Use of molecular biology in environmental toxicology

1. Use of molecular biology in environmental toxicology Joe Staton University of South Carolina, Columbia

2. Overview: • Classical methods • Molecular methodology • Applications to toxicological studies

3. What are the new moleculartechnologies?

4. Tracking a chosen gene— • DNA cloning and sequencing (E. coli) • Polymerase chain reaction (PCR) • Reverse Transcriptase PCR (RT-PCR) • Expression (bacterial) vectors • In situ hybridization

5. Methods for finding new genes: • cDNA libraries • Expressed sequence tags (ESTs) • Microarrays • Subtractive libraries • Serial Analysis of Gene Expression • Genome Projects—high-throughput sequencing • Gene chips

6. Good candidate genes? Considerations: • Population marker? • Direct effect on enzyme? • Main link in pathway? • Indicator of organism life history?

7. Getting the data... Introduction to Polymerase Chain Reaction and DNA Sequencing

8. Polymerase Chain Reaction(PCR)1993 Nobel prize in Chemistry to Kerry Mullis

9. Recovering DNA Conserved DNAUnknown DNAConserved DNA 5’-CGTCGGATGTAAGAGACTCTCACAAACGTCCGATCGGCGT-3’ 3’-GCAGCCTACATTCTCTGAGAGTGTTTGCAGGCTAGCCGCA-5’

10. Recovering DNA Conserved DNAUnknown DNAConserved DNA 5’-CGTCGGATGTAAGAGACTCTCACAAACGTCCGATCGGCGT-3’ 3’-GCAGCCTACATTCTCTGAGAGTGTTTGCAGGCTAGCCGCA-5’ GTC CAG

11. Making Primers Primer a 5’-CGTCGGATGTA-3’ 5’-CGTCGGATGTAAGAGACTCTCACAAACGCTCCGATCGGCGT-3’ 3’-GCAGCCTACATTCTCTGAGAGTGTTTGCGAGGCTAGCCGCA-5’ 3’-GCTAGCCGCA-5’ Primer b

12. Temperature Profile of PCR Melting 94  Temp increasing (° C) 72 Extension 50 Annealing (=stringency) One cycle Time increasing

13. Mechanics of PCR Cycle 1 Melt - 94C Anneal - 45-60C Extend - 72C Repeat 30 times Cycle 30 1.07x109 copies! Primers Synthesized DNA

14. Net effect of PCR What Goes In What Comes Out Total DNA PCR primers dNTP’s (A,C,G,T) DNA Polymerase Buffer, etc. What went in + ~1 Billion Copies of the Amplified Fragment

15. 1 1 2 3 Larger Fragments 2 SmallerFragments 3

16. DNA Sequencing Sanger random termination method (enzymatic)

17. Model of the chemistry-- ddATP G A T C T G G G C T A C T C G G G C G T C G C A A G C C C G C A A T G A G C C C G C A A C C C G A T G A G C C C G C A A G A C C C G A T G A G C C C G C A

18. Demo of the autosequencer ddATP ddGTP ddCTP ddTTP Origin G C C A G T G C A G C T G G A FINISH! G C T A G T C G A C T A T C C T

19. Demo of the autosequencer ddATPddGTPddCTPddTTP Origin

20. Demo of the autosequencer 1 2 3 4 Origin

21. Actual gel image

22. Tracked gel image

23. Single scanned sequence

24. How does RT-PCR differ? • Uses RNA as a start template • No introns • Can quantify amount • Uses a retroviral enzyme to make cDNA from RNA • PCR as usual

25. What if you want to measure protein, too?

26. Use antibodies: • Purified protein from subjects injected into vertebrate (e.g., rabbits) • Rabbits produce antibodies • Antibodies from purified from rabbit blood • Used for detection of proteins (ELISA, in situ hybridization, etc.)

27. What if protein is difficult to get? • Get RNA for gene • Put in a genetically engineered plasmid (expression vector) • Use bacteria to generate protein in super-large quantities • Inject into vertebrate host to create antibodies

28. Tracking multiple genes to whole genomes: Prospects for the future or Gordian Knot?

29. Multigene technologies: • cDNA libraries • Expressed sequence tags (ESTs) • Microarrays (hybridization) • Subtractive libraries • Serial Analysis of Gene Expression (SAGE) • Genome Projects—high-throughput sequencing • Gene chips (hybridization)

30. ESTs and Microarrays: • Make cDNA library of tissues of interest • Sequence all unique cDNAs and identify • Create unique DNA fragments for each gene • Bind cloned fragments onto a solid support (e.g., nylon filter) • Label RNA from test subject and bind to DNA array • Use “brightness” of color at each spot as data

31. Microarray mechanics

32. What if you are unsure about the genes affected?

33. Subtractive libraries • Concept: control cDNAs “block” tester cDNAs from amplification in PCR • Use: will amplify only differentially expressed cDNAs in a reaction, which can then be used for in situ hybridization, microarrays, etc.

34. Serial Analysis of Gene Expression (SAGE) • Measures amount of mRNA products in a tissue • Compare levels in control vs. test organisms • Relate treatment to specific genes or suites of genes

35. Make cDNA from isolated mRNA with poly-T Strept- avidin Beads Step 1: Isolating mRNA fragments e.g., AE = NlaIII

36. Step 2: Add linkers to each half for reaction

37. Step 3: Release from beads with TE (BsmFI)

38. Step 3: Increasing amount of product

39. Step 4: Purify and clone

40. Sequencing clones • Sequence many DITAGS • 26-bp units • 23 to 30 DITAGS per clone • Sequence ~1000-1500 clones! • Normalize data and perform statistical analysis

41. Microarrays vs. SAGE • Chip-less • Large post-sequencing effort • Digital data of copy number in library • Must have developed chip • Large pre-sequencing effort • Analog data of relative binding to site

42. Genome projects: • Method: Brute force cloning and sequencing of entire genome of an organism • Use: whole genome microarray or gene chip

43. Drawbacks: • Expensive • Limited organisms • Bacteria (Escherichia coli) • Virus (Lambda) • Fly (Drosophila melanogaster) • Round worm (Caenorhabditis elegans) • Cabbage relative (Arabidopsis thaliana) • Frog (Xenopus laevis) • Human (Homo sapiens)