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Chapter 4- Genes and development. _______________ - expression of traits ________________-transmission of traits. Embryology. Embryology. Embryology. 1920s Hostility. Genetics. Genetics. 1970s. Genetics. 1870s- What controls inheritance- ___________ or _______________?. - PowerPoint PPT Presentation
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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