Genetic dissection 1) isolate a mutant in the process 2) study phenotype 3) genetic analysis reveals logic I. MUTATION Mutation- change in DNA sequence that can change information (protein or how and where to express protein) it encodes Types: 1) chromosomal mutations- 2) smaller insertions/deletions 3) point mutations A->T transition A->G transversion Amino acid effects of point mutations: tyrosine TAT, TAC TAT-> CAT tyr -> his misense TAT -> TAA tyr -> stop nonsense TAT -> TTT tyr -> phe neutral in many cases TAT -> TAC tyr-> tyr silent Genetic effects of mutation: a) null mutation- complete absence of activity b) loss of function - loss of most of activity c) gain of function- new function of gene d) suppressors- compensate for other mutations e) enhancer- enhances phenotype of a mutation II.MUTAGENESIS Spontaneous Mutations: Tautomers- mispairing Chemical changes transposons Environmental- UV Induced Mutations: x-rays, gamma rays- chromosome breakage- very penetrating UV light Chemical mutagens base analogs- 5 bromo U deamination by nitrous acid alkylation reagents intercalating agents Insertion mutations transposons T-DNA Target of mutagenesis somatic mutation- not inheritable gamete mutation- inheritable 2 problems in mutagenesis: 1) formation of mosaic plants from seed mutagenesis 2) plant is dipoloid- many mutations are hidden III. MUTANT SCREENING Screening- looking through mutagenized plants for one with an altered phenotype Screens- Visual screening Survival in certain conditions Biochemical stains- cyclic hydroximates in corn- turn blue with FeCl3 Biochemical intermediates that give fluorescence chlorismate->anthranilate-[trp1]>PKanthranilate->CDRP->indolGP-> trp -e.g. trpthophane biosynthesis- anthranilate is a fluorsecent Artificial reporter Luc gene inserted, controlled by promoter - mutants stop fluorescing Selection- making non-mutated die -antibiotic selection -herbicide resistance chlorosulfon -toxic intermediate nitrate reductase: NO3 -> NO2 trp pathway: 5-methylanthranilate ADH- alchohol dehydrogenase allyl alchohol -> acrolein aldehyde -auxotrophies mutation in biosynthetic pathway Lethal mutants housekeeping genes solutions: 1) maintain as heterozygotes - see dead seeds as homozygotes 2) look for a weak loss of function 3) look for conditional mutants- temperature sensitive IV. ANALYSIS OF MUTANTS 1) Recessive vs. Dominant alleles Test: cross heterozygote a + x a + -> aa, a+,+a, ++ if 1:3 mut:wt, then mutation is recessive if 3:1 mut:wt, then mutation is dominant significance: 1) interpretation dominance- often gain of function 2) recessive loss of function -mutant represents broken form 2) Allelism- does similar mutations represent same gene or multiple genes -Analyze from crossing two plants with independent mutations a) if mutation is recessive: complementation b) if mutations are dominant: segregation A) Complementation: -make homozygotes A1 x A2 plants - if F1 is mutant, then are allelic - if F1 is wt, then non-allelic B) Segregation test: Again cross homozygotes of mutants, however, in first generation
- always see mutant phenotype -second generation (F2) if see wt (expect in 1/16), then mutations are non-allelic
e.g. Maize kernel mutants screen for loss of red color c= colorless, recessive +=wt red female c1c1 c2c2 c3c3 c4c4 c5c5 c6c6 female Mutant wt wt Mutant wt wt c1c1 Mutant Mutant wt wt wt c2c2 Mutant wt wt wt c3c3 Mutant wt wt c4c4 Mutant Mutant c5c5 Mutant c6c6 Result: Complementation groups: c1, c4 c2, c3 c5, c6 3) Epistasis: Gene interactions Example- anthocyanin synthesis in maize kernels (click here for pathway) Mutations in biosynthetic pathway wt: red c2, a1, a2: colorless bz1, bz2: bronze double mutants: c2/a1 colorless- but uninformative bz1/a1 colorless - a1 comes before bz1 bz2/a1 colorless - a1 comes before bz2
-for biosynthetic pathway mutants, the phenotype of the earlier gene in the pathway shows in the double mutant
-feeding experiments:
add flavone (naringenin): c2=colorless; c2 +naringenin=red
a1=colorless; a1 + naringenin=colorless
Mutations in Regulatory pathway
many genes are controlled by a regulatory pathway
signal -> A -> B -> C -| D -> E -> expression
-> : positive action- stimulates next step
null mutation makes insensitive to signal
-| : negative action- represses next step
null mutation makes gene turn on at all times
double mutants
of B and C
see C phenotype, not B phenotype
in regulatory pathways, see downstream component in double mutants
Additive pathways
additive effect double mutants of dissimilar phenotypes produce a combination of phenotypes rather than one or another
-indicates that pathways are separate
V. MODEL SYSTEMS
Requirements:
grow in small space
short generation time
large number of progeny
small genome size
low amount of repeated DNA
easy to mutagenize
ability to self fertilize
ability to manipulate- transformation
Models:
Yeast
haploid and diploid stages
grow and select on medium
can transform easily
traits:
cell cycle
housekeeping processes
Chlamydomonas- green algae
haploid genetics
unicellular
grows synchronously
transformable- glass beads
traits:
photosynthesis
motility, light response
nitrate assimilation
Ceratopteris- fern
haploid and diploid stages
120 days generation time
prothallus grows in small space 106/m2
traits:
developmental mutants- sex determination
tolerance to salt, herbicides, toxins, O2 stress
Arabidopsis- mouse-eared cress
diploid only
dozens per 5-6 cm pot
5-6 weeks generation time
10,000 seeds per plant
self fertilization
Genome Sizes
N genome size Mb
E. coli 4.5
Yeast 15
Neurospora 42
C. elegans 80
Drosophila 160
Chlamydomonas 100
Arabidopsis 100
Tomato 714
Rice 970
Tobacco 1,600
Potato 1,900
Ceratopteris 5,000
Corn 5,000
Wheat 5,900
VI. EXAMPLE: Analysis of Flower Development
Diversity of flowers- shapes, numbers height of parts determine pollinator-plant interactions
-specialization for pollinator
-sexual dimoprphism- prevents outbreeding
-Darwin’s dilemma - why flowering plants diversified so quickly- 100 mill yrs.
-what determines pattern?
Arabidopsis- model
-small genome easier to study, hopefully simpler
- bonifide flower FLOWER MODEL?
A. Initial analysis- Meyerowitz
1. structure of arabidopsis flower
whorl 1: 4 sepals- protect developing flower
whorl 2: 4 petals
whorl 3: 6 stamens—male part of flower
whorl 4: 2 fused carpels - female part of flower
-focused on homeotic mutants - segments made in wrong place
-as will see- complex process so really messed up flowers are broken earlier in pathway so are harder to interpret
-homeotic are mutants that determine identity rather than ability to make it
2. 1st screen: 3 classes of mutants
Class A: APETELA 2 (AP2), APETALA 1 (AP1)- has carpels instead of sepals in whorl 1,
stamens instead of petals in whorl 2
Class B: APETELA 3 (AP3) and PISTALATA (PI) - sepals in whorl 2 instead of petals,
carpels in whorl 3 instead of stamens
Class C: AGAMOUS (AG) - Pattern: SEPALS PETALS PETALS SEPALS ......
so whorl 1 has sepals instead of stamens
whorl 4 has sepals instead of carpels
ABC Model to explain:
-seem to act on pairs of neighboring whorls -so not just one gene one whorl
-overlaps- so they may act together
A- act in whorl 1 +2
B- act in whorls 2 + 3
C- act in whorls 3 +4
Whorl 1: A only- sepal
Whorl 2: A + B function - petal
Whorl 3: B + C function- stamen
Whorl 4: C function only - carpel
to explain wide identity shifts in A and C mutants
need A+C to be mutually exclusive - mutation in one allows other to spread
3. Tests:
Double Mutants
AP2/AP3 - takes out A and B action- -carpels only in all whorls
AP3/AG - leaves only A function -sepals only
AP2/AG - leaves only B function-leaves found in whorls 1 +4
-organ intermediate between stamen and petals in whorls 2 + 3
Triple mutant
AP2/AP3/AG -only leaves in all whorls
B. Further dissection of complex pathway
1. Find more screwed up mutants reflecting earlier steps
- had to ignore in first model, otherwise could not include
-simplification good if pulls together in end
shoot or flower ® whorl or spiral ® organ identity
Superman: 4th whorl extra stamens (thus name)
-wt prevents B expression in 4th whorl (thru stimulating or stopping cell proliferation)
-Superman/AP3 double- looks like AP3- so supports model it only acts to stop B function
Leafy: lacks any petals or stamens- replaced by somewhat carpel
-also see arranged in spiral (like leaves) rather than whorls
-wt tells whorls 2 and 3 they are in flower = flower identity
-wt makes flower-like whorls instead of leaf-like spirals
Unusual Flower Organs (UFO): makes petal sepal intermediates and aborted leaves (filaments)
-also spiral organization instead of whorl
-partial conversion of flowers to shoots
-additive effects- different genes work together
-e.g. UFO and Leafy similar- leafy more global, UFO more specific to defining borders
-single genes function in multiple parts of pathway
- play role in controlling cell proliferation
2. Use current mutants to find enhancer or suppressor mutants- represent other genes in pathway
-Cauliflower1 (CAL1) isolated as making AP1 phenotype in meristerm identity more extreme
-UFO enhancers
C. With gene cloning- can address more specific parts of model:
1. specific tests
In Situ expression of each gene
all except AP2 found expressed in those whorls predicted to act in
Exogenous expression- overexpressing AG suppresses AP2
2. Determine function-
-sequence of genes suggest that they are transcription factors - MADS box proteins
-see localization of proteins to nucleus
3. Find more genes by using expression of cloned gene as a probe
4. Model applicable to other flowers as well? - Ultimate test of Arabidopsis as model
Snapdragon, many similar mutants
-homologs of all genes identified in arabidopsis
arabidopsis snapdragon
AP1 SQUAMOSA
AP3 DEFICEINS
PI GLOBOSA
AG PLENA
-can substitute for each other AP3 for DEFICEINS in snapdragon
-broad: AG homologs in Rape (Brassica napa) BAG1, petunia pMADS3, Rice OSMADS3 express in tobacco similar function
-interesting- more redundant genes in other plants
-maize, tomato, snapdragon- 2 AG homologs- one does not have all function of AG
-dioecious plants: sorrel- see modification in AP3 and AG to explain sex determination
-not seen in all dioecious plants
-Leafy constituitively expressed in Arabidopsis- turns shoots into flowers
-constituitive expression in aspen tree- get quicker flowering (usually have to wait 8-20
yrs for flowering)
LINKS:
Model Systems:
Chlamydomonas
information- Yahoo
Arabidopsis-Yahoo
list of sites
Ceratopteris
Arabidopsis Genome
Center- U. Penn
Arabidopsis Stock Center- location
which provides seeds of ecotypes, mutants etc.
Arabidopsis
Molecular genetics Lab Manual-Cold Spring Harbor
Lotus spp.
- genetic system of legumes (picture of plant and newsletter)
Mutation, screening and analysis
Protocol
for mutagenesis of seed with EMS- Cold Spring Harbor Protocols
Images of
Maize Mutants- Maize genome database
Images of tomato
mutants -Tomato genomics research center
Images
of Arabidopsis mutants-(loads faster than maize above)
Mutant screening lab
exercise- JMU
Epistasis-
Great article in PLSC431 NDSU
Virtual
Fly Lab- shows crosses with fruit flies
Flower Development
Yanofsky Lab
Flower page- lots of pictures of mutants
Genes
controlling flower development- PLSC731 NDSU
Last revised: Jan 2000- Straney