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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?
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.
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.
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
George Wells Beadle
Edward Lawrie Tatum
Born in Boulder
Grwoth medium
Mutant
Minimal
Minmal
+ Arginine
arg 1
-
+
-
-
arg 2-3
-
+
+
-
arg 4-7
-
+
+
+
arg 4-7
Minmal
+Ornithine
arg 1
arg 2-3
ornithine
A
Minmal
+citulline
citulline
B
C
arginine
Srb and Horowitz, 1944
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
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.
Mutation:
heritable change in the nucleotide sequence of a cell’s DNA
Spontaneous mutation
Depurination
Depyrimidination
cytosine deamination
mutation
Induced mutation (by mutagen)
radiation (ionizing, nonionizing)
chemicals (Base analogs,
intercalating agents)
Substitution
Same sense
missense
nonsense
Point mutation
Deletion
frameshift
insertion
inframe
Mutation
Transposable elements
P element, TC, sleeping beauty etc
Deletion
Duplication
Chromosome change
Inversions
Translocation
Fission and fusion
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.
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
Before talking about morphs
Let 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.
Null, KO, amorph
Loss-of-function
lof or lf
Reduction-of-function, KD
or partial loss-of-function
= hypomorph
- Recessive ?
- What situation is dominant?
- What is hyploid-insufficiency?
- Caused by what type lesions?
Nonsense
missense
deletion
insertion
chromosomal
rearrangement
Narrow definition and often used:
Gain-of-function mutation = hyperactive mutation
Broader definition that fits the meaning of the word:
Hyperactive = hypermorph
Gain-of-function
Misexpression = neomorph/hypermorph
dominant negative < antimorph
Neomorphic
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 control
translational control
RNA or protein stability
Genotype
gf/gf
gf/+
gf/null
gf/df
gf/gf/+
gf/+/+
Phenotype
mutant
may be mutant
?
?
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/+
Dominant negative, antimorphic
The mutant gene has a negative effect in the same direction as
loss-of-function mutations. Its product is toxic to the wild-type
protein in a dn/+ heterozygote. It competes with wild type.
Null/+ wild type phenotype
Dn/+
mutant phenotype
dn/dn > dn/null > +/dn > +/null ~ +/+
having dn is worse than having null
Mechanisms:
1. Competes with wt for another positive factor - common
2. Forms a non-functional multimers with wt.
Please read Herskowitz’s review in 1987. He made the proposal
without experiments
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 different
Protein binds to another protein that the wt does not bind
Exercise
Gene 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?
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.
Deficiency (Df) = deletion of a segment of chromosome
Key: Df reduces the dosage of many genes
Df/+ is not exactly the same as null/+ because
dosage effects of other genes in Df likely exist.
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.
Ras biochemistry
-GTPase : cycle betwen GTP and GDP
-functional switch
- Activator SOS for the exchange reaction
- Negative: GAP
- Effector region
GDP
SOS
RAS
GDP
Inactive
GTP
RAS
GTP
GAP
Pi
target
Active
Three-d structure
QuickTime™ and a
GIF decompressor
are needed to see this picture.
QuickTime™ and a
GIF decompressor
are needed to see this picture.
QuickTime™ and a
GIF decompressor
are needed to see this picture.
QuickTime™ and a
GIF decompressor
are needed to see this picture.
QuickTime™ and a
GIF decompressor
are needed to see this picture.
Ras oncogene always on. Lack of GTPase
v12
E13
E61
Not dependent on Sos,
GAP has no role on it.
Still binds to target
Still binds to GAP
GDP
SOS
RAS
GDP
GTP
RAS
GTP
target
X
Inactive
GAP
Pi
Active
Question: are Ras oncogenes gf alleles?
Caehorhabditis elegans.
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.
Figure 8.2. Life cycle of C. elegans
Fertilized egg
egg laid
~14 hrs
Hatching/L1 larva
~11.5 hrs
gonadogenesis
starvation
L2 larva
~7hrs
dauer larva
(many months)
L3 larva
~7.5hrs
L4 larva
~9.5hrs
adult
eggs
food
Figure.8.3. The sexes of self-fertilized and cross-fertilized C. elegans progeny.
Hermaphrodite
Male
XO
XX
meiosis
Sperm
gametes
Oocyte
Sperm
X
X
X
O
100%
100%
50%
50%
fertilization
XX
100%
Self-progeny
XX
XO
50%
50%
cross-progeny
1, precursor cell s
dorsal
P1
cell migration
hox genes
gonad
P12
2, vulval induction
RTK/Ras/MPK signaling
Notch Signaling etc.
Cell fate
Lineage
3, morphogenesis
cell division, fusion,
migration, etc.
3°
3°
2°
1°
2°
3°
AC
WT
3°
3°
2°
1°
2°
3°
3°
3°
X
- AC
3°
3°
3°
3°
Indicating:
- AC is required for vulval induction
- AC may send a signal to induce vulval cells
signal
pathway
function
anchor cell
inductive signal
E
E
V
V
Wild type
E
E
E
ras(lf)/ras(lf)
ras(dn)/+
V
V
signal
V
E
100% induction
E
E
E
Vulvaless 0%
V
Multivulva
V
200%
ras(gf)/ras(gf)
V
V
Ras
function
Let us work on things
1. Isolated a Vulvaless mutant, called mutant sy94
sy94/+ Vulvaless
sy94/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?
1. Isolated a lf mutant.
Revertant screen dn/+ becomes null/+
2. Determined the phenotype for lf alleles
Making the null/sy94 strain
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.
3. Df and Dp test.
ras genotyhpe Under
induction
dn/dn
Lethal
dn/Df
Lethal
dn/dn/Dp
96%
dn/+
59%
dn/+/dp
0%
Phenotype severity
Dosage analysis of dominant mutations in the ras gene in C. elegans.
Mechanism of dn of Ras
GDP
GTP
GNEF
Ras
GDP
GAP
inactive
GAP
Pi
Ras GNEF
dominant negative (dn)
Ras
GTP
active
Ras
GTP
gain-of-function (gf)
or oncogenic
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.
gf/gf/Dp
100%
gf/gf
93%
gf/+/Dp
53%
gf/+
23%
gf/Df
8%
ras genotyhpe
Under induction
dn/dn
Lethal
dn/Df
Lethal
dn/dn/Dp
96%
dn/+
59%
dn/+/dp
0%
Phenotype severity
Multivulva
Phenotype severity
ras genotyhpe
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 mutation
B: m is gf allele
C: m is a lf allele
question
Regarding the mutant a phenotype caused by a
gf mutation
A: gf/gf/+ is always less severe than gf/gf
B: gf/gf/+ is always more severe than gf/gf
C: can be either