Differences in dinucleotide frequencies of thermophilic genes encoding water soluble and membrane proteins

Differences in dinucleotide frequencies of thermophilic genes encoding water soluble and membrane proteins first-author †E-mail:naka@kenroku.kanazawa-u.ac.jp †E-mail:naka@kenroku.kanazawa-u.ac.jp naka@kenroku.kanazawa-u.ac.jp Department of Clinical Laboratory Science, Graduate Course of Medical Science and Technology, School of Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan Department of Clinical Laboratory Science, Graduate Course of Medical Science and Technology, School of Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan The occurrence frequencies of the dinucleotides of genes of three thermophilic and three mesophilic species from both archaea and eubacteria were investigated in this study. The genes encoding water soluble proteins were rich in the dinucleotides of purine dimers, whereas the genes encoding membrane proteins were rich in pyrimidine dimers. The dinucleotides of purine dimers are the counterparts of pyrimidine dimers in a double-stranded DNA. The purine/pyrimidine dimers were favored in the thermophiles but not in the mesophiles, based on comparisons of observed and expected frequencies. This finding is in agreement with our previous study which showed that purine/pyrimidine dimers are positive factors that increase the thermal stability of DNA. The dinucleotides AA, AG, and GA are components of the codons of charged residues of Glu, Asp, Lys, and Arg, and the dinucleotides TT, CT, and TC are components of the codons of hydrophobic residues of Leu, Ile, and Phe. This is consistent with the suitabilities of the different amino acid residues for water soluble and membrane proteins. Our analysis provides a picture of how thermophilic species produce water soluble and membrane proteins with distinctive characters: the genes encoding water soluble proteins use DNA sequences rich in purine dimers, and the genes encoding membrane proteins use DNA sequences rich in pyrimidine dimers on the opposite strand. Water soluble and membrane proteins Purine/pyrimidine dimers Thermophilic and mesophilic species Dinucleotide frequencies Introduction 1 The G+C content of bacterial genomes varies among species from 25% to 75%, but is relatively constant within a bacterial genome (Muto and Osawa, 1987 1987 ; Lawrence and Ochman, 1997 1997 ). The nucleotide sequences of genes of bacterial genomes have species-specific dinucleotide compositions (Karlin and Burge, 1995 1995 ; Karlin et al., 1997 1997 ; Nakashima et al., 1998 1998 ). Comparative studies of the DNA and protein sequences of thermophilic and mesophilic species have revealed differences in their compositions. The synonymous codon usage in genes of thermophiles is different from that of mesophiles (Lynn et al., 2002 2002 ). Kawashima et al. ( 2000 2000 ) reported that in archaea a simple combination of purine (R) and pyrimidine (Y) dinucleotides, RR+YY-RY-YR, is linearly correlated with optimal growth temperature (OGT). An increased frequency of purine nucleotides in the coding strands contributes to thermostability (Paz et al., 2004 2004 ). It has been reported that a simple summation of the purines adenine and guanine (A+G) is correlated with OGT (Lambros et al., 2003 2003 ; Zeldovich et al., 2007 2007 ). Studies of thermophilic and mesophilic proteins have shown differences in their amino acid compositions (Kumar et al., 2000 2000 ; Kreil and Ouzounis, 2001 2001 ; Farias and Bonato, 2003 2003 ; Yokota et al., 2006 2006 ; Zhou et al., 2008 2008 ). We previously reported that the ten symmetrical components of the dinucleotide compositions of genes encoding water soluble proteins showed a linear relationship with OGT based on regression analysis (Nakashima et al., 2003 2003 ). The purine/pyrimidine dimers were positive, but purine-pyrimidine or pyrimidine-purine dimers were roughly negative factors for the thermal stability of DNA. The dinucleotide AA pairs with TT and we cannot distinguish AA from TT in a double-stranded DNA. The dinucleotide AT pairs with AT. Therefore, ten symmetrical components are enough to study the character of a double-stranded DNA. When we consider a coding sequence, it is important to recognize on which strand the gene is located. In this case, we have to consider 16 kinds of dinucleotides. It has been estimated that more than a quarter of all known proteins are membrane proteins (Anson, 2009 2009 ). These proteins have different amino acid compositions from water soluble proteins. Apolar amino acid residues are suitable for membrane proteins because such proteins have membrane-spanning regions which have hydrophobic characteristics. As there is a difference in amino acid composition between water soluble and membrane proteins, the dinucleotide compositions of their genes must be different. This raises the issue of how the species prepare two different kinds of DNA sequences. To address this question, we investigated the dinucleotide compositions of membrane proteins from both thermophilic and mesophilic species and compared them with those of water soluble proteins. Materials and methods 1 Sequence retrieval 2 Three thermophilic archaea ( Sulfolobus tokodaii (Kawarabayasi et al., 2001 2001 ), Archaeoglobus fulgidus (Klenk et al., 1997 1997 ), Methanopyrus kandleri (Slesarev et al., 2002 2002 )), three thermophilic eubacteria ( Thermoanaerobacter tengcongensis (Bao et al., 2002 2002 ), Thermotoga maritima (Nelson et al., 1999 1999 ), Thermus thermophilus HB8 (GenBank: AP008226.1)), three mesophilic archaea ( Methanosphaera stadtmanae (Fricke et al., 2006 2006 ), Methanocorpusculum labreanum (Anderson et al., 2009 2009 ), Halobacterium sp. NRC-1 (Ng et al., 2000 2000 )), and three mesophilic eubacteria ( Haemophilus influenzae Rd KW20 (Fleischmann et al., 1995 1995 ), Escherichia coli K12 MG1655 (Blattner et al., 1997 1997 ), Pseudomonas aeruginosa PA01 (Stover et al., 2000 2000 )) were surveyed in this study. The species were selected arbitrarily, taking into consideration only the coverages of a wide range of genomic G+C contents. Their genome sequences were retrieved from the web ftp site ( ftp://ftp.ncbi.gov/genomes ftp://ftp.ncbi.gov/genomes ) of the National Center for Biotechnology Information (NCBI). The protein-coding nucleotide sequences and amino acid sequences were retrieved from NCBI as ffn and faa files. Selection of genes encoding water soluble and membrane proteins 2 The proteins were classified as water soluble or membrane proteins according to the annotation of the Genome to Protein Structure and Function (GTOP) database (Kawabata et al., 2002 2002 ). The SOSUI program (Hirokawa et al., 1998 1998 ) was used in the GTOP database to predict the transmembrane regions. Those proteins without transmembrane regions were considered to be water soluble proteins. Proteins which had more than two transmembrane regions were employed in the calculation of the dinucleotide compositions of genes for membrane proteins. This is because if a protein has a signal peptide it might be counted as a transmembrane region. The membrane proteins were divided into 100 sections and one protein was randomly selected from each section. The water soluble proteins were similarly selected. The water soluble and membrane proteins were examined for their amino acid sequence similarities using the BLAST program (Altschul et al., 1990 1990 ). Those proteins which had more than 30% sequence identity between water soluble proteins or between membrane proteins were replaced, to keep the sequence identity below 30%. Proteins longer than 100 residues, and their corresponding genes, were employed. Calculation of expected dinucleotide composition 2 The expected dinucleotide composition was calculated as the product of the mononucleotide composition for each gene. The averages of the expected dinucleotide compositions for 100 genes encoding water soluble and membrane proteins were calculated. Then the ratios of the average of the observed to the expected dinucleotide compositions were calculated. Results 1 Dinucleotide composition 2 The average dinucleotide compositions of the genes for 100 water soluble proteins and 100 membrane proteins in 12 species are listed in Table 1 1 with their genomic G+C contents. In T. maritima , dinucleotides such as AA, GA, and AG were enriched in the water soluble protein-coding genes, whereas TT, TC, and CT were enriched in the membrane protein-coding genes. It is interesting that AA, GA, and AG are purine dimers and TT, TC, and CT are pyrimidine dimers, and they are counterparts in a double helix DNA. To show the difference in dinucleotide composition more clearly, the ratios of the dinucleotide compositions of water soluble protein-coding genes to those of membrane protein-coding genes were calculated. In T. maritima , the three highest ratios of dinucleotide compositions were AA (1.56=11.20/7.20), AG (1.50=8.55/5.69), and GA (1.46=10.99/7.54). This result indicated that the genes encoding water soluble proteins were rich in AA, AG, and GA dinucleotides compared to the genes encoding membrane proteins. The three lowest ratios of dinucleotide compositions were TT (0.58=5.92/10.23), CT (0.68=5.38/7.92), and TC (0.72=6.71/9.29) in T. maritima . This result indicated that the dinucleotides TT, CT, and TC were frequently observed in the genes encoding membrane proteins compared to the genes encoding water soluble proteins (Table 2 2 ). The genes for water soluble proteins were biased toward purine dimers such as GA, AA, and AG, and the genes for membrane proteins were biased toward pyrimidine dimers such as TC, TT, and CT. This trend was observed in both the thermophiles and the mesophiles. 2 Species 2 G+C (%) 16 Dinucleotide composition (%) 2 Sum (%) AA TT AG CT GA TC GG CC AC GT CA TG AT TA GC CG RR YY Thermophilic archaea S. tokodaii 32.8  Soluble 13.00 9.29 8.69 5.03 8.26 4.02 5.04 2.64 4.40 5.02 4.43 5.65 9.79 10.13 2.84 1.77 34.99 20.98  Membrane 8.92 14.16 5.87 6.94 4.52 5.84 3.99 2.84 4.29 5.18 4.73 5.38 11.03 11.88 3.00 1.43 23.30 29.78 A. fulgidus 48.6  Soluble 8.58 6.51 9.42 5.57 10.30 4.93 8.34 4.12 4.89 4.59 5.74 7.00 5.39 3.60 6.27 4.75 36.64 21.13  Membrane 5.91 9.67 6.02 8.02 6.44 7.53 7.03 5.17 4.55 4.89 5.76 7.07 6.06 4.35 6.63 4.90 25.40 30.39 M. kandleri 61.2  Soluble 4.52 2.77 7.42 4.66 10.13 6.59 10.05 7.06 6.09 6.58 4.09 5.40 3.76 3.01 6.99 10.88 32.12 21.08  Membrane 3.03 3.93 4.79 6.72 6.75 7.98 10.05 7.64 5.73 7.19 4.09 6.31 4.18 3.83 7.45 10.33 24.62 26.27 20 Thermophilic eubacteria T. tengcongensis 37.6  Soluble 13.40 8.94 9.19 4.83 9.02 3.24 6.16 2.86 4.19 4.48 5.08 6.80 8.29 7.54 4.23 1.75 37.77 19.87  Membrane 9.67 13.02 6.65 6.47 5.98 4.36 5.69 3.04 4.30 4.68 5.21 7.07 8.79 8.49 4.81 1.77 27.99 26.89 T. maritima 46.2  Soluble 11.20 5.92 8.55 5.38 10.99 6.71 6.65 3.96 5.85 5.19 5.73 6.22 5.75 3.38 3.55 4.97 37.39 21.97  Membrane 7.20 10.23 5.69 7.92 7.54 9.29 6.27 4.14 4.78 5.98 5.44 7.14 6.17 3.62 3.96 4.65 26.70 31.58 T. thermophilus 69.5  Soluble 2.94 2.67 6.17 6.83 6.75 6.09 15.32 14.60 4.84 3.49 4.71 4.74 1.77 1.25 9.25 8.58 31.18 30.19  Membrane 1.77 3.95 3.79 10.10 4.07 8.88 14.08 15.53 4.00 3.73 4.12 5.40 1.80 1.35 9.40 8.03 23.71 38.46 20 Mesophilic archaea M. stadtmanae 27.6  Soluble 15.76 8.35 6.75 3.93 7.31 3.42 3.43 1.92 5.24 4.74 6.53 7.11 12.26 10.38 2.34 0.53 33.25 17.62  Membrane 11.73 12.33 5.48 4.45 4.91 3.99 3.50 1.62 4.79 4.99 6.22 6.31 13.96 13.06 2.28 0.38 25.62 22.39 M. labreanum 50.0  Soluble 8.43 4.83 5.76 5.08 8.75 7.48 6.69 6.01 5.78 4.86 6.45 6.18 6.88 3.14 6.00 7.68 29.63 23.40  Membrane 5.47 8.06 3.82 6.75 6.64 9.20 7.02 5.50 4.28 5.74 5.31 7.19 7.73 3.81 6.04 7.44 22.95 29.51 Halobacterium 67.9  Soluble 2.51 1.72 4.49 4.14 8.66 6.50 9.14 9.00 8.18 5.29 5.32 4.33 2.48 1.08 10.99 16.17 24.80 21.36  Membrane 1.82 3.00 2.79 5.66 5.18 8.17 10.06 7.90 5.88 7.54 4.62 6.25 2.76 1.52 11.56 15.29 19.85 24.73 20 Mesophilic eubacteria H. influenzae 38.1  Soluble 13.15 10.39 5.39 4.27 6.14 4.53 4.25 3.01 4.87 5.35 6.27 7.25 8.88 6.68 5.36 4.20 28.93 22.20  Membrane 9.01 14.92 4.01 5.21 4.30 4.84 4.85 2.86 4.22 5.78 5.67 7.91 9.57 7.76 5.47 3.63 22.17 27.83 E. coli 50.8  Soluble 7.72 5.89 4.94 5.28 6.74 5.25 6.55 5.23 5.69 5.55 6.25 8.00 6.45 4.00 8.53 7.92 25.95 21.65  Membrane 5.25 8.85 3.44 6.50 4.79 5.86 7.48 4.82 4.46 6.63 5.23 9.42 6.82 4.64 8.62 7.19 20.96 26.03 P. aeruginosa 66.6  Soluble 3.36 2.30 5.13 5.98 6.51 5.68 8.66 10.06 5.65 4.06 6.03 6.20 3.24 1.40 13.24 12.49 23.66 24.02  Membrane 2.09 2.97 3.38 8.39 4.62 7.14 9.18 9.67 4.37 4.87 5.18 8.24 3.49 1.36 13.59 11.45 19.27 28.17 2 Species 2 Dinucleotide Water soluble proteins Membrane proteins Thermophilic archaea   S. tokodaii GA, AG, AA TT, TC, CT   A. fulgidus GA, AG, AA TC, TT, CT   M. kandleri AG, GA, AA CT, TT, TA Thermophilic eubacteria   T. tengcongensis GA, AA, AG TT, TC, CT   T. maritima AA, AG, GA TT, CT, TC   T. thermophilus GA, AA, AG CT, TT, TC Mesophilic archaea   M. stadtmanae GA, CG, AA TT, TA, TC   M. labreanum AA, AG, AC TT, CT, TC   Halobacterium GA, AG, AC TT, TG, GT Mesophilic eubacteria   H. influenzae AA, GA, AG TT, CT, TA   E. coli AA, AG, GA TT, CT, GT   P. aeruginosa AA, AG, GA CT, TG, TT The occurrence frequency of dinucleotides was dependent on G+C content. For example, H. influenzae with a genomic G+C content of 38.1% showed a higher occurrence frequency for the dinucleotides AA and TT, and a lower occurrence frequency for the dinucleotides CC and GG in the genes for both water soluble and membrane proteins. P. aeruginosa , with a genomic G+C content of 66.6%, showed a lower occurrence frequency for the dinucleotides AA and TT, and a higher occurrence frequency for the dinucleotides CC and GG. To analyze the difference between thermophilic and mesophilic genes, the sums of purine/pyrimidine dimers were calculated. The sum of purine dimers for the water soluble protein-coding genes of T. maritima was 37.39% and that for the membrane protein-coding genes was 26.70% (Table 1 1 ). So the deviation of the two sets of genes was 10.69%. The sum of pyrimidine dimers for the water soluble protein-coding genes of T. maritima was 21.97% and that for the membrane protein-coding genes was 31.58%. In this case, the deviation was 9.61%. The corresponding deviations were 4.99% and 4.38% in E. coli . Thus, the deviation of the sum of purine/pyrimidine dimers between the water soluble and the membrane proteins-coding genes was generally larger in the thermophiles than in the mesophiles. The larger deviation implied that the protein-coding genes in thermophiles are more biased towards purine/pyrimidine dimers than those in mesophiles. Favorable purine/pyrimidine dimers in thermophiles 2 The ratios of the observed to the expected dinucleotide compositions among species were calculated. Ratios greater than 1.1 were considered favorable and those less than 0.9 were considered unfavorable. To simplify the result, only purine/pyrimidine dimers were considered here. In T. maritima , TC, GA, CT, TT and AA were favorable dinucleotides for the genes encoding water soluble proteins and no unfavorable dinucleotides were observed. The dinucleotides TC, GA, AA, CT, TT, and GG were favorable and the dinucleotide CC was unfavorable for the genes encoding membrane proteins in T. maritima . Thus, there were five favorable purine/pyrimidine dimers in the genes encoding water soluble proteins, and six in those encoding membrane proteins. One pyrimidine dimer was observed as unfavorable for the genes encoding membrane proteins. The favorable dinucleotides were almost identical for the genes encoding both protein types. This is consistent with the result of Karlin’s group that the dinucleotide relative abundance values of different DNA sequences from the same organism are generally much more similar to each other than those from different organisms (Karlin and Burge, 1995 1995 ; Karlin et al., 1997 1997 ). The dinucleotide relative abundance values from Karlin’s group correspond to the ratios of the observed to the expected dinucleotide compositions in our study. In E. coli , the dinucleotides AA and TT were favorable for the genes encoding both water soluble and membrane proteins. The dinucleotides AG, CC, and GG were unfavorable for the genes encoding water soluble proteins and the dinucleotides AG, CC, TC, and GA were unfavorable for the genes encoding membrane proteins. So, for both protein types, there were two favorable purine/pyrimidine dimers. There were three unfavorable purine/pyrimidine dimers for the genes encoding water soluble proteins and four for those encoding membrane proteins (Table 3 3 ). There are eight purine/pyrimidine dimers in total. In T. thermophilus , almost all purine/pyrimidine dimers were classed as favorable. This indicates that purine/pyrimidine dimers were favorable in the thermophiles, except M. kandleri , but not in the mesophiles. n f : number of favorable purine/pyrimidine dimers; n u : number of unfavorable purine/pyrimidine dimers 2 Species 2 Water soluble proteins 2 Membrane proteins n f n u n f n u Thermophilic archaea   S. tokodaii 6 0 4 0   A. fulgidus 5 0 7 0   M. kandleri 2 2 3 2 Thermophilic eubacteria   T. tengcongensis 7 1 6 1   T. maritima 5 0 6 1   T. thermophilus 8 0 7 0 Mesophilic archaea   M. stadtmanae 3 0 1 3   M. labreanum 3 2 4 2   Halobacterium 2 5 2 4 Mesophilic eubacteria   H. influenzae 2 4 3 4   E. coli 2 3 2 4   P. aeruginosa 4 2 4 4 Sum of purine/pyrimidine dimers along a nucleotide sequence 2 The dinucleotides TT, TC, and CT were frequently observed in the genes encoding membrane proteins. To analyze the occurrence of pyrimidine dimers, the sum of the dinucleotides CT+TC+TT+CC was counted in a frame of 30 nucleotides, sliding the frame without overlapping along a nucleotide sequence. Similarly, the sum of the dinucleotides AG+GA+AA+GG was counted. The plot of the sum of purine/pyrimidine dimers along the nucleotide sequence which encodes the ABC transporter permease protein of T. maritima is shown in Fig. 1 1 . The peaks of the sum of pyrimidine dimers correspond to the transmembrane regions and the local minima of the sum of purine dimers. Thus the sum of pyrimidine dimers corresponds to the transmembrane region. This result suggests that multi-spanning transmembrane proteins have more pyrimidine dimers than single-spanning transmembrane proteins. The dinucleotides CT, TC, and TT were frequently observed as components of codons such as Leu (CTN, TTA, TTG), Ser (TCN), and Phe (TTC, TTT) in transmembrane regions. The nucleotide sequence is the ABC transporter permease protein of T. maritima Discussion 1 As expected, the dinucleotide compositions of genes encoding water soluble and membrane proteins were different (Tables 1 1 and 2 2 ). The genes encoding water soluble proteins were rich in the dinucleotides AG, AA, and GA, and their counterparts CT, TT, and TC were abundant in the genes encoding membrane proteins. The above purine/pyrimidine dimers were favorable in the genes from the thermophiles, but not in the genes from the mesophiles (Table 3 3 ). We tried to understand how the organisms prepare two kinds of different DNA sequences. The organisms use a simple strategy: the genes for water soluble and membrane proteins use DNA sequences on different DNA strands (Fig. 2 2 ). The protein-coding genes from the thermophiles were richer in purine/pyrimidine dimers than those from the mesophiles. This is consistent with our previous study which suggested that the purine/pyrimidine dimers were positive factors that increased the thermal stability of DNA (Nakashima et al., 2003 2003 ). The dinucleotides AA, AG, and GA are components of the codons of charged residues of Glu, Asp, Lys, and Arg, and these residues are known to stabilize proteins at higher temperatures (Nakashima et al., 2003 2003 ). The dinucleotides TT, CT, and TC are components of the codons of hydrophobic residues of Leu, Ile, and Phe. This is consistent with the suitability of these amino acid residues for membrane proteins. Water soluble and membrane proteins are likely to sit in a series on a DNA strand We showed the results from three thermophilic and three mesophilic species of both archaea and eubacteria in this study. We examined the dinucleotide compositions of genes from other thermophilic and mesophilic species, and obtained a similar trend for the dinucleotide composition. The dinucleotide composition is thought to consist of two parts: one is attributable to mononucleotide composition, and the other is a deviation from expectation given by the multiplication of mononucleotide contents. In a double-stranded DNA, the amount of adenine is equal to the amount of thymine and the amount of guanine is equal to the amount of cytosine. This is known as Chargaff’s first parity rule (Chargaff et al., 1951 1951 ; 1952 1952 ). This rule also applies to single-stranded DNA and is called Chargaff’s second parity rule (Karkas et al., 1968 1968 ; Runder et al., 1968 1968 ). Mitchell and Bridge ( 2006 2006 ) tested Chargaff’s second parity rule over 3 400 genomic sequences and the validity of this rule has been confirmed for genome sequences from archaea, eubacteria, eukaryotes, and viruses. Therefore, the mononucleotide composition is represented simply as G+C content. This is why we selected species showing a wide range of G+C contents. The present study indicated that the character of the dinucleotide composition held for genes of a wide range of G+C contents. The occurrence frequencies of the dinucleotides GG and CC in the genes were low compared to those of other purine/pyrimidine dimers. In the thermophiles, the occurrence frequency of the dinucleotide GG was higher in the genes encoding water soluble proteins than in those encoding membrane proteins. However, the opposite trend was found in the mesophiles (Table 1 1 ). The dinucleotide GG is a component of the codon of the Gly residue (GGN). The character of Gly might be related to the above occurrence frequency of GG. The degree of localization of the dinucleotide CC in the transmembrane regions was low compared to that of other pyrimidine dimers. The dinucleotide CC is a component of the codon of the Pro residue (CCN). Transmembrane regions are composed of α-helices, and the Pro residue is a strong helix-breaker (Chou and Fasman, 1978 1978 ), so this might be the reason why the dinucleotide CC is not favored in transmembrane regions. The DNA strand in which the genes for water soluble proteins were located was different from the strand carrying the genes for membrane proteins. Motivated by this result, we investigated the distributions of genes encoding water soluble and membrane proteins. The genes were divided into two types, so four types of gene pairs were possible. The occurrence of the four different types of gene pairs was counted whenever the two genes were located successively on an identical strand. Then the observed number of gene pairs was divided by the calculated number to obtain a ratio, i.e., observed/calculated. The calculated number of gene pairs was estimated by the product of the frequency of the genes. This procedure was followed for both strands separately. The two strands were represented by a top strand and a bottom strand. The ratios (observed/calculated) of the four types of gene pairs on both strands in 12 species are listed in Table 4 4 . The numbers of genes encoding water soluble and membrane proteins on both strands are also listed in Table 4 4 . The ratios of the gene pairs corresponding to two membrane proteins were greater than 1, indicating that they were favorable in all species, whereas those of gene pairs corresponding to a water soluble and a membrane protein (and the reverse) were less than 1, indicating that these were unfavorable in all species. This result indicates that genes encoding membrane proteins are likely to sit in a series on a DNA strand. In E. coli , the genes encoding membrane proteins such as the cytochrome o ubiquinol oxidase subunit, the nicotinamide adenine dinucleotide (NADH) ubiquinone oxidoreductase subunit, and the adenosine triphosphate (ATP) synthase subunit, were located successively as components of operons. This result agrees with the empirical knowledge that functionally-related genes are clustered in an operon. This is also consistent with Chargaff’s cluster rule that purine/pyrimidine nucleotides tend to cluster in a DNA sequence (Forsdyke and Mortimer, 2000 2000 ). S-S: soluble-soluble; S-M: soluble-membrane; M-S: membrane-soluble; M-M: membrane-membrane 2 Species 2 Strand 2 No. of proteins 4 Ratio of protein pairs Soluble Membrane S-S S-M M-S M-M Thermophilic archaea   S. tokodaii Top 1035 383 1.11 0.79 0.79 1.34 Bottom 1065 343 1.12 0.64 0.69 1.88   A. fulgidus Top 913 265 1.03 0.82 0.79 1.95 Bottom 923 306 1.06 0.78 0.75 1.85   M. kandleri Top 671 172 1.08 0.64 0.75 2.16 Bottom 676 168 1.03 0.78 0.82 2.08 Thermophilic eubacteria   T. tengcongensis Top 919 333 1.03 0.93 0.90 1.24 Bottom 954 382 1.10 0.80 0.81 1.34   T. maritima Top 723 280 1.08 0.80 0.77 1.59 Bottom 634 209 1.03 0.85 0.88 1.52   T. thermophilus Top 674 217 1.00 0.92 0.86 1.62 Bottom 834 248 1.04 0.78 0.92 1.54 Mesophilic archaea   M. stadtmanae Top 586 163 1.06 0.78 0.80 1.79 Bottom 616 169 1.06 0.85 0.69 1.83   M. labreanum Top 667 199 1.12 0.73 0.67 1.71 Bottom 650 223 1.11 0.70 0.73 1.76   Halobacterium Top 973 270 1.07 0.82 0.79 1.55 Bottom 1059 303 1.01 0.79 0.95 1.76 Mesophilic eubacteria   H. influenzae Top 656 194 1.01 0.79 0.92 1.83 Bottom 659 200 1.01 0.86 0.89 1.70   E. coli Top 1448 593 1.04 0.87 0.91 1.30 Bottom 1596 546 1.04 0.93 0.84 1.37   P. aeruginosa Top 2010 732 1.05 0.78 0.90 1.53 Bottom 2065 759 1.04 0.90 0.84 1.42 Conclusions 1 The genes encoding water soluble proteins use DNA sequences rich in purine dimers, and the genes encoding membrane proteins use sequences rich in pyrimidine dimers on the opposite strand. 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Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins 10.1631/jzus.B1000331 2010-09-13 2011-05-09 2011-05-05 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2011 2011 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 12