your view on genetics
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Your view on genetics. A: I am pretty good at it. B: I am not too familiar with genetics, but I am eager to learn more. C: I am not too familiar with genetics and I do not like it much. Just for fun. Which statement is closer to your belief?. - PowerPoint PPT PresentationTRANSCRIPT
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Your view on genetics
A: I am pretty good at it.
B: I am not too familiar with genetics, but I am eager to learn more.
C: I am not too familiar with genetics and I do not like it much.
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Just for funWhich statement is closer to your belief?
A: Biochemistry and Genetics are two distinct research fields. Every graduate student needs to choose between them for learning and research.
B: Biochemistry and Genetics interact closely in today’s research, but each lab should stay with one discipline and just collaborate with others.
C: Genetics and Biochemistry are two different research approaches that are no longer clearly separated. If needed, today’s students should use both to tackle biological problems.
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Using genetic alterations to dissect functions of gene products
- Development of one gene one enzyme concept.
- molecular lesions, biochemical defects and genetic natures
- The nature of mutations ("morphs").
- Deficiencies and duplications
- Genetic mapping.
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Development of one gene one enzyme concept
1900, Archibold Garrod recognized that absence of a functional enzyme causes certain inherited disorders in humans.
1911. Bateson recognized the link between genes and enzymes. Genes are inherited and enzyme is for phenotypes
1941. George Beadle and Edwin Tatun: one gene one polypeptide
- set up by earlier work in Drosophila work on eye colors in 1935 at Caltech and Europe. - Genetic control of biochemical reactions in Neurospora.
(PNAS 27: 499-506). Work done at Stanford.
- Nobel Prize in 1958. Credit to Garrod
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George Wells BeadleEdward Lawrie Tatum Born in Boulder
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Mutant
arg 1
Grwoth medium
Minimal
-
Minmal+ Arginine
+
Minmal+citulline
-
Minmal+Ornithine
-
ornithine citulline argininearg 1arg 2-3arg 4-7
Srb and Horowitz, 1944
arg 2-3 - + + -
arg 4-7 - + + +
A B C
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Gene is a stretch of DNA
1926 Fredric Griffith showed that hereditary substance could be transferred from dead bacteria to living bacteria
1944. Oswald Avery et al. show that the Griffith’s substance is DNA
1953. DNA structure. Watson and Crick deduced the structure
1953. S. Benzer demonstrated intragenic recombination in phage: gene is a segment of DNA.
1960s. Charles Yanofsky: linear relationship between mutations in the NT sequence and changes in AA sequence of the protein
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Using genetic alterations to dissect the functions of gene products
- Development of one gene one enzyme concept.
- molecular lesions, biochemical defects and genetic natures
- The nature of mutations ("morphs").
- Deficiencies and duplications
- Genetic mapping.
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Mutation: heritable change in the nucleotide sequence of a cell’s DNA
mutation
Spontaneous mutationDepurinationDepyrimidinationcytosine deamination
Induced mutation (by mutagen) radiation (ionizing, nonionizing)
chemicals (Base analogs, intercalating agents)
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Mutation
Point mutation
Chromosome change
Transposable elementsP element, TC, sleeping beauty etc
Same sensemissensenonsense
Substitution
Deletion
insertion
frameshift
inframe
DeletionDuplicationInversionsTranslocationFission and fusion
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Using genetic alterations to dissect functions of gene products
- Development of one gene one enzyme concept.
- molecular lesions, biochemical defects and genetic natures
- The nature of mutations ("morphs").
- Deficiencies and duplications
- Genetic mapping.
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The nature of mutations ("morphs")
- loss-of-function mutations
- hyperactive mutations
- dominant negative mutations
- Change-of-function (neomorphic) mutations.
- phenotypes created by over-or misexpression
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Before talking about morphsLet us first make sure we understand:
Recessive mutations m/m with phenotype
Dominant mutations m/+ with phenotype
Statement 1: most human diseases are recessive.A: yes. B: no. C: not sure.
Statement 2:Most of oncogenes contain dominant mutations.A: yes. B: no. C: not sure.
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Loss-of-function lof or lf
Null, KO, amorph
Reduction-of-function, KDor partial loss-of-function = hypomorph
- Recessive ? - What situation is dominant? - What is hyploid-insufficiency? - Caused by what type lesions?
Nonsensemissense
deletioninsertion
chromosomalrearrangement
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Gain-of-function mutation = hyperactive mutation
Narrow definition and often used:
Broader definition that fits the meaning of the word:
Gain-of-function
Hyperactive = hypermorph
dominant negative < antimorph
Neomorphic
Misexpression = neomorph/hypermorph
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Hyperactive mutations = hypermorph, let us call it gf
- Protein (enzyme) is more active than wt- Protein activity can no longer be turned off- Protein was expressed at a higher level
transcriptional controltranslational controlRNA or protein stability
Genotype Phenotype gf/gf mutantgf/+ may be mutantgf/null ?gf/df ? gf/gf/+ gf/+/+
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Exercise
1. Compare the phenotype severity between gf/null mutants and gf/gf mutations. gf/gf is more severe. A: yes, B: no.
2. Compare gf/null with gf/+A: gf/null is less severe than gf/+B: gf/null is more severe than gf/+
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Dominant negative, antimorphic
The mutant gene has a negative effect in the same direction asloss-of-function mutations. Its product is toxic to the wild-typeprotein in a dn/+ heterozygote. It competes with wild type.
dn/dn > dn/null > +/dn > +/null ~ +/+
having dn is worse than having null
Mechanisms:1. Competes with wt for another positive factor - common2. Forms a non-functional multimers with wt.
Please read Herskowitz’s review in 1987. He made the proposal without experiments
Null/+ wild type phenotypeDn/+ mutant phenotype
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Neomorphic: the mutant gene generate a new function that
is different from its normal role.
Key: adding normal gene copy neither enhance its phenotype nor reduce its phenotype.
neo/+ = neo/+/+ (regarding the new phenotype)
Protein changed its activity to do something differentProtein binds to another protein that the wt does not bind
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ExerciseGene A is normally expressed only in muscle cells. Gene A(lf) cause muscle reduction. Gene A (gf) causes over production of muscle.
A mutation in gene A’s promoter, cause it to be expressed at a high level in skin and abnormal skin development.
Is this mutation a gf allele, or Neo allele?
How do we determine that? Do we need to?
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Using genetic alterations to dissect functions of gene products
- Development of one gene one enzyme concept.
- molecular lesions, biochemical defects and genetic natures
- The nature of mutations ("morphs").
- Deficiencies and duplications
- Genetic mapping.
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Deficiency (Df) = deletion of a segment of chromosome
Duplication (Dp) = duplication of a segment of chromsome 1. Free duplication = small extra chromosome 2. Attached duplication, more stable.
Regarding a particular gene in dp, dp is not same as adding a copy of the gene. However, the side effect is smaller than df.
Key: Df reduces the dosage of many genesDf/+ is not exactly the same as null/+ because dosage effects of other genes in Df likely exist.
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Ras biochemistry
-GTPase : cycle betwen GTP and GDP -functional switch
- Activator SOS for the exchange reaction- Negative: GAP- Effector region
RASGDP
RASGTP
Pi
GTPGDP
target
ActiveInactive
SOS
GAP
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QuickTime™ and aGIF decompressor
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Three-d structure
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QuickTime™ and aGIF decompressor
are needed to see this picture.
QuickTime™ and aGIF decompressor
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QuickTime™ and aGIF decompressor
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QuickTime™ and aGIF decompressor
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Ras oncogene always on. Lack of GTPasev12E13E61
Not dependent on Sos,GAP has no role on it.Still binds to targetStill binds to GAP
RASGDP
RASGTP
Pi
GTPGDP
target
ActiveInactive
SOS
GAPX
Question: are Ras oncogenes gf alleles?
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1. ~ 1000 cells, small, easy to do genetics.
2. Entire lineage and nerve system mapped.
3. Entire genome has been sequenced.
4. A very popular model system.
Caehorhabditis elegans.
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Figure 8.2. Life cycle of C. elegans
Fertilized egg
egg laid
Hatching/L1 larvae
mb
ryo
ge
ne
s is
~11.5 hrs
L2 larva
L3 larva
~7hrs
~7.5hrs
L4 larva
~9.5hrs
adult
go
na
do
ge
ne
sis
Sp
erm
toa
ge
ne
sis
oo
ge
ne
s is
dauer larva
food
starvation
(many months)
eggs
~14 hrs
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Figure.8.3. The sexes of self-fertilized and cross-fertilized C. elegans progeny.
Sperm Oocyte Sperm
gametes X X OX
50% 50%100%100%
XX
100%
XX
50%
XO
50%
Self-progeny cross-progeny
XX XO
meiosis
Hermaphrodite Male
fertilization
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cell migrationhox genes
gonadP1 P12dorsal
3, morphogenesis cell division, fusion,
migration, etc.
Cell fate 3° 3° 2° 1° 2° 3°
Lineage
1, precursor cells
2, vulval induction RTK/Ras/MPK signaling Notch Signaling etc.
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AC
3° 3° 2° 1° 2° 3°WT
X
3° 3° 3° 3° 3° 3°- AC
Indicating:
- AC is required for vulval induction
- AC may send a signal to induce vulval cells
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signal pathway function
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anchor cellinductive signal
E E V V V E
Wild type 100% induction
signal Ras function
E E EEEE
Vulvaless 0%ras(lf)/ras(lf)ras(dn)/+
Multivulva 200%
V V V V V V
ras(gf)/ras(gf)
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Let us work on things
1. Isolated a Vulvaless mutant, called mutant sy94
sy94/+ Vulvalesssy94/sy94 more severe, die early
Question: lf (A) gf (B) Dn (C) or Neo (D) ?
Df/+ is wild type, so it is not haploid insufficient (lf).
What is the key to make the distinction?
What do we do?
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1. Isolated a lf mutant. Revertant screen dn/+ becomes null/+
2. Determined the phenotype for lf alleles
Making the null/sy94 strain
3. Df and Dp test.
If sy94 is a gf, is sy94/null more severe than sy94/+ ?A: yes, B: no.
If sy94 is a dn, is sy94/null more severe than sy94/+ ?A: yes, B: no.
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ras genotyhpe Under induction
dn/dn
dn/Df
dn/dn/Dp
dn/+
dn/+/dp
Lethal
Lethal
96%
59%
0% Ph
eno
typ
e s
eve
rity
Ge
ne
ac
tiv
ity
/do
sag
e
Dosage analysis of dominant mutations in the ras gene in C. elegans.
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Mechanism of dn of Ras
RasGDP
RasGTP
GNEF
GAP
GDP GTP
inactive active
RasGTP
gain-of-function (gf) or oncogenic
GAP
Pi
Ras GNEF
dominant negative (dn)
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gf story:
1. Determine that it is in the same gene as that in dn
2. Determine that it is gf, not dn, not neomorphic
3. Oncogene connection.
ras genotyhpe Multivulva
gf/gf/Dp
gf/gf
gf/+/Dp
gf/+
gf/Df
ras genotyhpe Under induction
dn/dn
dn/Df
dn/dn/Dp
dn/+
dn/+/dp
Lethal
Lethal
96%
59%
0%
100%
93%
53%
23%
8%Ge
ne
ac
tiv
ity
/do
sa
ge
Ph
en
oty
pe
se
ve
rity
Ph
en
oty
pe
se
ve
rity
Ge
ne
ac
tiv
ity
/do
sa
ge
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Question
John is studying the nature of a mutation in gene A in the fly. He found that m/m has a severe mutant phenotype. m/+ has a very weak phenotype.
He introduced an additional copy of the wild type gene (using transposible element) into the m/m mutant and found the m/m/+ animals are significantly less severe in the phenotype.
A: m is dn mutationB: m is gf alleleC: m is a lf allele
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question
A: gf/gf/+ is always less severe than gf/gfB: gf/gf/+ is always more severe than gf/gfC: can be either
Regarding the mutant a phenotype caused by a gf mutation