Research Article

Bioinformatics Tools Help to find out the Role of Unknown SRFK Initiation on Egg Activation at the Time of Fertilization

Shatarupa Dey¹ and Pranab Roy²*

¹Florida Institute of Technology, Melbourne, USA

²Department of Biotechnology, Institute of Child Health, India

Submission: May 17, 2019; Published: August 08, 2019

*Corresponding author: Pranab Roy, Institute of Child Health, 11, Biresh Guha Street, Kolkata - 700017, India

How to cite this article: Shatarupa D, Pranab R. Bioinformatics Tools Help to find out the Role of Unknown SRFK Initiation on Egg Activation at the Time of Fertilization. Adv Biotechnol Microbiol. 2019; 14(3): 555889. DOI: 10.19080/AIBM.2019.14.555889

Abstract

Release of Ca2+ ions from endoplasmic reticulum indicates egg activation at fertilization time [1]. SRC family kinase protein is directly or indirectly involved Phospholipase C Gamma (Plcγ) in this activation that leads to Ca2+ release [1]. Ca2+elevation in egg helps to block polyspermy and helps embryonic development [2]. It is already known that SFK is present in starfish Asterina miniata eggs and SFK1, SFK2 are main SRC kinases which are responsible for egg activation [3]. There are several SFK proteins present and perhaps they might have roles in ca2+ signalling pathway and egg activation. In this work, our main goal was to find out gene sequences of other unknown SRC kinases those are mainly responsible for egg activation at the time of egg fertilization. To achieve our goal, we use different type of bioinformatics tools from where we come to know gene alignment position, exon part of gene, phylogenic relationship of query protein and function of proteins according to their domain.

Introduction

Ca2+ release in all species at fertilization time is a hallmark of egg activation. Ca2+ helps to block polyspermy that initiate egg activation. Evidence that Src-type tyrosine kinase activity is necessary for initiation of calcium release at fertilization. SRC kinase is an important component which produces IP. From recent studies, it is found in some lower animals like starfish, sea urchin and frogs that SFK directly activate Plcγ and that produces IP3. CA2+ is released from IP3 receptor of ER. By using different inhibitor of SRC kinase it is proved that ca2+ release is delayed when SRC is inhibited and increase of rapidly activation of eggs is happened when kinase activity is increased. SFKs is expressed in starfish (Asterina miniata) egg and mainly SFK1, SFK2 and SFK3 are involved egg activation and ca2+ release. There are several SFK proteins are present and perhaps they might have role in Ca2+ signalling pathway and egg activation [4]. It is possible that different SFKs might have same overlapping role in Ca2+ pathway. Here, our main target is to identify gene sequence of SFK s and other unknown protein which might be involved in egg activation by using different bioinformatics tools. From previous study it is known that marine protostomes worm treated with either PP2 to inhibit SFKs or with U73122 to block PLCγ activity. By using different mechanism, it is known that inhibitor significantly reduce post insinuation of polar body. Confocal imaging techniques proof that Ca2+ wave block by U73122 but not by PP2, though immunoblot gives false result about PP2 inhibitor [5].

Methodology

Blast

Blast (Basic Local Alignment Search Tool) is a sequence similarity search tool that can be used to a quickly search sequence database for matches to a query sequence. Several types of blast exits to compare all combination of nucleotide or protein queries against a nucleotide or protein Blast on query sequence. Blast provides e value, statistical information about alignment. Here, we run blast against Patricia miniata model organism. Mainly whole genome shotgun coatings database is used here for optimizing program for blastn of somewhat similar sequence [6].

Spidey

Spidey is an active alignment and splicing NCBI toolkit. Two main objective of this software are

Alignment regardless intron size

Avoid pseudogenes and paralogs

It first aligns sequences and then collect exons only. It can take any genomic or FASTA mRNA sequence. It helps to determine the identity per exon, no of gaps per exon, Overall percentage identity, donor and acceptor site of exon. Here we use this software to delete intron part from aligning mRNA sequence and shot out the exon part that is coding sequence.

Smart Blast

Smart blast is more specific than blast. Smart blast summery provides a unique combining phytogenic tree and graphical overview between query and five matching sequences. Matching is represented by colored coating. Green indicates match from reference species, blue represents matches from no redundant database and yellow indicates query. Smart blast also gives information of conserved domain matches for query. If there are both superfamily matches and specific hit, then it will show specific hit only. It does not represent matches from multi domain model. Here it is mainly used to find phylogenic relationship with query sequence [7-8].

Interpro

Interpro helps to do functional analysis of protein according to their domain, families. It is mainly used in large-scale analysis of proteomes, genomes and metagenomes and it is very helpful to characterise individual protein sequences. Here it is tried to analysis of unknown function of SRC kinase gene sequences after changing it into Fasta protein format by using X blast toolkit. Additional information such as a description, consistent names and Gene Ontology (GO) terms are associated with each entry, where possible that would be helpful in future gene sequence analysis.

Results

Result1

Blast for checking alignment of our query sequence with other reference sequence

BLAST Result for Asterina miniata Src family tyrosine kinase (SFK1) mRNA, complete cds (Gene Bank: AY518774.1) (Figure 1a, 1b, & Figure 2).

Misc. feature: 882...1703

/gene=”SFK1”

/note=”Region: tyrosine kinase domain”

Result 2

Scanning exon part of aligned sequence after using Spidey tool (Figure 3a, 3b,3c, 3d).

Result 3

Smart blast of accession AY518774.1for find out photogenic relationship of our query protein SRFk1(Figure4a,4b&Figure5).

Result 4

Result from Interpro (Figure 6a,6b).

Discussion

In this work Asterina miniata Src Family Tyrosine Kinase (SFK1) mRNA, complete cds and Asterina miniata Src Family Kinase (SFK3) mRNA, complete cds are mainly used. We already know SFK1 and SFK3 both are important Ca2+ signalling pathway and egg activation at fertilization time. By using different bio informatics tool and method we try to find out coding gene sequence, unknown function, alignment range, phytogenic tree and graphical overview of known sequence. This data will be helpful to scan specific gene sequences from other unknown SRC kinase protein that might have role for egg activation and Ca2+ signalling pathway at fertilization time.

References

1. O Neill FJ, Gillett J, Foltz KR, (2004) Distinct roles for multiple Src family kinases at fertilization. J Cell Sci 117(Pt 25): 6227-6238.
2. Bates RC, Fees CP, Holland WL, Winger CC, Batbayar K, et al. (2014) Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization. Dev Biol 386(1): 165-180.
3. Stricker SA, Carroll DJ, Tsui WL (2010) Roles of Src family kinase signaling during fertilization and the first cell cycle in the marine protostome worm Cerebratulus. Int J Dev Biol 54(5): 787-793.
4. Bates RC, Fees CP, Holland WL, Winger CC, Batbayar K, et al. (2014) Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization. Dev Biol 386(1): 165-180.
5. Giusti AF, Xu W, Hinkle B, Terasaki M, Jaffe LA (2000) Evidence that fertilization activates starfish eggs by sequential activation of a Src-like kinase and phospholipase cgamma. J Biol Chem 275(22): 16788-16794.
6. https://www.ebi.ac.uk/interpro/search/sequence-search.
7. https://ncbiinsights.ncbi.nlm.nih.gov/2015/07/29/smartblast/
8. https://blast.ncbi.nlm.nih.gov/Blast.cgi.