Factors influencing synonymous codon and amino acid usage biases in Aromonas salmonicida phage 44RR

A.P.Ghosh, A. Deb, and K. Sau* Department of Biotechnology, Haldia Institute of Technology, PO-HIT, Dist-Purba-Medinipur, Haldia, W. B. 721657. To reveal the structure-function of the genes of A. salmonicida phage 44RR, synonymous codon and amino acid usage biases in this phage have been investigated at length. As expected for an AT-rich phage, third codon position of the synonymous codons of 44RR carries mostly A and/or T base. Analyses on the RSCU values of 44RR genes reveal that synonymous codon usage bias in 44RR is strongly dictated by the mutational pressure. Further analysis reveals that 44RR-specifc tRNAs preferentially influence the codon usage of putatively lowly expressed genes of 44RR, whereas, codon usage of its putatively highly expressed genes are influenced mainly by the abundant cellular tRNAs. The data suggest that translational selection also plays a role in shaping the codon usage variation in 44RR. Additional analysis reveals that the three factors such as hydropathy, aromaticity and cysteine content are mostly responsible for the variation of amino acid usage in 44RR proteins. Key words: Relative synonymous codon usage (RSCU); correspondence analysis; amino acid usage; phage 44RR. .

Introduction

Synonymous codon and amino acid usages in living organisms had been shown to vary interas well as intra-genomically. While mutational pressure (Levine et al., 2000; Jenkins et al., 2001; Jenkins and Holmes, 2003), translational selection (Grantham et al., 1981; Ikemura, 1985; Sharp and Cowe, 1991; Lesnik et al., 2000; Ghosh et al., 2000; Gupta and Ghosh, 2001), secondary structure of proteins (Oresisc and Shalloway, 1998; Xie and Ding, 1998; Chiusano et al., 2000; Gupta et al., 2000; DOnofrio et al., 2002), replicational & transcriptional selection (McInerney, 1998; Romero et al., 2000) environmental factors (Lynn et al., 2002; Basak et al., 2004) etc influence the codon usage in various organisms, amino acid usage

*Corresponding author: (K. Sau): keya_sau@yahoo.co.in Tel: +91-3224-252900. Ext. 234. Fax: +91-3224-252800

was shown to be governed by hydrophobicity, aromaticity, mean molecular weight, cysteine content, etc. (Lobry and Gautier, 1994; Garat and Musto, 2000; Zavala et al., 2002; Banerjee et al., 2004; Naya et al., 2004). Apart from elucidating the structure-function and evolution of genes / genomes, codon usage in particular has number of practical applications. Synonymous codon and amino acid usage biases had been studied in details in a few phage genomes mainly belonging to different coliphages, mycobacteriophages, S. aureus phages, and P. aeruginosa phages (Sharp et al., 1984-85; Gouy, 1987; Holm, 1987; Kunisawa, 1992; Kunisawa et al., 1998, Sahu et al., 2004; Sahu et al., 2005, Sau et al., 2005a; Sau et al., 2005b). Synonymous codon usage of highly and lowly expressed genes in T4 were shown to be influenced by the abundant host tRNAs and own tRNAs, respectively, (Kunisawa, 1992). In phage T7, codon usage was also suggested to be influenced mainly by the abundant host tRNAs (Sharp et al., 1984-85). It was described that codon usage in both T4 and T7 are influenced mainly by mutational pressure (Kunisawa et al., 1998).

Table 1. Overall codon usage analysis in 44RR. 44RR HEG* RSCU 0.77 1.23 0.66 1.1 0.62 0.72 0.31 2.59 1.1 1.5 0.4 1 2.2 0.54 0.72 0.54 1.33 1.42 1 0.67 0.71 0.88 1.15 0.2 0.85 1.8 1.46 1.76 0.46 0.33 1.98 0.65 1.02 0.35 0.91 1.09 0.88 A.salmonicida HEG RSCU 0.46 1.54 0.03 0.31 0.17 0.65 0.00 4.84 0.54 2.36 0.11 1 0.94 1.11 0.62 1.34 1.04 2.78 0.11 0.16 0.38 1.53 0.48 0.85 0.24 2.42 0.62 3.01 0.29 0.07 1.11 1.90 0.36 0.63 0.52 1.48 0.37 1 1 1 1 1 2 1 1 1 tRNA copy 44RR

Amino Acid Phe Leu Codon UUU UUC UUA UUG CUU CUC CUA CUG Ile AUU AUC AUA Met Val AUG GUU GUC GUA GUG Ser UCU UCC UCA UCG AGU AGC Pro CCU CCC CCA CCG Thr ACU ACC ACA ACG Ala GCU GCC GCA GCG Tyr His UAU UAC CAU

Overall RSCU 0.88 1.12 0.58 1.39 1.32 0.92 0.39 1.4 1.19 1.27 0.54 1 2.02 0.77 0.75 0.45 1.31 0.74 1.49 0.89 0.68 0.89 1.04 0.25 1.43 1.29 1.25 1.18 1.06 0.51 1.5 0.74 1.09 0.67 1.15 0.85 1.12

LEG** RSCU 0.94 1.06 0.72 1.43 1.39 0.85 0.49 1.12 1.12 1.08 0.8 1 1.98 0.8 0.78 0.44 1.26 0.52 1.7 1.04 0.62 0.86 1.01 0.19 1.59 1.21 1.22 0.92 1.31 0.55 1.5 0.66 1.05 0.79 1.22 0.78 1.11

CAC Gln Asn Lys Asp Glu Cys Trp Arg CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG UGU UGC UGG CGU CGC CGA CGG AGA AGG Gly GGU GGC GGA GGG

0.88 1.29 0.71 0.93 1.07 1.38 0.62 1.15 0.85 1.5 0.5 0.79 1.21 1 1.89 1.63 1.16 0.64 0.49 0.19 1.68 1.3 0.74 0.28

1.12 1.45 0.55 0.84 1.16 1.37 0.63 1.01 0.99 1.7 0.3 0.85 1.15 1 1.89 1.89 0.94 0.67 0.33 0.28 2.1 1.31 0.44 0.15

0.89 1.18 0.82 0.94 1.06 1.34 0.66 1.18 0.82 1.45 0.55 0.9 1.1 1 1.63 1.37 1.31 0.68 0.78 0.23 1.38 1.33 0.93 0.36

1.63 0.51 1.49 0.24 1.76 0.78 1.22 0.79 1.21 1.31 0.69 0.73 1.27 1 3.04 2.39 0.07 0.29 0.07 0.14 1.75 1.98 0.05 0.22

1 1

Note- HEG and LEG denote highly and lowly expressed genes. The * and ** indicate putatively highly and lowly expressed genes of phage 44RR, which have been categorized respectively on the basis of lowest 10% and highest 10% of the genes according to their Nc values.

In phages lambda, N15, P2, and P4, synonymous codon usage patterns were found nearly similar to that of the lowly expressed genes of E. coli (Holm, 1987; Gouy, 1987; Kunisawa, 1992; Kunisawa et al., 1998). Contrary to above, codon usages of mycobacteriophages and S. aureus phages were found almost identical to their respective bacterial hosts (Kunisawa, 2000; Sahu et al., 2004; Sahu et al., 2005; Sau et al., 2005a). It was shown that mutational pressure and translational selection mainly influence the codon usages in mycobacterial and S. aureus phages (Sahu et al., 2004; Sahu et al., 2005; Sau et al., 2005a). While

tRNAs of mycobacteriophage Bxz1 was suggested to regulate the expression of both its highly and lowly expressed genes (Sahu et al., 2004), tRNAs present in phages D29 and L5 were suggested to affect their amino acid usage to some extent (Kunisawa, 2000). Very recently, it was shown that in AT-rich P. aeruginosa phage PhiKZ, codon usage bias is dictated mainly by mutational bias and, to an extent, by translational selection. Analysis also revealed that amino acid usage in PhiKZ proteins are mainly dictated by mean molecular weight, aromaticity and cysteine content (Sau et al., 2005b).

Complete genome sequences of three Aeromonas phages, namely, 44RR (synonym 44RR2.8t), phage 31, and Aeh1, are available in public databases at present. While former two phages grow in A. salmonicida, Aeh1 grows in A. hydrophila (Ttart et al., 2001). The %GC content is nearly identical in all three phages. Thus far, codon and amino acid usage biases have not been studied in Aeromonas phages at length though such

Fig. 1. Nc plot of phage 44RR genes. See text for details.

study may reveal the structure-function of protein-coding genes, evolution etc. In this communication, We have studied both synonymous codon and amino acid usage biases in A. salmonicida phage 44RR (Ttart et al., 2001), determined the factors responsible for codon and amino acid usage biases in 44RR, and discussed the data elaborately. My data show that synonymous codon usage of 44RR genes are influenced by both mutational bias and translational selection, whereas, amino usage of the 44RR proteins are mainly dictated by hydropathy, aromaticity and cysteine content. Materials and Methods The genome sequence of bacteriophage 44RR (synonym 44RR2.8t) was down loaded

from GenBank (USA) and its two hundred forty nine protein coding sequences (carrying 50 or more codons) have been extracted from the genome by coderet (http:bioweb.Pasteur.fr/seqanal/interfaces/cod ret.html) program. The relative synonymous codon usage (RSCU) in all protein coding sequences was determined to study the overall codon usage variation among the genes. RSCU is defined as the ratio of the observed frequency of codons to the expected frequency if all the synonymous codons for those amino acids are used equally (Sharp and Li, 1987). RSCU values greater than 1.0 indicate that the corresponding codon is more frequently used than expected, whereas the reverse is true for RSCU values less than 1.0. RSCU values in seven putatively highly expressed genes (e. g. genes encoding chaperonins, detoxification, and outer membrane proteins etc.) of A. salmonicida were also determined in this paper for comparison. GC3S is the frequency of (G+C) and A3S, T3S, G3S, and C3S are the frequencies of A, T, G and C at the synonymous third positions of codons. Nc, the effective number of codons used by a gene, is generally used to measure the bias of synonymous codons and independent of amino acid compositions and codon number (Wright, 1990). The values of Nc range from 20 (when one codon is used per amino acid) to 61 (when all the codons are used with equal probability). Nc values were calculated according to the method of Banerjee et al. (2005). The putatively highly and lowly expressed genes have been categorized respectively on the basis of lowest 10% and highest 10% of the genes according to their Nc values. The program CodonW 1.3 (available at www.molbio.ox.ac.uk/cu) was used for calculating most of the parameters including correspondence analysis (CA) on the relative synonymous codon and amino acid usages.

In correspondence analysis, the data are plotted in a multidimensional space of 59 axes (excluding Met, Trp and stop codons) and then it determines the most prominent axes contributing the codon usage variation among the genes. In the present study RSCU values have been used for CA in order to minimize the amino acid composition. Results and Discussion Overall codon usage analysis in A. salmonicida phage 44RR. The relative synonymous codon usage (RSCU) values determined in all the 249 protein coding genes of 44RR shows that A and / or T ending codons are predominant in this phage (Table 1). This is expected as %GC content in 44RR is 43.66. As analysis of overall RSCU alone is not sufficient to reveal the heterogeneity of codon usage in 44RR genes, WE also determined the effective numbers of codons used by gene (Nc) and (G+C) percentage at the synonymous third positions of codons (GC3s). It was observed that in 44RR, Nc values range from 26.486 to 55.241 with a mean of 40.025 and standard deviation (s.d.) 6.248, whereas, GC3s ranges from 0.234 to 0.577 with a mean of 0.414 and s.d. 0.056. Taking together the results suggest that apart from the mutational bias, other factors might have some influences in the codon usage variation among 44RR genes. Effect of mutational pressure in codon usage variation in 44RR. It was suggested that a plot of Nc vs GC3s could effectively be used to explore the codon usage variation among the genes (Wright, 1990). According to Wright (1990), the comparison of actual distribution of genes, with the expected distribution under no selection, could be indicative if codon usage bias of genes has some other influences other than mutational bias. If the codon usage bias is completely dictated by GC3s, the values of

Nc should fall on the expected curve between GC3s and Nc. In other words it can be said that if codon usage bias is completely dictated by

Fig. 2. Positions of the 44RR genes along the two major axes of variation in the correspondence analysis on RSCU values. The genes presented by the open circles.

GC3s composition the difference between observed and expected Nc values should be very small in majority of genes. To explore the possible influence of natural selection and mutational bias on synonymous codon usage on 44RR genome we calculated (NcExpected- NcObserved)/NcExpected. The frequency distributions of (NcExpected- NcObserved)/NcExpected shown in Fig. 1 demonstrate that majority of genes have large deviation of NcObserved from NcExpected. This suggests that the majority of genes in 44RR have additional codon usage bias, which is independent of mutational bias.

Table 2. Relative synonymous codon usage (RSCU) values for each codon for the two groups of genes in phage 44RR. The asterisk denotes the codons whose occurrences are significantly (p < 0.01) higher in the extreme left side of axis 1 than the genes present on the extreme right of the first major axis. Superscript "a" denotes for genes of extreme left of axis 1 and "b" for extreme right genes. Each group contains 10% of sequences at either extreme of the major axis generated by correspondence analysis. N is the number of codons, AA represents amino acid. AA Phe Leu Codon UUU UUC* UUA UUG CUU CUC CUA CUG* Ile AUU AUC* AUA Met Val AUG GUU GUC GUA GUG Tyr UAU UAC* His Gln Asn Lys Asp Glu CAU CAC CAA CAG AAU AAC* AAA AAG GAU GAC GAA GAG RSCUa 0.50 1.50 0.02 0.66 0.66 0.77 0.07 3.82 0.94 2.02 0.04 1.00 2.07 0.78 0.92 0.23 Na ( 46) (137) ( 1) ( 36) ( 36) ( 42) ( 4) (208) ( 92) (199) ( 4) (145) (171) ( 64) ( 76) ( 19) RSCUb Nb 1.10 ( 67) 0.90 ( 55) 1.20 ( 36) 1.47 ( 44) 1.17 ( 35) 0.67 ( 20) 0.80 ( 24) 0.70 ( 21) 1.07 ( 69) 0.82 ( 53) 1.10 ( 71) 1.00 ( 83) 1.87 ( 77) 0.70 ( 29) 0.68 ( 28) 0.75 ( 31) 1.20 ( 71) 0.80 ( 47) Trp 0.88 1.12 0.59 0.59 1.41 1.24 0.76 1.22 0.78 1.58 0.42 ( 26) ( 33) ( 58) ( 63) 1.18 ( 36) 0.82 ( 25) 1.38 ( 64) 0.62 ( 29) 1.06 ( 74) Ser Arg Gly Arg Cys Ala Thr Pro AA Ser Codon UCU* UCC* UCA UCG CCU CCC CCA CCG ACU* ACC* ACA ACG GCU* GCC GCA GCG UGU UGC UGG CGU* CGC* CGA CGG AGU AGC AGA AGG GGU* GGC GGA GGG RSCUa 1.57 1.91 0.43 0.37 1.36 0.28 0.71 1.64 1.46 2.13 0.26 0.14 1.92 0.85 0.87 0.36 0.40 1.60 1.00 2.71 2.38 0.54 0.27 0.60 1.12 0.06 0.03 2.21 1.54 0.19 0.05 Na RSCUb Nb ( 22) ( 9) ( 51) ( 38) ( 25) ( 9) ( 36) ( 30) ( 37) ( 42) ( 70) ( 19) ( 47) ( 26) ( 48) ( 24) ( 10) ( 19) ( 54) ( 16) ( 19) ( 30) ( 14) ( 23) ( 21) ( 37) ( 15) ( 57) ( 55) ( 39) ( 8)

( 73) 0.80 ( 89) 0.33 ( 20) 1.87 ( 17) 1.39 ( 44) 1.00 ( 9) 0.36 ( 23) 1.44 ( 53) 1.20 ( 83) 0.88 (121) 1.00 ( 15) 1.67 ( 8) ( 86) ( 88) ( 36) ( 7) ( 28) ( 48) ( 90) ( 79) ( 18) ( 9) 0.45 0.72 1.32 0.66 0.69 1.31 1.00 0.73 0.87 1.37 0.64 (194) 1.30

0.73 ( 47) 1.27 ( 82)

1.41 (138)

( 28) 0.84 ( 52) 0.77 ( 2) 1.69 ( 1) 0.69 (162) 1.43 (113) 1.38 ( 14) 0.98 ( 4) 0.20

(151) 0.94 ( 66) (210) 1.33 (110) (129) 0.67 ( 55) (171) 1.06 ( 75) (110) 0.94 ( 66) (289) 1.40 (125) ( 76) 0.60 ( 54)

Next, we carried out correspondence analysis (CA) on the RSCU values of the 249 proteincoding genes of 44RR phage in order to determine the factors influencing the codon usage bias in 44RR. Figure 2 shows the distributions of 44RR genes on the first two major axes of the correspondence analysis. It is found that the first major axis is accounted for 10.13% of the total variation and the second major axis accounted for 5.20% of the total variation. The position of the genes along the first major axis is negatively correlated with the A3s (r = -0.575, p<0.01) and positively correlated with T3s (r = 0.154, p<0.01), and C3s (r = 0.555, p<0.01). Interestingly, first major axis does not correlate with G3s. It is also found that the positions of the genes along the second major axis is significantly correlated only with G3s (r = 0.125, p<0.05). As the above results did not clearly demonstrate how the A, T, G, and C ending codons are distributed along the first major axis, we compared the codon usage in 10% of the 44RR genes located at the extreme right of axis 1 with that of the 10% of the 44RR genes located at the extreme left of the axis 1. To estimate the codon usage variation between these two sets of genes we performed chi square tests taking p<0.01 as significant criterion. Table 2 shows RSCU values for each codon for the two groups of genes. The asterisk represents the codons whose occurrences are significantly higher in the genes situated on the extreme left side of axis 1, compared to the genes present on the extreme right of the first major axis. It is important to note that out of 13 codons that are statistically over-represented in genes located on the extreme left side of axis 1 there are 7 C ending-, 5 T ending-, and 1 G ending codons. This actually represents 53.85% C ending-, 38.46% T ending, and 7.69% G ending codons. The data in fact supports the data presented in Fig. 1 and clearly shows that mutational pressure is strongly operative in shaping the codon usage bias in 44RR genes.

This type of mutational pressure was also shown to influence the codon usage bias in many bacterial and non-bacterial viruses (Kunisawa et al., 1998, Gu et al., 2004; Sahu et al., 2004; Sahu et al., 2005; Sau et al., 2005). Effect of translational selection on synonymous codon usage variation in 44RR. Most bacteriophages do not carry any tRNA encoding gene and completely depends on hosts tRNAs for their gene expression. Some phages like T4, BxZ1, PhiKZ etc. however carry some tRNA encoding genes and analyses showed that the phage-specific tRNAs either influence the expression of their lowly- or both the lowly- as well as highly expressed genes of phages (Kunisawa, 1992; Sahu et al., 2004; Sau et al., 2005). Phage 44RR also harbors 17 tRNA encoding genes within its genome. These tRNAs if expressed may incorporate all the amino acids except Ala, Gly, Val, Glu, Gln, and Cys. To see whether 44RR-specific tRNAs influence the synonymous codon usage of its putatively highly and / or lowly expressed genes, We carried out an in depth analysis and the data has been presented in Table 1. It is found that there are 32 over-represented codons in lowly expressed genes of 44RR in comparison with that of its highly expressed genes. Out of 32 over-represented codons, 15 are recognized by the anticodons of 44RR-specific tRNAs. This indicates that nearly 47% overrepresented codons of lowly expressed genes are recognized by 44RR-specific tRNAs. In contrast, about 41% over-represented codons of highly expressed genes of 44RR are recognized by its own tRNAs. Thus, the data suggest that 44RR-specific tRNAs preferentially recognizes the codons of lowly expressed genes of 44RR. Function exactly similar to that of phage 44RR-specific tRNAs has been reported before (Kunisawa, 1992).

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Fig. 3. Correlation between GRAVY scores of each amino acid residues versus the axis 1 values of correspondence analysis. Single letter codes are used to show the positions of 20 amino acid residues. Amino acid residues are presented closed circles.

The cellular tRNA abundance is positively correlated with over-represented codons of the highly expressed genes in several organisms (Grantham et al., 1981; Sharp et al., 1984-85; Gouy, 1987; Ikemura. 1992; Zhou et al., 1999; Kanaya et al., 1999; Kanaya et al., 2001). In coliphage T4, synonymous codon usage of the highly expressed genes was also shown to be positively correlated with the abundant cellular tRNAs (Kunisawa, 1992). This may also be true for the highly expressed genes of 44RR. As the status of tRNA copy number is not known in A. salmonicida at present, we determined the RSCU values of the putatively highly expressed genes of A. salmonicida and compared the resulting data with that of highly expressed genes of 44RR (Table 1). It was found that among the 19 over-represented codons present in each of the highly expressed genes of 44RR and A. salmonicida, 13 are common in both species. This indicates that nearly 68% over-represented codons of highly expressed genes of 44RR are recognized by abundant host tRNAs. Amino Acid Usage in 44RR. To identify the factors influencing the amino acid composition in 44RR, we also

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carried out CA on the relative amino acid usage of its 249 proteins. Analysis showed that the first and second major axes of CA are accounted for 18.66% and 10.91% of the total variation of the amino acid composition of 44RR proteins, respectively. Further analysis revealed that first major axis is positively correlated with GRAVY (r = 0.410, p < 0.01) of each 44RR protein. A plot of GRAVY score of each amino acid residue versus first major axis in fact shows that charged residues like lys, arg, his, asp, glu are located on the negative side of first axis (Fig. 3). Further analysis has shown that the first major axis is also significantly correlated (r = -0.404, p <0.01) with the Cys content of 44RR proteins (data not shown). Interestingly, among the 249 44RR proteins, 48 proteins do not carry any Cys residue, whereas, 5 proteins which are located at the extreme right side in Fig. 4, are found to harbor more than 5 % Cys Acknowledgement
We thank Dr. T. C. Ghosh and Dr. S. Sau, both from Bose Institute, Kolkata, for critically reading and modifying this manuscript. We also like to thank Dr. N. Bhattacharya, Head, Department of Biotechnology, Haldia Institute of Technology for his help and encouragement during the entire work. References: Banerjee, T., Basak, S., et al. 2004. J Biomol Struct Dyn, 22: 13-23 Banerjee, T., Gupta, S.K., et al. 2005. Biochem. Biophys.Res Commun. 330: 1015-8. Basak, S.K., Banerjee, T., et al. 2004. J. Biomol. Struct. & Dynamics. 22: 205-214. Chiusano, M. L., Alvarez-valin, F., Di Giulio, M., et al. 2002. Gene. 261: 63-69. DOnofrio, G., Ghosh, T.C., and Bernardi, G. 2002. Gene. 300: 179-187 Garat, B. and Musto, H. 2000. Biochem.Biophys.Res.commun. 279: 996-1000. Grantham, R., Gautier, C., Gouy, M., et al.. 1981Nucleic Acids Res. 9: r43 r74. Ghosh, T. C., Gupta, S. K., and Majumdar, S. 2000. Int. J. Parasitol. 30: 715 722. Gouy, M. 1987. Mol Biol Evol 4: 426 444. Gu, W., Zhou, T., Ma, J., et al., 2004. Virus Res. 101: 155- 161.

residue. It would be interesting to explore the contribution of these Cys-rich as well as Cysless proteins towards the temporal gene regulation as well as the development of 44RR in its host. In contrast, second major axis is significantly correlated (r = 0.650, p < 0.01) with aromaticity of each 44RR protein (data not shown). From amino acid frequency analysis, it is also found that all the aromatic amino acids are not very frequent in 44RR proteins (data not shown). Incidentally, aromatic amino acid were found rare in E. coli, T. maritama, G. lamblia and phage PhiKZ proteins and it was suggested that these amino acids are not incorporated preferentially in proteins as their biosynthesis is energetically expensive for organisms (Lobry et al., 1994; Garat and Musto, 2000; Zavala et al., 2002; Sau et al., 2005b).
Gupta, S.K., Majumdar, S., et al. 2000. Biochem Biophys Res Commun. 269: 692-696. Gupta, S.K. and Ghosh, T.C. 2001. Gene. 273: 63 70. Holm, L., 1986. Nucleic Acids Res. 7: 3075 - 3087. Ikemura, T. 1985. Mol. Biol. Evol. 2: 13 -34. Ikemura, T. 1992. Transfer RNA in protein synthesis Ann Arbor, London, Tokyo, CRC Press Jenkins, G. M., PageL, M., et al. 2001. J Mol Evol. 52: 383 - 390. Jenkins, G. M. and Holmes, E. C. 2003. Virus Res. 92: 1-7. Kanaya, S., Yamada, Y., Kudo, Y., and Ikemura, T. 1999. Gene. 238: 143-155. Kanaya, S., Yamada, Y., Kinouchi, M., et al., 2001. J. Mol. Evol. 53: 290-298. Kunisawa, T. 1992. J Theor Biol. 159: 287-98 Kunisawa, T., Kanaya, S., et al.. 1998. DNA Res. 5: 319 326 Kunisawa, T. 2000. J Theor Biol. 205:167-70 Lesnik, T., Solomovici, J., et al., 2000. J. Theor. Biol. 202: 175 185 Levine, D. B. and Whitemore, B. 2000. J. Gen. Virol. 81: 2313 2325 Lobry, JR. and Gautier, C. 1994. Nucleic Acids Res. 22: 3174-80 Lynn, D. J., Singer, G. A., et al. 2002. Nucleic Acids Res. 30: 4272-7 Mclnerney, J. O., 1998. Proc. Natl. Acad. Sci. USA. 95: 10698 10703

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Naya, H., Zavala, A., et al., 2004. Biochem. Biophys. Res. Commun. 325: 1252-7 Oresisc, M. and Shalloway, D. 1998. J. Mol. Biol. 281: 31 48 Romero, H., Zavala, A., et al.. 2000. Nucl. Acids Res. 28: 2084-2090 Sahu, K., Gupta, S. K., et al. 2004. J. Biochem. Mol. Biol. 37: 487 492 Sahu, K., Gupta, S. K., Sau, S., Ghosh, T. C. 2005. J Biomol Struct Dyn 23:63-71 Sau, K., Gupta, S. K., et al., 2005a. Virus Res. 113:123-31 Sau, K., Sau, S., et al., 2005b. Acta Biochim Biophys Sin (Shanghai). 37:625-33

Sharp, P. M., Rogers, M. S. et al., 1984-85. Nucl. Acids Res 21: 150 60 Sharp, P.M. and Li, W. H. 1987. Nucleic Acids Res. 15: 1281 1295 Sharp, P.M. and Cowe, E., 1991. Yeast. 7: 657-678 Tetart, F., Desplats, Cet al. 2001. J Bacteriol. 183: 358-66 Wright, F. 1990. Gene. 87: 23 29 Xie, T. and Ding, D.F. 1998.FEBS Lett. 434: 93-96. Zhou, J., Liu, W. J., et al. 1999. J. Virol. 73: 49724982. Zavala, A., Naya, H., Romero, H. and Musto, H. 2002.. J. Mol. Evol. 54: 563-8.

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