
GTCAGAAGAT
GCTGGAAGAT
GTTGGAAGAT
GTTGGAAGAT
GTTGGAAGAT
2765
GTGAATTTTA
GCAAATTTTA
GCAAATTTTA
GCAAATTTTA
GCAAATTTTA
GCAAATTTTA
2865
GGACACTGTC
GGATACTATC
GGATGCTGTC
GGATGCTGTC
GGATGCTGTC
GGATGCTGTC
2965
TGTTAAAAAG
TGTTAAGAAG
TGTTAAGAAG
TGTTAAGAAG
TGTTAAGAAG
TGTTAAGAAG
3065
ATTAATGGTT
ATTAATAGTT
ATTAATAGTT
ATTAATAGTT
ATTAATAGTT
ATTAATAGTT
3165
GAACACTGTT
GAACATTATT
GAACATTGTC
GAATATTGTC
GAATATTGTC
GAATATTGTC
3265
GCCTGAA--G--AGAACGG
G--AGAACGG
G--TGA---G--AGAACGG
GCCAGAA---
TTGCTTTGGA
TTGCTTTGGA
2675
GTGGGAAATC
GTGGGAAATC
GTGGGAAATC
GTGGGAAACC
GTGGGAAACC
GTGGGAAACC
2775
ACCTTACCTT
ATTTAACCTT
ATTTAACATT
ATTTAACATT
ATTTAACATT
ATTTAACATT
2875
ACCTGATGAT
ACCCAGTGAT
ACCCAGTGAT
ACCCAGCGAT
ACCCAGCGAT
ACCCAGCGAT
2975
TTTTATATCC
TATTATATCC
TATTATGTCC
TATTATGTCC
TATTATGTCC
TATTATGTCC
3075
TCACCAAAGA
TCACTCAAGA
TTACTCAAGA
TTGCTCAAGA
TTGCTCAAGA
TTGCTCAAGA
3175
AATTTCACAC
AATATCACAC
AGTATCACAT
AGTGTCACAT
AGTGTCACAT
AGTGTCACAT
3275
ACAT--TT-G
ACATTCTTTG
ACATACTTTG
---------ACATTCTTTG
ACAT--TT-G
AGCCCCTGAT
AGCCCCTGAT
2 685
GGACCAATCT
ACACGAAATT
ACACTAAACT
ACACTAAACT
ACACTAAACT
ACACTAAACT
2785
CTCATGGCCC
TTCATGGCCT
ATCACGGTCT
CTCACGGGCT
CTCACGGGCT
CTCACGGGCT
2885
TATAGTCTGT
TACAAGCTAA
TACAATCTGA
TACAATCTGA
TACAATCTGA
TACAATCTGA
2985
ACGATAATTT
ATGATCATTT
ATGATCATTT
ATGATTATTT
ATGATTATTT
ATGATTATTT
3085
TGCTATCGAC
TGATATTGAA
TG--AT-GAA
TG--AT-GAA
TG--AT-GAA
TG--AT-GAA
3185
CAGAGAATGA
CAAAGAATGA
CAAAGAATGA
CAAAGAATGA
CAAAGAATGA
CAAAGAATGA
3285
AGCA-TCTTT
A--AGTCTAA
A-----------------A--------AGCA-TCTTT
GAAAAATGAC
GAAAAATGAC
2695
CACTTTCCTG
CACTTTCCTG
CACTTTCCTT
CACTTTCCTT
CACTTTCCTT
CACTTTCCTT
2795
ATGAGTAA AG
AT GAGCAAAG
ATCAGCAA AG
ATCAGCAAAG
ATCAGCAA AG
ATCAGCAAAG
2895
TATATCTGGT
TGTATTTT AT
TGTATTTTAT
TGTATTTT AT
TGTATTTTAT
TGTATTTT AT
2995
TATTCCCA TC
TATCCCCATT
TATCCCCA TT
TATCCCCATT
TATCCCCA TT
TATCCCCATT
3095
AAGCAGCAGA
AAACACCA GA
AAACACCAGC
AAACACCAGC
AAACACCAGC
AAACACCAGC
3195
AAAAGTTGTT
AAAAGCTA TT
AA AAGTTGTT
AAAAGTTG TT
AAAAGTTGTT
AAAAGTTG TT
3295
TTACCAAGCA
TCAT-GATCA
---------------------------TTATCAAGCA
SAMPLE COLLECTION:
Bowhead whale tissue samples were all obtained by Dr. Hans Thweissen
(NEOUCOMP) with permission from the National Marine Fisheries Services (permit
#814-1899-01) and with cooperation from native Alaskan Inuit hunters. These hunts
happened in Barrow, Alaska twice yearly, during spring and fall hunts. The bowhead
whales most likely came from Beaufort-Bering Sea population. The number of whales
caught during each hunt change from hunt to hunt and year to year. Race was performed
of sample #35, a kidney sample from 09B9, an adult, male bowhead whale. The tissue
was stored in RNA later® solution (Ambion®) for molecular data.
17
RNA EXTRACTIONS Ambion TRI Reagent®
RNA extractions were completed on tissue stored in RNA-later from the bowhead
hunts. All RNA extractions were completed using TRI Reagent® (Ambion®).
Methodology followed manufacturer’s suggested protocols with recommended additional
steps for extraction of RNA from fatty tissues. In brief, tissue placed in an RNase free
tube was homogenized using a sonicator (Fisher Scientific 60 Sonic Disembrator)
machine XuL of TRI Reagent® solution. Next, the homogenate was incubated for 5
minutes at room temperature then centrifuged at 12,000 xg for 10 minutes at 4°C cite the
machine you used as well-Rich’s lab. The supernatant was transferred to a fresh RNase
free tube and 100 µl of BCP was added per 1 mL of TRI Reagent solution. The tubes
were then capped tightly, vortexed and incubated at room temperature for 10 minutes.
Next the tubes were centrifuged at 12,000xg for 15 minutes at 4°C. The colorless top
aqueous layer was then transferred again to a new RNase free tube. In the tube, 500 µl of
isopropanol was added per mL of TRI Reagent solution. The mixture was vortexed and
then incubated at room temperature for 10 minutes. Again, the tube was centrifuged at
12.000xg for 8 minutes at 4°C. The supernatant liquid was discarded leaving a gel like
white pellet on the side and bottom of the tube. Then 1mL of 75% ethanol was added to
each extraction (the previous wording was too close to the exact protocol directions).
Tubes were then centrifuged at 7,500 xg for 5 minutes at 4°C to wash the pellet. The
ethanol wash was next discarded without disturbing the pellet and RNA was left to air
dry for 5 minutes. The samples were eluted in 25 µl of molecular water, nano-dropped to
18
determine RNA concentration and samples were stored at -80°C in the department UltraLow freezer (VWR Scientific Products).
PRIMERS
Primers were made based on Bos taurus sequences for leptin receptor in highly
conserved areas near the 3’ end. These primers contained not more than 50% cytosine or
guanine due to the possibility of the primers folding on top of themselves. An alignment
was made from NCBI on the computer program Sequencher® 4.10.1 (Gene Codes
Corporation). Other primers used in the charts below that are not gene specific came as
part of an Applied Biosystems Fist Choice RLM-RACE Kit.
Base Pair
Length
Primer
Sequence
5' RACE Adapter
5'-GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA-3'
44
5' RACE Outer Primer
5'-GCTGATGGCGATGAATGAACACTG-3'
24
5' RACE Inner Primer
5'-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3'
35
410 OBR Reverse
5'-AAGTCCTCTTTCATCCAGCACTGTATGTT-3'
29
1469 OBR Reverse
5'-CATCAGAACAGTACAGGCTGCTCCT-3'
25
Table 1: 5’ RACE Primers
Primer
Sequence
Base Pair Length
3' RACE Adapter
5'-GCGAGCACAGAATTAATACGACTCACTATAGGT12VN-3'*
57
3' RACE Outer Primer
5'-GCGAGCACAGAATTAATACGACT-3'
23
3' RACE Inner Primer
5'-CGCGGATCCGAATTAATACGACTCACTATAGG -3'
32
2502 OBR Forward
5'-CACCAGCATGATGCAGATCT-3'
20
2520 OBR Forward
5'-CCCCATTGAGAAATATCAGTTCAGTC-3'
26
Table 2: 3’ RACE Primers
* (V=G+A+C, N=A+C+G+T)
19
RACE 5’
Rapid amplification of cDNA ends, or RACE, was performed on the RNA
extractions from the bowhead blubber tissue. A kit from Applied Biosystems was used,
entitled Fist Choice RLM-RACE Kit. RACE is a variation of a polymerase chain reaction
(PCR) designed to help clone cDNA segments. Methodology followed manufacturer’s
protocols. In brief, RNA was extracted, and 5’ RACE reaction was attempted.
A. RNA Processing
In an RNase-free micro-centrifuge tube the following was assembled: 10
µg of RNA, 2 µl of 10X CIP buffer, 2 µl Calf Intestine Alkaline Phosphatase
(CIP), and 6 µl of Nuclease-free water. Multiple attempts were completed with
varying amounts of starting RNA (see chart below). The tube was vortexed, spun
briefly and incubated at 37°C for one hour. Next 15 µl of ammonium acetate, 115
µl of nuclease free water and 150 µl of acid phenol chloroform was added to the
tube. Reaction mixture was vortexed and centrifuged at room temperature for 5
minutes at 10,000 xg. The top aqueous layer was then transferred to a new RNase
free tube. 150 µl of chloroform was then added, the tube was vortexed and
centrifuged again at room temperature and 10,000 xg. The top aqueous layer
again was transferred to a new RNase free tube and 150 µl of isopropanol was
added. The reaction was then vortexed and chilled on ice for 10 minutes. After
chilling on ice the reaction was centrifuged for 20 minutes. A pellet at the bottom
of the tube was rinsed with 0.5 mL cold 70% ethanol and centrifuged for 5
minutes. Ethanol was removed carefully, not disturbing the washed pellet and the
20
pellet was allowed to air dry. Pellet was resuspended in 11 µl of nuclease free
water and the sample was placed on ice. The following was then assembled in an
RNase free tube: 5µL CIP’d RNA from previous reaction, 1 µl of 10X TAP
buffer, 2 µl of Tobacco Acid Pyrophosphatase, and 2 µl of nuclease free water.
Reaction was vortexed, spun briefly and incubated at 37°C over a heat block for 1
hour. After incubation the following was assembled in an RNase free tube: 2 µl of
CIP/TAP-treated RNA from previous reaction, 1 µl 5’Race adapter, 1 µl of warm
10XRNA Ligase buffer, 2 µl of T4 RNA ligase (2.5U/µL), and 4 µl of Nuclease
free water. The reaction mixture was mixed and spun briefly, then incubated at
37°C. The reaction was stored at-20°C.
Trial #
Sample Tissue Type Nano-drop value
1
#35
Kidney
345.6 µg/µL
2
#35
Kidney
345.6 µg/µL
Table 3: 5’ RACE Sample trial values
1 µg RNA
~10 µl
~16 µl
B. Reverse Transcription
For the reverse transcription reaction, the following was assembled in an
RNase free microfuge tube on ice: 2 µl of Ligated RNA, 4 µl of dNTPMix, 2 µl
of Random Decamers, 2 µl of 10X RT Buffer, 1 µl of RNase Inhibitor, 1 µl of MMLV Reverse Transcriptase and 8 µl of Nuclease free water. The reaction
mixture was mixed gently, spun briefly and incubated at 42°C for one hour.
C. Nested PCR for 5’ RLM-RACE
21
First the following components were combined in a PCR tube on ice: 1 µl
of RT reaction, 5 µl of 10XPCR Buffer, 4 µl of dNTP Mix, 2 µl of 5’ Race genespecific outer primer (10 µM), 2 µl of 5’ Race outer primer, 36 µl of Nucleasefree water, and 1.25 U of thermostable DNA polymerase (0.25 µl of 5 U/µl)
(Takara LA PCR kit version 2.1) Next, the tube was mixed gently, spun briefly
and following the conditions shown bellow was placed in a thermocycler
(Eppendorf Mastercycler Personal, model #22331 Hamburg).
Stage
Reps
Initial Denaturation
Amplification
1
2
Final extension
3
Temp
1 94°C
35 94°C
60°C
72°C
1 72°C
Time
3 min
30 sec
30 sec
2 min
7 min
Table 4: 5’ RACE PCR reaction specifics.
Once the previously mentioned cycle was complete, the following was
combined in a PCR tube on ice: 2 µl of Outer PCR product, 5 µl of 10X PCR
Buffer, 4 µl of dNTP Mix, 2 µl of 5’ RACE gene specific inner primer (10µM), 2
µl of 5’ RACE Inner Primer, 35 µl of Nuclease-free water and 1.25 U of
thermostable DNA polymerase (0.25 µl of 5 U/µl) (Takara). The same cycle
parameters were used as in Table X in the thermocycler.
D. Gel Analysis of Products
After the second PCR was complete, gel electrophoresis was performed. A
1% agarose gel was poured in to a horizontal electrophoresis system (OWL model
22
B1A). The gel was allowed to set with a plastic comb inserted to make wells for
the DNA ladder and samples. After the gel was set, it was submersed in pcr buffer
(TAE 1x) and the ladder (Axygen Biosciences 100bp Ladder DNA market) and
samples with 3 µl of load dye (Promega Blue/Orange 6x Loading Dye) were
loaded. Running conditions for each gel followed 70 mA for approximately 30
minutes. Once ran the gel was submerged for three minutes in ethidium bromide
in darkness to develop. Next the gel was rinsed in water and visualized using Gel
Snap® computer program and G: Box gel imager (both by Syngene)
RACE 3’
A. Reverse Transcription
The following was combined in an RNase-free tube: 2 µl RNA, 4µl of
dNTP Mix, 2 µl 3’ RACE Adapter, 2 µl 10X RT Buffer, 1 µl RNase Inhibitor, 1
µl M-MLV Reverse Transcriptase, 8 µl Nuclease-free water. The reaction mixture
was mixed gently spun briefly and incubated at 42°C for one hour.
B. PCR for 3’ RLM-RACE
First, the following was added to a PCR tube on ice: 1 µl of the RT reaction, 5
µl 10X PCR buffer, 4 µl dNTP mix, 2 µl of 3’ RACE gene specific outer primer
(to uM), 2 µl of 3’ RACE outer primer, 36 µl Nuclease-free water, and 1.25 U of
thermostable DNA polymerase (0.25 µl of 5 U/µl) (Takara). Multiple attempts
were completed with varying amounts of starting RNA (see chart below). The
23
tube was flicked gently and spun briefly. The same cycle perameters were used as
in Table X in a thermocycler. Second, First, the following was added to a PCR
tube on ice: 1 µl of the RT reaction, 5 µl 10X PCR buffer, 4 µl of dNTP mix, 2 µl
of 3’ RACE gene specific inner primer (to µM), 2 µl of 3’ RACE inner primer, 36
µl Nuclease-free water, and 1.25 U of thermostable DNA polymerase (0.25 µl of
5 U/µl) (Takara). The tube was flicked gently and spun briefly. The same cycle
perameters were used as in Table X in a thermocycler. Once PCR was complete
results were visualized using gel electrophoresis.
Trial #
Sample Tissue Type Nano-drop value
1
#35
Kidney
345.6 µg/µL
2
#35
Kidney
345.6 µg/µL
3
#35
Kidney
345.6 µg/µL
Table 5: 3’ RACE sample trial values
1 µg RNA
~3.5 µL
~7 µL
~3 µL
Gel Purification
Purification of RACE products were done using spin tubes from a Millipore
Ultrafree®-DA kit. Product bands that were cut out of the 1% agarose gels using a sterile
razor blade in the Kodak Gel Logic 2200 Imaging System. Once cut out of the gel, the
small fragments were placed into spin columns included in the kit that have a mesh to let
the cDNA products slip to the bottom of the tube and catch the agarose medium. Tubes
were placed into the centrifuge for 10 minutes so that the product would collect at the
bottom for cloning.
24
CLONING TA TOPO Kit
In an RNase free tube the following was assembled: 2 µl of RACE PCR products
from gel cleanups, 0.5 µl of TOPO Kit salt solution, and 0.5 µl of TOPO® vector. This
mixture was incubated at room temperature for 12 minutes and then placed on ice. While
on ice, X-gal (Shelton Scientific, Inc.) was applied to pre-poured Petri dishes and set in
the incubator (Fisher Scientific Isotemp Incubator) at 37°C to dry. Bacteria (One Shot®
Chemically Competent E.coli) were also removed from the ultra-low freezer and placed
on ice to thaw at this time. Once on ice, 25 µl of bacteria were added to each reaction and
the reactions were mixed gently (not by pipetting up and down so as not to kill the
bacteria due to friction). The reactions were then heat shocked for 30 seconds at 42°C
then immediately placed on ice. Next the 125 µl of S.O.C. medium was added to the
reactions and they were placed on the shaker at 37° (Orbit Lab-Line Environ Shaker) for
one hour. After being incubated in the shaker, between 40 µl and 80 µl of each reaction
was spread onto the pre-warmed perti dishes with the X-gal coating. Bacteria were left in
the incubator over night to grow colonies.
Colonies formed overnight, and ones which did not turn blue were selected using
a pipette tip. A culture was formed using 1 mL of LB/amp broth and selected colonies.
The cultures were placed in the shaker overnight.
Clones were checked (Qiagen PCR Kit) to insure an insert was present in the
vector inserted in the bacteria. The following was assembled in a PCR tube: 3.5 µl of
nuclease-free water, 6.0 µl Q-Master Mix, 0.25 µl of T7 primer, 0.25 µl of T3 primer and
0.1 µl of the bacterial culture. Reaction mixtures were placed into a thermocycle Results
were visualized using gel electrophoresis to identify insert size.
25
Primer Sequence
T7
5'-TAATACGACTCACTATAGGG-3'
T3
5'-ATTAACCCTCACTAAAGGGA-3'
Table 6: Vector Primer Sequences
Stage Reps Temp
Initial denaturation
1
1 95°C
Amplification
2
25 95°C
49°C
72°C
Final extention
3
1 72°C
Table 7: PCR reaction specifics for clone check
Base Pair Length
20
20
Time
1.5 min
45 sec
45 sec
1.5 min
4 min
PLASMID PREP (Qiagen MiniPrep Kit)
Once clones were found to have proper insert size for the Lepr product, in an
RNase free tube, 1.5 mL of bacterial culture was added and tube was placed in the
centrifuge for 8 minutes at 8000 rpm. After spinning, a bacterial pellet remained at the
bottom of the tube and the liquid broth was discarded. Next 250 µl of P1 solution was
added and was pipetted up and down to resuspend the pellet. Then 250 µl of P2 solution
was added and the tube was closed and inverted several times. Following, 350 µl of N3
solution was added and again the reaction mixture was inverted to mix, this time turning
cloudy. The reaction mixture was next centrifuged on high for ten minutes. All of the
reaction liquid was then transferred to a pre-labeled QIAprep spin column (membrane
and clear catch tube), being careful not to transfer any precipitate at the bottom of the
reaction tube. The spin column was placed in the centrifuge on high for 60 seconds. Next,
26
the liquid was discarded and 725 µl of PE buffer was added and again the reaction
mixture was centrifuges for 60 seconds. The liquid was discarded and the spin column
was centrifuged for 60 seconds on high to dry. The clear catch tube was discarded and the
spin column membrane was inserted into a new RNase free 1.5 mL tube. 40 mL of
nuclease-free water was added on top of the spin column membrane and the reaction
mixture was centrifuged on high for 60 seconds. The spin column was finally discarded
and the purified DNA in water was then used for sequencing.
SEQUENCING
A sequencing reaction was assembled in an RNase free tube containing the
following: 2.3 µl of Big Dye Reaction Mix (specifics), 0.5 µl of primer (~3.2 pmol) and
5.0 µl of DNA. The reaction was then placed in the thermocycler and ran under the
following condition.
Stage
Gradient Flux
Reps
1
Temp
29 96°C
44°C
60°C
Time
20 sec
10 sec
4 min
Table 8: Sequencing reaction specifics
ETHANOL SEQUENCE REACTION CLEAN-UPS
In a clean 1.5 mL tube 1.7 µl of 125 mm EDTA was added. In the sequencing
reaction tubes from the following reaction, 19 µl of 100% ethanol was added to the
sequencing mixture and the entire contents were moved to the clean 1.5 mL tube
containing the EDTA. The reaction tube was allowed to sit at room temperature for 15
27
minutes then centrifuged on high for 25 minutes with the hinges facing outward. Facing
the hinges in the same direction allowed for the pellet to form in a predicted area. Once
centrifuged, the liquid in the reaction tube was removed by pipetting up opposite of
where the pellet should have formed. This liquid was discarded. Next 30 µl of 70%
ethanol was added to the reaction tube containing only the pellet and this was allowed to
sit for one minute before returning to the centrifuge to spin on high for another 15
minutes, with hinges facing out. Once spun, again, the liquid was carefully removed
trying not to disturb the pellet, and the reaction was set in the preheated DNA Speed
Vac® (Savant) at medium setting for 10 minutes to dry the sample. Once reaction
mixture was dried in the Speed Vac®, 28 µl of HiDi formamide™ (Applied Biosystems)
was added and the reaction was placed ina preheated heat-block (Fisher Scientific, Dry
Bath Incubator®) at 95°C for 2 minutes. Next the tube was cooled over ice for 2 minutes
and the reaction mixture was transferred to a 96 well plate for Senger sequencing analysis
in the 3130x Genetic Analyzer (Applied Biosystems).
SEQUENCE ANALYSIS
Once sequences were obtained, results were BLAST searched on the national
database, NCBI. BLAST searching told if the fragments we obtained were ungulate Lepr
(most closely associated reported sequence). Sequences which when BLAST searched
were identified as ungulate sequence were place in the Sequencher® 4.10.1 (Gene Codes
Corporation) program and aligned with previously looked at Lepr sequences to determine
where our results were in relation to known exons.
28
CHAPTER III
RESULTS
RNA was extracted following a TRIReagent® protocol from RNAlater®
preserved tissue of a male bowhead (B9) kidney. The extracted RNA was then quantified
using a nano-drop. This analysis yielded 345.6 ng/µl of RNA. First strand cDNA
synthesis was performed using recommended protocols for 5’ RACE (Applied
Biosystems) and 3’ RACE and results were evaluated on agarose gels (see below). Gene
specific primers for the RACE PCR reactions were designed based upon a Lepr
alignment from sequence found on Genbank (see figure 5 in Materials and Methods).
5’ RACE:
First results of attempts at 5’ RACE seemed promising. Smears were seen
indicating possible capture of differently spliced isoforms. The gel image below (Figure
6) shows smears indicating possible isoforms of OBR in whale kidney using designed
gene-specific and RACE supplied primers. Attempts were made to clone the products
into bacterial vectors, but the resultant sequences were not shown to be similar to any
leptin receptor sequences in Genbank.
29
Figure 6: 5’ PCR results from first attempt to amplify procucts
from 5’ RACE. The first lane is ladder and the three other lanes are
three alloquates of 5' RACE reaction using 2750R primer.
3’ RACE:
The RNA extracted from whale kidney was also used to attempt 3’ RACE. Genespecific and RACE supplied primers were used in first strand cDNA synthesis. Results
were visualized on an agarose gel (see Figure 7). Smears again were seen indicating
possible different Lepr isoforms. These were cut out of the gel and gel purified
(Millipore Ultrafree®-DA kit). Inner primer 3’RACE was performed (see Materials and
Methods for details) using supplied inner primers and the same gene-specific primers.
Bands were seen, gel purified (Millipore Ultrafree®-DA kit) and cloned (TOPO® vector
kit) into a bactieral vector. Clone checks were performed (Figure 8) and bands were seen
indicating that different isoforms were cloned into bacterial vectors. Attempts at
30
sequencing these clones were unsuccessful. A small portion of the sequence was
acquired from a cloned PCR product (see alignment Figure 9) and results from this
demonstrate that expected Lepr sequence in whales shares a high degree of similarity
with ungulate taxa.
Figure 7: 3’ RACE PCR #1 smears show possible isoforms
31
Figure 8: 3’ Agarose gel showing PCR products from clones of products of 3’ RACE
PCR in figure 7. The left lane is molecular ladder and bands in other lanes indicate
inserts are present in individual plasmids from those colonies.
2305
2315
2325
2335
2345
2355
2365
2375
2385
2395
B53205
~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~CTGTTAAGA AGTATTATGT CCATGATTAT
Bos NM
GATGTATTTT ATTCTTGAGT GGAAAATTCT TAATGAAGAC AGTGAAATTA AATGGCTTAG AATCCCTTCC TCTGTTAAGA AGTATTATGT CCATGATTAT
2405
2415
2425
2435
2445
2455
2465
2475
2485
2495
B53205
TTTTTCCCCA TTGAGAAATT TCAGTTCAGT CTTTACCCAA TATTTATGGA AGGAGTGGGG AAACCGAAGA TAATTGATAG TTTCACCCCC TCTGATGAAA
Bos NM
TTTATCCCCA TTGAGAAATA TCAGTTCAGT CTTTACCCAA TATTTACGGA AGGAGTAGGG AAACCGAAGA TAATTAATAG TTTTGCTCAA GATGATGAAA
B53205
AAACCAGCAT GATGCAGGAT ATATATGTAA TTGTGCCAAT AATTATTTCC TCCTCAATCT TATTGCTTGG AATATTGTCA GTGTCACATC AAAGAATGAA
Bos NM
AACACCAGCA TGATGCAGAT CTATATGTAA TTGTGCCAAT AATAATTTCC TCCTCAATCT TATTGCTTGG AATATTGTCA GTGTCACATC AAAGAATGAA
B53205
AAAGCTGTTT TGGGAAGATG TTCCAAACCC CAAGAACTGT TCCTGGGCAC AAGGTCTTA. ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~ ~~~~~~~~~~
Bos NM
AAAGTTGTTT TGGGAAGATG TTCCAAACCC CAAGAACTGT TCCTGGGCAC AAGGACTTAA TTTTCAGAAG CCAGAAACAT TTGAGCATCT TTTTATCAAG
2505
2605
2515
2615
2525
2625
2535
2635
2545
2645
2555
2655
2565
2665
2575
2675
2585
2685
2595
2695
Figure 9: Alignment of Bos taurus Lepr and PCR Clone check of kidney tissue from
Bowhead whale. Base pairs that differ are highlighted in yellow.
32
CHAPTER IV
DISCUSSION AND CONCLUSION
This project employed a number of bioinformatic and molecular DNA methods to
attempt to sequence and identify known and possible unknown isoforms of Lepr in
bowhead whale tissue samples. Previous studies suggested that RACE is an efficient
method of obtaining large sections of sequence such as seen in the Kawachi lab study on
Bos taurus.
The 5’ RACE protocol focused on making elongated cDNA copies of the present
RNAs in the bowhead whale kidney tissue sample used. In the first attempt 5’ RACE
products appeared as smears on the gels (figure 6) suggesting possible isoforms of Lepr
with the gene specific primers made using the alignment shown above had worked.
However, subsequent cloning of these products and then sequencing of those products
did not confirm the presence of Lepr sequences. The lack of success could be due to
possible low Lepr RNA copy number in the kidney sample used, RNA degradation so
that the 5’ “cap” could not attach properly, or human error when following the protocol.
Lacking success for 5’ RACE, 3’ RACE was performed. For the 3’ RACE, RNA
is copied from the poly-A 3’ end found on mRNAs. Results here were more hopeful as
smears (figure 7) again were seen once PCR took place, suggesting capture of multiple
isoforms. Though some 20 base pair fragments were identified from the sequencing
reactions, BLAST search and comparisons to our primers revealed that only our primer
sequence was obtained in return with the larger sequences. Again, some possible issues
33
could have caused by the bacteria only up taking a portion of the cDNA into vector,
possible to much or too little starting RNA, or human error in following protocol.
Though problems still persist, using RACE to obtain sequence of the Lepr
isoforms is still a possibility. More trouble shooting with primers and RACE protocol still
could provide answers. In lieu of finding full length Lepr sequences an attempt to directly
amplify a portion of the Lepr sequence from genomic DNA was attempted. This resulted
in a positive PCR product which was cloned and sequenced resulting in a ~230 base pair
sequence of bow head Lepr was obtained. This short sequence was aligned and found to
be most similar to Bos taurus Ob-Rb, confirming that Bowhead whales do have Lepr. If
whales make leptin, but the leptin is signaling in these animals the way it does in other
mammals, then how is it possible that they can sustain such large fat deposits in the form
of blubber. Is it possible something with their receptor? Comparisions of this 230 base
pair sequence reveal only a single nucleotide sequence difference with ungulates and so it
does not appear that there is a significant difference in their primary sequence but this
sequence only represents a small portion of the total Lepr gene sequence.
34
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