Indrani Datta Maria Gutierrez Jessica Blicharski Mentors : Dr. Shailesh Lal and Dr. Gautam Singh

1. Bioinformatics and Molecular Processes for the Discovery, Identification, and Cataloguing of Matrix Attachment Regions(Mars) & Helitrons Indrani Datta Maria Gutierrez Jessica Blicharski Mentors : Dr. Shailesh Lal and Dr. Gautam Singh

2. Cloning and Characterization of Sh2 gene from Zea mays ssp. parviglumis & nicaraguensis Maria Gutierrez SIBHI Mid-Term Presentation Oakland University

3. Zea mays. ssp parviglumis (Teo1) & nicaraguensis (Teo2): Ancestor of Modern Day Maize • Teosinte: Wild grass domesticated by humans • All lineages of maize can be traced back to it’s primitive form Zea mays ssp. Parviglumis • Maize became distinct from Teosinte grass about 9,200 years ago • Cob size has fluctuated in growth 50x over from original 2 cm • Phenotype has changed, but has it’s genotype (as a result of domestication)

4. Shrunken2 gene • Sh2 gene encodes a key enzyme involved in starch biosynthesis Glucose-1 PO4 ADP- Glucose + ATP → +PP ADP- Glucose Pyrophosphorylase • Null mutation of Sh2 gene, starch is not produced, hence maize kernels appear shrunken and not plump

5. Shrunken2 gene • Has domestication altered the Sh2 gene of maize? • starch content of corn has increased (nutritional value) • Kernels are no longer covered by a stone-like, hard to digest fruitcase (quality) • Biggest corn ear found is almost 1 foot long (size) !!!

6. Gene Structure of Shrunken2 Zea Mays 3’ 5’ • Sh2 gene contains 16 exons and 15 introns • Introns range from 150bp to 1,852bp in length • The translation initiation (AUG) and stop codon (UAG) are located in exons 2 and 15, respectively.

7. A B E D F C The Main Objective of the Project AUG TAA 11 14 15 1 2 4 5 6 9 10 12 13 8 7 3 16 1 • Sh2 gene from Teosinte remains uncharacterized • Design PCR primers from the conserved region of Zea mays Sh2 gene and use them in PCR reactions on genomic DNA isolated from Teosinte (Teo1 and Teo2) • Amplify the cognate genomic sequence of Teo1 and Teo2 • Purify the band after electrophoresis • Sequence the band and compare with the sequence with Sh2 gene of Zea mays

8. The Project Amplification of Teo1 &Teo2 of 5’ Amplification of B73 sh2 gene Amplification of Teo1 3’ • Amplification of B73 using primers from 5’ and 3’ • Use same primers on Teo1 and Teo2

9. Expected Results • Sequence DNA of amplification from Teo1, compare it to sh2 sequence of B73 • Use same primer for Teo2 amplification • Determine gene structure of Teo1 and Teo2 by DNA sequencing

10. Molecular and Genetic Analysis of a Maize Mutant Caused by a Helitron Insertion in Sh2 gene Jessica Blicharski SIBHI Mid-Term Presentation Oakland University

11. Transposable Elements Gene Disruption Gene Restoration

12. Helitrons • No duplication of host sequence • Insert between A and T • No terminal repeats • Transposition by rolling circle mechanism • Novel Family of Transposable Elements • Believed to play important role in genomic content of maize • Distinct Features from Class I & II

13. A B E D F C Helitron insertion into Sh2 gene 3 11 14 15 1 2 4 5 6 9 10 12 13 8 7 1 AUG TAA • Helitron inserts into intron 11 of Sh2 gene • Knocks out gene function • No production of ADP- Glucose Pyrophosphorylase • “Shrunken” phenotype

14. Restoration of function • Excision of helitron restores wild type phenotype (plump) • Found 4 “revertant” kernels in the field • Planted kernels and extracted genomic DNA from the leaves • Analyzed the extracted DNA using primers flanking the insertion site of the kernels

15. PCR Analysis of Revertant Kernels • Helitron insertion in sh2 gene is >10,000 bp in length (will not amplify under our PCR parameters • Amplified wild type sized band in all 4 whole revertants • Sequence of the fragments indicated wild type sequence at the site of Helitron insertion • No footprints? 500bp

16. PCR Analysis of Revertant Kernels Wild Type Sequence at the Site of Helitron Insertion

17. Interpretation of the PCR Results -/+ sh2/sh2 Sh2/sh2 + Pollen Contamination + Sh2/sh2 PCR Contamination

18. SNP rules out pollen contamination • sh2-7527 has a single base deletion (SNP) in intron 13 • PCR amplifed and sequenced intron 13 from all the revertants • Rev2’s wild phenotype was due to pollen contamination, but rev’s 1,3, and 4 showed no pollen contamination (contained strain specific SNP in inton 13)

19. Experiment in Progress……. • Sequence gDNA containing both the site of helitron excision and the SNP in one fragment from the revertants • We are currently designing more PCR primers and optimizing PCR parameters to achieve this objective.

20. Bioinformatics Processes for Indentification & Cataloguing of Matrix Attachment Regions (Mars) Indrani Datta SIBHI Mid term-Presentation Oakland University

21. Chromosome: DNA Packing • Eukaryotic DNA is packed into Chromatin • Consists of chromosomal DNA and lots of proteins • Condense the DNA in smallest possible volume • Chromatin fibres are arranged in living cells as independent loops anchored to the nuclear matrix

22. The Nuclear Matrix • Only 3% of nuclear proteins are the Nuclear Matrix or Scaffold Proteins. • Loops of 11nm fiber are attached to these proteins. • Loop Lengths: 5-120 kb (Average = 50 kb)

23. Mar: A locus control element that engenders a position independent expression capability upon the associated gene(s) • Mars are 100-1000 bp sequences • Help to bind the chromatin loop to protein network known as nuclear matrix • Potential origin of replication • AT rich regions containing topoisomerase binding site • Sequences rich in transcription binding site • Contain curved DNA • Matrix Attachment Regions exist at the ends of expressed genes

24. Our Purpose • Search and Obtain Annoted Mars sequences • Trying to find features ( consensus sequences) • By pattern matching with regular expression • Sources can be experiment or Journal paper • Creating a Database of Mars & Helitrons • Display information in a publicly available web site Genebank Background sequence Get Annoted Mars sequences Feature in annoted sequences Count # of occurance of feature in each Mar

25. Obtaining Mars Sequences • Query the NCBI based on annotation • Retrieve records from Genbank • Sequences from Genomic DNA • Result • 59 records retrieved

26. Some of the Result

27. Database Schema • Contain five tables • Lrp ( contain information related to Mars or helitron sequence) • Sequence (contain informaion related to background sequence • Feature ( contain information related to patterns) • Featureloc (contain information related to feature start –stop in lrp) • Reference ( contain information related to journals)

28. Populate the Database • Populated Sequence table • Contains information of Background sequence • Populated Lrp table • Contain either Mars or Helitron sequence • From background sequence

29. Pattern matching • Patterns obtained from Journals • 39 regular expression • Tried pattern matching with all lrps • Search with regular expression with in annoted Mars • Result is shown in graph

30. Pattern Matching Result

31. Web site (http://genomecluster.secs.oakland.edu/patterns) Web site (http://genomecluster.secs.oakland.edu/patterns)

32. In progress…… • Normalizing the pattern search result • Insertion of Helitron information in database • Web site will contain more feature information related to Mars & Helitrons

33. Acknowledgement • Dr.Gautam B.Singh • Dr.Shaliesh Lal • Entire SIBHI Faculty • Guy Lima

34. References • Singh Gautam B et. Al .Data Mining Algorithm for Discovering Matrix Association Regions(Mars) Proceedings of SPIE, 2000 4057 330-341 • Nikolaev L G et al. Construction of a chromosome specific library of human Mars and mapping of matrix attachment regions on human chromosome 19. Nucleic Acid Res, 1996 24(7)1330:1336 • http://www.plantcell.org/cgi/reprint/11/6/1117.pdf • http://en.wikipedia.org/wiki/Topoisomerase

35. Thank You Questions ??