BACKGROUND
non-protein encoding dna performs a variety of important biological functions .
however, many of the functions of non-coding dna are poorly understood.
one such non-coding dna element is guanine-rich dna, which has been characterized in several functional domains: the telomeres, the ribosomal dna and, in mammals, the immunoglobulin heavy-chain switch regions  <cit> .
these g-rich dna elements all have stretches of consecutive guanines and have the capacity to form secondary structures such as g-quadruplex or g <dig> dna by hoogsteen bonding  <cit> .
g <dig> dna has been proposed to have multiple biological functions in vivo including the regulation of gene expression and chromosome dynamics  <cit> .
it has been hypothesised that the g <dig> conformation in rna transcripts derived from g-rich dna may be targets of transcriptional regulation based on the findings that many factors associated with rna processing, including hnrnp d, hnrnp a <dig>  and nucleolin, bind g <dig> dna through their conserved rna-recognition motif and rna-binding domain  <cit> .
more direct evidence for g-rich dna affecting the transcription regulation was the finding that the g-rich dna segment in the human c-myc promoter, which could form g <dig> dna in vitro, functions as a repressor element  <cit> .
many studies have also suggested a possible role for g-rich motifs in chromosome dynamics.
sen and gilbert originally proposed that the g-rich telomeres and internal chromosomal motifs in homologous chromosomes may participate in pairing during meiotic prophase, based on their observation that g-rich motifs in dna can form parallel four-stranded g <dig> dna  <cit> .
many proteins involved in chromosome synapsis and recombination were also found to interact with the g-rich dna motifs  <cit> .
furthermore, the synaptonemal complex lateral element component hop <dig> plays key roles in meiotic chromosome pairing, promoting synapsis of double-stranded dna helices in vitro via the formation of g <dig> dna  <cit> .
in addition to the in vitro evidence, g <dig> dna structures formed by g-rich dna in the immunoglobulin switch region were shown to be present in vivo, and are proposed to play a role in the immunoglobulin class switch recombination  <cit> .
these findings support the possibility of g-rich dna being involved in chromosome dynamics.
the probability of one stretch of  <dig> guanines occurring by chance in the  <dig> mb at-rich c. elegans genome   <cit>  is approximately 1/ <dig> to the 18th power,  <dig> in  <dig> trillion.
in the caenorhabditis elegans genome there are approximately  <dig> such homopolymeric poly-g/poly-c tracts .
thus, these tracts are greatly over-represented in the genome.
a study by denver and colleagues on homopolymeric nucleotide  runs in c. elegans reported that the observed number of the g/c tracts is much greater than expected.
while the number of a/t tracts declines steadily with the increase of the length as expected, g/c tracts do not display that trend  <cit> .
furthermore, retention of the tracts was found to be dependent on enzymatic activity as disruption of dog- <dig>  a protein with similarity to the human fancj helicase, caused deletions that initiated in g/c tracts with no less than  <dig> guanines  <cit> .
cheung et al.
proposed that dog- <dig> may prevent deletions of g/c tracts by unwinding g-rich secondary structures arising during lagging strand dna synthesis  <cit> .
youds et al.
demonstrated involvement of the homologous recombination repair pathway in the prevention of g/c tract deletions in the dog- <dig> mutant  <cit> .
based on the observations that g/c tracts are over-represented in the c. elegans genome and protected by enzymatic activity, it seems unlikely that the occurrence and maintenance of g/c tracts are by chance.
in this study, we characterize the frequency and distribution of g/c tracts in two species of nematode, c. elegans and c. briggsae and explore possible biological roles of these tracts in these two organisms.
RESULTS
g/c tracts are over-represented in the c. elegans genome
although statistically no g/c tracts containing  <dig> or more consecutive gs are expected in the  <dig> mbp c. elegans genome,  <dig> g/c tracts were found.
they are over-represented in the c. elegans genome, especially compared to the human  and yeast  genomes.
these g/c tracts range in size from  <dig> to  <dig> base pairs and the frequency decreases with increased length .
the tracts are distributed throughout the genome along all six chromosomes.
the five autosomes have approximately  <dig> to  <dig> tracts each, while the x chromosome has more than  <dig> tracts.
the density of g/c tracts on lgx is  <dig>  per mbps compared to  <dig>  per mbps of lgv  .
a: there is no gene cluster center and arm differentiation on the x chromosome  <cit> .
in this study, the lgx was divided into three parts based on its physical length.
g/c tracts are conserved in c. elegans hawaiian  isolate
given that g/c tracts are unstable in the c. elegans dog- <dig> mutant, we wanted to test if g/c tracts were conserved in other wild type isolates of c. elegans or whether g/c tracts are inherently unstable.
recent studies using array comparative genomic hybridization  demonstrated that there is approximately 2% gene content variance between the hawaiian  and bristol  isolates, and that these differences are primarily deletions in the cb <dig> hawaiian strain  <cit> .
furthermore there are a large number of single nucleotide polymorphisms  between the two intraspecific isolates,  <dig>  for  <dig> % covered sequence  <cit> .
we assayed the conservation of g/c tracts in the cb <dig> hawaiian strain by pcr and sequencing.
one random chosen g/c tract on each chromosome was analyzed and the sequencing results showed that the presence, length and orientation of g/c tracts were conserved in the hawaiian strain.
this result demonstrated the preservation of the tested g/c tracts in these two wild type isolates of c. elegans.
g/c tracts are also over-represented in caenorhabditis briggsae
to determine if the location and orientation of g/c tracts are conserved in closely related nematode species, we investigated the g/c tract conservation in c. briggsae, a closely related species of c. elegans.
in total,  <dig> g/c tracts in size from  <dig> to  <dig> base pairs were found in the c. briggsae genome based on the sequence release cb <dig> agp <dig>  <cit> , of which  <dig> g/c tracts are intragenic and  <dig> g/c tracts are intergenic.
although there are fewer g/c tracts in the c. briggsae genome compared to the c. elegans genome, the tracts are still over-represented as the c. briggsae genome is similar to that of c. elegans in size and gc content  <cit> .
we found that the positions of g/c tracts are not well conserved between two species.
the assay on the conservation of the g/c tracts was performed using the following procedure: 1) find the gene  or the nearest gene to a g/c tract  in c. briggsae genome; 2) identify the corresponding c. elegans ortholog; 3) screen for g/c tracts in the gene or surrounding sequence.
three intragenic g/c tracts are located within the same genes in two species: a  <dig> bp tract in c. elegans gene vab- <dig> and an  <dig> bp tract in its c. briggsae ortholog cbg15813; a  <dig> bp tract in hmr- <dig> and an  <dig> bp tract in cbg07964; and a  <dig> bp tract in h10d <dig>  with its counterpart a  <dig> bp tract in cbg <dig>  however, only the tracts in vab- <dig> and cbg <dig> could be considered conserved when the facts such as tract orientation and position specificity were taken into account .
for intergenic g/c tracts,  <dig> g/c tracts were found to be located in similar locations in c. elegans and c. briggsae.
however, using a strict criterion , only  <dig> g/c tracts could be classified as conserved .
g/c tracts considered conserved between the two species under strict criterion were highlighted with bold font.
# intergenic g/c tracts were defined by closest gene in this table.
*there are two g/c tracts in both cbg <dig> of c. briggsae and ceh- <dig> of c. elegans.
the fact that the c. briggsae genome also possesses abundant g/c tracts but fewer than c. elegans led us to question whether g/c tracts are protected by a c. briggsae dog- <dig> ortholog in a manner similar to c. elegans.
based on the results of amino acid reciprocal best blast hits, there is a predicted ortholog of dog- <dig> in c. briggsae named cbg <dig>  rt-pcr showed that it was transcriptionally active, producing an mrna of  <dig> base pairs.
we sequenced the c. briggsae dog- <dig> cdna and found that there was an additional exon that was not included in the predicted c. briggsae gene model .
the corrected gene model showed 71% amino acid identity and 87% protein similarity with its c. elegans ortholog.
since there were no mutant alleles of cbg <dig> available we tested whether the c. briggsae dog- <dig> ortholog had a role in maintaining g/c tracts.
we introduced the full length cbg <dig> gene with its promoter region into the dog- <dig> knockout mutant to see if the c. briggsae dog- <dig> ortholog could rescue the g/c tract deletion phenotype.
deletions were observed in the vab- <dig> g/c tract in 12/ <dig>  dog- <dig> animals.
deletions of the same site were observed in only 2/ <dig>  dog-1; hex <dig> transgenic animals, significantly lower than non-transgenic animals .
this result clearly demonstrates that the c. briggsae dog- <dig> ortholog cbg <dig> can protect the integrity of g/c tracts in c. elegans, rescuing the g/c tract deletion phenotype of the dog- <dig> mutant.
therefore, cbg <dig> could be protecting the  <dig> g/c tracts in c. briggsae just as dog- <dig> does in c. elegans.
although the positions of most g/c tracts are not conserved, the g/c tracts are protected by the c. briggsae dog- <dig> ortholog.
g/c tracts are distributed non-randomly on c. elegans chromosomes
although the gc content in the c. elegans genome is similar on all the chromosomes  <cit> , the g/c tracts are not distributed uniformly across the chromosomes.
c. elegans autosomes can be divided into three genetically defined compartments of the left arm , the central gene cluster region , and the right arm   <cit> .
we found that more g/c tracts are located in the chromosome arms than in the central regions .
while this pattern is fairly subtle on lgiii, it is more obvious on the other chromosomes especially on lgv where only 9% of g/c tracts are located in the central region while 91% reside on the arms of lgv .
this non-random distribution of g/c tracts on the autosomes correlates with both the meiotic cross-over distribution  <cit>  and negatively with gene density  <cit> .
an enrichment of g/c tracts on the chromosome arms of the x chromosome was observed even though the x chromosome does not have a central gene cluster or a meiotic cross-over pattern.
the distributions of the intragenic and intergenic g/c tracts do not differ from the overall distribution pattern with more and longer g/c tracts on the arms.
nor is there any distinct pattern with regard to the orientation of g/c tracts on either dna strand.
g/c tracts are distributed uniformly in c. briggsae genome
in order to graphically demonstrate the distribution of g/c tracts in the genome, a "marey map"  <cit>  approach was used.
as shown in figure  <dig>  abundant g/c tracts in a given region result in a steeper slope than areas that lack of g/c tracts.
when the distribution of g/c tracts in c. elegans was represented in this way, the plot resulted in "s" curves, subtle on lgiii but more obvious on the other chromosomes , indicating the abundance of g/c tracts on chromosome arms compared to the clusters.
the curves on our map were similar to those on the genetic/physical marey map reported by barnes et al.
<cit> , which reflected the increased meiotic crossing-over in the arm regions compared to the crossing-over in the central gene clusters.
based on the knowledge that the two genomes exhibit extensive colinearity  <cit> , we used the genomic positions of the c. elegans orthologs of the g/c tracts' associated genes in c. briggsae to create a predicted genomic distribution map of g/c tracts in c. briggsae.
in the c. briggsae plots , g/c tracts are also dispersed across every chromosome in the c. briggsae genome.
because there are fewer g/c tracts on each chromosome, the curves are beneath their c. elegans counterparts.
the slope of the c. briggsae line illustrates that the g/c tracts are distributed evenly across the chromosomes.
both the analysis presented here  and a more recent analysis based on the chromosome-based assembly of c. briggsae genome  result in a similar pattern .
thus, although there is no specific patterning to the position or the orientation of the tracts, the number and dispersed location of them in these two species is suggestive of a biological role.
g/c tracts mostly occur between genes or in introns
although 27% of c. elegans genome is in predicted exons  <cit> , only  <dig> out of  <dig> g/c tracts  were in gene exons .
consecutive guanines or cytosines would encode strings of glycines or prolines, an unlikely sequence combination.
the four genes containing g/c tract sequences are located near the ends of the chromosome arms.
the predicted gene products are not currently assigned to any kog  category  <cit>  and as there is no est or sage data, it is possible that they are not expressed.
it is possible that these genes were predicted incorrectly and that the g/c tracts are not in coding regions.
for the remaining  <dig> g/c tracts,  <dig> of them were found to be in non-coding regions of genes , and the other  <dig> g/c tracts are located between genes.
the genes containing tracts vary in length, from less than  <dig> bp to more than  <dig> kb, with an average length of  <dig>  kb, much longer than the average gene size for the whole genome   <cit> .
this was true for all chromosomal regions: autosome central,  <dig>  kb; autosome arm,  <dig>  kb; and x chromosome,  <dig>  kb.
the location of the tracts within introns was unbiased, but not the orientation.
almost twice as many intragenic g tracts are located on non-coding strands as on coding strands .
this preference might be due to catastrophic problems caused by higher structures formed by g/c tracts during the transcription on the non-coding strand.
the number of intragenic g/c tracts on each chromosome is very similar.
thus, the high number of g/c tracts on the x chromosome is largely due to a greater number of tracts between genes on that chromosome .
eddy and colleagues previously reported that g-rich dna motifs with potential to form g <dig> dna are highly represented in proto-oncogenes in human genome  <cit> .
this result prompted us to investigate whether or not the g/c tract bearing genes in c. elegans are functionally related.
sixty-nine percent of the genes containing g/c tracts could be assigned to a kog classification .
the most abundant category "poorly characterized genes"  combined with the number of unassigned genes  account for more than half of the total g/c tract bearing genes, indicating that many g/c tract bearing genes are not well understood.
"signal transduction mechanisms" and "transcription" are the two most abundant categories with specified functions, and contain  <dig> and  <dig> genes respectively.
however, they are not statistically over-represented based on the c. elegans whole genome kog classification  <cit>  .
overall, there is no evidence that the g/c tract bearing genes are functionally related.
*the g/c tract in t06a <dig> on lgiv was located in two overlapping genes t06a <dig>  and mel- <dig>  both genes were analyzed in this study.
the average distance from an intergenic g/c tract to the nearest gene is  <dig>  kb.
although some of the  <dig> intergenic g/c tracts are located as far as nearly  <dig> kb from the nearest gene, most intergenic g/c tracts are found to be close to genes.
most intergenic g/c tracts  are found to be within  <dig> kb of the nearest gene and almost one third of intergenic g/c tracts  are within  <dig> bp of the nearest gene.
thirteen g/c tracts were found to reside between two genes that are closer than  <dig> kb.
interestingly, although it is known that the gene density in the central gene cluster regions on autosomes is higher than autosome arms  <cit> , the average distance of the intergenic g/c tracts from a gene in autosome central gene cluster regions is actually slightly larger than that on the arms .
g/c tracts are associated with the levels of regional gene expression
both introns and the flanking regions of genes are known to affect gene expression.
we thus suspected that the abundant g/c tracts in these areas are associated with the transcriptional regulation of corresponding genes.
we investigated the relationship between the position of the g/c tract and the level of transcription using c. elegans sage  data  <cit> .
sage data for genes containing intragenic g/c tracts and for various distances away from a g/c tract was assembled and collated .
only the specific tags assigned to genes  were used.
genes with a g/c tract within an intron  had on average  <dig>  sage tags .
similarly, genes within  <dig> bp of a g/c tract had  <dig>  tags.
genes 500– <dig> bp away from a g/c tract had on average  <dig>  tags and those  <dig> – <dig> kb had  <dig>  tags.
genes further away, 3– <dig> kb, exhibited higher levels of gene expression.
the average of  <dig>  tags per gene differed significantly from the averages for genes closer to tracts .
for genes  <dig> kb away from g/c tracts the average sage tag number is  <dig> .
thus, genes located distantly from g/c tracts have higher levels of transcription than genes close to g/c tracts, which may reflect a relationship to chromatin domains.
while genes on the autosome arms in c. elegans tend to be poorly expressed  <cit> , average number of sage tags of genes associated with g/c tracts located on autosome arms does not significantly differ from that of those in the central gene clusters .
there is no significant difference in sage tag number whether the g tract is on the coding strand or not, and whether the g/c tract is upstream  or downstream .
spearman correlation coefficient analysis revealed correlation between the sage tag numbers and gene distance from a g/c tract for genes that are no more than  <dig> kb away from a g/c tract .
this correlation was not observed on genes further away.
*the g/c tract in t06a <dig> on lgiv was located in two overlapping genes t06a <dig>  and mel- <dig>  both genes were analyzed in this study.
discussion
both the c. elegans and c. briggsae genomes contain many more g/c tracts than expected as not even one tract greater than  <dig> bps is expected to be found by chance in a genome of  <dig> mb of dna.
most g/c tracts are located in intergenic or intronic areas.
genes containing tracts are much larger than average and more g/c tracts are located on the gene-poor chromosomal arms.
thus, the tracts are located in regions with a low density of coding dna.
however, the fact that both the c. elegans and c. briggsae genomes contain a large number of these unusual homopolymeric tracts distributed across each chromosome and the existence of a conserved protein, dog- <dig>  that prevents the deletion of g/c tracts support the possibility that g/c tracts have biological significance.
initially it would appear that the x chromosome in c. elegans is significantly different than the autosomes with regard to g/c tracts distribution.
the x chromosome contains almost twice as many g/c tracts as the autosomes whereas both inverted and tandem repetitive sequences are more frequent on the autosomes than on the x chromosome  <cit> .
for example, cerep <dig>  with  <dig> copies distributed over the autosomes, has only one copy located on the x chromosome  <cit> .
although the x chromosome has many more g/c tracts than the autosomes, it has approximately the same number of intragenic g/c tracts  compared to the autosomes , which means the large number of g/c tracts on the x are due to a larger number of intergenic g/c tracts  .
furthermore, the overall gene density of the x chromosome is lower than that of the autosomes  <cit> .
taken together with regard to the distribution of g/c tracts on x chromosome, it would appear that the x chromosome is organized like an autosomal arm.
more g/c tracts were found to be located on the arms of the x chromosome even though the x chromosome does not have a meiotically defined central gene cluster, suggesting that there is no correlation between the meiotic pattern and g/c tracts.
recent study on c. briggsae genetic mapping using snps showed that c. briggsae has a similar meiotic pattern as c. elegans  <cit> .
this finding, along with our observation of the uniform distribution of g/c tracts in c. briggsae, also suggests that g/c tracts are not correlated with meiotic recombination.
we examined in c. elegans the distribution of the tracts in the context of the level of gene expression.
based on sage analysis, we observed a variation of transcription levels that correlates with distance from the g/c tract.
the average number of sage tags associated with g/c tract flanking genes is significantly lower than the number of sage tags associated with genes further from g/c tracts.
this effect is similar to the suppression of human c-myc gene by its g-rich promoter  <cit> .
although no evidence for direct regulation by g/c tracts on specific genes was found in c. elegans, our observation showed that there is a correlation with the presence of a g/c tract and the level of expression.
if g/c tracts played important gene specific cis-regulatory functions they should be conserved in c. briggsae but few are.
thus, we believe g/c tracts are unlikely to be regulating transcription in a gene specific manner.
on the other hand, previous studies on regulatory elements in c. elegans muscle genes showed that regulatory elements are also highly represented in c. briggsae muscle genes but the conservation of individual sites is weak  <cit> .
one of their interpretations was that a specific site in c. elegans could disappear over evolutionary time and reappear at a different position and retain its regulatory activity.
similarly, g/c tracts in c. elegans, although not conserved in position in c. briggsae, could be affecting regional gene expression.
this may be due to changes in the local chromatin environment, perhaps as a result of the g/c tract that affects gene transcription, or that these chromatin environments may be more conducive to the generation or maintenance of g/c tracts.
the capability of g-rich secondary dna structures to interact with each other using non watson-crick base pairing makes it an excellent candidate to coordinate chromosome dynamics such as pairing during meiosis.
sen and gilbert originally proposed that g <dig> dna might facilitate the pairing of homologous chromosomes without the need for testing homology at the sequence level  <cit> .
there are several lines of evidence suggesting that the g/c tracts in c. elegans might have a role in meiotic chromosome pairing.
many pairing or recombination related components can interact with g-rich dna secondary structures  <cit> .
for example, the mismatch repair protein mutsα binds g <dig> dna and promotes synapsis and recombination in the mammalian immunoglobulin switch regions  <cit>  and in yeast, the meiotic synapsis protein hop <dig> can promote the formation of g <dig> dna and the synapsis of double-stranded dna helices through the generation of g <dig> dna  <cit> .
the distribution of g/c tracts across the chromosomes in two species is compatible with a role for the tracts in chromosome alignment for pairing.
in many organisms, homolog pairing is achieved by double-stranded dna breaks and rad51-mediated strand invasion.
subsequent strand invasion can then align and pair homologous chromosomes before the formation of the synaptonemal complex.
in c. elegans and d. melanogaster, this is not the case as alignment and synapsis occurs without the generation of dsbs .
although the specific mechanisms that drive homologous chromosome pairing while preventing the pairing of non-homologous chromosomes are unknown, dna domains in c. elegans chromosomes have been proposed to play key roles in homolog pairing.
genetic analysis of c. elegans chromosome rearrangements identified cis-acting regions for pairing  <cit>  which occur at one 'end' of each c. elegans chromosome which were called the homolog recognition region   <cit> .
recent reports showed that specific c2h <dig> zinc-finger proteins bind to these regions and mediate the chromosome synapsis  <cit> , however, they are not essential for homolog pairing  <cit> .
once synapsis initiates, it is likely that sequences distributed along the chromosomes promote proper alignment.
in the case of large deletions or insertions that disrupt alignment, pairing for recombination can reinitiate, thus, there must be some mechanism by which homologous chromosomes can be brought into register along the length of the chromosomes without stand invasion  <cit> .
abundant g/c tracts distributed along the chromosomes with the ability to generate secondary g-rich structures that can pair with other g-rich structures on the homologs could function to align homologous chromosomes.
the observation that yeast hop <dig> protein is able to promote synapsis of double-stranded dna helices by the formation of g <dig> dna raised the possibility that hop <dig> homologs in c. elegans  might have similar capabilities.
this speculation is supported by the fact that mutants of him- <dig> and him- <dig> paralogs htp- <dig>  htp- <dig> were reported to all have chromosome pairing defects  <cit> .
it will be interesting to investigate whether him- <dig> or the him- <dig> paralogs can interact with g/c tracts.
CONCLUSIONS
in this article, we reported our analysis on the g/c tracts in c. elegans genome and their conservation in another caenorhabditis species, c. briggsae.
overrepresented g/c tracts are dispersed throughout the genomes of these two species, but the specific positions and the overall distribution patterns do not appear to be conserved.
along with the finding that g/c tracts are correlated with the levels of regional gene expression in c. elegans, we proposed that g/c tracts have possible biological roles in chromosome dynamics and/or gene expression regulation.

 non-protein encoding dna performs a variety of important biological functions. however, many of the functions of non-coding dna are poorly understood. one such non-coding dna element is guanine-rich dna, which has been characterized in several functional domains: the telomeres, the ribosomal dna and, in mammals, the immunoglobulin heavy-chain switch regions. these g-rich dna elements all have stretches of consecutive guanines and have the capacity to form secondary structures such as g-quadruplex or g dna by hoogsteen bonding. g dna has been proposed to have multiple biological functions in vivo including the regulation of gene expression and chromosome dynamics. it has been hypothesised that the g conformation in rna transcripts derived from g-rich dna may be targets of transcriptional regulation based on the findings that many factors associated with rna processing, including hnrnp d, hnrnp a and nucleolin, bind g dna through their conserved rna-recognition motif and rna-binding domain. more direct evidence for g-rich dna affecting the transcription regulation was the finding that the g-rich dna segment in the human c-myc promoter, which could form g dna in vitro, functions as a repressor element. many studies have also suggested a possible role for g-rich motifs in chromosome dynamics. sen and gilbert originally proposed that the g-rich telomeres and internal chromosomal motifs in homologous chromosomes may participate in pairing during meiotic prophase, based on their observation that g-rich motifs in dna can form parallel four-stranded g dna. many proteins involved in chromosome synapsis and recombination were also found to interact with the g-rich dna motifs. furthermore, the synaptonemal complex lateral element component hop plays key roles in meiotic chromosome pairing, promoting synapsis of double-stranded dna helices in vitro via the formation of g dna. in addition to the in vitro evidence, g dna structures formed by g-rich dna in the immunoglobulin switch region were shown to be present in vivo, and are proposed to play a role in the immunoglobulin class switch recombination. these findings support the possibility of g-rich dna being involved in chromosome dynamics. the probability of one stretch of guanines occurring by chance in the mb at-rich c. elegans genome is approximately 1/ to the 18th power, in trillion. in the caenorhabditis elegans genome there are approximately such homopolymeric poly-g/poly-c tracts. thus, these tracts are greatly over-represented in the genome. a study by denver and colleagues on homopolymeric nucleotide runs in c. elegans reported that the observed number of the g/c tracts is much greater than expected. while the number of a/t tracts declines steadily with the increase of the length as expected, g/c tracts do not display that trend. furthermore, retention of the tracts was found to be dependent on enzymatic activity as disruption of dog- a protein with similarity to the human fancj helicase, caused deletions that initiated in g/c tracts with no less than guanines. cheung et al. proposed that dog- may prevent deletions of g/c tracts by unwinding g-rich secondary structures arising during lagging strand dna synthesis. youds et al. demonstrated involvement of the homologous recombination repair pathway in the prevention of g/c tract deletions in the dog- mutant. based on the observations that g/c tracts are over-represented in the c. elegans genome and protected by enzymatic activity, it seems unlikely that the occurrence and maintenance of g/c tracts are by chance. in this study, we characterize the frequency and distribution of g/c tracts in two species of nematode, c. elegans and c. briggsae and explore possible biological roles of these tracts in these two organisms.  g/c tracts are over-represented in the c. elegans genome although statistically no g/c tracts containing or more consecutive gs are expected in the mbp c. elegans genome, g/c tracts were found. they are over-represented in the c. elegans genome, especially compared to the human and yeast genomes. these g/c tracts range in size from to base pairs and the frequency decreases with increased length. the tracts are distributed throughout the genome along all six chromosomes. the five autosomes have approximately to tracts each, while the x chromosome has more than tracts. the density of g/c tracts on lgx is per mbps compared to per mbps of lgv. a: there is no gene cluster center and arm differentiation on the x chromosome. in this study, the lgx was divided into three parts based on its physical length. g/c tracts are conserved in c. elegans hawaiian isolate given that g/c tracts are unstable in the c. elegans dog- mutant, we wanted to test if g/c tracts were conserved in other wild type isolates of c. elegans or whether g/c tracts are inherently unstable. recent studies using array comparative genomic hybridization demonstrated that there is approximately 2% gene content variance between the hawaiian and bristol isolates, and that these differences are primarily deletions in the cb hawaiian strain. furthermore there are a large number of single nucleotide polymorphisms between the two intraspecific isolates, for % covered sequence. we assayed the conservation of g/c tracts in the cb hawaiian strain by pcr and sequencing. one random chosen g/c tract on each chromosome was analyzed and the sequencing results showed that the presence, length and orientation of g/c tracts were conserved in the hawaiian strain. this result demonstrated the preservation of the tested g/c tracts in these two wild type isolates of c. elegans. g/c tracts are also over-represented in caenorhabditis briggsae to determine if the location and orientation of g/c tracts are conserved in closely related nematode species, we investigated the g/c tract conservation in c. briggsae, a closely related species of c. elegans. in total, g/c tracts in size from to base pairs were found in the c. briggsae genome based on the sequence release cb agp, of which g/c tracts are intragenic and g/c tracts are intergenic. although there are fewer g/c tracts in the c. briggsae genome compared to the c. elegans genome, the tracts are still over-represented as the c. briggsae genome is similar to that of c. elegans in size and gc content. we found that the positions of g/c tracts are not well conserved between two species. the assay on the conservation of the g/c tracts was performed using the following procedure: 1) find the gene or the nearest gene to a g/c tract in c. briggsae genome; 2) identify the corresponding c. elegans ortholog; 3) screen for g/c tracts in the gene or surrounding sequence. three intragenic g/c tracts are located within the same genes in two species: a bp tract in c. elegans gene vab- and an bp tract in its c. briggsae ortholog cbg15813; a bp tract in hmr- and an bp tract in cbg07964; and a bp tract in h10d with its counterpart a bp tract in cbg however, only the tracts in vab- and cbg could be considered conserved when the facts such as tract orientation and position specificity were taken into account. for intergenic g/c tracts, g/c tracts were found to be located in similar locations in c. elegans and c. briggsae. however, using a strict criterion, only g/c tracts could be classified as conserved. g/c tracts considered conserved between the two species under strict criterion were highlighted with bold font. # intergenic g/c tracts were defined by closest gene in this table. *there are two g/c tracts in both cbg of c. briggsae and ceh- of c. elegans. the fact that the c. briggsae genome also possesses abundant g/c tracts but fewer than c. elegans led us to question whether g/c tracts are protected by a c. briggsae dog- ortholog in a manner similar to c. elegans. based on the results of amino acid reciprocal best blast hits, there is a predicted ortholog of dog- in c. briggsae named cbg rt-pcr showed that it was transcriptionally active, producing an mrna of base pairs. we sequenced the c. briggsae dog- cdna and found that there was an additional exon that was not included in the predicted c. briggsae gene model. the corrected gene model showed 71% amino acid identity and 87% protein similarity with its c. elegans ortholog. since there were no mutant alleles of cbg available we tested whether the c. briggsae dog- ortholog had a role in maintaining g/c tracts. we introduced the full length cbg gene with its promoter region into the dog- knockout mutant to see if the c. briggsae dog- ortholog could rescue the g/c tract deletion phenotype. deletions were observed in the vab- g/c tract in 12/ dog- animals. deletions of the same site were observed in only 2/ dog-1; hex transgenic animals, significantly lower than non-transgenic animals. this result clearly demonstrates that the c. briggsae dog- ortholog cbg can protect the integrity of g/c tracts in c. elegans, rescuing the g/c tract deletion phenotype of the dog- mutant. therefore, cbg could be protecting the g/c tracts in c. briggsae just as dog- does in c. elegans. although the positions of most g/c tracts are not conserved, the g/c tracts are protected by the c. briggsae dog- ortholog. g/c tracts are distributed non-randomly on c. elegans chromosomes although the gc content in the c. elegans genome is similar on all the chromosomes, the g/c tracts are not distributed uniformly across the chromosomes. c. elegans autosomes can be divided into three genetically defined compartments of the left arm, the central gene cluster region, and the right arm. we found that more g/c tracts are located in the chromosome arms than in the central regions. while this pattern is fairly subtle on lgiii, it is more obvious on the other chromosomes especially on lgv where only 9% of g/c tracts are located in the central region while 91% reside on the arms of lgv. this non-random distribution of g/c tracts on the autosomes correlates with both the meiotic cross-over distribution and negatively with gene density. an enrichment of g/c tracts on the chromosome arms of the x chromosome was observed even though the x chromosome does not have a central gene cluster or a meiotic cross-over pattern. the distributions of the intragenic and intergenic g/c tracts do not differ from the overall distribution pattern with more and longer g/c tracts on the arms. nor is there any distinct pattern with regard to the orientation of g/c tracts on either dna strand. g/c tracts are distributed uniformly in c. briggsae genome in order to graphically demonstrate the distribution of g/c tracts in the genome, a "marey map" approach was used. as shown in figure abundant g/c tracts in a given region result in a steeper slope than areas that lack of g/c tracts. when the distribution of g/c tracts in c. elegans was represented in this way, the plot resulted in "s" curves, subtle on lgiii but more obvious on the other chromosomes, indicating the abundance of g/c tracts on chromosome arms compared to the clusters. the curves on our map were similar to those on the genetic/physical marey map reported by barnes et al., which reflected the increased meiotic crossing-over in the arm regions compared to the crossing-over in the central gene clusters. based on the knowledge that the two genomes exhibit extensive colinearity, we used the genomic positions of the c. elegans orthologs of the g/c tracts' associated genes in c. briggsae to create a predicted genomic distribution map of g/c tracts in c. briggsae. in the c. briggsae plots, g/c tracts are also dispersed across every chromosome in the c. briggsae genome. because there are fewer g/c tracts on each chromosome, the curves are beneath their c. elegans counterparts. the slope of the c. briggsae line illustrates that the g/c tracts are distributed evenly across the chromosomes. both the analysis presented here and a more recent analysis based on the chromosome-based assembly of c. briggsae genome result in a similar pattern. thus, although there is no specific patterning to the position or the orientation of the tracts, the number and dispersed location of them in these two species is suggestive of a biological role. g/c tracts mostly occur between genes or in introns although 27% of c. elegans genome is in predicted exons, only out of g/c tracts were in gene exons. consecutive guanines or cytosines would encode strings of glycines or prolines, an unlikely sequence combination. the four genes containing g/c tract sequences are located near the ends of the chromosome arms. the predicted gene products are not currently assigned to any kog category and as there is no est or sage data, it is possible that they are not expressed. it is possible that these genes were predicted incorrectly and that the g/c tracts are not in coding regions. for the remaining g/c tracts, of them were found to be in non-coding regions of genes, and the other g/c tracts are located between genes. the genes containing tracts vary in length, from less than bp to more than kb, with an average length of kb, much longer than the average gene size for the whole genome. this was true for all chromosomal regions: autosome central, kb; autosome arm, kb; and x chromosome, kb. the location of the tracts within introns was unbiased, but not the orientation. almost twice as many intragenic g tracts are located on non-coding strands as on coding strands. this preference might be due to catastrophic problems caused by higher structures formed by g/c tracts during the transcription on the non-coding strand. the number of intragenic g/c tracts on each chromosome is very similar. thus, the high number of g/c tracts on the x chromosome is largely due to a greater number of tracts between genes on that chromosome. eddy and colleagues previously reported that g-rich dna motifs with potential to form g dna are highly represented in proto-oncogenes in human genome. this result prompted us to investigate whether or not the g/c tract bearing genes in c. elegans are functionally related. sixty-nine percent of the genes containing g/c tracts could be assigned to a kog classification. the most abundant category "poorly characterized genes" combined with the number of unassigned genes account for more than half of the total g/c tract bearing genes, indicating that many g/c tract bearing genes are not well understood. "signal transduction mechanisms" and "transcription" are the two most abundant categories with specified functions, and contain and genes respectively. however, they are not statistically over-represented based on the c. elegans whole genome kog classification. overall, there is no evidence that the g/c tract bearing genes are functionally related. *the g/c tract in t06a on lgiv was located in two overlapping genes t06a and mel- both genes were analyzed in this study. the average distance from an intergenic g/c tract to the nearest gene is kb. although some of the intergenic g/c tracts are located as far as nearly kb from the nearest gene, most intergenic g/c tracts are found to be close to genes. most intergenic g/c tracts are found to be within kb of the nearest gene and almost one third of intergenic g/c tracts are within bp of the nearest gene. thirteen g/c tracts were found to reside between two genes that are closer than kb. interestingly, although it is known that the gene density in the central gene cluster regions on autosomes is higher than autosome arms, the average distance of the intergenic g/c tracts from a gene in autosome central gene cluster regions is actually slightly larger than that on the arms. g/c tracts are associated with the levels of regional gene expression both introns and the flanking regions of genes are known to affect gene expression. we thus suspected that the abundant g/c tracts in these areas are associated with the transcriptional regulation of corresponding genes. we investigated the relationship between the position of the g/c tract and the level of transcription using c. elegans sage data. sage data for genes containing intragenic g/c tracts and for various distances away from a g/c tract was assembled and collated. only the specific tags assigned to genes were used. genes with a g/c tract within an intron had on average sage tags. similarly, genes within bp of a g/c tract had tags. genes 500– bp away from a g/c tract had on average tags and those – kb had tags. genes further away, 3– kb, exhibited higher levels of gene expression. the average of tags per gene differed significantly from the averages for genes closer to tracts. for genes kb away from g/c tracts the average sage tag number is. thus, genes located distantly from g/c tracts have higher levels of transcription than genes close to g/c tracts, which may reflect a relationship to chromatin domains. while genes on the autosome arms in c. elegans tend to be poorly expressed, average number of sage tags of genes associated with g/c tracts located on autosome arms does not significantly differ from that of those in the central gene clusters. there is no significant difference in sage tag number whether the g tract is on the coding strand or not, and whether the g/c tract is upstream or downstream. spearman correlation coefficient analysis revealed correlation between the sage tag numbers and gene distance from a g/c tract for genes that are no more than kb away from a g/c tract. this correlation was not observed on genes further away. *the g/c tract in t06a on lgiv was located in two overlapping genes t06a and mel- both genes were analyzed in this study. discussion both the c. elegans and c. briggsae genomes contain many more g/c tracts than expected as not even one tract greater than bps is expected to be found by chance in a genome of mb of dna. most g/c tracts are located in intergenic or intronic areas. genes containing tracts are much larger than average and more g/c tracts are located on the gene-poor chromosomal arms. thus, the tracts are located in regions with a low density of coding dna. however, the fact that both the c. elegans and c. briggsae genomes contain a large number of these unusual homopolymeric tracts distributed across each chromosome and the existence of a conserved protein, dog- that prevents the deletion of g/c tracts support the possibility that g/c tracts have biological significance. initially it would appear that the x chromosome in c. elegans is significantly different than the autosomes with regard to g/c tracts distribution. the x chromosome contains almost twice as many g/c tracts as the autosomes whereas both inverted and tandem repetitive sequences are more frequent on the autosomes than on the x chromosome. for example, cerep with copies distributed over the autosomes, has only one copy located on the x chromosome. although the x chromosome has many more g/c tracts than the autosomes, it has approximately the same number of intragenic g/c tracts compared to the autosomes, which means the large number of g/c tracts on the x are due to a larger number of intergenic g/c tracts. furthermore, the overall gene density of the x chromosome is lower than that of the autosomes. taken together with regard to the distribution of g/c tracts on x chromosome, it would appear that the x chromosome is organized like an autosomal arm. more g/c tracts were found to be located on the arms of the x chromosome even though the x chromosome does not have a meiotically defined central gene cluster, suggesting that there is no correlation between the meiotic pattern and g/c tracts. recent study on c. briggsae genetic mapping using snps showed that c. briggsae has a similar meiotic pattern as c. elegans. this finding, along with our observation of the uniform distribution of g/c tracts in c. briggsae, also suggests that g/c tracts are not correlated with meiotic recombination. we examined in c. elegans the distribution of the tracts in the context of the level of gene expression. based on sage analysis, we observed a variation of transcription levels that correlates with distance from the g/c tract. the average number of sage tags associated with g/c tract flanking genes is significantly lower than the number of sage tags associated with genes further from g/c tracts. this effect is similar to the suppression of human c-myc gene by its g-rich promoter. although no evidence for direct regulation by g/c tracts on specific genes was found in c. elegans, our observation showed that there is a correlation with the presence of a g/c tract and the level of expression. if g/c tracts played important gene specific cis-regulatory functions they should be conserved in c. briggsae but few are. thus, we believe g/c tracts are unlikely to be regulating transcription in a gene specific manner. on the other hand, previous studies on regulatory elements in c. elegans muscle genes showed that regulatory elements are also highly represented in c. briggsae muscle genes but the conservation of individual sites is weak. one of their interpretations was that a specific site in c. elegans could disappear over evolutionary time and reappear at a different position and retain its regulatory activity. similarly, g/c tracts in c. elegans, although not conserved in position in c. briggsae, could be affecting regional gene expression. this may be due to changes in the local chromatin environment, perhaps as a result of the g/c tract that affects gene transcription, or that these chromatin environments may be more conducive to the generation or maintenance of g/c tracts. the capability of g-rich secondary dna structures to interact with each other using non watson-crick base pairing makes it an excellent candidate to coordinate chromosome dynamics such as pairing during meiosis. sen and gilbert originally proposed that g dna might facilitate the pairing of homologous chromosomes without the need for testing homology at the sequence level. there are several lines of evidence suggesting that the g/c tracts in c. elegans might have a role in meiotic chromosome pairing. many pairing or recombination related components can interact with g-rich dna secondary structures. for example, the mismatch repair protein mutsα binds g dna and promotes synapsis and recombination in the mammalian immunoglobulin switch regions and in yeast, the meiotic synapsis protein hop can promote the formation of g dna and the synapsis of double-stranded dna helices through the generation of g dna. the distribution of g/c tracts across the chromosomes in two species is compatible with a role for the tracts in chromosome alignment for pairing. in many organisms, homolog pairing is achieved by double-stranded dna breaks and rad51-mediated strand invasion. subsequent strand invasion can then align and pair homologous chromosomes before the formation of the synaptonemal complex. in c. elegans and d. melanogaster, this is not the case as alignment and synapsis occurs without the generation of dsbs. although the specific mechanisms that drive homologous chromosome pairing while preventing the pairing of non-homologous chromosomes are unknown, dna domains in c. elegans chromosomes have been proposed to play key roles in homolog pairing. genetic analysis of c. elegans chromosome rearrangements identified cis-acting regions for pairing which occur at one'end' of each c. elegans chromosome which were called the homolog recognition region. recent reports showed that specific c2h zinc-finger proteins bind to these regions and mediate the chromosome synapsis, however, they are not essential for homolog pairing. once synapsis initiates, it is likely that sequences distributed along the chromosomes promote proper alignment. in the case of large deletions or insertions that disrupt alignment, pairing for recombination can reinitiate, thus, there must be some mechanism by which homologous chromosomes can be brought into register along the length of the chromosomes without stand invasion. abundant g/c tracts distributed along the chromosomes with the ability to generate secondary g-rich structures that can pair with other g-rich structures on the homologs could function to align homologous chromosomes. the observation that yeast hop protein is able to promote synapsis of double-stranded dna helices by the formation of g dna raised the possibility that hop homologs in c. elegans might have similar capabilities. this speculation is supported by the fact that mutants of him- and him- paralogs htp- htp- were reported to all have chromosome pairing defects. it will be interesting to investigate whether him- or the him- paralogs can interact with g/c tracts.  in this article, we reported our analysis on the g/c tracts in c. elegans genome and their conservation in another caenorhabditis species, c. briggsae. overrepresented g/c tracts are dispersed throughout the genomes of these two species, but the specific positions and the overall distribution patterns do not appear to be conserved. along with the finding that g/c tracts are correlated with the levels of regional gene expression in c. elegans, we proposed that g/c tracts have possible biological roles in chromosome dynamics and/or gene expression regulation. 
analysis of sage data showed that g/c tracts correlate with the levels of regional gene expression in c. elegans. the abundance and genomic distribution of g/c tracts in c. elegans, the effect of g/c tracts on regional transcription levels, and the lack of positional conservation of g/c tracts in c. briggsae suggest a role for g/c tracts in chromatin structure but not in the transcriptional regulation of specific genes. g/c tracts are over-represented and dispersed across all chromosomes in another caenorhabditis species, c. briggsae. the frequency and distribution of g/c tracts in the genomes of c. elegans and the related nematode, c. briggsae were analyzed to investigate possible biological roles for g/c tracts. in c. elegans, g/c tracts are distributed along every chromosome in a non-random pattern. however, the positions and distribution of g/c tracts in c. briggsae differ from those in c. elegans. furthermore, the c. briggsae dog- ortholog cbg can rescue the mutator phenotype of c. elegans dog- mutants. most g/c tracts are within introns or are close to genes.
 in the genome of caenorhabditis elegans, homopolymeric poly-g/poly-c tracts exist at high frequency and are maintained by the activity of the dog- protein. the frequency and distribution of g/c tracts in the genomes of c. elegans and the related nematode, c. briggsae were analyzed to investigate possible biological roles for g/c tracts.  in c. elegans, g/c tracts are distributed along every chromosome in a non-random pattern. most g/c tracts are within introns or are close to genes. analysis of sage data showed that g/c tracts correlate with the levels of regional gene expression in c. elegans. g/c tracts are over-represented and dispersed across all chromosomes in another caenorhabditis species, c. briggsae. however, the positions and distribution of g/c tracts in c. briggsae differ from those in c. elegans. furthermore, the c. briggsae dog- ortholog cbg can rescue the mutator phenotype of c. elegans dog- mutants.  the abundance and genomic distribution of g/c tracts in c. elegans, the effect of g/c tracts on regional transcription levels, and the lack of positional conservation of g/c tracts in c. briggsae suggest a role for g/c tracts in chromatin structure but not in the transcriptional regulation of specific genes. 