The Origin and Possible Functional Role of Short Dispersed Repeats

Repetitive sequences are common not only in eukaryotic genomes but also in prokaryotic genomes. Bacterial genomes contain many types of repeats including tandem repeats and short dispersed repeats. This research aims to expand our understanding of short dispersed repeats (SDR), a novel type of repetitive sequences in cyanobacterial genomes. SDR are found in the genomes of Nostoc punctiforme as well as Anabaena PCC 7120 and Anabaena variabilis. The comparison of orthologs in which SDR elements are present revealed that SDR insertion was a recent evolutionary event. Evidences that suggest the mobility of these elements were seen, and a few different means of SDR propagation were proposed. The function of SDR is still unknown, but there is evidence that suggests the potential function of SDR in transcriptional processing, which was experimentally tested. Introduction Both eukaryotic and prokaryotic genomes are known to have repetitive DNA sequences. However, it was thought that repeats were rare in prokaryotic genomes due to their compact genomes and minimal non-coding regions. The advancement in computational genome analysis has permitted a more thorough examination of DNA sequences. As a result, it is now clear that repeat sequences are widespread in prokaryotic genomes. Short interspersed repeats are widely distributed within and among different bacterial species. One such sequence, REP (Repetitive Extragenic Palindrome), was first identified in E.coli. REP sequence is characterized as a 38-nt palindromic sequence capable of forming stem-loop or cruciform . Another dispersed sequence, ERIC (Enterobacterial Repetitive Intergenic Consensus), is preferentially located in non-coding transcribed regions and consists of conserved inverted repeats. The ubiquitous presence of these patterned sequences suggests a function that selects for their persistence and/or a rapid means of propagation. Several functional roles of these dispersed sequences have been hypothesized and tested, but no role is fully understood. The function of BIMEs (Bacterial Interspersed Mosaic Elements) is by far the most extensively studied. BIMEs are short repetitive sequence with conserved palindromic core called PU (palindromic units). Espéli et al. studied bacterial repeat elements called BIMEs (Bacterial Interspersed Mosaic Elements) for their potential involvement in transcription attenuation and mRNA stabilization. BIMEs are flanked by palindromic units (PUs) and either located on the 3’ end of transcriptional units or in between genes that belong to the same operon. Insertion of BIMEs between two genes on artificial operons resulted in the reduction of the level of full-length mRNA and in the accumulation of smaller mRNA that corresponds to the size of the upstream gene 4 . Short dispersed repeats (SDR) are a novel type of dispersed repeats found in Nostoc punctiforme genome. During the examination of heptameric tandem repeats in tRNA introns by Costa el al., it was discovered that a few of the strains contain a short, non-repetitive sequence within the repeat regions. These inserts vary in size and sequence, and a few of these sequences have been found in other parts of the genome. These repeated elements were examined in Nostoc genome as well as the genome of its closely related cyanobacteria. We studied the distribution of these elements and examined their origin and propagation. In addition, an experimental approach was taken to investigate the possible function of SDR. Materials and Methods Computational Survey of Short Dispersed Repeats The short insertion sequences from tRNA leu intron were collected and used to look for similar sequences. Three of those (Nos30, Nos37/Nos38, and Nos51) were considered for further study and were designated as reference sequences for SDR1, 2, and 3, respectively. The study of SDR1 revealed that there are other short repeated sequences in the genome as well. Collectively, we used eight reference sequences for this study. The genomes of N. punctiforme, Anabaena PCC7120, and Anabaena variabilis were examined to search for similar sequences to each reference sequence. The search was conducted using BioBIKE, a web-based bioinformatics tool that facilitates programming and integrated analysis of biological data 6 . Unlike traditional BLAST (Basic Local Alignment Search Tool), BioBIKE allows searches on the basis of sequence identity, near identity, and pattern. This allows more thorough search of the interested sequences. For this study, we allowed up to three mismatches in the subject sequences. The BioBIKE function SEQUENCE-SIMILAR-TO allows the user to set the number of mismatches to be allowed in the subject. Each returned subject in the initial query was used as the reference sequence for the second turn and on, also allowing up to three mismatches. The returned results were examined by hand to see the patterns and significance. Investigation of the Possible Functional Role of SDR pVCU303 was constructed by introducing KpnI and ClaI recognition sequences in the luxAB intergenic region of pRL559. pRL559 is a shuttle vector which bears glnA promoter (PglnA) upstream of luxAB gene (figure 1).