Transcript Chapter 4- Genes and development

Chapter 4- Genes and
development
_______________- expression of traits
________________-transmission of traits
Embryology
Genetics
Embryology
1920s
Hostility
Genetics
1870s- What controls inheritance___________ or _______________?
Embryology
Genetics
1970s
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
Evidence for
• _____________________ 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)
Fig. 4.8
Differential gene expression
Three tenets
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 _______
Techniques to know to understand differential gene
expression
1.
2.
3.
4.
___________- Detect DNA
_____________- Detect RNA
___________ Detect protein
____________________ Detect DNA or
RNA
5. __________________ to amplify copies
of DNA
1. Northern blot
1. ______ RNA
2. ______ on gel
3. _______ (blot) onto nylon membrane
4. _______ membrane
with radiolabled
RNA/DNA
Fig. 4-14
A developmental
Northern blot
2. Southern blot
Similar to __________ blot, except
1. Must chop up DNA to smaller pieces (restriction enzymes or
general shearing with strong acid)
Note- ______________________ are proteins that recognize specific
double-stranded DNA sequences and cleave the DNA
3. Western
1. Load isolated protein onto a ____.
2. _________ onto nylon membrane
3. _______ with _________ to specific proteins (these ________
can be conjugated to fluorescent molecule or an enzyme for
detection)
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 Dioxygeninlabeled DNA
Fig. 4.16
b. Add antibodies (alkaline
phosphatase linked)
to Dioxygenin
c. Add alkaline
phosphatase
substrate
Fig. 4.15
5. Polymerase
Chain Reaction
(PCR)
Fig. 4.17
One copy
many copies
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
3. Place modified
trophoblast
into uterus,
then cross the
___________
normal mice
Fig. 4.19
2. Introduce modified ES cells
into new _____________
4. Cross two
___________
______
to generate a
homozygote
Gene targeting (______________)
3. Generate heterozygote, then
______________ mice
1. Isolate _________________ cells
from trophoblast, expand and _________
with __________________ gene
2. _________and expand ES cells with integrated transgene
Other techniques
• _______________ to inhibit functionantisense RNA produced binds to mRNA
and cause degradation
– __________________________- short RNA
fragments result in degradation of specific
mRNA
Exampleantisense
Kruppel affects
fly
development
Fig. 4.23
Human ES cell transplantation therapy- ethics
From fertilized _______
ES cells from
developing embryo
coaxed into _______
cell types
Fig. 4.22
ES cell lines established –
___ lines available
Chapter 5- Differential Gene
Expression
DNA wrapped around histones
Overview of eukaryotic transcription/translation
Chromosome- 1.5 x 108 base pairs containing about 3000 genes
mRNA
Also see figs.
5.2 and 5.3
0.4% of a chromosome, containing 10 genes
1. Exon
5’
3’
ATG B
C
D
E
F
TAA
AATAAA
+1
3’
5’
DNA
2. Intron
3.Transcription
_____________
AUG
Pre-mRNA
Promoter
4._________
RNA
Processing
Cap
5. ______
AB
7mG
AUG
NH2
CD
UAA
PolyA
6. ____________
E
F
AAAAAA…..
UAA
Translation
7. ________________
COO-
Protein
Chromatin and transcription
A. Levels of organization
• DNA must be compact- each mammalian
cell has _______________ of DNA
a._______________- 700 nm
b. ______________- 200 nm
c. ______________-30 nm
d. ________________ -10 nm- histones + DNA
Chromatin and transcription (Chapter 13) (continued)
B. ________________
• Large amount of ________ residues, positive charge
• can interact with DNA
• five types of histones
•________________________
•H1 in _______ regions (8-14 bp)
H2A, H2B, H3 and H4 form
_________ around ________ of
DNA
Peptide-CH-CH2-CH2-CH2-CH2-NH3
Histone
tails
Ac
Ac
Ac
Ac
Ac
Ac
H4
H3
H4
H3
H2A H2B
H2A H2B
Ac
Ac
Peptide-CH-CH2-CH2-CH2-CH2-NH-C-CH3
O
Acetylation of lysine
•_______________ of histone tails ________________ as gene activity increases
How do histones get acetylated?
By ________________________________)
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 Histone deacetylases (HDACs)
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
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)
+1
Enhancer
-110
GCGC
-70
CCAAT
-30
TATA
A typical eukaryotic Pol II promoter
2. _____________- orientation /position independent
• affects level of transcription, not determine _______ status
•contains DNA sequences which _______ transcription factors
•A major determinant of ____________ gene transcription
Activating transcription
_______ binds TATA box via
______, followed by ordered
binding of other factors
RNA Polymerase
________________ now binds
and initiates transcription
Fig. 5.4
Function of __________
1. interaction with core ___________
TFIID =
•TAFs extend area of protection to +35 bp
•Question- Which other TAFs interact with DNA?
2. Enable TBP to bind _________________ promoters.
TBP + several TAFs
3. Interact with upstream _____________
4. Chromatin remodeling- Example: TAF250 is histone acetyl transferase (HAT)
How do we achieve differential
transcription?
Transcription activators in eukaryotes
___________________ (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
Three domains (domain- a cluster of amino acids that carry out specific functions)
1. _______________ domains• Zinc-containing (e.g. zinc finger)
• homeodomain- 60 AA
• b-barrels
• b-ZIP and bHLH motifs
2. _______________________ domains
• acidic domains
• Glutamine-rich domians
• Proline-rich domains
Trans-acting factor
NH3
DNA binding domain
3. ________________________ domain
e.g, Leucine zipper
Protein #1
Protein #2
• Leucines are spaced
7 AA apart
ZIP
domain
DNA binding region
(“b” domain)
Fig. 12.14- Model of Leucine Zipper
Protein-protein
Activation domain
interaction domain (30-100 amino acids)
COOH
An example of a developmental
transcription factor
• MITF is a transcription factor that activates
pigment genes
CBP
__________________ domain
___________________ interaction domain
___________________ domain
DNA
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
Big themes in regulation
1. Hepatic gene regulation occurs primarily at
the level of ___________________________
2. Tissue-specific expression is due to __________________
of transcription factors
HNF3a
HNF4
HNF1a
C/EBP
HNF3ab
Liver-specific genes
Transcription factors in early liver development
Anterior-posterior axis
HNF3b
Primitive streak
HNF3a
Endoderm differentiation
HNF3, HNF4
Foregut endoderm migration
into mesenchyme
HNF1a, c-jun
Organ formation
C/EBPa
Liver
Hepatic
Determination
Onset of liver
gene expression
Amplification of
liver gene expression
Other forms of gene regulation
1. What activates expression of the activators?
The Pax6 gene has 4 distinct ______________, each utilized
in four distinct_________ to drive Pax6 transcription
Pancreas Lens
Neural tube
Retina
2. ____________- sequences that block ________________ Fig. 5.15
Albumin gene promoter
Albumin expression silenced by inhibitor until birth
Fig. 5.16
Fig. 5.17
L1 promoter is silenced in all
tissues except neuronal due to
silencer element NRSE
Silencer elements are rare!
Delete NRSE
sequences
Other forms of gene regulation
3. _________________- Human b-globin gene cluster
•
•
•
•
Five erythroid-specific genes
Arranged in _____________________
LCR is upstream cluster of 5 (actually 7) _______
Each HS site binds numerous _______
Proposed LCR functions
•
•
•
•
•
•
•
Open ______________
prevent variegated ______________________
Affects timing of _________________
Keeps promoters ___________-free
Change subcellular localization of b locus
LCR transcription affects rest of locus expression
Recruit ________
Other forms of gene regulation
4. ___________-a major method of transcriptional
regulation in vertebrates
Globin gene cluster ________
Fig. 5.20
Model- Methylation
represents a biochemical
specialization
of large genomes that
participate in allele-specific
expression,
whereas differentiation
does not depend on
covalent modification.
4. Methylation-continued
Interesting methylation facts
•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 methylated
patterns 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.
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
female
male
•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.
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
“________________________”
Other forms of gene regulation
7. Dosage compensation
X chromosome inactivation
• 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
Dosage compensation comparisons
1X
1X
2X
2X
2-fold ________
in males
2-fold ________
in females
Stably inactivate ___ X
chromosome
X-inactivation- observations
 Xist is necessary and
sufficient for X inactivation
(using 450kb YAC)
 insert Xist transgene on
autosome results in
inactivated autosome
13
p
12
11
12
13
14
q
21
24
mouse autosome
Inactivated X chromosome
Xist RNA
X-inactivation
Xist Gene
Mechanism in
mammals
 If mutate Xist promoterpreferential X inactivation
on chromosome with
mutation
Random inactivation
Blocking factors
Preferential inactivation
– possibly due to failure to
compete with blocking factor
 Delete Xist exons 1-5mutant chromosome
chosen but not
inactivated
Prevent inactivation
delete
Other forms of gene regulation
8. Differential RNA ____________
a. RNA selection-only certain RNAs are exported
to _________
b. Differential RNA ____________-
Different spliced forms of a RNA
Other forms of gene regulation
9. RNA ____________
a. mRNA longevity- minutes to _____
b. Selective_______ of
translatione.g. the C. Elegans lin-4
RNA binds lin-14 mRNA
to ________ translation
Lin-14 RNA
Lin-4 RNA
Lin-14 mRNA
Untranslated
region
Fig. 5.32