one of the current challenges facing biologists is the discovery of novel functional elements in noncoding genomic sequence . with the rapidly increasing number of genomes being sequenced , a comparative genomics approach called 
the idea underlying phylogenetic footprinting is that selective pressure causes functional elements to evolve at a slower rate than the nonfunctional surrounding sequence .
therefore the most conserved motifs in a collection of homologous regions are excellent candidates as functional elements .
this note focuses on phylogenetic footprinting for the discovery of novel cis - regulatory elements in prokaryotic genomes .
a web tool for this purpose has been implemented in a program called microfootprinter , available at .
one reason to focus on prokaryotes is that over 300 prokaryotic genomes are completely sequenced at the time of this writing , making this by far the richest current medium for phylogenetic footprinting .
microfootprinter is actually a front end for the footprinter phylogenetic footprinting program ( 1 ) , but specifically tailored to prokaryotic genomes .
microfootprinter automatically takes care of the laborious tasks of ( i ) finding homologous genes in related prokaryotes , ( ii ) inferring their phylogenetic gene tree , ( iii ) extracting the noncoding cis - regulatory regions of each of these homologous genes , ( iv ) setting the most difficult of footprinter 's parameters and ( v ) running footprinter on these regulatory regions .
the result is the identification of motifs that are well conserved across the cis - regulatory regions of these homologous genes .
[ the reader is referred to earlier work ( 1,2 ) for details on footprinter and examples of its applications to biological data ] .
all search fields are considered , and any partial or complete match found is included in the results .
for instance , if the user enters  coli for the species search ; microfootprinter offers the list of all escherichia coli strains available . after choosing a species , if the user enters 
pyrim for the gene search , microfootprinter offers a list of all genes with this text in their gene product descriptions , notably genes involved in processing of pyrimidines . after choosing a species and gene ,
the user is asked to supply a few simple parameters ( or leave them at their default values ) .
these are the length of the desired motif ( in base pairs ) , the target number of motifs for microfootprinter to display , the target number of species in which to locate homologous genes , and the maximum parsimony score ( number of mutations ) to allow among the instances of each displayed motif .
if desired , the search for other species can also be restricted to any taxonomic clade containing the user 's chosen species , for instance , restricted to just proteobacteria . for each of these user inputs
after the user has set the parameters , it typically takes 1 to 2 min of elapsed time for microfootprinter to perform all its computations and display footprinter 's output . for a description and interpretation of footprinter 's output ,
microfootprinter uses protein - level blast to find the closest homologs to the user 's chosen gene .
specifically , it uses ncbi 's blink facility , which provides the results of blast searches that have been done for every protein sequence in the entrez proteins data domain .
if there are close homologs in multiple sequenced strains of the same species , microfootprinter will select only the single strain whose homolog 's protein sequence is most similar to the query sequence .
microfootprinter infers this phylogeny by using clustalw ( 3 ) to align the homologous protein sequences .
the guide tree returned by clustalw is used as a reasonable approximation of the true gene tree . for each of these homologous genes , microfootprinter
each of these regions consists of up to 500 bp of noncoding sequence upstream of the start codon .
( it may be shorter , if there is another coding region fewer than 500 bp upstream . )
note that these regulatory regions typically contain both 5 untranslated region ( 5 utr ) and promoter sequences .
the fact that 5 utr is included makes microfootprinter useful for discovery of cis - regulatory mrna elements such as riboswitches .
operons are contiguous collections of genes on the same dna strand that are transcribed together .
the complication in this case is that the desired regulatory region may be upstream of the entire operon rather than immediately upstream of the selected gene . for most prokaryotes ,
it is not known which genes comprise operons . to handle this complication in a conservative manner , microfootprinter extracts and concatenates the noncoding sequences upstream of the gene and upstream of its plausible operon .
specifically , if the next coding region upstream is in the same orientation and fewer than 100 bp upstream , this short intergenic sequence is concatenated with the result of applying this same procedure to the upstream gene .
this process continues until interrupted either by a coding region in the opposite orientation or an intergenic region longer than 100 bp .
up to 500 bp of this final intergenic region are also concatentated to the result .
these concatenated noncoding sequences are actually separated from each other by the sequence nnnnnnnnnn so that , when inspecting the ultimate footprinter output , the user can identify when such concatenation has taken place .
in addition to providing the user with footprinter 's output , microfootprinter also provides the protein sequences , cis - regulatory sequences and gene tree . with these ,
the user can rerun footprinter directly , adjusting footprinter 's parameters if desired , or use another motif discovery tool .
most of these are not intended for phylogenetic footprinting , as they implicitly assume that the input sequences are independent rather than homologous .
we believe that , for sequences as diverged as the prokaryotes that are currently sequenced , this approach is less effective than the use of footprinter , which searches for conserved motifs directly in unaligned sequences .
microfootprinter provides the microbiologist with a convenient front end for footprinter , whereby specification of only the species and gene of interest is sufficient for the extraction of all the data necessary for phylogenetic footprinting on that gene .
ultimately , we would like to extend this service to the eukaryotes , but this is still premature .
for the few eukaryotes that are currently completely sequenced , a static catalog of all regulatory elements discovered by phylogenetic footprinting ( 58 ) is probably more appropriate at this time .
another extension that could be very helpful is the ability to analyze multiple genes from a single species for common regulatory elements , using the homologs of each gene as well .
this is a more difficult problem than simple phylogenetic footprinting , one for which footprinter was not intended . for discussion of what makes this problem more difficult and some approaches to its solution ,
 phylogenetic footprinting is a method for the discovery of regulatory elements in a set of homologous regulatory regions , usually collected from multiple species . 
 it does so by identifying the most conserved motifs in those homologous regions . 
 this note describes web software that has been designed specifically for this purpose in prokaryotic genomes , making use of the phylogenetic relationships among the homologous sequences in order to make more accurate predictions . 
 the software is called microfootprinter and is available at . 
INTRODUCTION
USER INPUTS
METHODS USED BY MicroFootPrinter
DISCUSSION