This Project This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background Information DNA nucleotidylexotransferase, a protein found in the nucleoplasm, is a template-independent DNA polymerase, which catalyzes the random addition of deoxynucleoside 5'-triphosphate to the 3'-end of a DNA initiator. One of the in vivo functions of this enzyme is the addition of nucleotides at the junction (N region) of rearranged Ig heavy chain and T-cell receptor gene segments during the maturation of B- and T-cells. Unlike most DNA polymerases, this protein does not require a template. The preferred substrate of this enzyme is a 3’-overhang, but it can also add nucleotides to blunt or recessed 3' ends. Cobalt is a necessary cofactor; however, the enzyme catalyzes reaction upon Magnesium and Manganese administration in vitro.
Figure 1. Structure of DNA nucleotidylexotransferase. This figure demonstrates the shape of DNTT, a protein found in the nucleoplasm. This specifically shows amino acids 19-125 of human DNA nucleotidylexotransferase.
Methods Whale Shark Predicted Orthologs: The human protein sequence (ENSP00000360216) was entered as a query in a Blast search against the Predicted Whale Shark Protein database on whaleshark.georgiaaquarium.org. The top predicted protein hits full predicted sequences were in turn used as queries against the NCBI human protein database. The whale shark database was then searched using the elephant shark (closest ortholog) predicted DNTT sequence as a query.
Predicted Orthologs: DNTT predicted orthologs were identified in other species using the NCBI Blast server. Protein BLASTs were performed against databases for the mouse, zebrafish, yeast, and elephant shark genomes. The DNTT protein was used as query sequence for these searches.
Phylogenetic Tree: The highest matching ortholog with the lowest E-Value for each species listed above, as well as the closest matches for the whale shark blast hits, were used to make a phylogenetic tree and a multiple sequence alignment among the species.Clustal was used to create both the sequence alignment and tree.
Searching for DNTT in the Whale Shark: The human DNTT protein sequence was used as a query against the whale shark predicted protein database. The top five hits are shown below. The lowest e-value was 1e-15 and the highest percent identity was only 48.00%.
Whale Shark ID
E-value
Alignment Length
Predicted Protein Length
% Identity
g46047.t1
1e-15
242
384
21.49
g27824.t1
2e-11
110
109
30.00
g23246.t1
7e-5
91
167
25.27
g21210.t1
2e-4
65
117
30.77
g46742.t1
2e-4
25
511
48.00
Table 1. This table shows the results when DNTT was searched in the Whale Shark. It provides the ID of the protein, the E-value, alignment length, predicted protein length and percent identity. The explanation above shows which percent was highest and which e-value was lowest, which presents the most similar results to the original human DNTT protein.
Protein Domains DNA nucleotidylexotransferase is part of the BRCT superfamily and in the DNA polymerase type-X family. The polymerases in this family have different binding sites to the protein, but remain similar in domain. Figure 2. Putative domains of whale shark best hit predicted proteins. All of the best-hit whale shark predicted proteins contained putative BRCT and homeo domains as predicted by NCBI BLAST server analyses.
Orthologs
The human DNTT protein sequence was used as query in NCBI BLAST searches against individual species' protein databases. The sequence was blasted against the following species: mouse, chimpanzee, zebra fish, and yeast. Orthologues were found in mice and chimpanzees. All species returned homeodomain proteins but not necessarily DNTT orthologues.
Table 2. This table shows the species blasted against human protein DNTT. It presents the name of protein in that specific specie, the ID, length of protein and E-Value. The lower the E-value the more similar the specie is to the original, which was human in this study.
Phylogeny
The best hits from protein database searches using the human DNTT protein as query were used to create a phylogenetic tree. From this tree, it is clear that the human species and chimpanzees share the most similarity. They share traits that derived from a common ancestor and this relationship is seen for each branch of the phylogenetic tree.
Figure 3. This phylogenetic tree portrays the orthologues blasted against the DNTT human protein. The branch that says Zebra stands for the specie zebra fish. It is clear that humans and chimpanzees share a more similar DNTT sequence than mouse and human, zebra fish and human, and the yeast and human.
Conclusions We were not able to securely identify DNTT protein in whale sharks; however, we were able to find evidence for several DNTT domain polymerases and functions in other species. The DNA nucleotidylexotransferase that is originally found in humans can also be found in chimpanzees as well as in mice. The orthologues were researched further and a phylogeny tree could be formed and connections made based on the presence of DNTT in these organisms.
References
Mitchell, B. S., and K. N. Mahajan. "Role of Human Pso4 in Mammalian DNA Repair and Association with Terminal Deoxynucleotidyl Transferase."National Center for Biotechnology Information. U.S. National Library of Medicine, 16 Sept. 2003. Web. 14 Apr. 2015.
Peterson, R C et al. “Molecular Cloning of Human Terminal Deoxynucleotidyltransferase.” Proceedings of the National Academy of Sciences of the United States of America 81.14 (1984): 4363–4367. Print.
Peterson, RC, LC Cheung, FJ Bollum, ST White, and LM Chang. "Expression of Human Terminal Deoxynucleotidyl Transferase in Escherichia Coli." National Center for Biotechnology Information. U.S. National Library of Medicine, 5 Sept. 1985. Web. 14 Apr. 2015.
Yamashita, N., N. Shimazaki, S. Ibe, and A. Tanabe. "Terminal Deoxynucleotidyltransferase Directly Interacts with a Novel Nuclear Protein That Is Homologous to P65." National Center for Biotechnology Information. U.S. National Library of Medicine, 6 July 2001. Web. 14 Apr. 2015.
Hardy, Richard (2008). "Chapter 7: B Lymphocyte Development and Biology". In Paul, William. Fundamental Immunology (Book) (6th ed.). Philadelphia: Lippincott Williams & Wilkins. pp. 237–269.
This Project
This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background Information
DNA nucleotidylexotransferase, a protein found in the nucleoplasm, is a template-independent DNA polymerase, which catalyzes the random addition of deoxynucleoside 5'-triphosphate to the 3'-end of a DNA initiator. One of the in vivo functions of this enzyme is the addition of nucleotides at the junction (N region) of rearranged Ig heavy chain and T-cell receptor gene segments during the maturation of B- and T-cells. Unlike most DNA polymerases, this protein does not require a template. The preferred substrate of this enzyme is a 3’-overhang, but it can also add nucleotides to blunt or recessed 3' ends. Cobalt is a necessary cofactor; however, the enzyme catalyzes reaction upon Magnesium and Manganese administration in vitro.
Figure 1. Structure of DNA nucleotidylexotransferase. This figure demonstrates the shape of DNTT, a protein found in the nucleoplasm. This specifically shows amino acids 19-125 of human DNA nucleotidylexotransferase.
Methods
Whale Shark Predicted Orthologs:
The human protein sequence (ENSP00000360216) was entered as a query in a Blast search against the Predicted Whale Shark Protein database on whaleshark.georgiaaquarium.org. The top predicted protein hits full predicted sequences were in turn used as queries against the NCBI human protein database. The whale shark database was then searched using the elephant shark (closest ortholog) predicted DNTT sequence as a query.
Predicted Orthologs:
DNTT predicted orthologs were identified in other species using the NCBI Blast server. Protein BLASTs were performed against databases for the mouse, zebrafish, yeast, and elephant shark genomes. The DNTT protein was used as query sequence for these searches.
Phylogenetic Tree:
The highest matching ortholog with the lowest E-Value for each species listed above, as well as the closest matches for the whale shark blast hits, were used to make a phylogenetic tree and a multiple sequence alignment among the species.Clustal was used to create both the sequence alignment and tree.
Searching for DNTT in the Whale Shark:
The human DNTT protein sequence was used as a query against the whale shark predicted protein database. The top five hits are shown below. The lowest e-value was 1e-15 and the highest percent identity was only 48.00%.
Protein Domains
DNA nucleotidylexotransferase is part of the BRCT superfamily and in the DNA polymerase type-X family. The polymerases in this family have different binding sites to the protein, but remain similar in domain.
Figure 2. Putative domains of whale shark best hit predicted proteins. All of the best-hit whale shark predicted proteins contained putative BRCT and homeo domains as predicted by NCBI BLAST server analyses.
Orthologs
The human DNTT protein sequence was used as query in NCBI BLAST searches against individual species' protein databases. The sequence was blasted against the following species: mouse, chimpanzee, zebra fish, and yeast. Orthologues were found in mice and chimpanzees. All species returned homeodomain proteins but not necessarily DNTT orthologues.
Phylogeny
The best hits from protein database searches using the human DNTT protein as query were used to create a phylogenetic tree. From this tree, it is clear that the human species and chimpanzees share the most similarity. They share traits that derived from a common ancestor and this relationship is seen for each branch of the phylogenetic tree.
Figure 3. This phylogenetic tree portrays the orthologues blasted against the DNTT human protein. The branch that says Zebra stands for the specie zebra fish. It is clear that humans and chimpanzees share a more similar DNTT sequence than mouse and human, zebra fish and human, and the yeast and human.
Conclusions
We were not able to securely identify DNTT protein in whale sharks; however, we were able to find evidence for several DNTT domain polymerases and functions in other species. The DNA nucleotidylexotransferase that is originally found in humans can also be found in chimpanzees as well as in mice. The orthologues were researched further and a phylogeny tree could be formed and connections made based on the presence of DNTT in these organisms.
References
Mitchell, B. S., and K. N. Mahajan. "Role of Human Pso4 in Mammalian DNA Repair and Association with Terminal Deoxynucleotidyl Transferase."National Center for Biotechnology Information. U.S. National Library of Medicine, 16 Sept. 2003. Web. 14 Apr. 2015.
Peterson, R C et al. “Molecular Cloning of Human Terminal Deoxynucleotidyltransferase.” Proceedings of the National Academy of Sciences of the United States of America 81.14 (1984): 4363–4367. Print.
"DNA Nucleotidylexotransferase." DNTT. Uniprot, n.d. Web. 14 Apr. 2015.
Peterson, RC, LC Cheung, FJ Bollum, ST White, and LM Chang. "Expression of Human Terminal Deoxynucleotidyl Transferase in Escherichia Coli." National Center for Biotechnology Information. U.S. National Library of Medicine, 5 Sept. 1985. Web. 14 Apr. 2015.
Yamashita, N., N. Shimazaki, S. Ibe, and A. Tanabe. "Terminal Deoxynucleotidyltransferase Directly Interacts with a Novel Nuclear Protein That Is Homologous to P65." National Center for Biotechnology Information. U.S. National Library of Medicine, 6 July 2001. Web. 14 Apr. 2015.
Hardy, Richard (2008). "Chapter 7: B Lymphocyte Development and Biology". In Paul, William. Fundamental Immunology (Book) (6th ed.). Philadelphia: Lippincott Williams & Wilkins. pp. 237–269.