LESSON 3: GENES AND DEVELOPMENT Flashcards
Principles of gene control of cell differentiation
1.Genomic Equivalence
2. Selective Gene Expression
-all cells contain identical set of genes.
-Expts: Spemann, Briggs & King, & Wolffian regeneration
Genomic Equivalence
Different cells activate different genes at different times.
Selective Gene Expression
Short Regions of DNA double Helix
DNA
Beads on a string (form of chromatin)
Nucleosome
30 nm chromatin fibre of packed nucleosomes
Solenoid
Section of chromosome in an extended form
Looped chromosome
Condensed Section of Chromosome
Condensed chromosome
Eukaryotic Gene Structure is consist of:
1.Promoter Region
2.Cap Sequence (ACATTG)
3.ATG Codon
4. Exons
5.Introns
6.Translation Termination codon
7. 3’ untranslated Region
binding site of RNA polymerase and subsequent initiation of transcription
Promoter Region
– transcription initiation site. - represents the 5’ end of RNA, which will receive a “cap” of modified nucleotide soon after it is transcribed
- vary among genes; necessary for the binding of mRNA to ribosomes and its translation
Cap Sequence or ACATTG
– for initiation of translation
- Leader Sequence (intervening sequence between initiation points of transcription & translation)
-Determines the rate of translation initiation.
ATG Codon
base pairs coding for a protein.
Exons
non-coding sequences interspersed among the exons. Maybe longer and more numerous than exons
Introns
TAA
Translation termination codon
-transcribed but not translated into protein;
- AATAA sequence where a “tail” of adenylate residues (200-300) are added
3’ untranslated Region
confers stability and translatability on the mRNA
poly (A) tail
LEVELS OF GENE CONTROL
1.Transcription
2. Posttranscriptional
To become active protein the RNA must be:
A. processed into mRNA (removal of introns)
B. translocated from nucleus to cytoplasm
C. translated by the protein-synthesizing apparatus
D. Post-translationally modified to become active.
Two Types of Regulatory Elements
1.Cis-Regulators
Trans-Regulator
-represent specific DNA sequences on a given chromosomes which act only on adjacent genes.
-Found at 5’, 3’ ends and introns
CIS-REGULATORS
Types of CIS-Regulators (Influence which gene becomes transcribed in which cell)
1.Promoter
2. Enhancers
-TATA sequence (TATA box or Goldberg-Hogness box) about 30 base pairs upstream from the site where transcription begins and one or more promoter elements further upstream
- “functional anatomy” can be analyzed by determining which of its bases are necessary for efficient transcription
-required for the binding of RNA polymerase and accurate initiation of transcription
- specify the times and places of transcription
Promoters
- DNA sequence
- activate the utilization of the promoter
- control the efficiency and rate of transcript.
- functions by binding to transcription factors.
- activate only promoters on the same chromosomes
Enhanacers
CIS Regulators (process or explanation)
1.Eukaryotic RNA polymerases, however, will not bind to this naked DNA sequence.
2.Rather, they require additional protein factors to bind efficiently to the promoter.
3.The protein-encoding genes are transcribed by RNA polymerase II, and at least six nuclear proteins (basal transcription factors)have been shown to be necessary for the proper initiation of transcription by RNA polymerase II.
4. TFIID, recognizes the TATA box through one of its subunits, TATA-binding protein (TBP).
5.TFIID serves as the foundation of the transcription initiation complex, and it also serves to keep nucleosomes from forming in this region.
6.Once TFIID is stabilized by TFIIA, it becomes able to bind TFIIB.
7.Once TFIIB is in place, RNA polymerase can bind to this complex.
8.Other transcription factors (TFIIE, F, and H) are then used to release RNA polymerase from the complex so that it can transcribe the gene, and to unwind the DNA helix so that the RNA polymerase will have a free template from which to transcribe.
Function is critical for gene transcription, for if the TBP is not stabilized, it can fall off the small TATA sequence.
Transcription factors - TBP-associated factors, or TAFs
CIS Regulators: Requirement for Enhancers
-DNA sequence that can activate the utilization of a promoter, controlling the efficiency and rate of transcription from that particular promoter.
-primary elements responsible for tissue-specific transcription
-function by binding specific regulatory proteins called transcription factors
CIS Regulators: Function of Enhancers
-regulate the temporal and tissue-specific expression of all differentially regulated genes
-genes active in adjacent cell types have different enhancers (pancreas – exocrine protein genes have enhancers different for the endocrine protein insulin)
__________, the exocrine protein genes have enhancers different from that of the gene for the endocrine protein insulin.
pancreas
enhancers both lie in the _____________ of their genes.
5´ flanking sequences
_______________ transgenes by placing flanking regions from the genes for chymotrypsin and insulin onto the gene for bacterial chloramphenicol acetyltransferase (CAT), an enzyme that is not found in mammalian cells.
Walker and colleagues et al. 1983
CAT activity is easy to assay in mammalian cells, so the bacterial CAT gene can be used _________________________________________
as a reporter gene to tell investigators whether a particular enhancer is functioning
Walker and colleagues et al. 1983 transfected the transgenes into: ______,________, &________ measuring the activity of CAT in each of these cells)
1.ovary cells (which do not secrete either insulin or chymotrypsin)
2.an insulin-secreting cell line,
3. a chymotrypsin-secreting cell line
Walker and colleagues et al. 1983 transfected the transgenes results
-neither enhancer sequence caused the enzyme to be made in the ovarian cells
-insulin-secreting cell, however, the 5´ flanking region from the insulin gene enabled the CAT gene to be expressed, but the 5´ flanking region of the chymotrypsin gene did not.
- exocrine pancreatic cell line - the chymotrypsin 5´ flanking sequence allowed CAT expression, while the insulin enhancer did not.
-enhancers for 10 different exocrine proteins share a 20-base-pair consensus sequence - similar sequences play a role in activating an entire set of genes specifically expressed in the exocrine cells of the pancreas
-expression of genes in exocrine and in endocrine cells of the pancreas appears to be controlled by different enhancers.
CIS Regulators: Importance of Enhancers
-required by genes for their transcription
-major determinant of differential transcription in space (cell type) and time.
-enhancers at a relatively large distance from the promoter means that there can be multiple signals to determine whether a given gene is transcribed.
-interaction between the proteins bound to the enhancer sites with the transcription apparatus assembled at the promoter is thought to regulate transcription
-modular - the combination of transcription factors that causes particular genes to be transcribed.
-gene can have several enhancer elements, each turning it on in a different set of cells
-used to inhibit transcription.
-the same transcription factors that activate the transcription of one gene can be used to repress the transcription of other genes (“negative enhancers “ - silencers.)
-are sequences that act specifically to block transcription.
-useful in restricting the transcription of a particular gene to a particular group of cells or for regulating the timing of the gene’s expression.
Silencers
-soluble molecules from one gene and interact with genes on the same or different chromosomes
-With sequence specific DNA-binding domain
-Trans-activating domain enables that transcription factor to interact with proteins involved in binding RNS polymerase
-interaction enhances the efficient with which the basal transcription complex can be built and bind RNS polymerase II
trans-regulators
_________ enables that transcription factor to interact with proteins involved in binding RNS polymerase
Trans-activating domain
-proteins that bind to enhancer or promoter regions and interact to activate or repress the transcription of a particular gene.
-can bind to specific DNA sequences.
-can be grouped together in families based on similarities in structure
-family - share a common framework structure in their DNA-binding sites, and slight differences in the amino acids at the binding site can alter the sequence of the DNA to which the factor binds.
Transcription factors
Three major domains of transcription factors
1.DNA-binding domain
2.Trans-activating domain
3. Protein-protein interaction domain
recognizes a particular DNA sequence.
DNA-binding domain
activates or suppresses the transcription of the gene whose promoter or enhancer it has bound; enables the transcription factor to interact with proteins involved in binding RNA polymerase
trans-activating domain
allows the transcription factor’s activity to be modulated by TAFs or other transcription factors.
protein-protein interaction domain
Some major transcription factor families and subfamilies
1.HOMEODOMAIN PROTEINS
2.Zinc Finger Standard
3.Basic Helix-Loop Helix TF (bHLH)
4.Basic Leucine Zipper (bZip)–
5.Nuclear Hormone Receptors
6. Sox-2 TF
-critical for specifying the anterior-posterior body axes
- 60 aa arranged in a helix-turn-helix, such that the third helix extends into the major groove of the DNA it recognizes.
-aa in the amino-terminal portion also contact the bases in the minor groove
-Small changes in the aa composition of DNA-binding site – change the DNA sequence recognized by proteins.
HOMEODOMAIN PROTEINS
Types of Homodomain Proteins
1.Hox TF
2. Pou Domain
Some function is for axis formation (Helix 3 – major groove; NH2 - minor groove)
Hox TF
mutation transform body segment to anothe
Homeosis
region that comprises the homeodomain and the 2nd DNA-binding region
POU domain
Categories/ Type mentioned under POU Domain
1.Pit -1 (GHF1)
2.Oct1
3.Oct2
4. UNC-86
Pituitary-specific factor that activates the genes encoding GH, prolactin and other pituitary proteins.
Pit -1 (GHF1)
ubiquitous protein that recognizes a certain eight-base-pair sequence (octa box)
Oct1
B-cell specific factor recognizes a certain 8-base pair sequence called Octa box & activates Ig genes.
Oct2
a nematode gene product involved in determining the fates of neurons.
UNC-86
Sequence in Pit-1
ATATTCAT
Sequence in Oct2
ATTTGCAT
has two or more “DNA- binding Fingers” helical
domains . Zn ions link and stabilize the fingers, coordinated by 2 cysteines and 2 histidines.
Zinc Finger Standard
Example of Zinc Finger Standard -
WT and Krox 20
critical for kidney & gonads development
WT
for hindbrain development
Krox 20
binds to DNA via a region of basic AA (10-13 res.). May form a dimer with positive or negative regulators.
Basic Helix-Loop Helix TF (bHLH)
α- helix with several leucine residue that bind with other bZip proteins “Leucine Zipper” dimer.
Basic Leucine Zipper (bZip)
Example of Basic Helix-Loop Helix TF (bHLH)
E12 and E 47 complexed with Myo D or Id (inhibitor of different) proteins for muscle specification
Liver differentiation and adipogenesis
CCAAT enhancer - binding protein (C/EBP) (ex. Of bZIP)
-Sry-Sox - - bends DNA from “I” to “L”
-Important in mammalian 10 sex determinati
-Ex. Sry, SoxD, Sox2
Sox-2 TF
mediate the effect of hormones on genes.
Nuclear Hormone Receptors
a chain of 150-200 adenylate nucleotide is attached to the 3’ end of the pre-mRNA after transcription
Polyadenylation
TYPES OF RNA POLYMERASE
RNA Pol I, II and III
transcribes large ribosomal RNAs
RNA Pol I
Crucial to cell differentiation – determine which genes transcribed & produced
1.Capping
2.Polyadenylation
3. Splicing
addition of 7-methyl-guanylate to
the 5‘ end of pre-mRNA
CAPPING
Chemical modification (Transcruptional Control): __________of DNA can inactivate genes
Methylation
Function of Polyadenylation
Stabilize mRNA & allows its exit and translation
-removal of non-coding sequences (introns) from pre-mRNA to produce the mature mRNA.
-________ of mRNA precursors into different proteins by using different combinations of potential exons
Splicing
-might change the structure of the gene thus regulating its activity
-___________ of promoters of inactive genes at certain cytosine residues
DNA Methylation
Chemical modification (Transcriptional Control): __________ of histone allows DNA unpacking and transcription.
Acetylation
stabilizes nucleosomes and prevents transcription factors from binding
methylcytosine
three areas in which DNA methylation can contribute to differential gene activity
A. Methylation of promoter – temporal and spatial regulation on genes encoding tissue-specific proteins
B. Responsible for distinguishing between certain egg-derived and sperm derived genes in mammals – only one will be expressed during early development
C. Continued repression of the genes on one of the two X chromosomes in each female mammalian cell.
METHYLATION occurs exclusively on ___________ hence, called H3 – K9.
lysine 9 of histone 3,
Methylation of DNA maintains _________________
repression of chromatin
Methylation promotes binding of
HP1 Proteins (Heterochromatin proteins)
-male and female cells have approximately equal amounts of X-chromosome encoded gene products
-Transcription rates of X-chromosome has been altered – male and female cells transcribe the same amount of RNAs from their X chromosomes
Mammalian X- chromosome dosage compensation
– both X chromosomes in the female is active
- increased transcription in male X chromosome (binding of particular transcription factors to hundred sites)
Drosophila
one X chromosome is inactive in female (X chromosome inactivation)
Mammals
– chromatin of the inactive X chromosome
- condensed and replicates after most of the chromatin
Heterochromatin
-Ectodermal cell death
-Absence of mesoderm formation
-Embryonic death at day 10 gestation
mutant X chromosome in mice embryo
restores the positive charges and promotes close attraction between histone & DNA –> Condensed chromosome.
Deacetylation
-H4 Lysine 16
- removal of acetyl group
DEACETYLATION
removes the positive charge from histone reducing the force of attraction w/ DNA leading to wider opening of the chromatin
Acetylation of lysine
200/so adenyl groups added to the 3’ end
Polyadenylation
In a cell - ___ genes are repressed, ___ expressed
90% repressed, 10% expressed
initiates transcription producing 20-25 NT -5-triphosphorylated transcript
CTD- (Carboxyl Terminal Domain)
Transcription Proteins mentioned under CTD
1.TFs + TFs –> TIC
2. TBP-AF – Co-activators
3. Mediator Complex
affect nucleosome stability.
Chemical Modifications
Control of Gene Expression Before Transcription:
1.Selective Gene Amplification
2.Gene Rearrangement
3.Chemical Modifications
Chemical Modification type:
1.Methylation
2.Deacetylation
3. Polyadenylation
Selective RNA processing
POSTTRANSCRIPTIONAL
which nuclear transcripts are processed into cytoplasmic messages “Chosen few”
Censorship
the same nuclear RNA is spliced into different mRNAs. Protein Families
hnRNA Splicing
Example of Alternative 5’ splicing
1.Bc1 XL - inhibit apoptosis
2.bc1 Xs – promotes apoptosis (tumor cell)
Alternative mRNA Splicing
1.Splicing Regular protein + snRNPs & SF joined
2.Spliceosome complex w/c cuts the intron.
3. The two exons are joined by ligase to form mature mRNA.
-Also called RNA interference or RNAi
-Process result down-regulation of a gene at the RNA level
Post-Transcriptional Gene Silencing (PTGS)
-important source of protein diversity. 1 gene = many proteins
-Splicing is frequently in disrupted in human diseases.
Alternative Gene Splicing
a part of an exon may be included in the splicing at 5’ end
Alternative 5’ Splicing
an intron is included in the final mRNA. 2-5% human genes
Intron Retention
Example of Alternative 3’ Splicing
Chordin
part of an exon may be included in the splicing at the 3’ end
Alternative 3’ Splicing
-different exons found in 2 different mRNAs. Ex. FGFR2
Mutually Exclusive Exons
-the longer an mRNA persists, the more protein can be translated from it.
- stability of a message - length of its poly(A) tail - depends upon sequences in the 3´ untranslated region
-messenger RNAs can be selectively stabilized at specific times in specific cells
-polyadenylation confers stability Increase rate of translation
Differential mRNA Longevity
-Stored oocyte mRNA - informations are dormant until translated at /near fertilization
-Activated by ionic signals at fertilization/ovulation
-Other stored messages encode proteins that determine the fates of cells (bicoid and nanos - provide positional information in the Drosophila embryo)
Selective inhibition of mRNA translation
Function of Actin and which organism is it found?
F: Cell movement and contraction
O: Mouse and Starfish
Function of Cylins and which organism is it found?
F: Cell Division regulation
O: Sea Urchin, Clam, Startfish, frog
Function of Small subunit of ribonucleotide reductase and which organism is it found?
F: DNA Synthesis
O: Sea Urchin, Clam and Starfish
Function of Tubulin and which organism is it found?
F: Formation of mitotic spindles, cilia and flagella
O: Clam and Mouse
Function of Vg1 and which organism is it found?
F: Mesodermal determination
O: Frog
Function of Hypoxanthine phosphoribosyl-transferase and which organism is it found?
F: Purine Synthesis
O: Mouse
Function of Histones and which organism is it found?
F: Chromatin Formation
O: Sea Urchin, Frog, Clam
Function of Cadherins and which organism is it found?
F: Blastomere adhesion
O: Frog
Function of Metalloproteinases and which organism is it found?
F: Implantation in the Uterus
O: Mouse
Function of Growth Factors and which organism is it found?
F: Cell growth; uterine cell growth (?)
O: Mouse
Function of Sex determination factor FEM-3 and which organism is it found?
F: Sperm Formation
O: C. elegans
-time of mRNA translation regulated, but so is the place of RNA expression
-e.g. Vg1 mRNA is transported to vegetal pole of frog’s oocyte
-after fertilization, it is found only in vegetal blastomeres
Control of RNA expression by cytoplasmic localization
Post-Translational Modification relates:
-Phosphorylation
-Lipidation
-Ubiquitination
-Disulfide Bond
-Acetylation
-Glycosylation
Adds a phosphate to serine, theronine or tyrosins
Phosphorylation
Attaches a sugar, usually to an N or O atom in an amino acid side chain
Glycosylation
Adds an acetyl group to the N-terminus of a protein to increase stability
Acetylation
Attaches a lipid, such as a fatty acid, to a protein chain
Lipidation
Adds ubiquitin to a lysine residue of a target protein marking it for destruction
Ubiquitination
Covalently links the S atoms of two different cysteine residues
Disulfide Bond
