Chapter 4- Genes and development

Embryology

Genetics1920s

Hostility

Genetics

Genetics

EmbryologyEmbryology

1970s

_______________- expression of traits________________-transmission of traits

1870s- What controls inheritance- ___________ or _______________?

Huge conceptual problems(Early 1900’s)

1. Can identical chromosomes really result in distinct cell types?- defined as _______________________________

2. Do genes ___________ embryogenesis?

3. How can ___________________ affect sex in reptiles?

1. Can identical chromosomes really result in distinct cell types?- defined as genome equivalence

• _____________________ observed (transformation of one differentiated cell type to another, e.g. iris cells become lens cells (1950s)

• Amphibians can be cloned from

intestinal _________ cells (1960s) -nuclear transfer techniques developed

• Sheep cloned from ________ tissue– (1997)- “Dolly”, then cows and mice (1998)

Evidence for

Fig. 4.8

Differential gene expression

1. The DNA of all ______________ cells are identical in a given __________

2. All ____________ genes can be expressed

3. Only a ____________ of genes are expressed in a given _______

Three tenets

Techniques to know to understand differential gene expression

1. ___________- Detect DNA

2. _____________- Detect RNA

3. ___________ Detect protein

4. ____________________ Detect DNA or RNA

5. __________________ to amplify copies of DNA

1. Northern blot

1. ______ RNA2. ______ on gel

3. _______ (blot) onto nylon membrane

4. _______ membrane with radiolabled

RNA/DNA

A developmental Northern blotFig. 4-14

2. Southern blot

Note- ______________________ are proteins that recognize specific double-stranded DNA sequences and cleave the DNA

Similar to __________ blot, except 1. Must chop up DNA to smaller pieces (restriction enzymes or general shearing with strong acid)

3. Western

1. Load isolated protein onto a ____.2. _________ onto nylon membrane3. _______ with _________ to specific proteins (these ________

can be conjugated to fluorescent molecule or an enzyme fordetection)

4. ____________________- shows exactly where protein is expressed in organism

1. Mount thin slices of _______onto slide. 2. Incubate with _________ probe

Whole-mount in situ hybridization is also possible

a. Add Dioxygenin-labeled DNA

b. Add antibodies (alkaline phosphatase linked)

to Dioxygenin

c. Add alkaline phosphatase

substrateFig. 4.16

Fig. 4.15

5. Polymerase Chain Reaction

(PCR)

One copy

many copiesFig. 4.17

Genome-wide analysis- yeast

______________ organisms- a great way to study gene function

• Getting the DNA (a gene) into a cell-– ______________– __________________ (mix DNA with cells)– Retrovirus _____________ (infect a cell)

• P element in Drosophila- a transposable element that allows a gene to be inserted into specific positions in the fly genome

The _______________ mouse

1. Isolate _________________ cells from trophoblast, expand and transfect 2. Introduce modified ES cells

into new _____________3. Place modified

trophoblast into uterus, then cross the ___________ normal mice

4. Cross two _________________

to generate a homozygote

Fig. 4.19

Gene targeting (______________)

1. Isolate _________________ cells from trophoblast, expand and _________

with __________________ gene

2. _________and expand ES cells with integrated transgene

3. Generate heterozygote, then ______________ mice

Other techniques• _______________ to inhibit function-

antisense RNA produced binds to mRNA and cause degradation– __________________________- short RNA

fragments result in degradation of specific mRNA

Fig. 4.23

Example- antisense Kruppel affects fly development

Human ES cell transplantation therapy- ethics

Fig. 4.22

From fertilized _______ES cell lines established – ___ lines available

ES cells from developing embryocoaxed into _______cell types

Chapter 5- Differential Gene Expression

DNA wrapped around histones

UAA

AUGPre-mRNA

3. _____________

NH2 COO- Protein7. ________________

AUG

AAAAAA…..

D CA BE

7mG

UAA

F

4._________

Chromosome- 1.5 x 108 base pairs containing about 3000 genes

0.4% of a chromosome, containing 10 genes

ATGDNA

CB EDFTAA

5’3’

3’5’

+1 AATAAA

5. ______ 6. ____________

1. Exon

2. Intron

Promoter

Cap PolyA

Translation

Transcription

RNA Processing

mRNA

Overview of eukaryotic transcription/translation

Also see figs. 5.2 and 5.3

Chromatin and transcription

A. Levels of organization

• DNA must be compact- each mammalian cell has _______________ of DNA

a._______________- 700 nmb. ______________- 200 nmc. ______________-30 nmd. ________________ -10 nm- histones + DNA

Chromatin and transcription (Chapter 13) (continued)

B. ________________• Large amount of ________ residues, positive charge

•_______________ of histone tails ________________ as gene activity increases

Peptide-CH-CH2-CH2-CH2-CH2-NH3 Peptide-CH-CH2-CH2-CH2-CH2-NH-C-CH3

Histone tails

O

Acetylation of lysine

• can interact with DNA • five types of histones

•________________________

•H1 in _______ regions (8-14 bp)

H2A, H2B, H3 and H4 form _________ around ________ of DNA

H4

Ac

AcAc

AcH3

H2BH2A

H4

Ac

AcAc

AcH3

H2BH2A

Two type of HATs•________- in nucleus, involved in gene regulation e.g. p55 and Gcn5•________ - in cytoplasm, acetylates newly made histones

•Note- CBP (a coactivator) and TAFII250 (a TFIID subunit)and P/CAF also display HAT activity

-Three histone acetyltransferases

How do histones get acetylated? By ________________________________)

G. Histone deacetylation____________________ bind to _________________________ and to histone deacetylase to repress transcription

Examples of co-repressors - Sin3A and Sin3B (mammals) - NcoR/SMRT (mammals)

- Model for histone ______________________ in transcriptional repression

How do histones get acetylated? By Histone deacetylases (HDACs)

GCGC CCAAT TATA

A typical eukaryotic Pol II promoter

-30+1

Enhancer-70-110

2. _____________- orientation /position independent• affects level of transcription, not determine _______ status•contains DNA sequences which _______ transcription factors •A major determinant of ____________ gene transcription

Transcription inititation Regulatory elements- RNA Pol II

1. __________________•TATA box- initiate __________________, fixed position •________________ at -70 •_____________ at -110•_______________ promoters instead of TATA

•“________________” genes (constitutively active in most cells)• some “___________ “genes (tissue specific expression)

_______ binds TATA box via ______, followed by ordered binding of other factors

Activating transcription

RNA Polymerase ________________ now binds and initiates transcription

Fig. 5.4

Function of __________1. interaction with core ___________•TAFs extend area of protection to +35 bp•Question- Which other TAFs interact with DNA?2. Enable TBP to bind _________________ promoters.

4. Chromatin remodeling- Example: TAF250 is histone acetyl transferase (HAT)

3. Interact with upstream _____________

TFIID = TBP + several TAFs

How do we achieve differential transcription?

___________________ (DNA binding proteins, transacting factors) 1. ________________ proteins 2. bind to specific DNA sequences on promoter or enhancer 3. modify transcription of gene by altering _________________ loading

Transcription activators in eukaryotes

Trans-acting factor

DNA binding domain Activation domain(30-100 amino acids)

NH3 COOH

1. _______________ domains-• Zinc-containing (e.g. zinc finger)• homeodomain- 60 AA• -barrels• b-ZIP and bHLH motifs

2. _______________________ domains•acidic domains•Glutamine-rich domians•Proline-rich domains

3. ________________________ domaine.g, Leucine zipper

Protein-protein interaction domain

ZIP domainZIP domain

Protein #2 Protein #1

Fig. 12.14- Model of Leucine Zipper

Protein #2 Protein #1

Fig. 12.14- Model of Leucine Zipper

• Leucines are spaced 7 AA apart

• Leucines are spaced 7 AA apart

DNA binding region(“b” domain)

DNA binding region(“b” domain)

Three domains (domain- a cluster of amino acids that carry out specific functions)

An example of a developmental transcription factor

• MITF is a transcription factor that activates pigment genes

DNA

___________________ domain

___________________ interaction domain

__________________ domain

CBP

Fig. 5.8

How do we determine where a given enhancer/promoter is active?

Answer- Fuse enhancer/promoter to _______________ (B-gal) or __________________ (GFP) gene, then introduce the fusion gene into the organism

Muscle-spec. promoter B-Gal

Eye-specific promoter GFP

Fig. 5.7

HNF3

HNF4

HNF1C/EBP

HNF3

Liver-specific genes

2. Tissue-specific expression is due to __________________ of transcription factors

1. Hepatic gene regulation occurs primarily at the level of ___________________________

Big themes in regulation

Anterior-posterior axis

Primitive streak

Endoderm differentiation

Foregut endoderm migration into mesenchyme

Organ formation

Liver

Hepatic Determination

Onset of liver gene expression

Amplification of liver gene expression

HNF3

HNF3

HNF3, HNF4

HNF1, c-jun

C/EBP

Transcription factors in early liver development

Other forms of gene regulation1. What activates expression of the activators?

The Pax6 gene has 4 distinct ______________, each utilized in four distinct_________ to drive Pax6 transcription

Fig. 5.152. ____________- sequences that block ________________

Albumin expression silenced by inhibitor until birthAlbumin gene promoter

L1 promoter is silenced in all tissues except neuronal due to silencer element NRSE

Fig. 5.16

Fig. 5.17

Delete NRSE sequences

Silencer elements are rare!

Pancreas Lens Neural tube Retina

• Five erythroid-specific genes• Arranged in _____________________• LCR is upstream cluster of 5 (actually 7) _______• Each HS site binds numerous _______

3. _________________- Human -globin gene clusterOther forms of gene regulation

Proposed LCR functions

• Open ______________• prevent variegated ______________________• Affects timing of _________________• Keeps promoters ___________-free• Change subcellular localization of locus• LCR transcription affects rest of locus expression • Recruit ________

4. ___________-a major method of transcriptional regulation in vertebrates

Other forms of gene regulation

Model- Methylation represents a biochemical specialization of large genomes that participate in allele-specific expression, whereas differentiation does not depend on covalent modification.

Globin gene cluster ________

Fig. 5.20

•3% of Cs are methylated in mammals, 90% of these at CG

•As methylation increases, transcription decreases•GC-rich regions are preferentially found in 5’ regions•mice lacking methyl transferase die during embryogenesis•Model- methylation groups interfere with factor

binding on DNA•Importance of methylation question due to lack of methylation in Drosophila•CG sequence occurs at only 10% of expected frequency

70-80% of these are methylatedpatterns reset during gamete formation

•methylation status of a panel of tissue-specific genes could not be correlated with expression in tissues of fetal and newborn mice •Methylation deficient mice- observe biallelic expression of imprinted genes.

4. Methylation-continued

Interesting methylation facts

5. Genomic _______________

• Differential expression of two _______alleles

• Only occurs in ________(placental, nonmarsupial) mammals

• Not in other ___________

• Of 20-some identified genes

• Many involved in• _____________

– Igf2, IgF2r, H19, Grb1

• ________________– Prader-Willi syndrome PS)– Angelman syndromes (AS)– Peg1/Mest

A potential mechanism of genomic imprinting

•A single enhancer drives either the Igf2 or the H19 gene, but ______.

•____________ binding prevents enhancer from acting on Igf2 gene.

•CTCF cannot bind if region is __________; hence Igf2 is expressed.

male

female

6. X chromosome ___________-

A. Introduction- ____________ first described in females in 1949 _____ syndrome (45,X) are Barr body negative;

________syndrome (47, XXY) are Barr body positive

___hypothesis- one of the two X chromosomes in female is inactivated; all but one is inactivated if multiple X chromosomes - referred to as “________________________”

• Introduction- X-chromosome inactivation occurs at _______ of

embyrogenesis Inactivation process is _______ Inactivation state __________ throughout life

• A few genes remain active in the inactive X chromosome, including XIST at Xq13

X chromosome inactivation7. Dosage compensation

Other forms of gene regulation

Dosage compensation comparisons

2-fold ________in males

2-fold ________in females

Stably inactivate ___ Xchromosome

2X

1X

2X

1X

Xist is necessary and sufficient for X inactivation (using 450kb YAC)

insert Xist transgene on autosome results in inactivated autosome

X-inactivation- observations

mouse autosome

11

12

13

21

24

12

13

14

p

q

Xist RNA

Inactivated X chromosome

Mechanism in mammals

If mutate Xist promoter- preferential X inactivation on chromosome with mutation– possibly due to failure to

compete with blocking factor

X-inactivation

Preferential inactivation

Delete Xist exons 1-5- mutant chromosome chosen but not inactivated

Prevent inactivation

delete

Xist Gene

Blocking factors

Random inactivation

8. Differential RNA ____________

Other forms of gene regulation

a. RNA selection-only certain RNAs are exported to _________

b. Differential RNA ____________-

Different spliced forms of a RNA

9. RNA ____________

Other forms of gene regulation

a. mRNA longevity- minutes to _____

b. Selective_______ of translation-

e.g. the C. Elegans lin-4 RNA binds lin-14 mRNA to ________ translation

Fig. 5.32

Lin-14 RNA

Lin-4 RNA

Lin-14 mRNA Untranslated

region