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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
                     Protein Synthesis
DNA                         Transcription
          nucleus




                                                 ribosome

                                mRNA
      cytoplasm
                  Translation




                                       protein
Transcription (Gene expression)
                                   gene
• DNA double helix is
  unwound

                        ATG AAC TCC
• A strand of RNA is    TAC TTG AGG
  produced from the
  complementary DNA
  strand by RNA
  polymerase
                        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
     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.
          Genetic Disorders I
• A few rare disorders
  are caused by single
  mutations that cause
  result in a defective
  enzyme
  – Huntington’s Disease
          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
              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
        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
                    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
           Transcriptional Regulation

• Promoter sequence
• Expression of
  transcription factors
• Splicing
    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
The entire mRNA molecule is not
           translated
  Start codon
          AUG




                coding region   Stop codon
                                   UGA
                                   UAA
                                   UAG
  Somatostatin receptor: AY585720

        5’ untranslated




                                     coding region              3’ untranslated
                   Start codon


aca gac aca cac gtg acg aag atg aac tcc tcc tcg…..

                                                       Stop codon


                          …..aag acc ttc aca tcc tgc tga gag cct cct gct cct
        Untranslated mRNA
• 5’ and 3’ untranslated mRNA can affect
  stability of the mRNA molecule and also
  how it is translated
  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 Microarrays
  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
       Sequenced Genomes
• Mammals: Human, mouse, rat, cow, pig

• Birds: Chicken, zebra finch

• Fish: Zebrafish, Fugu (pufferfish), tilapia
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
           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
           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
      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
      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
       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
• Every ten-fold                            16

  increase in mRNA                          14




                          Cycle threshold
                                            12
  should lead to a 3.33                     10
  increase in cycle                          8
                                             6
  threshold                                  4

• A standard curve can                       2
                                             0
  be used to determine

                                                 1


                                                     10


                                                          10


                                                               1,


                                                                        10


                                                                                  10
                                                                   00
                                                           0




                                                                         ,0


                                                                                   0,
  how many copies of




                                                                    0


                                                                             00


                                                                                       00
                                                                                        0
                                                          Copies
  mRNA are present in
  a sample
Hormone Receptors in Cichlid Fish
• Cichlid fish
  Astatotilapia can
  have two
  phenotypes

• Dominant males are
  more aggressive
  than subordinate
  males
    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
               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
            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
        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
   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
       Behavioral Genomics
• Abbreviations
  – Young nurse (YN)
  – Young forager (YF)
  – Old nurse (ON)
  – Old foragers (OF)

• Yellow = increased gene
  expression
• Blue = decreased gene
  expression
Different Behavior, Different Genes
             When bees switch from nursing to foraging




                  These genes turn off




                  These genes turn on
  Gene Expression Signatures
• Changes in gene expression were so
  reliable that researchers could predict
  behavioral status based on gene
  expression (95% accurate)
Specific Genes
        Microarrays +s and -s
• Advantages
  – Can generate a mind blowing amount of data
  – Can identify gene networks
  – Can help identify unexpected new directions for
    research
• Disadvantages
  – Frequently yield false positives and false negatives
  – Large datasets can be overwhelming, sometimes
    hard to make sense of it all
  – Expensive
  Measuring Gene Expression
• Measuring gene expression is an
  important component of behavioral
  genetics research

• Like any correlational approach, its main
  weakness is that it cannot establish cause
  and effect relationships
Manipulating Gene
   Expression
            Knockout mice
• Breeding of knock-out mice is becoming
  routine. Many universities have facilities
  that will create lines of mice for a fee

• Key point: traditional knockout mice lose
  function of the gene of interest through
  development and as adults
  Transgenic Mice: “Knock-ins”
• Similar procedure as creating knock-out mice.

• Introduce a construct containing the gene of
  interest to mouse embryonic stem cells

• The construct should include a strong promoter,
  to ensure the gene will be expressed. This
  technique can also be used to determine if a
  given promoter is, in fact, strong
               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)
Does the Prairie Vole Promoter
       Affect Behavior?



Regulation of the transcription of the V1a
  RNA can influence the amount of V1a
                  protein
    Prairie Vole V1A Promoter in Mice

• Transgenic mice
  expressing the prairie
  vole V1a gene show
  patterns of receptor
  expression like prairie
  voles
   – White: wt mice
   – Striped: transgenics
   – Black: voles
  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
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
    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
Methylated DNA    Non-Methylated DNA




 Shipping crate      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
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
   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
                 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
• 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?

				
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posted:12/2/2011
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