Your view on genetics

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 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.

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 BeadleEdward Lawrie Tatum Born in Boulder

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

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





- Genetic mapping.

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)

Mutation

Point mutation

Chromosome change

Transposable elementsP element, TC, sleeping beauty etc

Same sensemissensenonsense

Substitution

Deletion

insertion

frameshift

inframe

DeletionDuplicationInversionsTranslocationFission and fusion






- 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 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.

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

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

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/+/+

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 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

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

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?






- Genetic mapping.

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.

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

QuickTime™ and aGIF decompressor

are needed to see this picture.

Three-d structure

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?

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.

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

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

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.

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

signal pathway function

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)

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?

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.

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.

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)

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

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

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

Your view on genetics

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