LIG4

This Project
This page is designed for Emory's Biology 142 Lab course. Groups of students were assigned proteins and asked to run sequence testing to find any homologs in similar species. Students were asked to determine if their proteins shared an homologs with the whale shark sequences.


Background Information
Ligase IV (LIG4) is a DNA ligase that joins single-stranded DNA breaks to double stranded DNA during an ATP-dependent reaction. The protein is essential in for recombination and for DNA double stranded break repairs(NCBI 2015). Also, it is plays a pivotal role in DNA non-homologous end-joining. LIG4 forms a complex with X-Ray repair cross complementing protein 4 (XRCC4) and interacts with DNA-dependent protein kinase (NCBI 2004)external image nrm3523-i1.jpgFigure 1: DNA damage and various proteins used for its repair. LIG4 is the protein used to help bring the the broken single stranded DNA back to a double stranded DNA with the help other various proteins.
Mutations in the Gene:LIG4 syndrome is an autosomal hereditary disorder that have a hypomorphic mutation in DNA Ligase IV (Girard 2004). This syndrome is an immunodeficiency disease that delays development and growth. Most patients with this syndrome displays skin and facial abnormalities,microcephaly, and developmental and growth delay.

MethodsWhale Shark Predicted Orthologs:The human protein LIG4 ENSP00000349393 was used as a query sequence against the whale shark predicted proteins database through Galaxy and the Georgia Aquarium. The top hits from this search were recored. The top hits from the first blast searches were run as reciprocal query sequences against the human protein database through NCBI.Predicted Orthologs:The predicted orthologs were identified in the other species through the NCBI Server. Protein blast, using human as a default query, were run against using protein databases for mouse, hippopotamus, beagle dog, fruit flies, true yeasts, and redeye piranha. Phylogenetic Tree:The hit with the lowest e-Value from each species was used to create a phylogenetic tree relating whale sharks, humans, and the other species based on the LIG4 protein along with the top 4 whale shark BLAST hits. The phylogenetic tree was created using ClustalW2.

Analyzing The Whale Shark GenomeThe Human LIG4 protein sequences was the query the predicted whale shark sequence used to perform the blast+. The top hits, based on lowest e-value, were recorded below in Table 1.
Whale Shark ID
E-value
Alignment length
% Identity
g45065.t1
6e-15
196
27.55
g26119.t1
4e-08
76
35.53
g25015.t1
3e-08
52
34.62
g26186.t1
4e-07
71
30.99

Table 1: Human LIG4 protein against predicted whale shark protein with the top hits according to lowest e-value.


Protein DomainsLIG4 is of the DNA Ligase superfamily, therefore, if the whale shark is orthologues to the human LIG4 protein, it would contain the DNA Ligase domain. The NCBI Blast was ran with the human LIG4 protein against the yeast LIG4 because it had the lowest e-value making it a viable orthologue. A study conducted showed that the yeast species has high amounts of DNA ligase in its DNA which explains the emergence of the DNA ligase family in the Blast (Cameron 1979). The DNA Ligase are a large family of evolutionary related proteins that play important roles in a wide range of DNA roles, including chromosomal DNA replication, DNA repair and recombination, in all three kingdoms of life (Martin 2002).
CD search result summary
CD search result summary


Figure 2: The protein domains seen when the human LIG4 protein was BLASTed against the yeast.


Orthologs
The humanLIG4 protein sequence [ENST00000035692] was used as query in the NCBI Blast search against individual protein sequences of the following species. As seen in Table 2, the orthologues of the Beagle Dog, mouse, yeast, and fruit fly were nearly identical to the protein LIG4 however, the hippopotamus returned with unlikely matches.

Query
Database
Description
Query Cover
E Value
Identity
Human
Hippo
__RecName: Full=Hemoglobin subunit beta; AltName: Full=Beta-globin; AltName: Full=Hemoglobin beta chain [Hippopotamus amphibius]__
2%
6.0
__P19016.1__
Human
Mouse
__DNA ligase 3 isoform a [Mus musculus]__
60%
2e-38
__NP_034846.2__
Human
Beagle Dog
__DNA ligase 3 isoform X3 [Canis lupus familiaris]__
59%
3e-40
__XP_005624871.1__
Human
Fruit Fly
__Ligase4 [Drosophila melanogaster]__
89%
1e-122
__NP_572907.1__
Human
Yeast
__YALI0D21384p [Yarrowia lipolytica] [Yarrowia lipolytica CLIB122]__
98%
2e-124
__XP_503109.1__
Human
Piranha
__hypothetical protein [Serrasalmus rhombeus]__
20%
1.7
__AGW00428.1__
Table 2: Best hits with the human LIG4 protein BLAST. The human LIG4 sequence was compared to individual species using protein BLAST searches. The query, database, description, query cover, e-value and identity were recorded for the top hits in each BLAST


Phylogeny
The best hits from the protein database were used to make the phylogenetic tree. From this tree, it is evident that the four whale shark predicted genes are very closely related as they are grouped together on the tree (Figure 2). It is surprising that the four predicted whale genes are closely related to the yeast and not the human gene. This most likely suggest divergence in the DNA ligase gene between the predicted genes and the human gene that occurred earlier in the transition.
external image PNeZQ_Wtjg5h2LD52uvCR4W0F5bxbb1CViNKZp1hegzEprAq9lEvTL-O_3hteLKuLJHsd2VaDf0uadhXtc2OpOFkSkZa7n8sPvZuXzG40pYLS9LqR7JZd-rbiNP9EpYEuqEUOw
Figure 2: Phylogenetic Tree of LIG4 best hits. The best hits inputed into the ClustalW2 program using their individual protein sequences. Branch lengths represent relative evolutionary time.

ConclusionsWith the results found in our queries of the human gene against the whale shark and the other predicted orthologs, a clear homologue to the LIG4 gene was not determined. Based off of Table 2, yeast and the beagle dog, mouse and fruit fly show that they are close homologues to the LIG4 human gene. However, the phylogenetic tree is does not give us a clear indicator and does not correlate well with Table 2. Since there were close indicators to the previously stated species, those species could have similar factors to the human LIG4 gene. This finding could be contributed to similar evolutionary patterns after deviating form the phylogenetic tree.

ReferencesCameron, John. "Evidence for Transposition of Dispersed Repetitive DNA Families in." ScienceDirect. Department of Biochemistry Stanford University School of Medicine Stanford, 5 Jan. 1979. Web. 13 Apr. 2015.

"Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine, 5 Apr. 2015. Web. 13 Apr. 2015.

Girard, Pierre. "Human Molecular Genetics." Analysis of DNA Ligase IV Mutations Found in LIG4 Syndrome Patients: The Impact of Two Linked Polymorphisms. Human Molecular Genetics, 13 Mar. 2004. Web. 13 Apr. 2015.

"LIG4 Gene." Genetics Home Reference. Genetics Home Reference, 6 Apr. 2015. Web. 13 Apr. 2015.

Martin, Ina V., and Stuart A. MacNeill. "ATP-dependent DNA Ligases." Genome Biology. BioMed Central, 19 Mar. 2002. Web. 14 Apr. 2015.

"Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, 11 June 2004. Web. 13 Apr. 2015.

Background Information
IL18The human protein Interleukin 18 (IL18) is an immuno-modulatory protein that contributes to interferon production in T-helper type cells, which are a kind of T cell that aid the adaptive immune system in regulating immune processes. It is also a proinflammatory cytokine that stimulates the production of killer cells in the spleen.
Mutations in the GeneG/C polymorphisms in the IL18 protein are related to the development and progression of oral squamous cell carcinoma, but that C/A polymorphisms in the gene are not associated with the development of this cancer. In another recent study from the University of Nebraska Medical Center, researchers demonstrated that IL18 levels in patients with type 1 diabetes are significantly elevated, and suggested that regulated IL18 levels might make diabetes therapies more effective.

MethodsThe human protein IL18 ENSP00000280357 was used as a query sequence against the whale shark predicted proteins database through Galaxy and the Georgia Aquarium. The top hits from this search were recored.The top hits from the first blast searches were run as reciprocal query sequences against the human protein database through NCBI.The whale shark sequences were blasted against human, mouse, hippopotamus, beagle dog, fruit flies, true yeasts, and redeye piranha databases to identify orthologs in other species.
The hit with the lowest e-Value from each species was used to create a phylogenetic tree relating whale sharks, humans, and the other species based on the IL18 protein.

Analyzing The Whale Shark GenomeWhen we searched for IL18 in Whale Sharks, we found very little similarity between the human protein and any proteins produced in whale sharks.Below is a table of the top three hits returned from the blast.

Sequence ID
Length
% Identical Matches
e-Value
g12092.t1
39
38.46
7x10^-5
g19248.t1
62
33.87
9x10^-5
g18309.t1
51
23.53
9x10^-5
Next, we ran these sequences agains the human protein database in order to perform a reciprocal search.The data from this blast is synthesized below.















Protein Name
E-Value
% Identity
Accession
Nuclear Receptor Corepressor 2 isoform 3
3.2
39%
NP_001193583.1
hCG1980378
4.8
29%
EAW62213.1
fos-related antigen 2 isoform X1
2x10^-9
42%
XP_006712039.1



Protein Domains

Orthologs
Phylogeny
Using the sequences found in the human, beagle dog, mouse, whale shark, fruit fly, yeast, hippopotamus, and piranha genome, and the online Clustal tool, a phylogenetic tree was created. According to the table in Orthologs, the greatest match of the whale shark protein was found in human, dog, mouse, and fruit fly organisms. It was surprising to find the lack of similarity between the whale shark protein and the hippopotamus and piranha proteins, however, and the greater similarity of that protein to land mammals. This finding may suggest that this protein was developed a long time ago from a common ancestor that was more related to humans and whale sharks than hippopotami and piranhas.
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Conclusions

Because we had such high E-vaues in the original synthesis of our protein, it seems unlikely that there is any homology between the IL18 protein in humans and in Whale Sharks.

References

Genes and mapped phenotypes. (2015, February 28). Retrieved March 30, 2015, from http://www.ncbi.nlm.nih.gov/gene/3606

Harms, R., Yarde, D., & Guinn, Z. (2015). Increased expression of IL-18 in the serum and islets of type 1 diabetics. <i>Molecular Immunology,</i> 306-312. Retrieved April 1, 2015, from http://www.ncbi.nlm.nih.gov/pubmed/25576800

Singh, P., Ahmad, M., & Kumar, V. (2014). Effects of interleukin-18 promoter (C607A and G137C) gene polymorphisms and their association with oral squamous cell carcinoma (OSCC) in northern India. Tumor Biology, 35(12), 12275-12284. Retrieved March 30, 2015, fromhttp://www.ncbi.nlm.nih.gov/pubmed/25398