Adam Biales NERL Postdoctoral Fellow

1. Genomics Training A Cooperative Endeavor between USEPA Region 5 -and- USEPA Office of Research and Development Adam Biales NERL Postdoctoral Fellow

2. Overview • DNA • RNA • Protein • PCR

3. The Central Dogma • Central dogma of molecular biology states that DNA carries the genetic information which is transcribed to RNA and subsequently translated to protein DNA RNA Protein

4. The Central Dogma DNA RNA Protein

5. Overview of DNA DNA stores information – Blue print • 1-5 billion base pairs in length • 2 meters in length • Very complex • Lots more DNA than genes • Non-coding • Repetitive (microsatellites, SINES, etc.) • Introns • Structural – telomeres, centromeres • Protein coding ≈ 1% • Complexity becoming clear • 1970 ≈ 100 million genes • 2002 ≈ 30,000 genes Mitochondrial vs. Nuclear

6. Nucleosome

7. Chromosomes

8. Overview of DNA DNA stores information – Blue print • Very complex • Lots more DNA than genes • Non-coding • Repetitive (microsatellites, SINES, etc.) • Introns • Structural – telomeres, centromeres • Protein coding ≈ 1% • Complexity becoming clear • 1970 ≈ 100 million genes • 2002 ≈ 30,000 genes Mitochondrial vs. Nuclear

9. DNA structure • DNA is a polymer of nucleotides • Purines (Adenine,Guanine) • Pyrimidines (Cytosine,Thymine) • Two strands twist around each other (double helix) • Bases of the two strands face each other • Forms base pairs • A pairs with T, G pairs with C

10. Nucleotide

12. DNA structure • DNA is a polymer of nucleotides • Purines (Adenine,Guanine) • Pyrimidines (Cytosine,Thymine) • Two strands twist around each other (double helix) • Bases of the two strands face each other • Forms base pairs • A pairs with T, G pairs with C

14. The Central Dogma DNA RNA Protein

15. Overview of RNA • Different types • mRNA, tRNA, rRNA • Carries message from DNA to ribosome – protein • Often quick turn over • Highly regulated • All functions not understood

16. RNA structure • Single stranded • DNA and RNA have structural differences • Pentoses (five-carbon sugar component) • In DNA, the pentose is deoxyribose • In RNA, it is ribose • Nitrogenous bases • thymine in DNA, uracil in RNA

17. RNA Structure

18. The Central Dogma DNA RNA Protein

19. Overview of Proteins • Made from amino acids strung together • Form regular 3-D structures • Structure dictates function • Subunits add specificity to function • Turned on and off by chemical modifications

20. Levels of Protein Structure

21. Overview of Proteins • Made from amino acids strung together • Form regular 3-D structures • Structure dictates function • Subunits add specificity to function • Turned on and off by chemical modifications

22. Proteins • Proteins are used for a variety of functions in the cell: • Structural support • Metabolism • Motion • Defense • DNA replication, RNA synthesis, etc. • And many more functions

23. DNA Polymerase

24. PCR

25. Polymerase Chain Reaction - PCR • An in vitro method for replicating DNA • Makes billions of copies of a DNA template in a few hours • Requires only a tiny amount of starting material • Highly specific: primers determine the sequence to be amplified

26. PCR reaction components • Template DNA strand • Nucleotides (dNTPS) • Heat stable DNA Polymerase – Taq • Same enzyme that replicates DNA in your cells • Isolated from hotsprings in Yellowstone • Primers (oligos) – 20ish bp

27. Primer specificity 4 nucleotides, 3.2 x 109 base pairs in the human genome… An oligo of 10 bases in length = 410 (an exact sequence match expected to occur ~ every 1 million bases) …expected to be represented in the genome 3,200 times An oligo of 10 bases in length = 415 (an exact sequence match expected to occur ~ every 1 billion bases) …expected to be represented in the genome 3 times An oligo of 10 bases in length = 420 (an exact sequence match expected to occur ~ every 1 trillion bases) …expected to be represented in the genome…well you get the idea

29. PCR Amplification

30. Expression Based Technology

31. Expression Based Technology • Immediate responses on cellular level • Biological information • Can be specific for a given stressor • Rapid

32. Regulation • Needs to be heavily regulated • Appropriate cell activation • If not regulated leads to pathology • Cancers • Cell-cycle • Signaling • Occurs on each level: • DNA, RNA, Protein

33. Gene Structure Promoter Cis element 1000 - 250000 bp

34. Regulation

35. GENE DNA Gene Structure Promoter Cis element 1000 - 250000 bp

36. Exon Intron Exon Intron Exon Intron mRNA AAAAAAN250 Transcription/RNA processing 5’ 3’

37. The Central Dogma Reverse Transcriptase cDNA DNA Protein RNA

38. QPCR • Realtime quantitative PCR • Advantages • Quantitative • Sensitive • Reproducible • Minimal tissue requirements • Rapid results

39. Dynamic range Able to QA 107 100 “Real-Time” visualization of amplified products Highly reproducible across entire range 12 replicates each DCt < 0.50 cycles

40. Microarrays • Monitor changes across 1000s of genes in 1 experiment • Available for rainbow trout • Zebrafish • FHM – 2006 • Daphnia • Signatures – 1 set of genes = 1 chemical • Systems biology • Information about mechanism • Cluster analysis

41. Control (reference sample) Treatment (ie. effluent) Isolate Tissue RNA Isolate Tissue RNA RT & label RT & label 1 1 1 1 2 2 3 3 2 1 2 1 Microarray

42. Message present only in control Message present only in treatment Message present at equal levels in both treatment and control Adapted from H. Hamadeh and C. Afshari, American Scientist 88:508-515

43. Genes Expression Comparison between High and Low Grade Cancer Lapointe, Jacques et al. (2004) Proc. Natl. Acad. Sci. USA 101, 811-816

44. Proteomics • Similar to microarrays – global changes • Protein data more biologically relevant • Higher biological level • Less variable • 2-D gel electrophoresis • Non-directed, no assumptions • Can see modifications Study of protein populations of one cell versus another

45. 2-D Gel Electrophoresis Control Treatment

46. Biological Effect Bio-assessment + Community Assessment Population Decline, Adaptation Population DNA Analyses Toxicity Testing Histopathology Cell death/Mitosis/Activation Molecular/RNA/Protein Changes The Exposure-Effect Hierarchy Biological Effect Biological Level Community Bio-assessment + Community Assessment Population Population Decline, Adaptation Population DNA Analyses Organism Toxicity Testing Tissue/Organ Histopathology Cellular Cell death/Mitosis/Activation Subcellular Molecular/RNA/Protein Changes

47. Current Molecular Assays • Vitellogenin • Egg protein – only females in breeding season • Males turn it on when exposed to estrogen • All kinds of fish • Fathead, trout, bass, gudgeon, shiners, roach, flounder • Can be used as an indicator to estrogen exposure

48. Estrogens • Estradiol, estrone, EE2 • Sources • Farm runoff • Contraceptives • Environmental levels - EE2 • Surface water 0.05 – 30.8 ng/L EE2 • Sewage effluents 0.05 – 62.0 ng/L EE2 • LOEC Vg protein 0.1 ng/L • LOEC sex interchange 0.6 ng/L • Can have additive effects • Potent responses at low levels • (4 ng/L EE2)

49. Estrogenic Effects • Male fish with ovatestes • Decreased male fitness • Decreased size/physical abnormalities • Decreased reproductive success (.32 ng/L) • NO EGGS PRODUCED at 3.5 ng/L • Skewed sex ratios • 0 males with secondary sex characters • 3.5 ng/L • May have long-term effect on reproductive success • 50% reduction in reproductive success 29 days after exposure • 5 months following treatment – decreased fertility

50. Issues • Fish swim • Not sitting by effluent for whole life • Level of real world exposure • Linkage between biological levels not completely established • Why are there any fish at all? • Needs field testing • Mixtures • Can we use gene expression as a metric in bioassessment