BACKGROUND
noncoding tandem-repetitive satellite dna  has long been known to constitute a large portion of any eukaryotic genome  <cit> .
recent studies in drosophila through underreplication documented a strong positive correlation between genome size and the amount of satdna  <cit> .
satdna is usually located in the heterochromatic parts of the chromosomes close to the centromeres and telomeres.
the high evolutionary turn-over of satdna and the frequent species-specific patterns have puzzled researchers for many years.
such data challenge the hypothesis that there is a general biological function of satdna.
the enormous diversity of satdna in nucleotide sequence, length of repeats, complexity, and genomic abundance, instead suggest that a specific function cannot be ascribed to satdna.
however, transcription of satdna and other non-coding dna has been found in many species .
the function of the vast majority of these transcripts is unclear, but recent studies using rna interference have documented the role of non-coding rnas in regulation of gene expression, chromatin organization, and genome functioning .
for example, it has been demonstrated that small non-coding rnas can play a crucial role in regulating heterochromatin formation  <cit>  and it has been found that transcripts of tandem repeated non-coding dna can give rise to dsrnas  <cit> .
the transcription of satdna was in some instances also linked to hammerhead  ribozyme activity which may affect certain regulatory mechanisms in the cell.
satdna-derived hh ribozyme structures have been detected in several organisms as different as schistosomes, cave crickets, and salamanders  <cit> .
the hh ribozyme is one of the smallest catalytic rnas and was first identified in viroid and viroid-like satellite rna sequences where they catalyze a specific phosphodiester bond isomerization reaction in the course of rolling-circle replication <cit> .
all hh ribozymes detected in animal satdna so far have been shown to self-cleave in cis long multimeric transcripts into monomers  <cit> .
the hh ribozyme from schistosoma mansoni is certainly the best characterized of natural hh ribozymes.
this particular ribozyme has also been shown to perform efficient ligation  <cit>  and trans-cleavage of an rna target  <cit> .
recently, an active hh ribozyme was also found in a mammalian messenger rna that self-cleaved both in vitro and in vivo and reduced protein expression of c-type lectin type ii  genes in mouse cells  <cit> .
this study showed that ribozymes might also act in post-transcriptional regulation of protein coding gene expression in a way that is similar to destabilizing protein factors.
hh ribozymes are characterized by a conserved central core which is mandatory for cleavage activity.
the consensus sequence for a hh ribozyme consists of three paired stems that branch from the core as defined by conserved nucleotides .
in addition, the natural hh ribozymes require additional sequence elements outside of the conserved catalytic core to facilitate intracellular activity.
these elements are variable and are therefore not phylogenetically conserved  <cit> .
the correlation between structure and function of the hh ribozyme became clear only recently  <cit> .
for many years studies of hh ribozymes were restricted to a minimal construct.
however, there have been major discrepancies between the crystal structure of this particular construct and the results of biochemical experiments  <cit> .
the biochemical data could only be explained if a large-scale rearrangement of the core region was assumed during catalysis  <cit> .
recently, the hh ribozymes found in schistosomes were characterized in more detail and loop-loop interactions were shown to have a large impact on hh ribozyme activity  <cit> .
previously, database searches identified a potential hh ribozyme  in dolichopoda cave crickets embedded within the pdo500satdna family and self-cleavage of satdna transcripts has been shown in vitro for rna from d. baccettii  <cit> .
the roughly  <dig> bp repeats of the pdo <dig> have been found in all dolichopoda species studied so far  <cit>  and constitute approximately 5% of the d. schiavazzii genome  <cit> .
phylogenetic analyses of the pdo <dig> sequences from  <dig> dolichopoda species showed that this satdna family evolved gradually at a rate of  <dig> % per one million year  <cit> .
thus, pdo500-based phylogenetic hypotheses were mainly congruent with the mitochondrial dna phylogeny  <cit> .
the hh ribozyme in dolichopoda performed self-cleavage in vitro and this was associated with processing of long multimer transcripts into monomers in vivo  <cit> .
for the hh ribozyme of dolichopoda two cleavage mechanisms were suggested: 1) cis cleavage by a single-hh  ribozyme which is the folding of the  <dig> bp hh sequence, and 2) trans cleavage by a double-hammerhead  which is a hybrid structure between two extended hh sequences of  <dig> bp.
we took a phylogenetic footprint approach to analyze in detail the pdo <dig> satdna family from  <dig> different populations representing  <dig> species of dolichopoda cave crickets with particular emphasis on the hh region.
we assessed whether the hh motif in the pdo <dig> satdna is more conserved than other parts of the pdo <dig> repeat unit.
hh stems should be more conserved than loops, due to their importance for stabilizing secondary structures.
furthermore, the core region was expected invariant.
rna folding patterns of the hh regions of pdo <dig> sequences from different populations and species may indicate which dolichopoda species are expected to utilize active hh ribozymes.
the results indicated that the putative hh motif in the pdo <dig> satdna is likely to be under selective constraints and may be functional in dolichopoda.
RESULTS
we analyzed  <dig> pdo <dig> satdna sequences from several populations of  <dig> dolichopoda species, each represented with 3- <dig> sequences.
the molecular characteristics of these sequences were described in detail elsewhere  <cit> .
the hh region sensu rojas et al.
<cit>  was present in all sequences except in sequence vat <dig> from d. geniculata which contains an extended deletion of  <dig> bp.
the pdo <dig> sequences in the data set ranged from  <dig> bp to  <dig> bp out of which  <dig> bp relate to the regular shh region  and  <dig> bp to the dhh region.
the  <dig> bp region is a  <dig> bp extension of the  <dig> bp region .
we used three different measures in order to assess the level of conservation in the hh and the nhh sections of the pdo <dig> satdna sequences.
two of these measures - the shannon-wiener index and a measure analogous to the homozygosity index - were subjected to a resampling procedure  and all comparisons yielded significant differences.
both the  <dig> bp shh sequence and the  <dig> bp dhh sequence were significantly more conserved than the nhh section .
for the histograms of the most frequent nucleotide at each position there is a slight trend of values being closer to  <dig> for the hh region than for the nhh region .
nevertheless, the mann-whitney u tests showed that this difference is statistically significant only for the  <dig> bp dhh region .
stems alone were also significantly different from the nhh region, both for the  <dig> bp and the  <dig> bp regions.
in contrast, loops were not significantly different.
according to the most frequent nucleotides found per position, the  <dig> bp shh region was not significantly different from the nhh region.
however, this is likely to result from the lower sample size of only  <dig> base pairs for the shh region compared with the dhh region with  <dig> bp.
there is a relatively high number of mutations in the stem i region, although the two positions closest to the core  <cit> , i.e.
c <dig>  and g <dig> , are almost invariant.
the positions g <dig>  and c <dig>  are altered in a number of sequences, but frequently these changes are compensatory mutations that sustain the watson-crick base pairing.
compensatory mutations were also found in stem ii where they affect the conserved positions g <dig>  and c <dig> .
stem iii contains a non-canonical base pair which in most sequences is g-u, which is the most common wobble found in rna secondary structures  <cit> .
the non-canonical base pair u-u was also found in this stem and was species specific to d. schiavazzii.
altogether, there are  <dig> compensatory mutations distributed in  <dig> sequences that can potentially restore the base pairing stems .
six of them fold into an active hh structure .
the remaining seven sequences cannot fold into an active hh structure due to other mutations.
the secondary structure predictions for the  <dig> pdo <dig> sequences with a hh ribozyme region are summarized in table  <dig>  the majority of sequences, i.e.
<dig> out of  <dig>  folded into active hh ribozyme structures according to the criteria specified above.
for  <dig> sequences, the hh ribozyme structure was the first choice, i.e.
the structure with the lowest free energy.
for the remaining  <dig> sequences the hh structure was not the structure with the lowest free energy, although the respective values were within the range of those described earlier for the active hh structures of the pdo <dig> sequences pst <dig>  and for6- <dig>   <cit> .
the hh structures that were energetically preferred all showed the pst <dig> structures, not the hh structures previously described for for6- <dig>
folding analyses were also performed for population specific consensus sequences as determined previously  <cit> .
again, the vast majority of sequences, i.e.
<dig> out of  <dig>  folded into active hh ribozyme structures.
the consensus sequences of d. schiavazzii and the pra and tus populations of d. geniculata formed the for6- <dig> structure, whereas all other consensus sequences folded into the pst <dig> structure.
discussion
the hh ribozyme is the best studied ribozyme structure to date  <cit> .
however, hh ribozymes have still not been found in a large number of species.
of the non-viroid organisms, the natural hh ribozyme has primarily been studied in schistosoma mansoni.
in the current study, we investigated the conservation of the hh ribozyme sequence, embedded in the pdo <dig> satdna repeats, in  <dig> species of dolichopoda cave crickets.
this is currently the most comprehensive dataset of hh ribozyme sequences in terms of the number of species involved.
here we applied a phylogenetic footprinting approach  <cit>  in order to assess the phylogenetic conservation of the hh ribozyme region.
using three different measures of sequence variation, we found a significantly higher level of sequence conservation for the hh region than for the nhh region of the pdo <dig> repeats.
this clearly suggests an evolutionary constraint on the hh structure in the pdo <dig> satellite family, which has been hypothetized earlier on the basis of self cleavage of pdo <dig> transcripts from d. baccettii  <cit> .
in particular pairing stem regions as well as the core of the hh ribozyme are highly conserved in the pdo <dig> satdna family.
satdna is known for its high evolutionary turnover; it may thus be surprising to find highly conserved hh ribozyme sequences within the pdo <dig> satdna family.
however, the pdo <dig> satdna was characterized earlier as relatively homogeneous.
bachmann et al.
<cit>  noted that the pdo <dig> satdna was relatively conserved and low in copy number in d. schiavazzi, at least compared to the two species-specific satdna families pdop <dig>  <cit>  and pdspv <dig>  <cit> .
two trends for the mode of evolution of the three satdna families were detected: 1) a positive correlation of sequence variability and copy number, and 2) a negative correlation between sequence variability and length of repeat.
while the first trend was considered in line with the theory on molecular evolution of satdna, the second trend was not , has been associated with a ribonucleoprotein  in the ovaries  <cit> ; an rna binding protein  has been characterized as a promising candidate as a constituent of this complex  <cit> .
the function of this complex is unclear; however, it has been associated with trans cleavage of an rna target  <cit> .
the sat <dig> with its hh ribozyme has been found in nine different species representing four families of amphibians  <cit> .
there are striking similarities between the hh ribozyme in amphibians and dolichopoda as they are both found in satellite dna transcripts and are conserved between large numbers of species.
in addition, the dolichopoda hh also has the potential to form an extended hh structure  as previously described for the amphibian hh ribozyme  <cit> .
whether the hh ribozyme in the pdo <dig> satdna from dolichopoda is involved in a similar rnp complex as in amphibians needs to be investigated.
neither the well-characterized hh ribozyme from s. mansoni, which is also derived from satdna transcripts, nor the hh ribozyme from a. thaliana, have been linked to a function.
on the other hand, the split hh ribozyme found in mrna from rodents has been implicated in regulation of gene expression  <cit> .
CONCLUSIONS
further functional analyses are certainly necessary to address the biological significance of the pdo <dig> hh ribozyme in dolichopoda cave crickets.
future experiments may focus on the transcription pattern of pdo <dig> sequences in the different dolichopoda species and on the self-cleavage of pdo <dig> repeats that carry nucleotide substitutions in the  <dig> bp and  <dig> bp regions described above.
however, the data presented herein strongly suggest that the hh ribozyme in dolichopoda cave crickets is an example of sequence conservation in non-coding dna due to functional importance to the organism.
nevertheless, this is certainly a special case similar to that described earlier in amphibians  <cit> .
coding for functional ribozymes, at present, cannot be considered a general function of satdna.
there are two major known functions related to satdna: 1) the proper functioning of the centromere  <cit> , and 2) processes related to non-coding rnas.
the hh ribozyme in dolichopoda is a special case of category two.
the encoding of the hh ribozyme may explain the slow evolutionary rate for the pdo <dig> satdna family.

 noncoding tandem-repetitive satellite dna has long been known to constitute a large portion of any eukaryotic genome. recent studies in drosophila through underreplication documented a strong positive correlation between genome size and the amount of satdna. satdna is usually located in the heterochromatic parts of the chromosomes close to the centromeres and telomeres. the high evolutionary turn-over of satdna and the frequent species-specific patterns have puzzled researchers for many years. such data challenge the hypothesis that there is a general biological function of satdna. the enormous diversity of satdna in nucleotide sequence, length of repeats, complexity, and genomic abundance, instead suggest that a specific function cannot be ascribed to satdna. however, transcription of satdna and other non-coding dna has been found in many species. the function of the vast majority of these transcripts is unclear, but recent studies using rna interference have documented the role of non-coding rnas in regulation of gene expression, chromatin organization, and genome functioning. for example, it has been demonstrated that small non-coding rnas can play a crucial role in regulating heterochromatin formation and it has been found that transcripts of tandem repeated non-coding dna can give rise to dsrnas. the transcription of satdna was in some instances also linked to hammerhead ribozyme activity which may affect certain regulatory mechanisms in the cell. satdna-derived hh ribozyme structures have been detected in several organisms as different as schistosomes, cave crickets, and salamanders. the hh ribozyme is one of the smallest catalytic rnas and was first identified in viroid and viroid-like satellite rna sequences where they catalyze a specific phosphodiester bond isomerization reaction in the course of rolling-circle replication. all hh ribozymes detected in animal satdna so far have been shown to self-cleave in cis long multimeric transcripts into monomers. the hh ribozyme from schistosoma mansoni is certainly the best characterized of natural hh ribozymes. this particular ribozyme has also been shown to perform efficient ligation and trans-cleavage of an rna target. recently, an active hh ribozyme was also found in a mammalian messenger rna that self-cleaved both in vitro and in vivo and reduced protein expression of c-type lectin type ii genes in mouse cells. this study showed that ribozymes might also act in post-transcriptional regulation of protein coding gene expression in a way that is similar to destabilizing protein factors. hh ribozymes are characterized by a conserved central core which is mandatory for cleavage activity. the consensus sequence for a hh ribozyme consists of three paired stems that branch from the core as defined by conserved nucleotides. in addition, the natural hh ribozymes require additional sequence elements outside of the conserved catalytic core to facilitate intracellular activity. these elements are variable and are therefore not phylogenetically conserved. the correlation between structure and function of the hh ribozyme became clear only recently. for many years studies of hh ribozymes were restricted to a minimal construct. however, there have been major discrepancies between the crystal structure of this particular construct and the results of biochemical experiments. the biochemical data could only be explained if a large-scale rearrangement of the core region was assumed during catalysis. recently, the hh ribozymes found in schistosomes were characterized in more detail and loop-loop interactions were shown to have a large impact on hh ribozyme activity. previously, database searches identified a potential hh ribozyme in dolichopoda cave crickets embedded within the pdo500satdna family and self-cleavage of satdna transcripts has been shown in vitro for rna from d. baccettii. the roughly bp repeats of the pdo have been found in all dolichopoda species studied so far and constitute approximately 5% of the d. schiavazzii genome. phylogenetic analyses of the pdo sequences from dolichopoda species showed that this satdna family evolved gradually at a rate of % per one million year. thus, pdo500-based phylogenetic hypotheses were mainly congruent with the mitochondrial dna phylogeny. the hh ribozyme in dolichopoda performed self-cleavage in vitro and this was associated with processing of long multimer transcripts into monomers in vivo. for the hh ribozyme of dolichopoda two cleavage mechanisms were suggested: 1) cis cleavage by a single-hh ribozyme which is the folding of the bp hh sequence, and 2) trans cleavage by a double-hammerhead which is a hybrid structure between two extended hh sequences of bp. we took a phylogenetic footprint approach to analyze in detail the pdo satdna family from different populations representing species of dolichopoda cave crickets with particular emphasis on the hh region. we assessed whether the hh motif in the pdo satdna is more conserved than other parts of the pdo repeat unit. hh stems should be more conserved than loops, due to their importance for stabilizing secondary structures. furthermore, the core region was expected invariant. rna folding patterns of the hh regions of pdo sequences from different populations and species may indicate which dolichopoda species are expected to utilize active hh ribozymes. the results indicated that the putative hh motif in the pdo satdna is likely to be under selective constraints and may be functional in dolichopoda.  we analyzed pdo satdna sequences from several populations of dolichopoda species, each represented with 3- sequences. the molecular characteristics of these sequences were described in detail elsewhere. the hh region sensu rojas et al. was present in all sequences except in sequence vat from d. geniculata which contains an extended deletion of bp. the pdo sequences in the data set ranged from bp to bp out of which bp relate to the regular shh region and bp to the dhh region. the bp region is a bp extension of the bp region. we used three different measures in order to assess the level of conservation in the hh and the nhh sections of the pdo satdna sequences. two of these measures - the shannon-wiener index and a measure analogous to the homozygosity index - were subjected to a resampling procedure and all comparisons yielded significant differences. both the bp shh sequence and the bp dhh sequence were significantly more conserved than the nhh section. for the histograms of the most frequent nucleotide at each position there is a slight trend of values being closer to for the hh region than for the nhh region. nevertheless, the mann-whitney u tests showed that this difference is statistically significant only for the bp dhh region. stems alone were also significantly different from the nhh region, both for the bp and the bp regions. in contrast, loops were not significantly different. according to the most frequent nucleotides found per position, the bp shh region was not significantly different from the nhh region. however, this is likely to result from the lower sample size of only base pairs for the shh region compared with the dhh region with bp. there is a relatively high number of mutations in the stem i region, although the two positions closest to the core, i.e. c and g, are almost invariant. the positions g and c are altered in a number of sequences, but frequently these changes are compensatory mutations that sustain the watson-crick base pairing. compensatory mutations were also found in stem ii where they affect the conserved positions g and c. stem iii contains a non-canonical base pair which in most sequences is g-u, which is the most common wobble found in rna secondary structures. the non-canonical base pair u-u was also found in this stem and was species specific to d. schiavazzii. altogether, there are compensatory mutations distributed in sequences that can potentially restore the base pairing stems. six of them fold into an active hh structure. the remaining seven sequences cannot fold into an active hh structure due to other mutations. the secondary structure predictions for the pdo sequences with a hh ribozyme region are summarized in table the majority of sequences, i.e. out of folded into active hh ribozyme structures according to the criteria specified above. for sequences, the hh ribozyme structure was the first choice, i.e. the structure with the lowest free energy. for the remaining sequences the hh structure was not the structure with the lowest free energy, although the respective values were within the range of those described earlier for the active hh structures of the pdo sequences pst and for6-. the hh structures that were energetically preferred all showed the pst structures, not the hh structures previously described for for6- folding analyses were also performed for population specific consensus sequences as determined previously. again, the vast majority of sequences, i.e. out of folded into active hh ribozyme structures. the consensus sequences of d. schiavazzii and the pra and tus populations of d. geniculata formed the for6- structure, whereas all other consensus sequences folded into the pst structure. discussion the hh ribozyme is the best studied ribozyme structure to date. however, hh ribozymes have still not been found in a large number of species. of the non-viroid organisms, the natural hh ribozyme has primarily been studied in schistosoma mansoni. in the current study, we investigated the conservation of the hh ribozyme sequence, embedded in the pdo satdna repeats, in species of dolichopoda cave crickets. this is currently the most comprehensive dataset of hh ribozyme sequences in terms of the number of species involved. here we applied a phylogenetic footprinting approach in order to assess the phylogenetic conservation of the hh ribozyme region. using three different measures of sequence variation, we found a significantly higher level of sequence conservation for the hh region than for the nhh region of the pdo repeats. this clearly suggests an evolutionary constraint on the hh structure in the pdo satellite family, which has been hypothetized earlier on the basis of self cleavage of pdo transcripts from d. baccettii. in particular pairing stem regions as well as the core of the hh ribozyme are highly conserved in the pdo satdna family. satdna is known for its high evolutionary turnover; it may thus be surprising to find highly conserved hh ribozyme sequences within the pdo satdna family. however, the pdo satdna was characterized earlier as relatively homogeneous. bachmann et al. noted that the pdo satdna was relatively conserved and low in copy number in d. schiavazzi, at least compared to the two species-specific satdna families pdop and pdspv. two trends for the mode of evolution of the three satdna families were detected: 1) a positive correlation of sequence variability and copy number, and 2) a negative correlation between sequence variability and length of repeat. while the first trend was considered in line with the theory on molecular evolution of satdna, the second trend was not, has been associated with a ribonucleoprotein in the ovaries; an rna binding protein has been characterized as a promising candidate as a constituent of this complex. the function of this complex is unclear; however, it has been associated with trans cleavage of an rna target. the sat with its hh ribozyme has been found in nine different species representing four families of amphibians. there are striking similarities between the hh ribozyme in amphibians and dolichopoda as they are both found in satellite dna transcripts and are conserved between large numbers of species. in addition, the dolichopoda hh also has the potential to form an extended hh structure as previously described for the amphibian hh ribozyme. whether the hh ribozyme in the pdo satdna from dolichopoda is involved in a similar rnp complex as in amphibians needs to be investigated. neither the well-characterized hh ribozyme from s. mansoni, which is also derived from satdna transcripts, nor the hh ribozyme from a. thaliana, have been linked to a function. on the other hand, the split hh ribozyme found in mrna from rodents has been implicated in regulation of gene expression.  further functional analyses are certainly necessary to address the biological significance of the pdo hh ribozyme in dolichopoda cave crickets. future experiments may focus on the transcription pattern of pdo sequences in the different dolichopoda species and on the self-cleavage of pdo repeats that carry nucleotide substitutions in the bp and bp regions described above. however, the data presented herein strongly suggest that the hh ribozyme in dolichopoda cave crickets is an example of sequence conservation in non-coding dna due to functional importance to the organism. nevertheless, this is certainly a special case similar to that described earlier in amphibians. coding for functional ribozymes, at present, cannot be considered a general function of satdna. there are two major known functions related to satdna: 1) the proper functioning of the centromere, and 2) processes related to non-coding rnas. the hh ribozyme in dolichopoda is a special case of category two. the encoding of the hh ribozyme may explain the slow evolutionary rate for the pdo satdna family. 
recent studies have shown that satellite dna can be transcribed and be involved in regulation of chromatin structure and gene expression. the core region of the hh ribozyme was affected by very few nucleotide substitutions and the cleavage position was altered only once among sequences. rna folding of the hh sequences revealed that a potentially active hh ribozyme can be found in most of the dolichopoda populations and species. in stems, several compensatory mutations were detected that maintain base pairing. we assessed the phylogenetic footprints of the hh ribozyme within the pdo sequences from different populations representing species of dolichopoda. however, the functional role of hh ribozymes in eukaryotic organisms is unclear. whether the hh ribozyme in dolichopoda is involved in similar functions remains to be investigated. some satellite dnas, such as the pdo sequence family in dolichopoda cave crickets, have a catalytic hammerhead ribozyme structure and activity embedded within each repeat. the phylogenetic footprints suggest that the hh region of the pdo sequence family is selected for function in dolichopoda cave crickets. future studies need to demonstrate how the observed nucleotide changes and evolutionary constraint have affected the catalytic efficiency of the hammerhead. the hh region was significantly more conserved than the non-hammerhead region of the pdo repeat. the possible functions have been related to trans cleavage of an rna target by a ribonucleoprotein and regulation of gene expression.
 the great variety in sequence, length, complexity, and abundance of satellite dna has made it difficult to ascribe any function to this genome component. recent studies have shown that satellite dna can be transcribed and be involved in regulation of chromatin structure and gene expression. some satellite dnas, such as the pdo sequence family in dolichopoda cave crickets, have a catalytic hammerhead ribozyme structure and activity embedded within each repeat.  we assessed the phylogenetic footprints of the hh ribozyme within the pdo sequences from different populations representing species of dolichopoda. the hh region was significantly more conserved than the non-hammerhead region of the pdo repeat. in addition, stems were more conserved than loops. in stems, several compensatory mutations were detected that maintain base pairing. the core region of the hh ribozyme was affected by very few nucleotide substitutions and the cleavage position was altered only once among sequences. rna folding of the hh sequences revealed that a potentially active hh ribozyme can be found in most of the dolichopoda populations and species.  the phylogenetic footprints suggest that the hh region of the pdo sequence family is selected for function in dolichopoda cave crickets. however, the functional role of hh ribozymes in eukaryotic organisms is unclear. the possible functions have been related to trans cleavage of an rna target by a ribonucleoprotein and regulation of gene expression. whether the hh ribozyme in dolichopoda is involved in similar functions remains to be investigated. future studies need to demonstrate how the observed nucleotide changes and evolutionary constraint have affected the catalytic efficiency of the hammerhead. 