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Genomics and Behavior “Central Dogma” “gene expression” DNA RNA Protein Behavior Outline • Transcription • Analyzing genes and gene expression – Early life experience and serotonin transporter • Manipulating gene expression – Pair bonding behavior • Epigenetics – Maternal behavior 1 Protein Synthesis DNA Transcription nucleus ribosome mRNA cytoplasm Translation protein 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 AUG AAC UC C TAC TTG AGG 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 2 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. Transcription • The production of messenger RNA (mRNA) from a sequence of DNA (gene) – Gene expression – “Turning on the gene” Important DNA Sequences • Promoter – Indicates where transcription should begin • Coding region – Sequence that codes for a protein • Terminator – Indicates where transcription should stop promoter coding region terminator 3 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 Transcription factors RNA polymerase Initiation of Transcription • Transcription factors bind to promoter region Transcription factors promoter Initiation of Transcription • Transcription factors allow RNA polymerase to bind to promoter region RNA polymerase promoter 4 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 Transcription RNA RNA polymerase promoter Termination of Transcription • When the RNA polymerase reaches the terminator sequence, it stops transcribing RNA RNA polymerase terminator 5 Transcriptional Regulation • Promoter sequence • Expression of transcription factors • Splicing 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 Measuring Gene Expression PCR and Next Generation Sequencing 6 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 Polymerase Chain Reaction (PCR) • Generally credited to Kary Mullis – 1993 Nobel Prize • One of the most widely used techniques in molecular biology 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 7 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 PCR animation • Run PCR.exe file Gel Electrophoresis • Run gel animation 8 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) Comparative Genomics: 5HTT • Individual variation in the length of the serotonin transporter promoter observed in rhesus monkeys Serotonin Transporter 9 Activity • The rhesus monkey short allele has reduced transcriptional activity compared to the rhesus monkey long allele 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) Serotonin metabolites • In monkeys raised in nursery, heterozygotes had reduced 5HIAA • In monkeys raised by mothers, there was no effect of genotype 10 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??? 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 Why is it called real time? • After each cycle, the instrument measures the amount of fluorescence in the reaction 11 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 Cycle thresholds 18 Cycle threshold 16 14 12 10 8 6 4 2 0 0 00 0, 10 00 ,0 10 0 10 0 00 1, 10 1 • 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 Copies Hormone Receptors in Cichlid Fish • Cichlid fish Astatotilapia can have two phenotypes • Dominant males are more aggressive than subordinate males 12 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. 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 New Frontiers: Mass Sequencing • New sequencing techniques allow for the ability to sequence all of the mRNAs in a single sample – Illumina – 454 Sequencing (Roche) • Can detect mutations in sequences • This technology is making microarrays obsolete 13 RNA Seq • • • • Extract and isolate mRNAs (poly A RNA) Reverse transcribe and fragment mRNAs Add sequencing adaptors Amplification of each sequence creates clusters • Each sequence read base by base • Align fragments and quantify RNAseq strategy • Analysis strategy based on counting number of transcripts in experimental gorups • Can identify splice variants – In come cases, part of a gene will not be included in the mRNA • Can identify single nucleotide polymorphisms and other mutations 14 Analysis • Sequencing reads need to be assembled so that the number of transcripts can be quantified – Typically done with a reference genome – Methods are available to assemble transcripts “de novo” using closely related species • Unless you can analyze data yourself, now it is cheaper to do the sequencing than to do the analysis 15 Manipulating Gene Expression Vasopressin • Vasopressin • Behavioral effects – Aggression – Parental behavior – Pair bonding/affiliation • Vasopressin receptors – V1a – V1b Prairie vole (Microtus ochrogaster) Vasopressin Receptor 1a (V1a) Gene • Prairie vole (Microtus ochrogaster) and meadow vole (M. montanus) genes are 99% identical • Biggest difference lies in the promoter region (more extensive in M. ocrhogaster) 16 Does the Prairie Vole Promoter Affect Behavior? Regulation of the transcription of the V1a RNA can influence the amount of V1a protein Behavioral Effect of Prairie Vole Transgene • Increased expression of the receptor alone had no effect on affiliative behavior • Injection of vasopressin (AVP) in transgenic mice increased sniffing and grooming of a female Complicating Issues • The prairie vole promoter gene is present in many species of voles that are not monogamous Fink et al. 2007, PNAS 103:10956 17 Partner Preference vs. Monogamy • Partner preference behavior has been examined in only a few species of voles • It may be that many non-monogamous species show some form of partner preference that is affected by vasopressin • The effects of the vasopressin promoter may depend on the expression of other genes Epigenetics Glossary • Epigenetic: modifications to genes that do not involve changing the DNA sequence itself and can be transmitted across generations 18 Levels of Organization coarse • Chromosome –Histone • Gene –DNA fine Packaging of DNA DNA Must be Unpackaged Before it is Read • Unwrapping DNA from around histones allow polymerases to read DNA for either transcription or replication 19 Methylated DNA Shipping crate Non-Methylated DNA Box closed with tape Methylation A specific kind of chemical change that can affect how easily DNA is unpacked from the histone DNA Methylation • Methylated DNA has a small methyl group attached to some of its bases • Usually methyl groups attach to cytosine (C) only 20 Methylated DNA Non-Methylated DNA Maternal Care, Methylation, and “Anxiety” • Previous studies showed that pups raised by high licking and grooming dams showed – Increased exploratory behavior – Reduced physiological responses to stress (namely corticosterone secretion) Methylation of Glucocorticoid Receptor Promoter • Cross-fostering experiment showed that rat pupss raised by low licking/grooming dams had increased methylation of the glucocorticoid receptor promoter 21 Effect of Methylation on Stress Response • Experimentally blocking methylation with a drug blocks the effect of low licking/grooming on corticosterone secretion during stress Methylation of Estrogen Receptor • Low parental care rats have low estrogen receptor expression • Estrogen receptor promotes parental care • Maternal behavior reduces methylation of estrogen receptor Cross Generational Effects • Pups raised by low licking/grooming dams grow up to lick their own pups at a low rate • Behavioral patterns are transmitted across generations Cross fostered 22 Question • Is this inheritance of an acquired characteristic? • Some people think so, others are not convinced • What do you think? “The Modern Synthesis” • Refers to the integration of ideas on genetics with theories of natural and sexual selection – Hardy-Weinberg Theorem – Genetical Theory of Natural Selection • Continuous variation has same method of inheritance (quantitative) as discontinuous variation (discrete) “The Modern Synthesis” • Populations contain genetic variation that arises randomly (mutation, recombination) • Evolution as changes in gene frequency • Most adaptive genetic variants have small effects on phenotype (evolution is slow) • Acquired traits are not inherited 23 • Until Ronald Fisher’s “Genetical Theory of Natural Selection”, many evolutionary biologists held views on inheritance that are generally not accepted today. One prominent theory was the “inheritance of acquired characteristics”, in which traits acquired by parents could be passed on to successive generations. Although usually attributed to Jean Baptiste Lamarck, it was a commonly accepted method of inheritance in the late 19th and early 20th centuries. The rise of the “modern synthesis” of evolution rejects this mode of inheritance, but recent findings in epigenetics raise some interesting issues. Do recent findings showing behavioral modification of DNA methylation constitute a challenge to the “modern synthesis” or do these results conform with existing ideas from the modern synthesis? 24