Báo cáo khoa học: Detection of nucleolar organizer and mitochondrial DNA insertion regions based on the isochore map of Arabidopsis thaliana ppt

Detection of nucleolar organizer and mitochondrial DNA insertion regions based on the isochore map of Arabidopsis thaliana Ling-Ling Chen 1 and Feng Gao 2 1 Laboratory for Computational Biology, Shandong Provincial Research Center for Bioinformatic Engineering and Techniques, Shandong University of Technology, Zibo, China 2 Department of Physics, Tianjin University, China From the 1970s onwards, Bernardi and coworkers began to investigate the organization of eukaryotic genomes using density gradient ultracentrifugation experiments. They concluded that the genomes of vertebrates [1–4] and many other eukaryotes [5,6] are organized with mosaics of isochores, i.e. long DNA segments relatively homogeneous in GC content com- pared to the heterogeneity throughout the whole gen- ome. For warm-blooded vertebrates, the length of isochore is 300 kb or longer [7] and for angiosperms, the isochore length is among the region of 50–150 kb [8]. Since then, many researchers have studied the characteristics of isochores and found that they are correlated with gene distribution, expression pattern [9], codon usage [10], the distribution of repeat sequences and other elements, etc. [11,12]. Although isochores have been intensively studied in recent years, two problems remain to be debated. The first problem is the boundary of isochores [7], and the other is the homogeneity of isochores [13]. It is difficult to solve the two problems using the traditional method, which utilizes an overlapping or nonoverlap- ping sliding window technique to calculate the GC content. A large window size leads to low resolution, Keywords Arabidopsis thaliana; GC content; isochore; mitochondrial insertion region; nucleolar organizer Correspondence L-L Chen, Laboratory for Computational Biology, Shandong Provincial Research Center for Bioinformatic Engineering and Techniques, Shandong University of Technology, Zibo, 255049, China Fax: +86 5332780271 Tel: +86 5332780271 E-mail: llchen@sdut.edu.cn (Received 7 January 2005, revised 23 April 2005, accepted 3 May 2005) doi:10.1111/j.1742-4658.2005.04748.x Eukaryotic genomes are composed of isochores, i.e. long sequences relat- ively homogeneous in GC content. In this paper, the isochore structure of Arabidopsis thaliana genome has been studied using a windowless technique based on the Z curve method and intuitive curves are drawn for all the five chromosomes. Using these curves, we can calculate the GC content at any resolution, even at the base level. It is observed that all the five chromo- somes are composed of several GC-rich and AT-rich regions alternatively. Usually, these regions, named ‘isochore-like regions’, have large fluctua- tions in the GC content. Five isochores with little fluctuations are also observed. Detailed analyses have been performed for these isochores. A GC-rich ‘isochore-like region’ and a GC-isochore in chromosome II and IV, respectively, are the nucleolar organizer regions (NORs), and genes located in the two regions prefer to use GC-ending codons. Another GC-isochore located in chromosome II is a mitochondrial DNA insertion region, the position and size of this region is precisely predicted by the cur- rent method. The amino acid usage and codon preference of genes in this organellar-to-nuclear transfer region show significant difference from other regions. Moreover, the centromeres are located in GC-rich ‘isochore-like regions’ in all the five chromosomes. The current method can provide a useful tool for analyzing whole genomic sequences of eukaryotes. Abbreviation NORs, nucleolar organizer regions. 3328 FEBS Journal 272 (2005) 3328–3336 ª 2005 FEBS whereas a small window size leads to large statistical fluctuations and the best window size does not exist in most cases. Oliver et al. developed an entropic segmen- tation method to determine the boundary of isochores [14]. Nekrutenko and Li proposed a compositional het- erogeneity index to compare the differences in compo- sitional heterogeneity between long genomic sequences [13]. The two problems can be converted to intuitive forms using a windowless technique based on the Z curve theory [15]. The GC content may be calculated at any resolution by using this method. Most import- antly, the related curve can display not only the local but also the global distribution of the GC content along the genomic sequences. Arabidopsis thaliana is the first plant genome to be completely sequenced. Its small size, short life cycle, prodigious seed production and a relatively small gen- ome of about 120 Mb make it a model plant for research [16]. The compositional organization of the A. thaliana genome has been studied by several groups [5,7]. Carels and Bernardi analyzed the contigs of A. thaliana and concluded that the GC level of genes and coding regions, as well as gene densities and expression level showed to be evidently higher in distal regions [5]. Oliver et al . systemically studied the whole A. thaliana genome using an improved segmentation method and concluded that no relationship between gene density and GC level was found in A. thaliana chromosomes II and IV [7]. There is significant distinc- tion between the conclusions of the two groups. Recently, Zhang and Zhang analyzed the A. thaliana genome by using the cumulative GC profile [17]. They concluded that the isochores in A. thaliana can be divi- ded into three types, GC-isochores, AT-isochores and centromere-isochores, respectively. They also found that the three types of isochores were distinct in the distribution of gene density, T-DNA insertion site and transposable element [17]. In this study, we also use the cumulative GC profile proposed by Zhang and Zhang [18,19] to investigate the isochore structure of A. thaliana genome. It is found that there are two GC- rich regions located in chromosome II, which show dif- ferent properties from other regions. The first GC-rich region is located in the nucleolar organizer region (NOR). The second region is a mitochondrial DNA insertion segment. The NOR in chromosome IV is a GC-isochore. It is also shown that the centromeres are located in GC-rich regions in all the five chromosomes and they have the lowest gene density, which are con- sistent with the result in [17]. All the five chromosomes show similar codon usage, codon preference and amino acid usage patterns, while these patterns are different in the identified isochores and the NORs. Results and Discussion The z¢ curves, isochore maps and some features of the five A. thaliana chromosomes Figure 1 shows the z¢ curves for five A. thaliana chro- mosomes. As can be seen clearly, each curve has dra- matic variations, indicating that the GC content along each chromosome is inhomogeneous. An up jump in the z¢ curve denotes a decrease of the GC content, while a drop in the curve indicates an increase of the GC content. The slope of the curve denotes the vari- ation rate of the GC content. According to the z¢ curve, each chromosome is composed of several GC- rich and AT-rich regions alternatively. The maximum, minimum and other turning points in the z¢ curves are borders of the regions. Within each region, there are several subregions, i.e. a self-similar structure with finite layers can be used to describe the real structures. Most of the regions have large fluctuations, indicating the GC content is inhomogeneous in these regions. Therefore, they are called ‘isochore-like regions’ in this paper. Some regions are approximately straight lines, indicating the GC content is nearly constant in these regions, which are considered to be isochores [2]. Through the intuitive z¢ curves, the two remaining questions can be converted to intuitive forms. For the first question, the border of each approximately straight line is thought to be the boundary of the iso- chores. Generally, isochores have relatively sharp bor- ders. Using an optimization method, the border can be pinpointed to a single base [20]. The homogeneity of isochore can be defined by an index h [17,20], which is defined as the variance of GC content of the region divided by that of the whole genome. If h ( 1, the variance of GC content of the region may be small enough to be considered as an isochore. It should be pointed out that the GC content of isochore is only relatively homogenous, unless h equals zero. No prior knowledge is available to define isochores based on h. In Zhang and Zhang [17], the threshold is arbitrarily chosen as h ¼ 0.2. There are many unassigned regions, as shown in [17]. If these regions are further segmented according to the turning points in the z¢ curves, most of these regions are identified to be isochores. In addi- tion, in [17], it is observed that there are still large fluc- tuations in the detected isochores, indicating the GC content is inhomogenous in these regions. So we choose a more stringent threshold h ¼ 0.05 and classify each base into an isochore or ‘isochore-like region’. Table 1 lists five identified isochores in the A. thali- ana chromosomes based on the threshold h ¼ 0.05. Three of them are GC-isochores and two are AT-iso- L L. Chen and F. Gao Isochore structure of A. thaliana genome FEBS Journal 272 (2005) 3328–3336 ª 2005 FEBS 3329 chores. They are indicated in Fig. 1 with black lines (the first isochore in chromosome IV is also a NOR, so it is indicated with orange dots). Table 2 shows all the ‘isochore-like regions’ in the five chromosomes based on the threshold h ¼ 0.05. The homogeneity index h-values of the ‘isochore-like regions’ are in the range of 0.06–0.67, which are higher than those of the isochores. As can be seen, the difference of GC content between two adjacent regions are relatively small, usu- ally in the range of 2–4%. The average gene density in each isochore and chromosome is calculated and the result shows that the gene density in AT-isochores is lower than that of GC-isochores, which is consistent with the results of [17]. Other h-values can also be chosen as the threshold of isochores. Table 3 lists three possible thresholds Fig. 1. The z n ¢ % n curves for the five A. thaliana chromosomes. A jump up in the z n ¢ % n curve denotes a decrease of the GC content, while a drop in the curve indicates an increase of the GC content. According to the z n ¢ % n curve, each chromosome is composed of several GC-rich and AT-rich regions alternatively. The identified isochores, centromeric regions and NOR in chromosome II and IV are indicated with black lines, red and orange dots, respectively. Isochore structure of A. thaliana genome L L. Chen and F. Gao 3330 FEBS Journal 272 (2005) 3328–3336 ª 2005 FEBS h ¼ 0.05, 0.1 and 0.2, respectively, the corresponding identified regions in Fig. 1 and the number of iso- chores using each threshold. If the h-value of a region is less than the defined threshold, it is recognized as an isochore, otherwise it is an ‘isochore-like region’. It can be seen that with the increase of the h-value, the number of identified isochores is increasing. From analyzing the z¢ curves, some interesting phe- nomena have been found. Firstly, the overall GC dis- tribution patterns of chromosomes I, III and V are very similar, and those of chromosomes II and IV are similar. But the two groups of patterns are highly different. We will discuss the reason for this pheno- menon. The centromeres are located in 14.6–14.8 Mb, 3.5–3.8 Mb, 13.5–13.9 Mb, 3.0–3.3 Mb and 11.7–11.9 Mb regions in chromosomes I to V, respectively [21]. For chromosomes I, III and V, cen- tromeres are metacentric or submetacentric, while for chromosomes II and IV, they are acrocentric. Fur- thermore, it is pointed out that the NORs juxtapose the telomeres of chromosomes II and IV, which com- prise uninterrupted 18 s, 5.8 s, 25 s RNA and 5 s RNA genes, and they form the structural and cata- lytic cores of cytoplasmic ribosomes [16]. The two NORs are marked with orange dots in Fig. 1, and they are located in 0–230 kb of chromosomes II and 0–350 kb of chromosomes IV, respectively. The sim- ilar genomic organization of chromosomes I, III and V makes their overall GC distribution patterns very similar, and the reason is the same for chromosomes II and IV. The function of centromere is very important in cell division. It mediate chromosome segregation during mitosis and meiosis by nucleating kinetochore forma- tion, providing a target for spindle attachment and maintaining sister chromatid cohesion [22]. Because centromere regions are heterochromatic and contain tandem repeats arrays, the genomic organization of centromere remains poorly characterized [23] and some gaps still exist in the complete sequence maps. Repetit- ive DNA sequences near the A. thaliana centromeres include 180 bp repeats, retroelements, transposons, microsatellites and middle repetitive sequences. The repeats are rare in the enchromatic arms and often most abundant in percentromeric DNA [16]. The unin- terrupted repeat arrays may up to more than 1 Mb in the centromere region of each chromosome [23] and the unsequenced regions of centromeres are mainly Table 2. The GC-rich and AT-rich ‘isochore-like regions’ in the five A. thaliana chromosomes with the threshold h ¼ 0.05. Chr. no. Type Start (Mb) Stop (Mb) Length (Mb) GC (%) h 1 GC 0 9.78 9.78 36.68 0.19 1 GC 13.50 15.88 2.38 37.30 0.07 1 AT 15.88 26.79 10.91 35.03 0.06 1 GC 26.79 30.43 3.64 36.56 0.08 2 GC 0 0.23 0.23 40.71 0.23 2 AT 0.23 2.42 2.19 33.94 0.08 2 GC 2.42 5.65 3.23 37.92 0.25 2 AT 5.65 13.38 7.73 34.99 0.14 2 GC 13.38 19.70 6.32 36.38 0.13 3 GC 0 7.50 7.50 37.03 0.15 3 AT 7.50 12.02 4.52 34.58 0.11 3 GC 12.02 15.61 3.59 37.81 0.12 3 AT 15.61 18.94 3.33 34.87 0.24 4 GC 0.36 2.29 1.93 36.26 0.21 4 GC 2.83 5.11 2.28 38.61 0.21 4 AT 5.11 12.51 7.40 35.10 0.67 4 GC 12.51 18.58 6.07 36.72 0.27 5 GC 0 7.15 7.15 36.78 0.06 5 AT 7.15 11.04 3.89 34.86 0.07 5 GC 11.04 13.45 2.41 38.73 0.09 5 AT 13.45 23.44 9.99 34.86 0.43 5 GC 23.44 26.99 3.55 36.56 0.12 Table 3. Three possible thresholds, the number of identified isochores and the corresponding regions in Fig. 1. h No. of isochores Region 0.05 5 Chromosome I: b Chromosome II: mtDNA insertion in region c Chromosome III: e Chromosome IV: a, c 0.1 12 Chromosome I: b, c, d, e Chromosome II: b, mtDNA insertion in region c Chromosome III: e Chromosome IV: a, c Chromosome V: a, b, c 0.2 19 Chromosome I: a, b, c, d, e Chromosome II: b, d, e, mtDNA insertion in region c Chromosome III: a, b, c, e Chromosome IV: a, c Chromosome V: a, b, c, e Table 1. Five identified isochores in the A. thaliana genome with the threshold h ¼ 0.05. No. Chr. no. Type Start (Mb) Stop (Mb) Length (Mb) GC (%) h 1 1 AT 9.78 13.50 3.72 34.67 0.03 2 2 GC 3.22 3.51 0.29 44.45 0.03 3 3 GC 18.94 23.47 4.53 36.84 0.05 4 4 GC 0 0.36 0.36 37.26 0.01 5 4 AT 2.29 2.83 0.54 34.51 0.05 L L. Chen and F. Gao Isochore structure of A. thaliana genome FEBS Journal 272 (2005) 3328–3336 ª 2005 FEBS 3331 composed of 180 bp repeats and 5 s rDNA [16]. Sequence from the central heterochromatic domain is characterized by a relatively low gene density, increased repeat density and pseudogene density [24]. The difference of genomic organization in heterochro- matin centromeres and euchromatic regions can be intuitively observed in the z¢ curves. All the centro- meres in the five chromosomes are located in GC-rich ‘isochore-like regions’. Because the gene density in centromere regions is much lower than that of other regions, the higher GC content in the centromere regions might be caused by the intergenic sequences. Secondly, there is an isochore located in 3220– 3510 kb in chromosomes II. The GC content of the isochore (44.45%) is much higher than that of the whole genome (35.86%). Detailed analysis shows that it is a mitochondrial DNA insertion region [25]. This insertion is much larger than any of the previously reported organellar-to-nuclear transfers, and it is 99% identical to the mitochondrial genome, suggesting that the transfer event was very recent [25]. The authenti- city of this insertion in the Columbia ecotype was con- firmed by PCR amplification across the junctions of mitochondrial and unique nuclear DNA, followed by the sequencing of the corresponding fragments [25]. This organellar-to-nuclear transfer isochore is indicated in Fig. 1, which can be easily detected because it is almost a ‘straight line’ region in the z¢ curve. The z¢ curve has successfully detected the integron island in Vibrio cholerae chromosome II [15]. So the present method is useful in finding the horizontal transfer regions of both prokaryotic and eukaryotic genomes. Some biological characteristics of isochores The genomic GC content of the five A. thaliana chro- mosomes is very similar (about 36%), which is much lower than that of vertebrates. The GC content map for five A. thaliana chromosomes can be obtained from http://genomat.img.cas.cz/draw_gc/tmp-gc/ [26]. Com- pared with vertebrates, the isochores in A. thaliana have small GC content variation. Isochores in human belong to five families covering a wide GC range, including GC-poor isochores of L1-L2 families (GC < 44%) and GC-rich isochores H1 (44% < GC < 47%), H2 (47% < GC < 52%) and H3 (GC > 52%) [7]. According to this classification, except the mitochondrial DNA insertion isochore in chromosome II, all other regions in A. thaliana belong to GC-poor families and most of the variation between two adjacent regions is less than 4%. Analysis from the Arabidopsis Genome Initiative shows that gene distribution patterns are very similar on each chromo- some. Figure 2 shows the z¢ curve of each ‘isochore- like region’ and the corresponding gene density in chromosome V. The GC content based on sliding win- dow technique (window size 100 Kb, step 1 Kb) is also shown. It can be observed that although centromere (region c) is located in GC-rich ‘isochore-like region’, its gene density is much lower than other regions, which is consistent with reference [17]. The gene den- sity of two AT-rich ‘isochore-like regions’ (regions b and d) are a little bit lower than that of two GC-rich ‘isochore-like regions’ (regions a and e). Other chro- mosomes have the similar gene density distributions. The codon usage, codon preference and amino acid usage are calculated for genes in each isochore and chromosome. Table 4 lists the results for the NOR and the mitochondrial DNA insertion isochore in chromo- some II and the whole chromosome. The results for other isochores and chromosomes are listed in supple- mentary Tables S1 and S2. Table 4 shows that the genes in NOR prefer amino acids encoded by GC-rich codons and GC-ending synonymous codons. The mitochondrial DNA insertion isochore does not show this preference and the amino acid usage is significantly different from that of the chromosome II, which might indicate the difference between the mitochondrial inser- tion genes and the nuclear genes. It also can be deduced that the higher GC content in NOR is caused by cod- ing and noncoding sequences, while for the mitochond- rial DNA insertion isochore, it is not caused by the genes, but for other elements in the sequences. Transposons in A. thaliana account for at least 10% of the genome, or about one-fifth of the intergenic DNA sequences [16]. The Arabidopsis Genome Initiat- ive figures the distribution of class I, II and Basho transposons in A. thaliana chromosomes. Class I retro- transposons are less abundant in A. thaliana than in other plants and primarily dominate the centromere regions. Class II transposons and Basho elements are clustered in the pericentromeric domains. All in all, transposons are more abundance in centromere GC-rich ‘isochore-like regions’ than other regions. Experimental procedures The complete sequences and annotation of genes in A. thaliana genome were downloaded from GenBank, Release 144.0. The length of the five chromosomes is 30 432 563, 19 705 359, 23 470 805, 18 585 042 and 26 992 728 bp, respectively. There are 163 560, 2451, 5433, 3030 and 13 823 undetermined bases in chromosome I to V, respectively, which are filtered in this calculation and marked in the z¢ curves. The information of RNA sequences, transposons and other control elements were Isochore structure of A. thaliana genome L L. Chen and F. Gao 3332 FEBS Journal 272 (2005) 3328–3336 ª 2005 FEBS obtained from the MIPS A. thaliana database [21] and TAIR (http://www.arabidopsis.org/). The Z curve method The Z curve is a three-dimensional space curve constitu- ting the unique representation of a given DNA sequence in the sense that for the curve and sequence each can be uniquely reconstructed from the other [18,19]. It is composed of a series of nodes P 0 , P 1 , P 2 , …, P N , whose coordinates x n , y n and z n (n ¼ 0, 1, 2, …, N, where N is the length of the DNA sequence being stud- ied) are calculated by the Z-transform of DNA sequence [18,19]: A B C Fig. 2. The z n ¢ curve and gene density for A. thaliana chromosome V. (A) The z¢ curve for A. thaliana chromosome V. (B) The GC content cal- culated based on a sliding window technique (window size 100 Kb, step 1 Kb). (C) Gene density calculated based on 100 Kb sliding windows along the chromosome. L L. Chen and F. Gao Isochore structure of A. thaliana genome FEBS Journal 272 (2005) 3328–3336 ª 2005 FEBS 3333 Table 4. The codon usage, codon preference and amino acid usage of the genes in NOR, the mitochondrial DNA insertion isochore in chro- mosome II and the whole chromosome II. CU, codon usage; CP, codon preference; AAU, amino acid usage. Amino acid Codon NOR (0–230 kb) Isochore (3220–3510 kb) Chromosome II CU CP AAU CU CP AAU CU CP AAU A GCT 2.74 0.38 7.11 2.49 0.39 6.37 2.77 0.43 6.39 A GCC 1.59 0.22 7.11 1.32 0.21 6.37 0.98 0.15 6.39 A GCA 1.88 0.26 7.11 1.58 0.25 6.37 1.78 0.28 6.39 A GCG 0.90 0.13 7.11 0.98 0.15 6.37 0.86 0.13 6.39 C TGT 0.81 0.54 1.51 0.87 0.57 1.52 1.10 0.60 1.84 C TGC 0.70 0.46 1.51 0.65 0.43 1.52 0.74 0.40 1.84 D GAT 3.58 0.65 5.48 2.45 0.66 3.70 3.70 0.69 5.40 D GAC 1.90 0.35 5.48 1.25 0.34 3.70 1.70 0.31 5.40 E GAA 2.84 0.47 6.00 2.99 0.58 5.14 3.49 0.52 6.72 E GAG 3.16 0.53 6.00 2.15 0.42 5.14 3.23 0.48 6.72 F TTT 2.04 0.50 4.09 3.36 0.56 5.99 2.26 0.53 4.28 F TTC 2.05 0.50 4.09 2.63 0.44 5.99 2.02 0.47 4.28 G GGT 2.04 0.29 7.11 1.93 0.29 6.74 2.18 0.34 6.48 G GGC 1.44 0.20 7.11 0.78 0.12 6.74 0.89 0.14 6.48 G GGA 2.21 0.31 7.11 2.42 0.36 6.74 2.38 0.37 6.48 G GGG 1.42 0.20 7.11 1.62 0.24 6.74 1.02 0.16 6.48 H CAT 1.33 0.58 2.29 1.57 0.68 2.29 1.46 0.63 2.32 H CAC 0.96 0.42 2.29 0.73 0.32 2.29 0.86 0.37 2.32 I ATT 1.83 0.35 5.28 2.56 0.39 6.53 2.16 0.41 5.27 I ATC 1.95 0.37 5.28 1.82 0.28 6.53 1.78 0.34 5.27 I ATA 1.50 0.28 5.28 2.14 0.33 6.53 1.33 0.25 5.27 K AAA 2.72 0.47 5.74 2.90 0.58 5.02 3.12 0.49 6.33 K AAG 3.01 0.53 5.74 2.12 0.42 5.02 3.21 0.51 6.33 L TTA 1.10 0.11 10.12 2.21 0.19 11.53 1.31 0.14 9.37 L TTG 2.16 0.21 10.12 2.20 0.19 11.53 2.12 0.23 9.37 L CTT 2.59 0.26 10.12 2.62 0.23 11.53 2.43 0.26 9.37 L CTC 2.04 0.20 10.12 1.41 0.12 11.53 1.55 0.17 9.37 L CTA 0.88 0.09 10.12 1.65 0.14 11.53 0.99 0.11 9.37 L CTG 1.34 0.13 10.12 1.42 0.12 11.53 0.98 0.10 9.37 M ATG 2.31 1.00 2.31 1.93 1.00 1.93 2.24 1.00 2.24 N AAT 1.70 0.46 3.66 2.08 0.62 3.35 2.32 0.53 4.39 N AAC 1.96 0.54 3.66 1.27 0.38 3.35 2.07 0.47 4.39 P CCT 2.04 0.42 4.82 2.08 0.37 5.59 1.90 0.39 4.90 P CCC 0.67 0.14 4.82 1.19 0.21 5.59 0.51 0.10 4.90 P CCA 1.29 0.27 4.82 1.39 0.25 5.59 1.66 0.34 4.90 P CCG 0.82 0.17 4.82 0.93 0.17 5.59 0.83 0.17 4.90 Q CAA 1.60 0.46 3.49 2.42 0.67 3.63 2.03 0.57 3.54 Q CAG 1.89 0.54 3.49 1.21 0.33 3.63 1.51 0.43 3.54 R CGT 0.92 0.16 5.71 1.18 0.17 6.97 0.89 0.16 5.42 R CGC 0.53 0.09 5.71 0.79 0.11 6.97 0.37 0.07 5.42 R CGA 0.57 0.10 5.71 1.02 0.15 6.97 0.64 0.12 5.42 R CGG 0.60 0.11 5.71 0.93 0.13 6.97 0.49 0.09 5.42 R AGA 1.80 0.32 5.71 1.82 0.26 6.97 1.94 0.36 5.42 R AGG 1.28 0.22 5.71 1.22 0.17 6.97 1.10 0.20 5.42 S TCT 2.81 0.30 9.32 2.20 0.25 8.90 2.56 0.28 9.10 S TCC 1.47 0.16 9.32 1.69 0.19 8.90 1.11 0.12 9.10 S TCA 1.63 0.17 9.32 1.44 0.16 8.90 1.88 0.21 9.10 S TCG 0.95 0.10 9.32 1.04 0.12 8.90 0.93 0.10 9.10 S AGT 1.16 0.12 9.32 1.47 0.16 8.90 1.46 0.16 9.10 S AGC 1.30 0.14 9.32 1.06 0.12 8.90 1.15 0.13 9.10 T ACT 1.52 0.31 4.91 1.52 0.32 4.70 1.75 0.34 5.15 T ACC 1.16 0.24 4.91 1.26 0.27 4.70 1.02 0.20 5.15 T ACA 1.40 0.29 4.91 1.30 0.28 4.70 1.60 0.31 5.15 Isochore structure of A. thaliana genome L L. Chen and F. Gao 3334 FEBS Journal 272 (2005) 3328–3336 ª 2005 FEBS x n ¼ðA n þG n ÞÀðC n þT n Þ; y n ¼ðA n þC n ÞÀðG n þT n Þ; n ¼ 0; 1; 2; :::; N; x n ; y n ; z n 2½ÀN; N; z n ¼ðA n þT n ÞÀðC n þG n Þ; 8 > < > : ð1Þ where A n , C n , G n and T n are the cumulative occurrence numbers of A, C, G and T from the first to the nth base in the above sequence, respectively. Note that we define x 0 ¼ y 0 ¼ z 0 ¼ 0 such that the Z curve always starts from the origin of the three-dimensional coordinate system. The three components of the Z curve, x n , y n and z n , represent three independent distributions that completely describe the DNA sequence being studied. The component x n , y n and z n displays the frequencies distributions of the purine ⁄ pyrimid- ine, amino ⁄ keto and weak H-bond ⁄ strong H-bond along the sequence, respectively. Calculation of the GC content using a window- less technique As mentioned above, z n displays the distribution of bases of GC ⁄ AT types along a sequence. Based on z n , the GC content can be calculated using a windowless technique [15]. Usually, for an AT-rich genome, z n is approximately a monotonously increasing linear function of n, whereas for a GC-rich gen- ome, z n is approximately a monotonously decreasing linear function of n. In both cases, it is convenient to fit the curve of z n % n by a straight line using the least square technique, z ¼ kn ð2Þ where (z, n) is the coordinate of a point on the straight line fitted and k is its slope. Instead of using the curve of z n % n, we will use the z n ¢ % n curve (abbreviated to z¢ curve) hereafter, where z 0 n ¼ z n À kn ð3Þ Let G þ C denote the average GC content within a region Dn in a sequence, we find from Eqns (1–3): G þ C ¼ 1 2 1 À k À Dz n 0 Dn   1 2 ð1 À k À k 0 Þð4Þ where k¢ ¼ Dz n ¢⁄Dn is the average slope of the z¢ curve within the region Dn. Both quantities of Dz n ¢ and Dn can be calculated using the z¢ curve. As we can see from Eqn (4) that a jump up in the z¢ curve, i.e. k¢ > 0, indicates a decrease of the GC content or an increase of the AT con- tent, otherwise, a drop in the curve, i.e. k¢ < 0 indicates an increase of the GC content or a decrease of the AT content. Acknowledgements We thank Prof. Chun-Ting Zhang for invaluable assistance. Discussions with Feng-Biao Guo, Hong-Yu Ou and Sheng-Yun Wen were very helpful. We also acknowledge all the referees for their constructive com- ments, which were very helpful in improving the qual- ity of the paper. This study was supported in part by the 973 Project of China (Grant 2003CB114400). References 1 Macaya G, Thiery JP & Bernardi G (1976) An approach to the organization of eukaryotic genomes at a macromolecular level. J Mol Biol 108 , 237–254. 2 Bernardi G, Olofsson B, Filipski J, Zerial M, Salinas J, Cuny G, Meunier-Rotival M & Rodier F (1985) The mosaic genome of warm-blooded vertebrates. 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Nature 402, 761–768. 26 Paces J, Zika R, Paces V, Pavlicek A, Clay O & Ber- nardi G (2004) Representing GC variation along eukar- yotic chromosomes. Gene 333, 135–141. Supplementary material The following material is available online Table S1. The codon usage, codon preference and amino acid usage of the genes in the five Arabidopsis thaliana chromosomes. Table S2. The codon usage, codon preference and amino acid usage of the genes in four isochores. Isochore structure of A. thaliana genome L L. Chen and F. Gao 3336 FEBS Journal 272 (2005) 3328–3336 ª 2005 FEBS . Detection of nucleolar organizer and mitochondrial DNA insertion regions based on the isochore map of Arabidopsis thaliana Ling-Ling Chen 1 and Feng Gao 2 1 Laboratory for Computational. are the nucleolar organizer regions (NORs), and genes located in the two regions prefer to use GC-ending codons. Another GC -isochore located in chromosome II is a mitochondrial DNA insertion region,. regions (regions b and d) are a little bit lower than that of two GC-rich isochore- like regions (regions a and e). Other chro- mosomes have the similar gene density distributions. The codon