BMC Genomics 2009, 10:101 http://www.biomedcentral.com/1471-2164/10/101 comprehensive search is necessary to assess their abun- dance. Experimental RNA analyses such as Northern blots needs to be done to determine if these sequences are tran- scribed, but in view of the strong evidence for evolution- ary conservation of secondary structure, they may function at the RNA level. In E. coli, many intergenic sequences are transcribed, which results in the presence of a large number of heterogeneous small RNAs [8]. These elements also have not been analyzed for function. Conclusion Small repeat sequences of Borrelia sp. linear plasmids show numerous changes in nucleotide sequence, never- theless, RNA-type motifs generated by these variable sequences are highly conserved evolutionarily. Two of the motifs may be candidates for non-coding RNAs. Two oth- ers appear linked to C-terminal ends of super families of protein genes/pseudogenes, but these genes display major changes in amino acid sequence and peptide chain length. Jacob Monod described evolutionary change in terms of "tinkering", a trial and error process in formation of new or modified genes with random mutations and/or ran- dom fusion of motifs [55]. Perhaps the variable super family virulence protein gene sequences show elements of "tinkering", however the interesting question is why the RNA motifs, which have also sustained mutations are well conserved when at least some of the associated protein genes are in a process of change or decay. We have men- tioned the possibility of these being reservoirs for forma- tion of variant or new proteins. Methods To search for conserved intergenic sequences, NCBI/ BLAST BLAST Assembled Genomes http:// blast.ncbi.nlm.nih.gov/Blast.cgi[56] and BLAST with microbial genomes http://www.ncbi.nlm.nih.gov/sutils/ genom_table.cgi[57] were used. Blast with microbial genomes used a value of 10 for expect and the default fil- ter. Nucleotide blast searches were optimized for both highly similar sequences megablast and discontiguous megablast. Default parameters were used. For similar sequence megablast the parameters were: maximum tar- get sequences, 100; automatically adjusted for short sequences; expect, 10; word size, 28. Discontinuous match/mismatch scores, 1,-2; gap costs, linear; filter, low complexity regions. Discontinuous megablast: same parameters as those of similar sequence megablast with the exception word size, 11; match/mismatch scores, 2, - 3; gap costs, existence: 5 extension: 2. The Swiss Institute of Bioinformatics SIB ExPASy Blast server [46] was used to find protein homologies. The blast program and data base used was: blastp – query against the UniProt Knowledgebase (Swiss-Prot + TrEMBL) and default parametes as shown under "Options" were used. The database was the complete database. Initial searches for repeat sequences and RNA motifs were performed by "walking" intergenic sequences from plas- mid lp28 of B. afzelii Pko. In addition, several regions that contain relatively large intergenic sequenes from B. burgdor- feri B31 and Borrelia garinii PB plasmids were also scanned. Intergenic regions were scanned at 200 bp at a time for con- served or partially sequences. These sequences were then modeled for conserved RNA stem loops. Cut offs in regions 5' and 3' of a determined stem loop(s) were made when the additional sequences failed to provide conserved stem- loops. Reverse transcript sequences as well as overlapping sequences at the 200 bp junctions were also structure mod- eled. Repeat sequences were found that displayed stem- loop structures, but these structures either were not found conserved in homologous sequences in other Borrelia spe- cies, or the nt sequence identity was too high and thus the structures did not show base-pair changes. These were dis- carded. The criteria for potential RNA identification were as follows: 1) presence of the sequence in three or more differ- ent plasmid regions and/or two or more Borrelia species, 2) presence of a conserved stem loop with at least 9 contigu- ous base-pairs, 3) two or more compensatory base changes that maintain a stem, 4) in some cases, the presence of con- served looped out or bulged positions. RNA secondary structure modeling of repeat nt sequences was performed with the Zuker and Turner mfold, version 3.2 [28,29]. Parameters used were: default window parameter, maximum interior/bulge loop size = 30, Max- imum asymmetry of an interior/bulge loop = 30, and no limit on maximum distance between paired bases. The RNAz Webserver: http://rna.tbi.univie.ac.at/cgi-bin/ RNAz.cgi[58] of Gruber et al [30] was used to detect thermo- dynamically stable and evolutionarily conserved RNA second- ary structures from multiple sequence alignments. The sequence alignment was ClustalW format. Default parameters were used, except for the Reading Direction set at forward. The ClustalW2 program http://www.ebi.ac.uk/[59] pro- vided by the EMBL-European Bioinformatics Institute [31] was used for amino acid and nucleotide sequence align- ments. Parameters were as set on the EMBL-EBI web page: http://www.ebi.ac.uk/Tools/clustalw2/index.html[32]. Random sequence analyses as described for Sequence #2 were performed using the Stothard mutagenesis program on webpage: http://molbiol.ru/eng/scripts/01_16.html[60]. Abbreviations nt: nucleotide; aa: amino acid; Ba lpX: B. afzelii PKo plas- mid lpX; Bb lpX: B. burgdorferi str. B31 plasmid lpX; Bg lpX: B. garinii PB plasmid lpX; CRASP-1: complement reg- ulator-acquiring surface protein_1; MITEs: miniature inverted repeat transposable elements. Page 10 of 12 (page number not for citation purposes)