2008 Spring Biological database Homework 1
This problem set is due by 2PM, March 25, 2008. You shall upload your answers to your web
site as instructed by your TA. For all questions, please make a reference such as screen-shot to
indicate the source of your answer.
1. Here is a nucleotide sequence:
CTCCAGGCCCGTGGGGCTGGCCCTGCACCGCCGAGCTTCCCGGGATGAGGGCCCCCGGTGTGGTCACCC
GGCGCGCCCCAGGTCGCTGAGGGACCCCGGCCAGGCGCGGAGATGGGGGTGCACGAATGTCCTGCCTGG
CTGTGGCTTCTCCTGTCCCTGCTGTCGCTCCCTCTGGGCCTCCCAGTCCTGGGCGCCCCACCACGCCTCAT
CTGTGACAGCCGAGTCCTGGAGAGGTACCTCTTGGAGGCCAAGGAGGCCGAGAATATCACGACGGGCTGT
GCTGAACACTGCAGCTTGAATGAGAATATCACTGTCCCAGACACCAAAGTTAATTTCTATGCCTGGAAGAG
GATGGAGGTCGGGCAGCAGGCCGTAGAAGTCTGGCAGGGCCTGGCCCTGCTGTCGGAAGCTGTCCTGCGG
GGCCAGGCCCTGTTGGTCAACTCTTCCCAGCCGTGGGAGCCCCTGCAGCTGCATGTGGATAAAGCCGTCA
GTGGCCTTCGCAGCCTCACCACTCTGCTTCGGGCTCTGGGAGCCCAGAAGGAAGCCATCTCCCCTCCAGA
TGCGGCCTCAGCTGCTCCACTCCGAACAATCACTGCTGACACTTTCCGCAAACTCTTCCGAGTCTACTCC
AATTTCCTCCGGGGAAAGCTGAAGCTGTACACAGGGGAGGCCTGCAGGACAGGGGACAGATGACCAGGTG
TGTCCACCTGGGCATATCCACCACCTCCCTCACCAACATTGCTTGTGCCACACCCTCCCCCGCCACTCCT
GAACCCCGTCGAGGGGCTCTCAGCTCAGCGCCAGCCTGTCCCATGGACACTCCAGTGCCAGCAATGACAT
CTCAGGGGCCAGAGGAACTGTCCAGAGAGCAACTCTGAGATCTAAGGATGTCACAGGGCCAACTTGAGGG
CCCAGAGCAGGAAGCATTCAGAGAGCAGCTTTAAACTCAGGGACAGAGCCATGCTGGGAAGACGCCTGAG
CTCACTCGGCACCCTGCAAAATTTGATGCCAGGACACGCTTTGGAGGCGATTTACCTGTTTTCGCACCTA
CCATCAGGGACAGGATGACCTGGAGAACTTAGGTGGCAAGCTGTGACTTCTCCAGGTCTCACGGGCATGG
Please use database mining tools of your choice to tell me as much as you can about
this sequence.
i.
What gene does this sequence represent in human? What is its GI number? GenBank
Accession number? Gene symbol? Unigene ID?
Ans :
Gene : Homo sapiens erythropoietin (EPO)
GI number : 62240996
Accession number : NM_000799
Gene symbol : Epo
Unigene ID (UGID) : 131206
ii.
What database(s) did you search, and what tool(s) did you use in your search? What
parameter settings did you use?
Ans.
databases : NCBI
tool : nucleotide blast
parameter settings :  enter the FASTA sequence
 choose search set - Human genomic + transcript
 program selection - Highly similar sequences (megablast)
iii.
Retrieve one ortholog of this gene’s complete mRNA sequence and Protein sequence in
FASTA format. Compare the results obtained by blastn vs. blastp.
Ans.
>gi|114615072|ref|XM_519268.2| PREDICTED: Pan troglodytes erythropoietin (EPO), mRNA
CCCGGAGCCGGAACGGGGCCACCGCGCCCGCTCTGCTCCGACACCGCGCCCCCTGGACAGCCGCCCTCTC
CTCCAGGCCCGTGGGGCTGGCCCTGCACCGCCGAGCTTCCCGGGATGAGGGCCCCCGGTGTGGTCACCCG
GCGCGCCCCAGGTCGCTGAGGGACCCCGGCCAGGCGCGGAGATGGGGGTGCACGAATGTCCTGCCTGGCT
GTGGCTTCTCCTGTCCCTGCTGTCGCTCCCTCTGGGCCTCCCAGTCCTGGGCGCCCCACCACGCCTCATC
TGTGACAGCCGAGTCCTGGAGAGGTACCTCTTGGAGGCCAAGGAGGCCGAGAATATCACGACGGGCTGTG
CCGAACACTGCAGCTTGAATGAGAATATCACTGTCCCAGACACCAAAGTTAATTTCTATGCCTGGAAGAG
GATGGAGGTCAGGCAGCAGGCCGTAGAAGTCTGGCAGGGCCTGGCCCTGCTCTCGGAAGCTGTCCTGCGG
GGCCAGGCCCTGTTGGTCAACTCTTCCCAGCCGTGGGAGCCCCTGCAGCTGCATGTGGATAAAGCCGTCA
GTGGCCTTCGCAGCCTCACCACTCTGCTTCGGGCTCTGGGAGCCCAGAAGGAAGCCATCTCCCCTCCAGA
TGCGGCCTCAGCTGCTCCACTCCGAACAATCACTGCTGACACTTTCCGCAAACTCTTCCGAGTCTACTCC
AATTTCCTCCGGGGAAAGCTGAAGCTGTACACAGGGGAGGCCTGCAGGACAGGGGACAGATGACCAGGTG
TGTCCACCTGGGCATATCCACCACCTCCCTCACCAACATTGCTTGTGCCACACCCTCCCCCGCCACTCCT
GAACCCCGTCGAGGAGCTCTCAGCTCAGCGCCAGCCTGTCCCTTGGACACTCCAGTGCCAGCAATGACAT
CTCAGGGGCCAGAGAAACTGTCCAGAGAGCAACTCTGAGATCTAAGGATGTCACAGGGCCAACTTGAGGG
CCCAGAGCAGGAAGCATTCAGAGAGCAGCTTTAAACTCAGGGACAGAGCCATGCTGGGAAGACGCCTGAG
CTCACTCGGCACCCTGCAAAATTTGATGCCAGGACACGCTTTGGAGGCGATTTACCTGTTTTCGCACCTA
CCATCAGGGACAGGATGACCTGGAGAACTTAGGTGGCAAGCTGTGACTTCTCCAGGTCTCACGGGCATGG
GCACTCCCTTGGTGGCAAGAGCCCCCTTGACACCGGGGTGGTGGGAACCATGAAGACAGGATGGGGGCTG
GCCTCTGGCTCTCATGGGGTCCAAGTTTTATGTATTCTTCAATCTCATTGACAAGAACTGAAACCACCAA
>gi|114615073|ref|XP_519268.2| PREDICTED: erythropoietin [Pan troglodytes]
MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPD
TKVNFYAWKRMEVRQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALG
AQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
by Blastn :
by Blastp :
Blastn : Search translated nucleotide database using a protein query
Blastp : Search protein databases using a protein query
iv.
Retrieve at least 5 homologenes of this gene. Perform a multiple sequence alignment?
The human sequence is most similar to what organism?
Seq1.
Seq2.
[Homo sapiens]
[Pan troglodytes]
Ans.
The sequence is similar to Pan troglodytes.
v.
Is the secondary structure of this protein known? If so, how many “helical fold”are there
in its 3D protein structure? How did you determine the exact amino acid number of each
helical region?
Ans.
Yes.
Four helical fold
The exact amino acid number of each helical region is showing under the DSSP secondary structure in the
schematic drawing.
vi.
Is the function of this protein known? If so, what does it do?
Ans.
Yes.
Erythropoietin is the principal hormone involved in the regulation of erythrocyte differentiation and the maintenance
of a physiological level of circulating erythrocyte mass.
vii.
Which normal human tissues is this gene mainly expressed in? How did you determine
this?
Ans.
Eye and prostate.
The expression profile is suggested by analysis of EST counts, and we can determine the mainly expressed
tissues by TPM (Transcripts per million) level.
viii.
Is this protein involved in any biological pathway(s)? If so, what does the pathway do?
Ans.
Epo involved in many biological pathways, for example, Epo regulate the erythrocyte differentiation in Erythrocyte
Differentiation Pathway.
ix.
Do any other databases contain information about the superfamily of this target gene
product? Which superfamily? How did you find out?
Ans.
Yes, we can find superfamily information of Epo by PDB website. It belongs to 4-helical cytokines.
x.
Look for publications relevant to the function(s) of this protein in the biomedical literature.
Show one abstract of a relevant article.
Ans.
Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy. New Eng. J. Med. 353:
782-792, 2005.
Erythropoietin as a Retinal Angiogenic Factor in Proliferative Diabetic Retinopathy
Daisuke Watanabe, M.D., Ph.D., Kiyoshi Suzuma, M.D., Ph.D., Shigeyuki Matsui, Ph.D., Masafumi Kurimoto, M.D., Junichi Kiryu, M.D., Ph.D.,
Mihori Kita, M.D., Ph.D., Izumi Suzuma, M.D., Ph.D., Hirokazu Ohashi, M.D., Ph.D., Tomonari Ojima, M.D., Tomoaki Murakami, M.D., Toshihiro
Kobayashi, Ph.D., Seiji Masuda, Ph.D., Masaya Nagao, Ph.D., Nagahisa Yoshimura, M.D., Ph.D., and Hitoshi Takagi, M.D., Ph.D.
ABSTRACT
Background Although vascular endothelial growth factor (VEGF) is a primary mediator of retinal angiogenesis, VEGF inhibition alone is
insufficient to prevent retinal neovascularization. Hence, it is postulated that there are other potent ischemia-induced angiogenic factors.
Erythropoietin possesses angiogenic activity, but its potential role in ocular angiogenesis is not established.
Methods We measured both erythropoietin and VEGF levels in the vitreous fluid of 144 patients with the use of radioimmunoassay and
enzyme-linked immunosorbent assay. Vitreous proliferative potential was measured according to the growth of retinal endothelial cells in vitro
and with soluble erythropoietin receptor. In addition, a murine model of ischemia-induced retinal neovascularization was used to evaluate
erythropoietin expression and regulation in vivo.
Results The median vitreous erythropoietin level in 73 patients with proliferative diabetic retinopathy was significantly higher than that in 71
patients without diabetes (464.0 vs. 36.5 mIU per milliliter, P<0.001). The median VEGF level in patients with retinopathy was also significantly
higher than that in patients without diabetes (345.0 vs. 3.9 pg per milliliter, P<0.001). Multivariate logistic-regression analyses indicated that
erythropoietin and VEGF were independently associated with proliferative diabetic retinopathy and that erythropoietin was more strongly
associated with the presence of proliferative diabetic retinopathy than was VEGF. Erythropoietin and VEGF gene-expression levels are
up-regulated in the murine ischemic retina, and the blockade of erythropoietin inhibits retinal neovascularization in vivo and endothelial-cell
proliferation in the vitreous of patients with diabetic retinopathy in vitro.
Conclusions Our data suggest that erythropoietin is a potent ischemia-induced angiogenic factor that acts independently of VEGF during retinal
angiogenesis in proliferative diabetic retinopathy.
xi.
Show the protein 3-D structure if there is any.
1. Find the zebrafish homolog of the above gene. And answer the following
questions:
i.
The zebrafish homolog is located on which chromosome? And in Human?
Ans.
Zebrafish : Chromosome 7
Human : Chromosome 7
ii.
Perform a cDNA and Polypeptide sequence alignment between human and zebrafish of
this gene.
iii.
How many exons does this gene have in zebrafish? How did you determine this?
Ans.
5 exons
By Eesembl
iv.
What is the expression pattern of this gene in zebrafish? In human? In mouse?
expression pattern in zebrafish
expression pattern in human
No expression pattern in mouse can be found.