Computational Survey of nis (Nostoc Iterated Sequence) in Cyanobacterial Genomes Introduction




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BBSI Research Proposal – Academic Year 2005/2006

Michiko Kato


Computational Survey of NIS (Nostoc Iterated Sequence) in

Cyanobacterial Genomes

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 regions1. 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 2, 3. Another dispersed sequence, ERIC (Enterobacterial Repetitive Intergenic Consensus), is preferentially located in non-coding transcribed regions and consists of conserved inverted repeats3. 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 but no role is fully understood.

NIS (Nostoc Iterated Sequence) is a novel type of dispersed repeats found in Nostoc genome. During the examination of heptameric tandem repeats in tRNAleu introns by Costa el al., it was discovered that a few of the strains contain a short, non-repetitive sequence within the repeat regions4. Currently, NIS is known to be 24-, 42-, 45-, or 48-nt sequence and some have tetranucleotide inverted repeats within the sequence. Six different sequence patterns have been categorized, and the distribution of each type in the genome varies.

So what is the significance of the presence of NIS? The existence of these sequences throughout Nostoc genome does not seem to be coincidental. The questions we hope to be able to answer are their origins, how they spread themselves in the genome, how common they are in prokaryotic genomes, and what their functions are. To start this long quest, the first thing to do is to characterize all of the NIS sequences and compare them in other related cyanobacterial genomes. Nostoc punctiforme ATCC29133 has phylogenetic relationship with Anabaena PCC 7120 and its close relative Anabaena variabilis ATCC29413. Comparative examination of these genomes would provide clues to the evolutionary origin of NIS. My research entails the computational survey of the NIS sequences in these three cyanobacterial genomes. The trend in the occurrence and distribution of all NIS groups will be examined.
Progress Report (Summer 2005)

The extensive search for all NIS sequences in Nostoc genome as well as Anabaena PCC 7120 and Anabaena variabilis was conducted and nearly completed. NIS is distributed throughout the genome, and occasionally found in plasmids.

One of the goals for this summer was to establish whether NIS insertion was a recent event in evolution. If NIS is present in Nostoc but absent in the same location in Anabaena, it would indicate that insertion took place after Nostoc had diverged from Anabaena. In order to compare NIS occurrence in different species, orthologous genes were examined in the instances where NIS was found inside of genes in Nostoc (figure 1). The result supports the hypothesis that NIS has recent evolutionary origin.


Figure 1: An example of orthologs comparison. The difference in the length of genes between N.punctiforme and Anabaena PCC 7120 corresponds to the length of NIS 1.

These small elements were introduced to Nostoc genome sometime during evolution, creating genetic variation between species. Then the next question is how these elements were introduced into the genome and replicate. NIS itself cannot be a transposon, for its size is too small to encode a gene. The presence of inverted repeats in many of NIS sequences suggests their propagation occur via RNA intermediate. Transposition via RNA intermediate is known in retrotransposons. However, NIS does not have the characteristics commonly described as retrotransposons. Recombination is another possibility of NIS propagation.

If the propagation of NIS is governed by a particular manner, we should see a pattern in the sequences surrounding NIS. For example, NIS-1 is known to be flanked by 7-nt tandem repeats on either side (Figure 2).

Figure 2: A simplified illustration of NIS 1 flanked by 7-nt repeats

Is the presence of these tandem repeats significant in the movement of NIS? I examined the flanking sequences in order to look for a pattern that might provide clues to the mystery of NIS mobility. Observation revealed that 7-nt flanking sequences are not common in NIS families other than NIS 1. However, there is a trend within some of NIS families. For example, 19 of NIS-4 share the same upstream flanking sequence, which later discovered to be NIS-6. Further examination revealed that NIS 6 is almost always associated with NIS-4. Furthermore, I discovered numerous instances where different NIS elements are clustered. Four of such cases involve NIS-1, -6, and -4 with short sequences in between (figure 3). Same 7-nt sequence was found at either side of this NIS cluster. The unknown short sequences were later found to beNIS-1 sequence that somehow split up in pieces.




Figure 3: An instance where NIS sequences were found in a cluster, sharing a few nucleotides with each other.

The occurrence of NIS does not seem to be random or independent. One of the possibilities is that the presence of one NIS sequence affects the mobility of other NIS sequences. In other words, NIS-1 that is flanked by 7-nt tandem repeats may have been interrupted by other NIS elements later, creating the sequence pattern above.


Research Plan: 2005-2006 academic year

I have found the pattern of NIS clusters described above by examining the sequences by eye. The evidence is enough to establish that the relationships between these NIS elements are not coincidental. I would like to develop a method to search these sequence patterns more efficiently. First, I need to overcome the challenge of finding NIS sequences in pieces. The relationships between NIS and 7-nt tandem repeats as well as among different NIS families will be clear after thorough analysis of the genome.

I also plan to investigate the possible role of NIS. During the summer, I made an observation that NIS commonly occur downstream of genes. This indicates possible function of NIS in transcriptional regulation.

The computational analysis will be performed primarily on BioLingua, a web-based programming environment.


Reference

  1. Rocha E.P.C, Danchin A, and Viari A (1999). Functional and evolutionary roles of long repeats in prokaryotes. Res. Microbiology. 150: 725-733

  2. Stern MJ, Ferro-Luzzi Ames G et al. (1984). Repetitive Extragenic Palindromic Sequences: A major component of the bacterial genomes. Cell. 37: 1015-1026

  3. Lupski JR and Weinstock GM (1992). Short, interspersed repetitive DNA sequences in prokaryotic genomes. Journal of bacteriology. 174: 4525-4529.

  4. Costa JL, Paulsrud P, and Lindblad P (2002). The cyanobacterial tRNAleu (UAA) intron: Evolutionary paterns in a genetic marker. Mol. Biol. Evol. 19: 850-857.


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