ABSTRACT
In the current era of evolutionary biology several species have undergone change in their genetic code distribution. Here a very simple way follows to describe how a species has gone under mutation at genetic level. The idea implemented on the species Euplotes with the help of comparative genomics analysis and some techniques like Clustal X and Phylip package. The purpose of analysis to find out the number of UGA codon in many Euplotes species and generate a phylogenetic tree with bootstrap value to show how the numbers of UGA varying among the species. Implementation of ClustalX and Phylip program to generate a tree with specific bootstrap values. The study shows the clear significance of changes in the numbers of Euplotes species during molecular evolution.
Keywords: - Euplotes, genetic code, bootstrap values, phylogenetic tree, Seqboot, etc.
Introduction
It was initially believed that genetic codes are universal, as in most of the organism genetic code coded for same protein, but as the availability of complete genome increases, studies shows in some organism genetic code showed deviation from their universality i.e. the same genetic code may code for different amino acid in other organism. Changes involve the reassignment of codon from one amino acid to another or from stop codon to sense codon or vice-versa. It is expected that changes causing the codon reassignment to be strongly disadvantageous and to be eliminated by selection. Disappearance of a codon from its original position and reappearance of a codon in the position where new amino acid preferred is possible due to mutation. Codon reassignment is caused by changes in the tRNA or release factor. Various mechanisms have been proposed for codon reassignment, these mechanisms are depends on gain and loss framework. Gain refers to a new tRNA gene, which is responsible of reassignment of a codon as a different amino acid while loss refers to the deletion of an existing tRNA for the reassignments of codon. In codon disappearance (CD) mechanism for a single amino acid coded by several genetic codes is replaced by synonym codon while in Ambiguous mechanism disappearance of codon doesn’t need. In this mechanism during a transition period one codon is ambiguously translated into to different amino acid. It corresponds to occurrence of gain before loss. In unassigned mechanism during transition period no tRNA is available that can translate into an amino acid. Here losses occur before gain.
Codon Disappearance (CD) mechanism (Osawa and Jukes 1989, 1995) - this mechanisms describes that for an amino acid (or stop codon) with more than one codon,it is possible that synonymous codons could be able to replace previous know codons. Means now it has been proved that all species are not using universal codons to translate it into an amino acid or stop codon. According to this mechanism the codon disappears entirely form the genome. After this the gain and loss in the translation system are neutral changes that do not affect the organism. After the gain and loss occur, the codon may reappear in the genome by mutations at sites where the new amino acid is preferred.
Ambiguous intermediate (AI) mechanism (Schultz and Yarus 1994,1996)- according to this mechanism the codon does not need to disappear in order to be reassigned and they have proposed that there is a transient period when the codon become ambiguous and thansalted into two different amino acids ambiguously.
Unassigned Codon (UC) mechanism (Sengupta and Paul Higgs, 2005) - this mechanism corresponds to the case where the loss occurs before the gain. There is an ibtermediate period where there is no tRNA available that can efficiently translate the codon; hence we say the codon is unassigned. Here the new codon becomes establishes when the gain in function of the new tRNA occurs and the codon is reassigned to a new amino acid. The other part of this mechanism is if a codon were truly unsassigned, and no tRNA could translate it at all, and then the loss of the original tRNA would be lethal if the codon ha not previously disappeared. Several cases are known where an alternative tRNA is able to translate; a codon after the tRNA that was specific to that codon has been deleted (Yokobori et al. 2001).
Compensatory Change (CC) mechanism â€" this mechanism that occurs in the gain loss framework is referred to as compensatory change because of its analogy with compensatory mutations in molecular evolution. (Kimura 1985) considered a pair of mutations such that each is deleterious when it occurs alone, but when both occur together they are neutral.
According to Sengupta and Higgs, 2005 the predictions from their theory and stimulations were that stop codon are most likely to be reassigned via codon disappearance mechanism because they are rare in the first place (and change disappearance is therefore relatively likely), and also because, if the codon do not disappear the penalty for read through of a stop codon is likely to be larger than the penalty for mis translation of an amino acid.UGA is rare in almost species used as stop codon. All of these have high AU content genome,which probably the reason UAA is preferred over UGA as a stop codon.
Reassignment of UGA codon from stop to Cysteine
Reassignment of UGA codon to Cystein is reported in various ciliates. In some Euplotes in which UGU & UGC codes for cysteine and the tRNA cysteine has UCA anticodon. Mutation of the wobbles positions U to G creates a GCA anticodon that can pair with UGU, UGC and UGA hence the reassignment UGA to Cysteine.
Here we have analysed that all 18s rRNA sequences of all available genomes to determine in Euplotes species this mutation has occurred. We have also noticed that all Euplotes species have UGA is stop have a UCA anticodon when UGA is stop ha
The UGA stop to cysteine and UAA and UAG for termination occurs in some Euplotes species (Meyer F, 1991). This analysis over 18s rRNA subunits sequences of some Euplotes,Ciliates and close species like Plasmodium. The reassignment has occurred in some Euplotes species and some other ciliates like Haltheia, Nyctotherus. In Euplotes only UGA codon codes for Cysteine. The tRNA Cys has a UCA anticodon. Mutation of the wobbles position G to U creating a GCA anticodon that can pair with all cysteine codons like UGU and UGC also pair with UGA. Hence the reassignment of UGA to Cysteine. We have analysed the 18s rRNA sequences of some Euplotes in this species mutation has occurred we have noticed that all species in which UGA is stop have a UCA anticodon but in some Euplotes UGA is Cysteine have a GCA anticodon.
All the studies done with the help of some phylogenetic techniques and softwares like Clustal X and Phylip.These softwares could be able to find out and help to understand and analyse the of phylogenetic relationship between Euplotes and some other ciliates species. These comparative genomics techniques are available online and offline. This study done by with the help of softwares Clustal X and Phylip. As per our knowledge NCBI has large collection of sequences like nucleotide and protein respectively. The phylogenetic analysis show how the Euplotes species, different from other ciliates at genetic code level. Many studies have been already done to shows the mutation in the tRNA cysteine of Euplotes species.
This study shows phylogenetic analysis with tree construction through Phylip program. Phylip program could be able to generate tree with higher bootstrap values among all Euplotes species. By the help of SEQBOOT program followed by DNAPARS and CONSENSE program of Phylip, we get a phylogenetic tree with maximum bootstrap value. The branches of phylogenetic tree with high bootstrap value helps to understand the evolutionary relationship among Euplotes and some other ciliates species like other Spirotrichs and Nyctotherus. To understand the complexity of the codon reassignment UGA stop to cysteine,we have taken a very close species like Plasmodium(Babesia microti) as outgroup species.
The phylogenetic tree could be able to describe that which species are close to each other in the evolution. The genetic code changes in Euplotes UGA as stop to cysteine could be understand by numbers of UGA codons in all species. The comparative analysis of all UGA codon, which is collected by CODON USAGE database. In the methods section, explanation has been given with each and every step to understand how the variations noticed in the numbers of the all UGA in target species.
MATERIAL & METHODS
All the study materials were collected from several scientific sites and databases like National Centre for Biotechnology Information (NCBI), Codon Usage Database.
18s rRNA nucleotide sequences analysis
18s rRNA nucleotide sequences from Euplotes, other Spirotrichs, Nycotherus ovalis and a close species of Plasmodium, Babesia microti. We have aligned all 18s rRNA sequences available from NCBI in fasta format.
Aligned sequences using Clustal X (version 1.83) for multiple sequence alignment and identified the regions where the misspairing (G:A) occurred due to mutation in tRNA cysteine. The mutation occurred we have noticed that it is common to all Euplotes species. Thus the genetic codes found in Euplotes species referred as non-standard genetic codes.
We considered another phylogenetics analysis program; Phylip package (3.67 versions) to generate a phylogenetic tree with high bootstrap value the tree showed the branches are common in many datasets. Means bootstrap values shows the species branches come together maximum time. Those branches have high bootstrap value are more close to each other in the evolution. We have generated a phylogenetic tree with the use of an out-group species Babesia microti to analyse the comparison among all Euplotes and other close species.
Sequences:-
>gi|51490773|emb|AJ811016.1| Euplotes harpa partial 18S rRNA gene, strain FC1
>gi|16943647|emb|AJ310491.1| Euplotes eurystomus 18S rRNA gene
>gi|16943646|emb|AJ310490.1| Euplotes minuta 18S rRNA gene
>gi|156636507|gb|EU103618.1| Euplotes aediculatus strain Lahorensis 18S ribosomal RNA gene, complete sequence
>gi|18307473|emb|AJ305251.1| Euplotes raikovi macronuclear partial 18S rRNA gene, strain 39W
>gi|38146187|gb|AY361895.1| Moneuplotes crassus strain SfaqsB*1 18S ribosomal RNA gene, partial sequence
>gi|38146169|gb|AY361856.1| Euplotes vannus strain SML 18S ribosomal RNA gene, partial sequence
>gi|18307470|emb|AJ305248.1| Euplotes rariseta macronuclear partial 18S rRNA gene, strain GRH5
>gi|18307476|emb|AJ305254.1| Euplotes muscicola macronuclear partial 18S rRNA gene, strain Bag12
>gi|28874736|emb|AJ549210.1| Euplotes magnicirratus macronuclear partial 18S rRNA gene, strain CAMP4.4
>gi|18307471|emb|AJ305249.1| Euplotes charon macronuclear partial 18S rRNA gene, strain Liv31
>gi|13162624|gb|AY007441.1| Halteria grandinella clone 1 18S ribosomal RNA, partial sequence
>gi|13491584|gb|AY007450.1| Metopus palaeformis clone 1 18S ribosomal RNA gene, partial sequence
>gi|3660532|emb|AJ222678.1| Nyctotherus ovalis (isolated from Periplaneta americana var. Amsterdam) 18S rRNA gene
>gi|163943657|gb|EU168705.1| Babesia microti isolate MM-1 18S ribosomal RNA gene, partial sequence
Clustal X
Figure: 3- Clustal X Software
In the above figure the gray region show the clear identification of mutation in tRNA cysteine anticodons for Euplotes and other ciliates species. If we will compare the gray along with Babesia micoti sequence no 15, region we will defiantly notice the mutation occurred at the first anticodon positon G: A misspairing.
The phylogenetic analysis with a combine alignment of 18s rRNA subuit sequences of Euplotes species. The 18s rRNA sequences were aligned initially using ClustalX (Thompson et al. 1997)
The lengths of the sequences were 1823 to 1898 bps.
RESULTS
After the successful running the phylip program we could be able to notice following results:
1-The phylogeny tree construction of Euplotes and other species, here Babesia microti is an out group species.
The outfile contain following information and an unrooted tree followed by bootstrap values.
Figure: 9- Edited tree file with Number of Codons present in the species
Here all the results are summarized in a table:
Codon Table
Euplotes and some other close species
Stop codons
Translation table
S.No.
Species
UAA
UAG
UGA
TT
1.
Halteria grandinella
0
0
2
6
2.
Euplotes minuta
2
0
0
10
3.
Euplotes vannus
20
5
0
10
4.
Moneuplotes crassus
48
5
61
10
5.
Euplotes harpa
?
?
?
10
6.
Euplotes aediculatus
28
2
84
10
7.
Euplotes eurystomus
4
0
0
10
8.
Euplotes rariseta
?
?
?
10
9.
Euplotes muscicola
?
?
?
10
10.
Euplotes magnicciratus
?
?
?
10
11.
Euplotes charon
?
?
?
10
12.
Euplotes raikovi
7
0
0
10
13.
Nyctotherus ovalis
44
9
35
10
14.
Metopus palaeformis
17
4
7
6
15.
Babesia microti
20
2
3
1
SUMMARY AND CONCLUSIONS
Summary of the whole work could be understood by following steps:
Collection of sequences of Euplotes and other 18s rRNA form NCBI database in FASTA format.
A complete text files preparation with all sequences for CLUSTAL X program to generate phylip format file.
After the generation of phylip alignment file for the SEQBOOT program followed by DNAPARS and CONSENSE.
Phyilp file loaded into the program SEQBOOT, a bootstap progam for the generations of outfile to construct a most reliable phylogenetic tree.
DNAPARS are able to load outfile into the program to generate an out file again.
Outfile generated by DNAPARS program now used by CONSENSE program to generate a outtree file this outtree file contain all the information about in a form of an unrooted tree of our given species.
The outfile could be open with notepad and now we have a desired output in the phylogenetic tree format.
After the completion of the methodology procedure, we could be able to notice the following details shown by analysis that we have found how could one species gone under the changes at genetic level due to the mutation or due to any other circumstances. Here We have analyzed that the UGA codon in several Euplotes species are code as either stop codon or cysteine.Through out the above phylogeny analysis we could be able to say that some species those are close to Euplotes have little change at their stop codon level they are close to the Euplotes species in the evolutionary relationship that why they define how an Euplotes species gone under the mutation stop codon to cysteine. Several new mechanisms shows the clear evidence of codon reassignment mechanism in Euplotes species the UGA codes as Cysteine due to presence of new reassigned tRNA cysteine,the codon reassignment process UGA stop to cysteine follows gain loss frame work. The tRNA cysteine anticodon UCA has been deleted before the appearance of new tRNA Cys GCA in the Euplotes species. There is an intermediate period where there is no tRNA available that could be efficiently translate the codon,during that period we can say that the UGA codon is unassigned. And UGA become established when the gain in the function of the new tRNA Cysteine occurs and the codon is reassigned to the amino acid Cysteine.
With the use of phylogeny and codon usage we can notice that the many of the closest species to the Euplotes species where UGA is reassigned have generally low usage of UGA, e.g. Halteria grandinella close relative of Euplotes has 2. But in a Plasmodium Babesia micrroti has 3 UGA. These evidences clearly show that the disappearance of UGA is possible and in many cases there is clear evidence that UGA was absent or almost absent, at the time of when it was reassigned. As we knows that stop codon are most likely to be reassigned via the codon disappearance mechanism (S. Sengupta and Higgs 2005).
