Genomics and Behavior

1. Genomics and Behavior

2. “gene expression” RNA Protein Behavior “Central Dogma” DNA

3. Outline • Transcription • Analyzing genes and gene expression • Early life experience and serotonin transporter • Manipulating gene expression • Pair bonding behavior • Epigenetics • Maternal behavior

4. DNA Transcription nucleus ribosome mRNA cytoplasm Translation protein Protein Synthesis

5. Transcription (Gene expression) gene • DNA double helix is unwound • A strand of RNA is produced from the complementary DNA strand by RNA polymerase ATG AAC TCC TAC TTG AGG A U G A A C U C C TAC TTG AGG

6. Translation • mRNA migrates out of the nucleus to the ribosome • Protein synthesis takes place based on the genetic code • A three base codon codes for an amino acid

7. Mutation can Change Protein Structure • A mutation in DNA can change physiology or behavior by changing properties in the resulting protein • Siamese cats have a mutation that makes the tyrosinase enzyme heat sensitive J. Hered.21: 309-318, 1930.

8. Genetic Disorders I • A few rare disorders are caused by single mutations that cause result in a defective enzyme • Huntington’s Disease

9. Gene Regulation • Although mutations causing changes in protein can have important effects on behavior, most mutations are either silent or harmful • Usually it is a difference in gene expression that influences a behavior

10. Transcription • The production of messenger RNA (mRNA) from a sequence of DNA (gene) • Gene expression • “Turning on the gene”

11. Important DNA Sequences • Promoter • Indicates where transcription should begin • Coding region • Sequence that codes for a protein • Terminator • Indicates where transcription should stop promoter terminator coding region

12. Transcription machinery • RNA Polymerase • Transcribes DNA into RNA • Transcription factors • Help RNA polymerase bind to DNA • There are hundreds of different transcription factors • Steroid hormone receptors RNA polymerase Transcription factors

13. Initiation of Transcription • Transcription factors bind to promoter region Transcription factors promoter

14. Initiation of Transcription • Transcription factors allow RNA polymerase to bind to promoter region RNA polymerase promoter

15. Transcription • RNA polymerase begins to move down the strand of DNA and transcribe it into RNA. It unwinds the DNA as it moves down the strand. RNA RNA polymerase promoter

16. Transcription RNA RNA polymerase promoter

17. Termination of Transcription • When the RNA polymerase reaches the terminator sequence, it stops transcribing RNA RNA polymerase terminator

18. Transcriptional Regulation • Promoter sequence • Expression of transcription factors • Splicing

19. Last point on transcription • Gene regulation doesn’t mean much unless a change in protein occurs • Translating the genetic code of RNA into a string of amino acids

20. The entire mRNA molecule is not translated Start codon AUG coding region • Stop codon • UGA • UAA • UAG

21. Somatostatin receptor: AY585720 5’ untranslated coding region • 3’ untranslated Start codon aca gac aca cac gtg acg aagatg aac tcc tcc tcg….. Stop codon …..aag acc ttc aca tcc tgc tga gag cct cct gct cct

22. Untranslated mRNA • 5’ and 3’ untranslated mRNA can affect stability of the mRNA molecule and also how it is translated

23. Coding vs. Noncoding region • Across species, usually it is the coding region of a gene that is more strongly conserved than the noncoding region • Most mutations affecting the function of a protein are harmful, so they tend to be selected against

24. Measuring Gene Expression PCR and Microarrays

25. Using a sequenced genome for behavioral research • Most methods for measuring gene expression require knowing the sequence in advance • It’s much easier to go to a database to retrieve the sequence that have to sequence it yourself

26. Sequenced Genomes • Mammals: Human, mouse, rat, cow, pig • Birds: Chicken, zebra finch • Fish: Zebrafish, Fugu (pufferfish), tilapia

27. Polymerase Chain Reaction (PCR) • Generally credited to Kary Mullis • 1993 Nobel Prize • One of the most widely used techniques in molecular biology

28. PCR Steps • Melting • Heat up sample so that double stranded DNA unwinds • Annealing • Cool down the sample so that gene specific primers anneal to gene of interest • Extension • Heat up sample so that DNA polymerase synthesizes new strand of DNA

29. Materials for PCR • Taq polymerase • buffers • dNTP: raw materials • Specific primers: short sequences of DNA that flank the region of interest • Template: a source of DNA • Can be genomic or cDNA

30. PCR animation • Run PCR.exe file

31. Gel Electrophoresis • Run gel animation

32. PCR Applications • Amplifying specific sequences of DNA used for • Detecting genetic differences (mutations, deletions, etc.) • Genetic fingerprinting and paternity testing • DNA sequencing • Manipulation of DNA (recombination)

33. Comparative Genomics: 5HTT • Individual variation in the length of the serotonin transporter promoter observed in rhesus monkeys

34. Serotonin Transporter

35. Activity • The rhesus monkey short allele has reduced transcriptional activity compared to the rhesus monkey long allele

36. Serotonin function • Researchers examined a serotonin metabolite (5HIAA) in cerebral-spinal fluid (CSF) • Examined monkeys raised in two different environments • Peer-reared (nursery) • Parent-reared (mother)

37. Serotonin metabolites • In monkeys raised in nursery, heterozygotes had reduced 5HIAA • In monkeys raised by mothers, there was no effect of genotype

38. Serotonin and Behavior • Individuals with the lowest CSF 5-HIAA levels are more likely to show aggressive behavior and engage in risk taking behavior (making extreme jumps, also drinking excessive amounts of alcohol) • What’s missing from the story???

39. Real-time PCR • A method for measuring the relative amount of gene expression in tissue samples • Extremely sensitive, often referred to as quantitative PCR or qPCR • All methods depend on increasing fluorescent signal as target gene is amplified

40. Why is it called real time? • After each cycle, the instrument measures the amount of fluorescence in the reaction

41. Cycle Threshold (Ct) • Ct: the number of cycles needed to reach a certain level of fluorescence • The more copies of an mRNA present, the lower the Ct

42. Cycle thresholds • Every ten-fold increase in mRNA should lead to a 3.33 increase in cycle threshold • A standard curve can be used to determine how many copies of mRNA are present in a sample

43. Hormone Receptors in Cichlid Fish • Cichlid fish Astatotilapia can have two phenotypes • Dominant males are more aggressive than subordinate males

44. Measuring hormone receptor mRNA • Dominant males had more androgen receptor mRNA in forebrain • Suggested that dominant males may be more sensitive to androgens Burmeister et al. 2007, Horm. Behav: 51 164-170.

45. Real time PCR +s and -s • Advantages • Relatively easy • Very sensitive • Gold standard of measuring gene expression quantitatively • Often used to confirm microarray results • Disadvantages • Usually poor spatial resolution • Gene expression does not always reflect protein expression

46. Microarrays • Allow for the measurement of tens of thousands of genes simultaneously • Generally requires that the species’ genome sequenced • Sometimes can use a closely related species • Human, mouse, rat, zebrafish commercially available • Zebra finch, honey bee, and cichlid fish also exist

48. Microarray data • Can be overwhelming • Many microarrays have probes from more than 10,000 genes • Data are usually more useful if the scientist has an idea of what to look for

49. Honey Bee Behavior • Younger bees stay inside the hive and take care of brood (nurses) • Older bees forage for nectar and pollen outside the hive

50. Age vs. Behavior Confound • In a normal colony age is confounded with behavior • To address this confound, researchers created colonies in which all the bees were the same age • In this environment, some young bees will become foragers