Professor Mark A. Tanouye April 16, 2000

1

LEARNING AND MEMORY IN ANIMALS:LEARNING MUTANTS,FORWARD GENETICS

Reading: p.189-198

NATURE VS NURTURENATURE Biological - usually called "genetic", but

actually more complicated, ill-defined.Component of intelligence that is inherited.

NURTURE Social - usually considered family background,education, socio-economic influences, ill-defined

IMPLICATIONSSocial programsHead StartSchool curriculaChild-rearing practices

"FORWARD" GENETICS(mutational analysis, functional analysis)

FUNCTION GENE PROTEIN(learning) (loss-of-function)

(stupidity)

clonemutation

LEARNING TESTMUTANT SCREEN

MAP GENECLONE GENE(positional cloning)PROTEIN SEQUENCE

IDENTIFY MUTANTMUTANT STRAIN

MAKING AND CHARACTERIZING MUTANTS

MUTAGENESIS

SCREEN

(overview)

NUMBER OF GENES ? complementation tests

damage DNA of one gene(single gene mutation)(somatic - cancer)(germline - pass on to progeny)

identify mutants with learning test or screen(dominant mutations, recessive mutations)

FUNCTIONAL PATHWAYS ?double mutant analysis

MAPPING

a) linkage (which chromosome) ?b) distance from other genesc) linear order of genes "Genetic model" organisms

bacteria, yeast, Drosophila, C. elegans, zebrafish, Arabidopsis, mouse, human recombination

LEARNING THAT ODORMEANS ELECTRIC SHOCK

(classical conditioning)

ODOR+

SHOCK

training

association

AVOIDODORALONE

testing

Odor "symbolizes"electric shock

PsychologyShock = unconditioned stimulus (US)Odor = conditioned stimulus (CS)

DROSOPHILA LEARNING APPARATUS

Professor Mark A. Tanouye April 16, 2000

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RESULTS OF LEARNING TESTSLearning in normal flies dunce flies: no learning

(all go toward light)

MUTATIONS(radiation)

MUTATIONS(radiation)

IONIZING RADIATION Ultraviolet X-rays Gamma rays

IONIZING RADIATION Ultraviolet X-rays Gamma rays

X

xxx

***

DELETIONDELETION

TRANSLOCATIONTRANSLOCATION

MUTATIONSchemical

MUTATIONSchemical

Mutagen/Carcinogenethylmethanesulfonateethylnitrosourea(alkylating drugs)

Mutagen/Carcinogenethylmethanesulfonateethylnitrosourea(alkylating drugs)

REPLICATION

G*

A

GENE

normal

mutation

AMINO ACID SUBSTITUTION GGT AGT gly ser

STOP CODON SUBSTITUTION TGG TAG trp stop

AMINO ACID SUBSTITUTION GGT AGT gly ser

STOP CODON SUBSTITUTION TGG TAG trp stop

PATHWAYS DEFINEDBY DROSOPHILA

LEARNING GENESAVOID ODOR

INITIALLEARNING

linotte

SHORT-TERMMEMORY

duncerutabaga

MEDIUM-TERMMEMORY

amnesiac

ANESTHESIA-RESISTANTMEMORY radish

LONG-TERMMEMORYdCREB2

DROSOPHILACHROMOSOMEMAPS

1st (X) 2nd 3rd 4th

3 major chromosomes (1,2,3)1 minor chromosome (4th)

180Mb13,600 genes

1889–1938, b. Schuyler Falls, N.Y., grad.Columbia (B.S., 1912; Ph.D., 1916)discoverer of white mutant; most mutantspolytene chromosome maps; free love

Professor Mark A. Tanouye April 16, 2000

3

which chromosome is the dnc mutation on?

CHROMOSOMAL LINKAGE

Possibilities for two mutations (genes)(dnc + marker mutation)

Linkage(+ recombination)

Linkage(+ recombination)

independent assortment (common)(uncommon, "closely-linked")

No linkage(different chromosomes)

No linkage(different chromosomes)

Linkage(no recombination)

Linkage(no recombination)

dnc

1(X)

3Sb

dnc

1(X)

rb B

dnc

1(X)

which chromosome is the dnc mutation on?

CHROMOSOMAL LINKAGE

dnc

1(X)

B

FM, B TM, Sb

Sb

3

Cy

2SM, Cy

dnc/Y; 2/2; 3/3 (males) mated to FM,B/+; SM,Cy/+; TM,Sb (females)

mated to male: (dnc/Y; 2/2; 3/3)

(female)

Look for missing class of animals for linkage

dnc/dnc; Cy/+; Sb/+ (present) dnc/dnc flies w/ B (absent)

GENETIC MAPPING(Recombination mapping - distance)

dncdnc

X

(female) (male)

ww

dnc = stupid (exptl)w = white eyes (marker)

(phenotypes?)

dnc

w

+

+(female double heterozygote)

Xdnc

w

+

+Xdnc

w

+

+

dnc

+

w

+

Xdnc+

w +

dnc+

w +

RECOMBINANTSdistance (eg. 2% = 2 map units)

GENETIC MAPPING

dncdnc

cv = missing wing vein(marker)

(Recombination mapping - direction)

dnc

w

+

+

dnc

w

+

+?

2 map units 2 map units

dnc

w

+

+ cv

+

THREE FACTOR CROSS

w-dnc 2 map unitsdnc-cv 3 map unitsw-cv 5 map units(if w & cv are known, unique solution)

POSITIONAL CLONING

w dnc cvmap

position

map intervalcontaining dnc

overlappingDNA clones

(chromosome walk)

25kb

1. clone entire map interval2. identify all genes (transcription units)3. transgene rescue (final identification)

DROSOPHILALEARNINGMUTANTS

dunce cAMP phosphodiesterasenamed after John Duns Scotus, 13thCopponent of classical learning

rutabaga adenyl cyclase turnip unknownradish unknown cabbage unknown

The vegetable series: because they're as dumb as vegetables

CaMKII Ca/calmodulin-dependent protein kinase IIdCREB2 cAMP response element binding protein (transcription factor)

amnesiac neuropeptidesimilar to mammalian pituitary adenyl cyclase activating peptide (PACAP)

linotte novel productnamed fr French expression tete de linotte having the same idiomaticmeaning as "bird-brain"

latheo unknownGreek for "forgetful" related to the Lethe River of Hades, a drink and youforget your former life

Professor Mark A. Tanouye April 16, 2000

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dCREB2 duncerutabaga

PACAP (amnesiac)