1. This Week: Mon—Omics Wed—Alternate sequencing Technologies and Viromics paper Next Week No class Mon or Wed Fri– Presentations by Colleen D and Vaughn Discussion of Environmental Community Phylogeny paper (led by John) Finals week Monday 9-noon Presentations by rest of class Wed 5pm—Final Papers Due

2. Other Kinds of “Omics” Can we assay RNA and proteins on a genome wide scale in the environment? RNA----sequencing --detection via hybridization Proteins—separation and identification Why would we want to assay RNA and proteins on a genome wide scale in the environment?

3. Goal: RNA  DNA Ingredients: RNA (total, or poly A) Enzyme—reverse transcriptase (viral in origin) Nucleotides Primers—poly T or random hexamers RNA Detection : Reverse Transcription

4. Reverse Transcription G A C G T AC G T A C T A C G Reverse Transcriptase 5’3’ RNA TTTTTTTT or N6mers Result is a pool of single stranded DNA Complementary to RNA (cDNA) 3’5’

5. cDNA can then be used as a template in PCR, using specific primers for gene of interest (RT- PCR) cDNA could be cloned to form a cDNA library cDNA can also be stored for future experiments—more stable than original RNA Reverse Transcription

6. Too much rRNA is problem for prokaryotes Often results in incomplete fragments; Heavily 3’ biased in eukaryotes Issues with Reverse Transcription

7. Environmental Transcriptomics Poretsky et al 2005 Identities of cDNA fragments 16S rRNA clone library 400 clones from Georgia Salt Marsh

8. Global Transcriptional Profiling: Microarrays Principle is similar to Northern Blot Northern Blot: total RNA separated by size 1 gene is labeled-probe Signal indicates hybridization-> expression Microarray: like doing thousands of Northerns simultaneously Thousands of known genes separated by space Total mRNA (or cDNA) is labeled Signal indicates hybridization-> expression

9. Northern Blots Isolate RNA Hybridize with labeled probe Wash and detect Run on gel to separate by size Transfer to membrane

10. Examining gene expression using DNA microarrays Step 1---Construct gene chip Each spot represents a single gene PCR products or 70mers are physically spotted onto a glass substrate (using a robot) From whole genome sequence or sequenced cDNA library either: PCR each individual gene or synthesize 70mer specific for each gene

11. Examining gene expression using DNA microarrays Alternative Step 1---Purchase gene chip Several spots for each gene From whole genome sequence or sequenced cDNA library Design short oligos (15- 22mers) tiled along the length of the genome/clones Chip with desired oligos is commercially synthesized

13. Step 2 ---the experiment Test condition Reference condition Observe relative changes in gene expression Isolate mRNA apply to chip RT to cDNA and label Pool Fluorescence detection

14. Microarrays--issues $$$$$ both to synthesize the chips (ordering thousands of primers or 70mers) and to buy the dyes to label the cDNA for each experiment Genes should be spotted in duplicate or triplicate Need to do reverse label experiments to confirm results reference sample biological issues statistical issues Sensitivity—highly dependent on background Total amount of mRNA needed can be high (esp. for prokaryotes) May not be quantitative—genes of particular interest often confirmed to be differentially expressed via Northern blot or RT-PCR

15. Microarrays—how to take into environment? Current environmental approaches tend to be taxonomic arrays: Many versions of a single gene (16S rRNA, nifH, amoA, nirS, nirK) spotted on the array Hybridization shows which types are expressing gene in sample

16. Jenkins et al 2004 NifH macroarray

17. Jenkins et al 2004 NifH macroarray shows expression of different types of nifH in the Chesapeake Bay

18. Proteomics Proteins have very different properties than nucleic acids Cellular localization Have 3d structure (active and inactive forms) Size, charge, hyrdrophobicity are different from NAs and from each other One principle is similar—in order to identify them from a mixture, need methods of separation

19. Proteins Two types of electrophoresis Non-denaturing: Preserves native protein conformation and activity Denaturing reducing agents (urea) or detergent (SDS) used to break intramolecular bonds and linearize protein, and impart uniform negative charge useful for determining size From Sigma total protein plant extraction kit SDS-PAGE = denaturing Polyacrylamide Gel Electrophoresis

20. Each amino acid has a characteristic pI (isoelectric point) the pH at which it carries no charge Combination of amino acid content and 2º and 3º strcuture give each protein a pI

21. Proteins---2 dimensional gels Stage 1- Isoelectric focusing — separates by native charge

22. Proteins---2 dimensional gels Stage 2----SDS-PAGE separates by size Often used to compare two different conditions/treatments to identify proteins unique to 1 condition Check out http://us.expasy.org/ for proteomics/2D gel data resources

24. Protein ID is heavily dependent on Database of potential peptides

25. Proteomics—can be quantitative