BACKGROUND
a prominent gc-compositional strand bias or gc-skew /), where c and g denote the numbers of cytosine and guanine residues, was reported recently around the transcription start sites  of arabidopsis genes  <cit> .
it is well known that gc-skews occur bi-directionally in circular bacterial genomes along the direction of replication, and that gc-skew is an effective index for predicting the replication origin in some bacteria  <cit> .
in this case the numbers of g and t  residues in the leading strand of these genomes exceed those of c and a .
several models have been proposed to explain this bias  <cit> .
a similar strand bias related to the direction of replication has also been observed in human mitochondrial genomes  <cit> .
also, mammalian and enterobacterial genomes have been reported to show a strand bias associated with transcribed regions  <cit> .
an excess of g+t over a+c was observed in mammals within the sense strand of genes.
a transcription-coupled dna-repair system might be involved in this bias  <cit> .
however, existing models cannot explain the excess of c over g in the sense strand around the tss in arabidopsis.
although slight gc-skews  were reported recently around the tss in metazoans  <cit> , it remains unclear whether the strong gc-skew of arabidopsis is similar to that observed in metazoans.
although large amounts of genomic and full-length cdna sequence data from plants are now publicly available, knowledge of the promoters and tss in plants is still limited compared to mammals, such as human and mouse.
it has been reported that the cpg island  <cit>  is the most effective index for predicting the promoter regions or tss in mammals <cit> .
however, the cpg islands are not specifically located in the promoter regions in arabidopsis, so they cannot be used for the prediction of tss or promoters  <cit> .
identifying another, more suitable, index for the prediction of plant-specific tss has therefore become a priority.
answers are required for two key issues.
first, which eukaryotic species, phyla or kingdoms have arabidopsis-like gc-skews around the tss?
second, what is the biological significance of these regions?
in the present study, we used sequences from various animal, fungus, protist and plant species to conduct comparative analyses.
we explored the potential value of gc-skew as an index for tss prediction in plants.
finally, we considered the biological meaning of the gc-skew around the tss of plant genes.
RESULTS
gc-skew around the tss of plant genes
the shift in gc-skew values around tss was assessed by calculating the gc-skew for regions between  <dig> -kb upstream and  <dig> -kb downstream of the tss in arabidopsis , rice , human and drosophila.
full-length cdnas and genomic sequences were exploited from the species with available tss data.
the sliding-window method was used, and a value for the gc-skew was computed at the central position for each  <dig> bp.
the average of the gc-skew values at each position was calculated for all of the genes.
the results confirmed that the mean gc-skew spectra of both arabidopsis and rice peaked at the same position as the tss .
the mean gc-skew values were approximately zero in regions upstream  from the tss, as the numbers of c and g residues were equal in both plant species.
the values increased from the proximal region  and peaked at the tss.
downstream from the tss, the mean gc-skew values were inversely low.
although a small gc-skew was detected around the tss in drosophila and an opposite skew  was observed in human, these were less prominent when compared to those in plants.
our findings for drosophila and human were consistent with previous data  <cit> , indicating that this is a plant- or angiosperm-specific phenomenon.
intriguingly, the mean gc-skew values at the tss were identical in arabidopsis and rice genes, which were  <dig>   and  <dig>  , respectively.
however, it should also be noted that the mean values for the g+c content in the tss region  in arabidopsis and rice were significantly different:  <dig> and 53%, respectively.
in contrast, the g:c ratios at the tss in arabidopsis and rice were identical, despite the considerable difference in g+c content.
we also determined the frequencies of the four nucleotides in the regions up- and downstream of the tss in plants .
high c-residue frequencies were observed in both arabidopsis and rice .
in contrast, g-residue frequencies decreased slightly around the tss .
these findings suggest that the peaks of gc-skew values observed near the plant tss were caused by an increased frequency of c residues and a slight reduction in the g-residue frequency.
no significant biases were observed in the frequencies of a and t residues, indicating a lack of at-skew at the tss .
the number of plant genes with a strong gc-skew in proximal regions of the tss, was calculated by assessing the distributions of gc-skew values in regions +/- <dig> bp of the tss.
in order to take into account the wobble of the tss, and to avoid experimental or mapping artifacts, only the maximum gc-skew values from these regions were used.
the distributions of the maximum gc-skew values in the proximal regions of the tss were similar in both plant species .
over 70% of arabidopsis genes and over 68% of rice genes showed a strong gc-skew  near the tss .
our results suggest that many plant  genes have strong gc-skews around the tss.
furthermore, this characteristic is common among both monocot and eudicot plants.
in contrast, the gc-skews  that were observed in human and drosophila were qualitatively and quantitatively different from those identified in plants.
gc-skew in eukaryotes
the existence of gc-skew peaks around the tss in various eukaryotes was examined by calculating gc-skew values 100-bp downstream of the 5' -ends of virtually assembled transcripts  <cit> .
although these did not represent the actual tss, we were able to approximate the gc-skew values in the downstream regions.
table  <dig> shows the mean gc-skew values in the downstream regions of the tss for several species.
a prominent gc-skew  was confirmed in the 5' -ends of the transcripts in seven out of the nine plant species examined, and in five out of seven species of fungi.
although opposite skews  were observed in several protist and animal species, no significant excess of c residues was detected in any of the  <dig> animal species or the  <dig> protist species analyzed.
the mean values and standard deviations  of the gc-skew values 100-bp downstream of the 5' -end were calculated in virtually assembled transcripts of nine plant species, seven species of fungus,  <dig> protist species and  <dig> animal species, which were downloaded from  <cit> .
the symbols + and ++ denote the predominance of c: ++  and + .
the symbols - and -- denote the predominance of g: --  and - .
we determined whether gc-skew peaks were actually present in the tss of fungal genes, by investigating the regions around the translation initiation sites  of fungal genomic sequences.
genomic sequence data and information on open reading frames  are publicly available for some fungi, although there is insufficient information about the tss.
nevertheless, it was possible to estimate the tendency towards gc-skew around the tss by analyzing sequences both up- and downstream of the tis in the available genomic sequences.
figure  <dig> shows the mean gc-skew values in regions between  <dig> -kb upstream and  <dig> -kb downstream of the tis in fungal species: aspergillus nidulans, fusarium graminearum, magnaporthe grisea, neurospora crassa, saccharomyces cerevisiae and schizosaccharomyces pombe.
gc-skew peaks similar to those of plants were observed in 50- to 100-bp upstream regions of the tis in all of these species, with the exception of s. cerevisiae.
correlation between the two nucleotide frequencies in plants
a possible mechanism causing the gc-skew around the tss is nucleotide substitution, raising the question of what kind of substitution could be responsible.
correlations between the two nucleotide frequencies in regions both up- and downstream of the tss were expected to indicate the answer .
if the substitution rate between two specific nucleotides in one region is higher than that in other regions, a larger negative correlation would be expected between the two nucleotide frequencies, compared to other regions.
in both plant species, the correlation coefficient  of a-t and g-c decreased dramatically around the tss: the values were - <dig>  to - <dig> , and  <dig>  to - <dig> , in arabidopsis ), respectively; and  <dig>  to - <dig> , and  <dig>  to - <dig> , in rice ), respectively.
in contrast, the r values of a-g and t-c increased significantly around the tss: values were - <dig>  to  <dig> , and - <dig>  to  <dig> , in arabidopsis ), and - <dig>  to  <dig> , and - <dig>  to - <dig> , in rice ).
gc-skew and gene-expression level
in order to examine the relationship between gc-skew at the tss and gene expression in plants, we conducted a statistical test using serial analysis of gene expression  data for arabidopsis   <cit> .
the mean gc-skew value in the tss of highly-expressed genes  was significantly higher  than in genes with low expression in arabidopsis: the mean values were  <dig>   and  <dig>  .
potential value of gc-skew as an index for tss prediction
many of the gc-skew peaks were preferentially located near the tss, therefore we assessed the potential value of gc-skew as a predictive index for tss in plants.
sequences 1-kb upstream of the predicted orf start positions in plant genomic sequences were used in the following analysis.
first, the gc-skew values were computed using the sliding-window technique, in which one window and the shift size were set to  <dig> and  <dig> bp, respectively.
gc-skew peaks satisfying a particular cut-off value were identified as primitive tss candidates from the noisy gc-skew spectrum using the savitzky-golay  filter  <cit> , which simultaneously smoothes and differentiates.
next, tss candidates that were located within  <dig> bp of another candidate were considered to be identical and were merged into the position with the highest gc-skew.
tss prediction was validated by counting as true positives , the candidates that were located within 100-bp up- or downstream of the actual tss.
if more than two candidates coexisted in the appropriate region, they were regarded as one tp.
using this method and the criteria described above, we validated the predictive performance of gc-skew under cut-off values ranging from - <dig>  to  <dig>  .
the specificity ) ranged from  <dig> to 87%, and the sensitivity ) ranged from  <dig> to 95% in arabidopsis.
the sp in rice ranged from  <dig> to 56%, and the sn ranged from  <dig> to 99%.
the false-positive rate ) varied with the cut-off values in a similar manner to sn.
the difference between our results and random cases was clarified using a receiver-operating characteristic  curve .
this is a plot of fpr versus sn, with each cut-off value corresponding to a point on the curve.
good roc curves lie closer to the top left-hand corner, whereas the random cases are represented as a diagonal line .
predictions made using the gc-skew appeared to differ from the random cases, and lay closer to the top left-hand corner in both arabidopsis and rice.
in addition, the correlation coefficient  corresponding to each gc-skew cut-off value was calculated in order to compare their predictive performances.
the gc-skew value that maximized φ was  <dig>  in both arabidopsis and rice .
only the tss of genes in which a tis was defined within the  <dig> -kb downstream region are included.
sequence data from  <dig> -kb upstream of the tis  were used for the tss prediction.
the asterisks denote maximum φ.
discussion
this study confirmed the presence of significant gc-skews  around the tss  of genes in some species of plants and fungi.
in contrast, our analysis revealed no significant excess of c residues in either animals or protists.
however, an opposite gc-skew  has been reported previously in several animal species   <cit> .
although small skews were detected for these species in our present analysis , they were not significant compared to those observed in plants.
aerts et al.
<cit>  reported a gc-skew  close to the tss in two nematode species  however, no significant gc-skew was detected in c. elegans in our analysis.
this inconsistency was probably due to the fact that our analysis targeted only regions downstream of the tss in c. elegans ; alternatively, the skew might have been too small to be detected using our method.
in either case, it is difficult to compare gc-skews between nematodes and plants, since trans-splicing at the 5' -ends of genes has been reported in nematodes  <cit> , as pointed out by aerts et al  <cit> .
as noted above, the gc-skews near the tss in plants and fungi differed from those of other species  in both quality and quantity.
to our knowledge, this is the first report to describe the prominent gc-skew  around the tss specific to plants and fungi.
we propose two possible explanations for the gc-skew peaks found close to the tss.
first, regulatory elements, such as transcription factor-biding sites , which are present in regions both up- and downstream of the tss, might contribute to  this phenomenon.
moreover, some tfbs have a strand preference .
therefore, if these types of tfbs are preferentially located around the tss of plant and fungal genes, they might influence the local gc-compositional strand bias.
second, the gc-skew might be involved in transcription-coupled events, such as transcription-associated mutational asymmetry  <cit> .
tatarinova and colleagues  <cit>  mentioned such transcription-associated mutational asymmetry and suggested that the gc-skew around the tss of genes might be caused by the substitution of c residues with t residues, due to c deamination in the template strand.
however, this hypothesis cannot fully explain all of the findings of our present study.
if the c-to-t transition occurred preferentially around the tss in the template strand, an at-skew would also be expected in these regions.
however, no significant at-skew  was observed at the tss of either arabidopsis or rice genes, indicating that the c-to-t transition was not the main cause of the gc-skew.
furthermore, in our analyses, significant changes in the correlation coefficient  were observed in both arabidopsis and rice .
assuming that the gc skew is caused by mutations during transcription initiation, changes in the correlation coefficient might be interpreted as an increase in the transversion ratio around the tss.
the increased negative correlation between c and g, coupled with the high gc-skew value in the same region, led us to speculate that g-to-c transversion occurred at relatively high rates in this region in plants and fungi, but not in animals and protists.
if mutations that yield a gc-skew occur mainly around the tss of single-stranded dna, highly-expressed genes would be expected to have a high gc-skew around the tss.
in fact, the mean gc-skew value in the tss of highly-expressed genes was significantly higher than in genes with low expression in arabidopsis.
this indicates that the gc-skew is associated with the level of gene expression, at least in arabidopsis.
strand-specific mutational rates are believed to be a by-product of transcription-coupled dna repair in mammals  <cit> , therefore the gc-skew observed around the tss of plant genes might result from the plant-specific dna lesion and repair system.
alternatively, if the gc-skew was caused by the higher frequency of strand-specific tfbs, the higher mean gc-skew value in highly-expressed genes might be interpreted as a greater effect of strand-specific tfbs for the transcription efficiency.
in either strand-specific tfbs or mutation, highly-expressed genes appear to have a high gc-skew around the tss.
as a gc-skew was also detected in the upstream regions of the tis in fungal genes, this feature might be generated by a common mechanism in both groups.
however, additional sequence data, and investigations into the patterns of nucleotide substitutions and their rates around the tss, both between and within groups, will be necessary to elucidate the origin and mechanism of gc-skew around the tss of plant and fungal genes.
tss prediction by the gc-skew peak was validated through a stepwise increase of cut-off values which demonstrated that the gc-skew could contribute to tss prediction.
although the optimal gc-skew cut-off value depends on the specific situation, our results will be helpful in determining the optimal cut-off.
figure  <dig> shows representative cases in which the gc-skew peaks are located near the tss in arabidopsis and rice genes.
the single index presented in this paper might not be sufficient to achieve accurate tss prediction.
however, our results indicate that gc-skew is a good candidate index for the tss, promoter or first exon in plants.
thus, the combined use of gc-skew and other indices, or the incorporation of this index into pre-existing programs appears to be a realistic and effective approach for tss prediction.
CONCLUSIONS
significant gc-skew  around the tss is strictly conserved among monocot and eudicot plants , and a similar skew is also seen in some fungi.
the mean gc-skew at the tss in the highly expressed genes was greater than that in the group with low expression.
we therefore propose that the gc-skew around the tss in some species of plants and fungi is associated with transcriptional activity.
this is probably a result of dna mutations during transcription initiation or the frequent use of strand-specific tfbs.
our findings also confirm that gc-skew has the potential to assist tss prediction in plant genomes, where there is a lack of correlation among cpg islands and genes.

 a prominent gc-compositional strand bias or gc-skew /), where c and g denote the numbers of cytosine and guanine residues, was reported recently around the transcription start sites of arabidopsis genes. it is well known that gc-skews occur bi-directionally in circular bacterial genomes along the direction of replication, and that gc-skew is an effective index for predicting the replication origin in some bacteria. in this case the numbers of g and t residues in the leading strand of these genomes exceed those of c and a. several models have been proposed to explain this bias. a similar strand bias related to the direction of replication has also been observed in human mitochondrial genomes. also, mammalian and enterobacterial genomes have been reported to show a strand bias associated with transcribed regions. an excess of g+t over a+c was observed in mammals within the sense strand of genes. a transcription-coupled dna-repair system might be involved in this bias. however, existing models cannot explain the excess of c over g in the sense strand around the tss in arabidopsis. although slight gc-skews were reported recently around the tss in metazoans, it remains unclear whether the strong gc-skew of arabidopsis is similar to that observed in metazoans. although large amounts of genomic and full-length cdna sequence data from plants are now publicly available, knowledge of the promoters and tss in plants is still limited compared to mammals, such as human and mouse. it has been reported that the cpg island is the most effective index for predicting the promoter regions or tss in mammals. however, the cpg islands are not specifically located in the promoter regions in arabidopsis, so they cannot be used for the prediction of tss or promoters. identifying another, more suitable, index for the prediction of plant-specific tss has therefore become a priority. answers are required for two key issues. first, which eukaryotic species, phyla or kingdoms have arabidopsis-like gc-skews around the tss? second, what is the biological significance of these regions? in the present study, we used sequences from various animal, fungus, protist and plant species to conduct comparative analyses. we explored the potential value of gc-skew as an index for tss prediction in plants. finally, we considered the biological meaning of the gc-skew around the tss of plant genes.  gc-skew around the tss of plant genes the shift in gc-skew values around tss was assessed by calculating the gc-skew for regions between -kb upstream and -kb downstream of the tss in arabidopsis, rice, human and drosophila. full-length cdnas and genomic sequences were exploited from the species with available tss data. the sliding-window method was used, and a value for the gc-skew was computed at the central position for each bp. the average of the gc-skew values at each position was calculated for all of the genes. the results confirmed that the mean gc-skew spectra of both arabidopsis and rice peaked at the same position as the tss. the mean gc-skew values were approximately zero in regions upstream from the tss, as the numbers of c and g residues were equal in both plant species. the values increased from the proximal region and peaked at the tss. downstream from the tss, the mean gc-skew values were inversely low. although a small gc-skew was detected around the tss in drosophila and an opposite skew was observed in human, these were less prominent when compared to those in plants. our findings for drosophila and human were consistent with previous data, indicating that this is a plant- or angiosperm-specific phenomenon. intriguingly, the mean gc-skew values at the tss were identical in arabidopsis and rice genes, which were and, respectively. however, it should also be noted that the mean values for the g+c content in the tss region in arabidopsis and rice were significantly different: and 53%, respectively. in contrast, the g:c ratios at the tss in arabidopsis and rice were identical, despite the considerable difference in g+c content. we also determined the frequencies of the four nucleotides in the regions up- and downstream of the tss in plants. high c-residue frequencies were observed in both arabidopsis and rice. in contrast, g-residue frequencies decreased slightly around the tss. these findings suggest that the peaks of gc-skew values observed near the plant tss were caused by an increased frequency of c residues and a slight reduction in the g-residue frequency. no significant biases were observed in the frequencies of a and t residues, indicating a lack of at-skew at the tss. the number of plant genes with a strong gc-skew in proximal regions of the tss, was calculated by assessing the distributions of gc-skew values in regions +/- bp of the tss. in order to take into account the wobble of the tss, and to avoid experimental or mapping artifacts, only the maximum gc-skew values from these regions were used. the distributions of the maximum gc-skew values in the proximal regions of the tss were similar in both plant species. over 70% of arabidopsis genes and over 68% of rice genes showed a strong gc-skew near the tss. our results suggest that many plant genes have strong gc-skews around the tss. furthermore, this characteristic is common among both monocot and eudicot plants. in contrast, the gc-skews that were observed in human and drosophila were qualitatively and quantitatively different from those identified in plants. gc-skew in eukaryotes the existence of gc-skew peaks around the tss in various eukaryotes was examined by calculating gc-skew values 100-bp downstream of the 5' -ends of virtually assembled transcripts. although these did not represent the actual tss, we were able to approximate the gc-skew values in the downstream regions. table shows the mean gc-skew values in the downstream regions of the tss for several species. a prominent gc-skew was confirmed in the 5' -ends of the transcripts in seven out of the nine plant species examined, and in five out of seven species of fungi. although opposite skews were observed in several protist and animal species, no significant excess of c residues was detected in any of the animal species or the protist species analyzed. the mean values and standard deviations of the gc-skew values 100-bp downstream of the 5' -end were calculated in virtually assembled transcripts of nine plant species, seven species of fungus, protist species and animal species, which were downloaded from. the symbols + and ++ denote the predominance of c: ++ and +. the symbols - and -- denote the predominance of g: -- and -. we determined whether gc-skew peaks were actually present in the tss of fungal genes, by investigating the regions around the translation initiation sites of fungal genomic sequences. genomic sequence data and information on open reading frames are publicly available for some fungi, although there is insufficient information about the tss. nevertheless, it was possible to estimate the tendency towards gc-skew around the tss by analyzing sequences both up- and downstream of the tis in the available genomic sequences. figure shows the mean gc-skew values in regions between -kb upstream and -kb downstream of the tis in fungal species: aspergillus nidulans, fusarium graminearum, magnaporthe grisea, neurospora crassa, saccharomyces cerevisiae and schizosaccharomyces pombe. gc-skew peaks similar to those of plants were observed in 50- to 100-bp upstream regions of the tis in all of these species, with the exception of s. cerevisiae. correlation between the two nucleotide frequencies in plants a possible mechanism causing the gc-skew around the tss is nucleotide substitution, raising the question of what kind of substitution could be responsible. correlations between the two nucleotide frequencies in regions both up- and downstream of the tss were expected to indicate the answer. if the substitution rate between two specific nucleotides in one region is higher than that in other regions, a larger negative correlation would be expected between the two nucleotide frequencies, compared to other regions. in both plant species, the correlation coefficient of a-t and g-c decreased dramatically around the tss: the values were - to -, and to -, in arabidopsis ), respectively; and to -, and to -, in rice ), respectively. in contrast, the r values of a-g and t-c increased significantly around the tss: values were - to, and - to, in arabidopsis ), and - to, and - to -, in rice ). gc-skew and gene-expression level in order to examine the relationship between gc-skew at the tss and gene expression in plants, we conducted a statistical test using serial analysis of gene expression data for arabidopsis. the mean gc-skew value in the tss of highly-expressed genes was significantly higher than in genes with low expression in arabidopsis: the mean values were and. potential value of gc-skew as an index for tss prediction many of the gc-skew peaks were preferentially located near the tss, therefore we assessed the potential value of gc-skew as a predictive index for tss in plants. sequences 1-kb upstream of the predicted orf start positions in plant genomic sequences were used in the following analysis. first, the gc-skew values were computed using the sliding-window technique, in which one window and the shift size were set to and bp, respectively. gc-skew peaks satisfying a particular cut-off value were identified as primitive tss candidates from the noisy gc-skew spectrum using the savitzky-golay filter, which simultaneously smoothes and differentiates. next, tss candidates that were located within bp of another candidate were considered to be identical and were merged into the position with the highest gc-skew. tss prediction was validated by counting as true positives, the candidates that were located within 100-bp up- or downstream of the actual tss. if more than two candidates coexisted in the appropriate region, they were regarded as one tp. using this method and the criteria described above, we validated the predictive performance of gc-skew under cut-off values ranging from - to. the specificity ) ranged from to 87%, and the sensitivity ) ranged from to 95% in arabidopsis. the sp in rice ranged from to 56%, and the sn ranged from to 99%. the false-positive rate ) varied with the cut-off values in a similar manner to sn. the difference between our results and random cases was clarified using a receiver-operating characteristic curve. this is a plot of fpr versus sn, with each cut-off value corresponding to a point on the curve. good roc curves lie closer to the top left-hand corner, whereas the random cases are represented as a diagonal line. predictions made using the gc-skew appeared to differ from the random cases, and lay closer to the top left-hand corner in both arabidopsis and rice. in addition, the correlation coefficient corresponding to each gc-skew cut-off value was calculated in order to compare their predictive performances. the gc-skew value that maximized φ was in both arabidopsis and rice. only the tss of genes in which a tis was defined within the -kb downstream region are included. sequence data from -kb upstream of the tis were used for the tss prediction. the asterisks denote maximum φ. discussion this study confirmed the presence of significant gc-skews around the tss of genes in some species of plants and fungi. in contrast, our analysis revealed no significant excess of c residues in either animals or protists. however, an opposite gc-skew has been reported previously in several animal species. although small skews were detected for these species in our present analysis, they were not significant compared to those observed in plants. aerts et al. reported a gc-skew close to the tss in two nematode species however, no significant gc-skew was detected in c. elegans in our analysis. this inconsistency was probably due to the fact that our analysis targeted only regions downstream of the tss in c. elegans; alternatively, the skew might have been too small to be detected using our method. in either case, it is difficult to compare gc-skews between nematodes and plants, since trans-splicing at the 5' -ends of genes has been reported in nematodes, as pointed out by aerts et al. as noted above, the gc-skews near the tss in plants and fungi differed from those of other species in both quality and quantity. to our knowledge, this is the first report to describe the prominent gc-skew around the tss specific to plants and fungi. we propose two possible explanations for the gc-skew peaks found close to the tss. first, regulatory elements, such as transcription factor-biding sites, which are present in regions both up- and downstream of the tss, might contribute to this phenomenon. moreover, some tfbs have a strand preference. therefore, if these types of tfbs are preferentially located around the tss of plant and fungal genes, they might influence the local gc-compositional strand bias. second, the gc-skew might be involved in transcription-coupled events, such as transcription-associated mutational asymmetry. tatarinova and colleagues mentioned such transcription-associated mutational asymmetry and suggested that the gc-skew around the tss of genes might be caused by the substitution of c residues with t residues, due to c deamination in the template strand. however, this hypothesis cannot fully explain all of the findings of our present study. if the c-to-t transition occurred preferentially around the tss in the template strand, an at-skew would also be expected in these regions. however, no significant at-skew was observed at the tss of either arabidopsis or rice genes, indicating that the c-to-t transition was not the main cause of the gc-skew. furthermore, in our analyses, significant changes in the correlation coefficient were observed in both arabidopsis and rice. assuming that the gc skew is caused by mutations during transcription initiation, changes in the correlation coefficient might be interpreted as an increase in the transversion ratio around the tss. the increased negative correlation between c and g, coupled with the high gc-skew value in the same region, led us to speculate that g-to-c transversion occurred at relatively high rates in this region in plants and fungi, but not in animals and protists. if mutations that yield a gc-skew occur mainly around the tss of single-stranded dna, highly-expressed genes would be expected to have a high gc-skew around the tss. in fact, the mean gc-skew value in the tss of highly-expressed genes was significantly higher than in genes with low expression in arabidopsis. this indicates that the gc-skew is associated with the level of gene expression, at least in arabidopsis. strand-specific mutational rates are believed to be a by-product of transcription-coupled dna repair in mammals, therefore the gc-skew observed around the tss of plant genes might result from the plant-specific dna lesion and repair system. alternatively, if the gc-skew was caused by the higher frequency of strand-specific tfbs, the higher mean gc-skew value in highly-expressed genes might be interpreted as a greater effect of strand-specific tfbs for the transcription efficiency. in either strand-specific tfbs or mutation, highly-expressed genes appear to have a high gc-skew around the tss. as a gc-skew was also detected in the upstream regions of the tis in fungal genes, this feature might be generated by a common mechanism in both groups. however, additional sequence data, and investigations into the patterns of nucleotide substitutions and their rates around the tss, both between and within groups, will be necessary to elucidate the origin and mechanism of gc-skew around the tss of plant and fungal genes. tss prediction by the gc-skew peak was validated through a stepwise increase of cut-off values which demonstrated that the gc-skew could contribute to tss prediction. although the optimal gc-skew cut-off value depends on the specific situation, our results will be helpful in determining the optimal cut-off. figure shows representative cases in which the gc-skew peaks are located near the tss in arabidopsis and rice genes. the single index presented in this paper might not be sufficient to achieve accurate tss prediction. however, our results indicate that gc-skew is a good candidate index for the tss, promoter or first exon in plants. thus, the combined use of gc-skew and other indices, or the incorporation of this index into pre-existing programs appears to be a realistic and effective approach for tss prediction.  significant gc-skew around the tss is strictly conserved among monocot and eudicot plants, and a similar skew is also seen in some fungi. the mean gc-skew at the tss in the highly expressed genes was greater than that in the group with low expression. we therefore propose that the gc-skew around the tss in some species of plants and fungi is associated with transcriptional activity. this is probably a result of dna mutations during transcription initiation or the frequent use of strand-specific tfbs. our findings also confirm that gc-skew has the potential to assist tss prediction in plant genomes, where there is a lack of correlation among cpg islands and genes. 
however, plant-like gc-skew in regions upstream of the translation initiation sites in some fungi was identified following analyses of the expressed sequence tags and/or genomic sequences from other species. our results confirm that the gc-skew can be used to assist the tss prediction in plant genomes. our study confirmed a significant gc-skew in the tss of oryza sativa genes. in addition, gc-skew is a good candidate index for tss prediction in plant genomes, where there is a lack of correlation among cpg islands and genes. on the basis of our dataset, we estimated that > and 68% of arabidopsis and rice genes, respectively, had a strong gc-skew in a 100-bp window. it might be caused by mutations during transcription initiation or the frequent use of transcription factor-biding sites having a strand preference. the gc-skew around the tss is strictly conserved between monocot and eudicot plants, and a similar skew has been observed in some fungi. a gc-compositional strand bias or gc-skew /), where c and g denote the numbers of cytosine and guanine residues, was recently reported near the transcription start sites of arabidopsis genes. many of the gc-skew peaks were preferentially located near the tss, so we examined the potential value of gc-skew as an index for tss identification. however, it is unclear whether other eukaryotic species have equally prominent gc-skews, and the biological meaning of this trait remains unknown. the full-length cdnas and genomic sequences from arabidopsis and rice were compared using statistical analyses. despite marked differences in the g+c content around the tss in the two plants, the degrees of bias were almost identical. although slight gc-skew peaks, including opposite skews, were detected around the tss of genes in human and drosophila, they were qualitatively and quantitatively different from those identified in plants. highly-expressed arabidopsis genes had overall a more marked gc-skew in the tss compared to genes with low expression levels. the mean gc-skew value in the tss of highly-expressed genes in arabidopsis was significantly greater than that of genes with low expression levels.
 a gc-compositional strand bias or gc-skew /), where c and g denote the numbers of cytosine and guanine residues, was recently reported near the transcription start sites of arabidopsis genes. however, it is unclear whether other eukaryotic species have equally prominent gc-skews, and the biological meaning of this trait remains unknown.  our study confirmed a significant gc-skew in the tss of oryza sativa genes. the full-length cdnas and genomic sequences from arabidopsis and rice were compared using statistical analyses. despite marked differences in the g+c content around the tss in the two plants, the degrees of bias were almost identical. although slight gc-skew peaks, including opposite skews, were detected around the tss of genes in human and drosophila, they were qualitatively and quantitatively different from those identified in plants. however, plant-like gc-skew in regions upstream of the translation initiation sites in some fungi was identified following analyses of the expressed sequence tags and/or genomic sequences from other species. on the basis of our dataset, we estimated that > and 68% of arabidopsis and rice genes, respectively, had a strong gc-skew in a 100-bp window. the mean gc-skew value in the tss of highly-expressed genes in arabidopsis was significantly greater than that of genes with low expression levels. many of the gc-skew peaks were preferentially located near the tss, so we examined the potential value of gc-skew as an index for tss identification. our results confirm that the gc-skew can be used to assist the tss prediction in plant genomes.  the gc-skew around the tss is strictly conserved between monocot and eudicot plants, and a similar skew has been observed in some fungi. highly-expressed arabidopsis genes had overall a more marked gc-skew in the tss compared to genes with low expression levels. we therefore propose that the gc-skew around the tss in some plants and fungi is related to transcription. it might be caused by mutations during transcription initiation or the frequent use of transcription factor-biding sites having a strand preference. in addition, gc-skew is a good candidate index for tss prediction in plant genomes, where there is a lack of correlation among cpg islands and genes. 