Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Summary

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

Abstract

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms' long evolutionary history or scarcity of fossilization.

In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene. It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

Introduction

DNA or RNA sequences carry genetic information for underlying phenotypes that can be analyzed through physiological and biochemical methods or observed through morphological and fossil evidence. In a sense, genetic information is more reliable than evaluating external phenotypes because the former is the basis for the latter. In evolutionary study, fossil evidence is very direct and convincing. However, many organisms, such as microorganisms, have little chance to form a fossil during long geologic ages. Therefore, molecular information such as nucleotide sequences and amino acid sequences from related extant organisms are of value for exploring evolutionary relations....

Protocol

1. Sequence Alignment

1. Collect amino acid sequences of eukaryotic SWEET and prokaryotic SemiSWEET in separate documents and list them in FASTA format. Download sequences from the National Center for Biotechnology Information (NCBI), European Molecular Biology Laboratory (EMBL), and the DNA Data Bank of Japan (DDBJ) databases by similarity search with the Basic Local Alignment Search Tool (BLAST) tool.
   1. In the example files, collect 228 putative SWEET protein sequences possessing two MtN3/saliva domains (7 transmembrane helices) of eukaryotes and 57 SemiSWEET protein sequences possessing a single MtN3/saliva domain (3 transmembran....

Results

Phylogenetic trees show that all of the first MtN3/saliva domains of the 35 SWEET sequences clustered as one clade and the second MtN3/saliva domains of the SWEET sequences clustered as another clade. In addition, alignment results of the SWEETs and SemiSWEETs show that some SemiSWEETs from α-Proteobacteria aligned with the first MtN3/saliva domain of the SWEET sequences, whereas SemiSWEETs from Methanobacteria (archaea) aligned with the second MtN3/saliva domain of the SWEET sequences. T.......

Discussion

It is becoming increasingly popular in biological research to make a phylogenetic tree based on nucleotide or amino acid sequences⁸. Generally, there are three critical stages of the practice including sequence alignment, evaluation of the aligned sequences with the proper method or algorithm, and visualization of the computational result as a phylogenetic tree. In the presented study, three rounds of sequence alignment were conducted: first, the SWEET protein sequences, including the first a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adobe Illustration			a graphical tool developed by Adobe Systems Software Ireland Ltd. Copyright © 2017
BioEdit			a biological sequence alignment editor written for Windows 95/98/NT/2000/XP/7. Copyright © Tom Hall
Clustal Omega			a package for making multiple sequence alignments of amino acid or nucleotide sequences.  http://www.clustal.org/
CorelDRAW			a graphic design software. Copyright © 2017 Corel Corporation
FigTree			a graphical viewer of phylogenetic trees designed by the University of Edinburgh
MEGA			MolecularEvolutionary Genetics Analysis version6.0 http://www.megasoftware.net/home
MrBayes			an Bayesian phylogenetic inference tool
NVIDIA			a company designs graphics processing units (GPUs) for the gaming and professional markets. Corporation Copyright © 2017
PAUP			Phylogenetic Analysis Using Parsimony. David Swofford's program implements the maximum likelihood method under a number of nucleotide models.
Photoshop			a raster graphics editor developed and published by Adobe Systems Software Ireland Ltd. Copyright © 2017
RHYTHM			a knowledge based prediction of hekix contacts. Charité Berlin – Protein Formatics Group - Copyright 2007-2009
TMHMM			a tool for prediction of transmembrane helices in proteins. http://www.cbs.dtu.dk/services/TMHMM/
Compter			4 GB memory, Core 2 or above CPU. Windows 7, Windows 10