Comparative analysis of human and mammalias genes of protein ortologs of cytochrome P450 2E1

Aim. To carry out a comparison analysis of nucleotide sequences for the cytochrome P450 2E1 protein ortologs genes and reestablish the connection of gene evolution with nucleotide content. Methods. In silico: BLAST, ClustalW, MEGA4, PHP. Results. A general phylogeny of CYP2E1 genes is described. The most affinity is found for human translated sequences with Pan troglodytes. The transition Ñ > T is the most often, it occurs in introns by 2.6 times more than in exons. The correlation of keto-amino skew of CYP2E1 genes and evolution age of species is stated. Conclusions. The analysis carried out in the paper allows us to assume that a common ancestor of the CYP2E1 protein isoform lived before the divergence between rodent and Primates orders, i.e. 70 million years ago. The single nucleotide substitution is accumulated in introns during evolution.

Introduction.The multigen family of cytochrome P450 is one of the most intensively studied issues, because it plays an important role in metabolism of endo-and exogenous substratum.The ethanol inducible isoform of P450 2E1 cytochrome (CYP2E1) is of a special interest [1].
The induction of Cyp2E1 expression leads to an increase in the level of oxygen radicals generated by CYP2E1, which are able to initiate the NADPH-depended lipid peroxidation.Because of this the cell acid-base balance is hereupon violated, and the oxidative stress develops [2].The interaction of the genetic and environmental factors results in the tissue-specific changes of synthesis and activity of Cyp2E1, which brings in a certain contribution to the pathogeny of liver, pancreas diseases and other.[3,4,5].
There are some data about the impact of exogenous factors on expression of Cyp2E1 [6], while the influence of genetic factors on expression of enzyme has not been studied enough.From the evolutional viewpoint, the cytochrome P450 is a unique protein [7], which survived from the primitive aerobes-prokaryotes to the human.This allows us to believe that most modern genes of cytochromes had a general predecessor, existed about 2 billions of years ago [8].
An opinion exists, that the primary evolutional function of cytochromes P450 in a cell was connected with its participation in the plastic and power metabolisms.The cytochrome P450 got the main specialization as xenobiotic biotransformator about 800 million years ago, which coincides with time of animals appearance [8].
In this paper, a comparative analysis of nucleotide sequences of mammalian and human genes of the Cyp2E1 orthology proteins is made for the restoration of evolutional history of the gene and mutational events.The ini tial align ment of nu cle o tide se quences was re al ized by BLASTN [10] and mul ti ple align ment was ex e cuted by ClustalW [11].The phylo gen etic anal y sis was car ried out by the MEGA4 packet [12].The analy sis of sin gle nu cle o tide sub sti tu tions was ful filled by us ing PHP com puter lan guage [13].The value of keto-amino skew (K skew ) was cal cu lated us ing the formula K skew = (NG+NT -NA -NC)/L, where NA, NG, NT, NC are ab so lute quan ti ties of cor re spond ing nucleo tides in a frag ment of length (L = NA + NG + NT + NC) [14].The ar ith met i cal mean val ues of K skew for exons and introns se quences were cal cu lated, and the stan dard er ror of sam ple was found us ing the for mula STD_ERR=RMSD/vN, where N is the size of a sample [15].

Materials and methods
Results and discussions The structure of the Cyp2E1 genes coincides for the majority of studied mammals.It consists of nine exons and eight introns.A unique variant of mRNA Cyp2E1 is described for all species, in which the translated sequence of gene begins in the first exon and terminates in the last one.In spite of identical number of exons, the length of Cyp2E1 differs for different species due to the varied introns length.
Thus, Homo sapiens has the longest sixth intron (due to the repetitive GGG sequence of 576 b.p.), and Mus musculus has the longest second intron.The structural likeness of the intron parts of CYP2E from Rattus norvegicus and Mus musculus is of special interest.They have around 86% similarity between the sequences of second and sixth introns, and 93%between the fourth introns.
To reconstruct the evolutional history of the P450 2E1 cytochrome gene the multiple alignments of the translated sequences were executed and a phylogenetic tree was built (Fig. 1).
It follows from Fig. 1, that the evolution of CYP2E1 gene undergoes a few divergences; in particular, there are independent ways of rodents, primates and other mammalian genes development.It is possible that the analyzed CYP2E1 genes had a common ancestor before the division of rodents and primates that is about 70 millions years ago.
The pairwise alignments of the CYP2E1 exonic and intronic sequences between each species and human were executed and frequencies of single nucleotide substitutions were analyzed in homologous sites to establish the nucleotide composition changes of the examined genes during their evolution.
Twelve possible types of single nucleotide substitutions were estimated.It should be noted that the substantial distinctions in frequencies of individual types of replacements in exonic and inronic parts of genes were found out.In particular, the A -G transitions were observed more frequent, than the T-C ones, and the G-A transitions were observed more frequent than the C-T ones.The final results are presented in Table .The substitutions are found to be more frequent in introns, than in exons.At the same time the transitions in exons are by 23% more frequent, than transversions.
Rather small predominance (about 5%). of transversions was observed in introns due to the transition G>C, which, possibly, related to the species features of CpG rich areas, where Cyp2E1 is located.This fact is of special interest and requires a more thorough research.
The fixed distinctions between nucleotide ratios in different functional portions of genes were shown in our previous work [14].Indeed, the high values of the keto-amine skew of nucleotide composition (K skew ) were registered in introns.This reflects predominance of the most frequent types of substituted nucleotides during the spontaneous mutagenesis.In order to check changes of K skew value in evolution, we calculated the value K skew for introns and exons of Cyp2E1 in each species and compared it with early obtained data [14].The changes in the K skew value depending on the evolutional affinity of nucleotide sequences is presented in Fig. 2.
Fig. 2A shows that the introns K skew values at evolution remote from Homo sapiens are negative due to the predominance of A and C.During evolution the K skew value increases from -0,041±0,021 (Mus Total frequencies of mutational events in gene CYP2E1 musculus ) and -0,042±0,032 (Bos taurus ) to 0,004±0,016 (Pan troglodytes), and for Homo sapiens it arrives 0,017±0,027.On the contrary, all the exons K skew values are negative (Fig. 2B) and similar for eight mammalian species.This fact can be explained by the accumulation of thymine and guanine in the loci of the least selection pressure.Thus, the K skew value can be of particular interest for further research, because it can serve as an additional criterion for the estimation of nucleotide sequence evolutional age.The presented numerical analysis of the gene sequences of P450 2E1 cythochrome orthologous proteins allowed us to describe general regularities of the Cyp2E1 phylogeny.We have shown that the frequencies of single nucleotide substitutions in the Cyp2E1 introns are by 2.62 times higher in comparison with the exons .The most frequent mutational events are transitions which come to 48% of all single nucleotide substitutions.

Fig. 1 .
Fig.1.Phylogenetic tree of the P450 2E1 cytochrome genes for human and seven mammals.Numerals mark evolutional distances in the reference units.