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M.K Chandra Prakash, Scientist (Computer Applications),

Computer program for identifying prospective DNA repair locations in whole chromosome for candidate gene identification associated with abiotic stress tolerance. M.K Chandra Prakash, Scientist (Computer Applications),

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M.K Chandra Prakash, Scientist (Computer Applications),

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  1. Computer program for identifying prospective DNA repair locations in whole chromosome for candidate gene identification associated with abiotic stress tolerance. M.K Chandra Prakash, Scientist (Computer Applications), ReenaRosy Thomas, Scientistand SukhadaMohandas, Emeritus Scientist, Indian Institute of Horticultural Research, Bangalore 560 089

  2. Computer program for identifying DNA repair locations • The present work is development of Computer program in Microsoft Visual Studio 2010 for windows platform, which can work with large files, like a full chromosome or genome sequences to locate the DNA repaired regions (Mobile genetic element excisions) in whole chromosome. • The program will read the large files (200 MB of input text data)of full chromosome using the file handler called stream reader which will avoid stack overflow/out-of -memory errors. • These errors was happening in earlier computer programs while reading large chunks of data.

  3. Mobile genetic elements in Tomato • Mobile genetic elements dominated large portion of many genomes and their abundance and movement remains a major force for shaping the genes and genomes in most of the organisms. • These are semi-parasitic DNA sequences which can replicate and spread through the host's genome. • These elements can insert within genes or regulatory sequences, which can disrupt gene function or expression, and later excises. However, a small sequence (8 bp foot prints) is left by the deleterious excised element after the DNA repair.

  4. Transposition • The Transposase, is an enzyme that binds to the ends of a Mobile genetic elements (MGE) and catalyzes the movement to another part of the genome by a cut and paste mechanism. • It also recognizes sequences within the genome, making 8bp staggered cuts for mobile genetic elements to insert. • When these deleterious element later excises from the site, the 8bp duplication remains at the site as a direct repeat to mark where the element has been excised.

  5. Mobil Genetic Elements in Eukaryotes • Nobel Prize in Physiology and Medicine 1983 • “for her discovery of Mobil genetic elements” • Studied transposable elements in corn 1940s-1950s • (formerly identified as mutator genes by Marcus Rhoades 1930s) • They provide a means for genomic change and variation, particularly in response to stress (McClintock’s "stress" hypothesis) Barbara McClintock (1902-1992) Cold Spring Harbor Laboratory, NY

  6. Mobile Genetic Elements in drosophila melanogaster

  7. Semi-parasitic DNA insertion • The likely reason why mobile genetic elements often insert into the genes / regulatory regions is their high expression levels. • Once these deleterious elements are excised, afoot prints#are left in excised regions. If these are located, this would enable us to locate the highly expressed regions in whole chromosome. • The tomato (Solanumlycopersicum) genome sequencing has been recently completed and whole genome sequences are available in public domain for researchers to identify useful coding regions # Chen and Wagner BMC Evolutionary Biology 2012, 12:25 http://www.biomedcentral.com/1471-2148/12/25

  8. Chromosome data reading • The developed computer program will read the entire chromosomeat one go irrespective of the chromosome size and also identifies the excisedlocations. • It generates a spread sheet output with footprints and their locations. It also extracts the sequence in FASTA format comprising 4000 bp before and after the excised locations respectively from whole chromosome and generates as text file. • These sequences are analyzed for potential coding regions for abiotic stress tolerance.

  9. Whole Genome Data sets • The whole genome sequences of Tomato are available in public domain. The latest whole genome data sets are downloaded from the solgenomics.net. • Approximately, the size of each chromosome sequences varies from smallest 45 Mega Bytes to largest 90 Mega Bytes of data. • All the chromosome data has been downloaded and converted to readable format.

  10. Whole Genome Data Distribution . . . • Total size of 12 chromosomes 772 MB of data. • Average 60 MB (approx. 25,000 pages) per chromosome. • Comprising of 950 million base pair for 12 chromosomes

  11. Program codes … • Microsoft Visual Basic 2010 program has been used for development of program. • A part program codes were displayed above. The stream reader was used to read large input files up to 200 MB to locate foot prints.

  12. Spread sheet output . . . The Program generates Microsoft Excel file output with footprints and its DNA repair (Excised) locations in whole chromosome.

  13. DNA repaired locations in chromosome 6 . . . 800 DNA repaired locations (footprints) are present in chromosome 6 of Tomato shown as small radial red lines

  14. Extracted regions of DNA repair locations as Text file . . . Once the locus is identified for the DNA repair locations, the program generates a text file by the Stream writer where 4000 bp before and after the DNA repair location will be written to the text file in FASTA format of potential coding regions which could be analysedusing various in silico methods for classifying abiotic stress gene coding regions.

  15. Conclusion : Mobile Genetic Element excision footprints : as a tool for Gene discovery • There was enough evidential support that the DNA repair and excision event occurred naturally. • The footprints has been searched using the Computer programin whole genome sequences of Solanumlycopersicum, approximately 800 -1200 locations these footprints are present in each chromosome. • This foot prints provides a clue for finding excised regions in whole chromosomes, which would be eventually an expressed regions associated with stress tolerant regions or defense responsive regions. • This approach would enable users to identify important regions abiotic stress tolerance regions in whole chromosomes using these excised locations.

  16. References • Barbara McClintock (1948) "Mutable Loci in Maize"Carnegie Institution of Washington Year Book 47, 155-169 • Capy, P., Langin, T., Higuet, D., Maurer, P., Bazin, C. (1997). Do the integrases of LTR-retrotransposons and class II element transposases have a common ancestor? Genetica 100(1-3): 63--72. • Chen and Wagner (2012). Hsp90 is important for fecundity, longevity, and buffering of cryptic deleterious variation in wild fly populations, BMC Evolutionary Biology 2012, 12:25; http://www.biomedcentral.com/1471-2148/12/25 • Grandbastien, M.-A. (1998). Activation of plant retrotransposons under stress conditions. Trends Plant Sci. 3, 181±187. • Grandbastien, M.-A., Lucas, H., Mhiri, C., Morel, J.-B., Vernhettes, S. and Casacuberta, J.M. (1997). The expression of the tobacco Tnt1 retrotransposon is linked to the plant defense responses. Genetica, 100, 241±252.

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