Genetics Midterm #2 Flashcards Preview

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Flashcards in Genetics Midterm #2 Deck (50)
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1

Transcriptional Regulation

a. Regulatory proteins and transcription factors bind to consensus DNA sequences (promoter regions) to facilitate transcription.
b. Additional regulatory DNA sequences (enhancers and silencers) bind regulatory proteins to facilitate transcription of specific genes in each cell type.
c. Open chromatin structure is favorable for transcription formed by protein action.
d. Alternative promoters are utilized in different cell types to produce different pre-mRNA molecules.
e. Methylation of DNA inhibits transcription.

2

mRNA Processing

a. Capping of the 5' end, polyadenylation of the 3' end, and intron splicing modify pre-mRNA.
b. Alternative capping and polyadenylation sites can be used in different cell types.
c. Alternative splicing produces different mature mRNA molecules from some cell types.
d. RNA editing modifies the base sequences of mRNA.

3

Regulation of mature mRNA

a. Translational regulatory proteins bind mature mRNA to delay translation initiation.
b. Small RNAs regulate the stability or translation initiation.
c. Transport of mature mRNA to cytoplasm is regulated.
d. RNA stability is regulated.

4

Cis-Acting Regulatory Sequences Bind Trans-Acting Regulatory Proteins to Control Eukaryotic Transcription.

- Activator proteins bind regulatory sequences to stimulate transcription.
- Repressor proteins bind other sequences to hinder transcription.
- Regulatory proteins are often found in large complexes in eukaryotes, unlike in bacteria.
- Individual transcription factors may regulate tens to hundreds of target genes.

5

Integration and Modularity of Eukaryotic Regulatory Sequences

- Enhancers and silencers typically contain binding sites (modules) for a number of transcription factors
- The modules allow the enhancers and silencers to integrate activities of different transcription factors to produce different outputs.
-Pioneer factors are the first to bind regulatory sequences, facilitating binding of additional transcription factors

6

Modularity of Regulatory Sequences:

- Multiple proteins bind, interact, and recruit additional proteins.
-Note: Identity of proteins bound can convert an enhancer into a silencer...
- Two proteins might bind cooperatively or, conversely, might sterically hinder one another's binding.

7

Transcription Factor Synthesis

- Transcription factor availability is dependent on their transcription and translation in the cell.
- Synthesis of transcription factors is tightly regulated and different cell types have distinct arrays of transcription factors.
- The availability of some transcription factors is controlled through signal transduction.

8

Signal Transduction

- Signal transduction pathways: events outside the cell stimulate sequential events inside the cell that result in new gene expression patterns.
- Involve transmembrane proteins that receive signals externally through an extracellular interaction domain.
-They transmit signals within the cell via a binding domain inside the cell.

9

Process of External Signaling into an Internal Response and the Effect on gene transcription

1. Stimulus
2. Release of transcription factor protein
3. Activation of transcription factor
4. Transportation to nucleus
5. Enhancer binding
6. Transcription initiation.

10

Identifying Eukaryotic Gene Regulatory Sequences: How do we identify control elements like enhancers?

Just like we did to identify promoter elements!
Example:
a. clone gene of interest into plasmid.
b. test the cloned gene's activity in appropriate (and inappropriate) cell types.
c. Delete blocks of DNA upstream and downstream coding sequences to ask if they are essential to appropriate expression.
d. Deletion-- loss of high expression? Now test if that region can act as an enhancer..

11

Have identified enhancer (by activity)

Now identify proteins that make it work:
- Gel shift
- Footprinting
- DNA sequence (look for conserved motifs already described in other enhancers/promoters)
-ChIP (chromatin immunoprecipitation)

12

Band Shift Assay

Identifies DNA fragments that bind nuclear proteins (usually extract from cell)
-Add antibody to the suspected Transcription Factor.
- "Super shift" results (if Ab binds TF bound to DNA)

13

DNA footprint protection Assay

Assay shows you WHERE on the fragment the protein is binding.

14

ChIP -Chromatin Immunoprecipitation

- You know a protein can bind a promoter/enhancer element in vitro.
- Does the protein bind that element in vivo? (in living cells)
- Isolate nuclei; isolate chromatin; Cross-link DNA-bound proteins to DNA; shear DNA; use antibody to precipitate protein; reverse cross-link to elute bound DNA; PCR to see if eluted DNA contains element.
- If gene is expressed only in fibroblasts, do you expect ChIP results will be the same in fibroblasts and lymphocytes?

15

Locus Control Regions

A locus control region (LCR) is a highly specialized regulatory sequence that acts to not only enhance transcription but also to insulate a locus from the effects of neighboring genes.
- First LCR was discovered in human beta-globin locus.

16

Hemoglobin has...

2 alpha-globin polypeptides
2 beta-globin polypeptides

- There are different "forms" of beta-like globin polypeptide made and incorporated into hemoglobin during development.

17

Beta-globin-gene complex

-Has "different" forms of beta-like globin polypeptides made during development.
- Each form has one gene
- Each gene has its own promoter...

18

Locus Control Region - How was it discovered?

- Thalassemias: loss/mutation of alpha-globin coding sequences. loss/mutation of beta-globin coding sequences. No mutation of either alpha or beta-globin coding sequences-- but still no hemoglobin???
- "Transgenic animals": introduce a gene into the genome of an embryo-- is incorporated into cells that become germ cells-- "transgene" is passed to next generation (in ALL progeny cells)
- "Transgenic animals" carrying beta-globin gene and its promoter. Should be expressed in reticulocytes. But.. NO.

19

Locus Control Regions

- In transgenes (expression vector with beta-globin gene and promoter injected into animal and incorporated into the genome of the recipient animal), presence of LCR resulted in:
- Tissue-specific transgene expression
-Genome integration-site independent expression
-Copy-number dependent expression ( increase in gene expression level as gene copies increase)
- Transgenes with enhancers often expressed in some animals and not in others-- due to integration site.

20

The Beta-Globin Locus Control Region (continued)

- The composition of enhanceosomes bound to the LCR varies at different developmental stages as do the proteins bound to the individual beta-like globin gene promoters.
- Result: appropriate "switches" in genes expressed during different stages in development.

21

Yeast Enhancer and Silencer Sequences

- In the yeast, transcription of genes in the galactose utilization pathway is carefully regulated by enhancer-like sequences.
- When galactose is the only sugar available, wild-type yeast induce transcription of four enzyme-producing genes, GAL1, GAL2, GAL7, and GAL10
- Together these import and then break down galactose.
- How to turn on all four genes at the same time? Common enhancer associated with each.
-Induction of group of enzymes: you will be studying the bacterial lac operon.

22

Using Galactose as an energy source in Yeast

-The enhancer element associated with each gene in this pathway is called the Upstream Activator Sequence (UASg)
-By the way, notice the direction of transcription of the four genes and the presence on more than one chromosome.

23

Regulation of GAL gene transcription (example of inducible expression)

a. Galactose is absent
- Gal 4 and Gal 80 are made from two constitutively-active genes.
- The UASg includes two copies of 17 base pair Gal4 binding sequence
- Note: Gal4 is bound by Gal80 and is unable to bind UASg in the absence of Galactose.
-There is no transcription of the GAL genes.

24

Gal 3 (constitutively expressed) binds galactose when galactose is present; this complex now binds Gal 80, causing Gal 80 to release Gal 4.

b. Galactose is present
- Gal4, Gal80, and Gal3 are constitutively expressed.
- Galactose is the inducer
-Gal 80 is bound by Gal3; Gal4 binds to UASg and activates transcription.

25

Gal 4 Activation of Transcription

- Gal 4 binding of UASg leads to the formation of a multiprotein complex called the Mediator.
- The Mediator is an enhanceosome that induces formation of a DNA loop, making contact with the general transcription apparatus at the GAL gene promoters in cis. Mediator-like complex conserved among all eukaryotes.
- Transcription of GAL genes is dependent on transcription activation by Gal4/UASg binding and subsequent Mediator formation.

26

Transcription Repression of the Yeast GAL1 gene (activation repression vs lack of activation)

- Lack of GAL1 expression is not due only to the inability of Gal 4 to bind UASg enhancer (when galactose is not present).
- In presence of glucose, GAL1 gene expression is actively repressed.
- Mig1 produced only when glucose is present in the cell.

27

Insulator Action

- Silencer: directly represses gene expression (as in Mig1/Tup1 repression of Gal1 expression)
- Insulator: prohibits gene activation by an enhancer by BLOCKING enhancer/promoter interaction
- Enhancer activity help initiate transcription
- Insulator sequence blocks enhancer action and can redirect enhancer actively to another gene.
- A particular enhancer activates a gene in preference over a nearby enhancer whose action if blocked.
- Insulators may direct the formation of DNA loops that contain enhancers and the genes they activate.
- The insulator is not really a "sheet". Cartoon of what is actually a protein complex.

28

Chromatin Structure Regulates Gene Expression

-Heterochromatin: highly condensed, usually inactive transcriptionally. Darkly stained regions of chromosomes.
-Constitutive: condensed in all cells. (most of the Y chromosome and all pericentromeric regions)
- Facultative: condensed in only some cells and relaxed in other cells. (position effect variegation, X Chromosome in female mammals)
- Euchromatin: relaxed, usually active transcriptionally. Light stained regions of chromosomes.

29

Heterochromatin: Position effect Variegation (PEV)

a. Wild type eye color
- w+ allele is expressed
b. Variegated eye color
- Inversion moves w+ near the centromere.
-Heterochromatin spread is variable.
- w+ allele is expressed in some spots and w+ is silenced in other spots.
- The eye is divided in specialized cells called ommatidia.

30

Discovery that PEV was DUE to heterochromatin spreading:

- Some Su(var) mutations are caused by defective expression of heterochromatin protein-1 (HP-1)
- HP-1 is associated with centromeres, telomeres, and other heterochromatic regions in Drosophila.
- HP-1 is a nucleosome-binding protein that targets lysine in position 9 of histone H3 if it is methylated (H3-K9 methyl)- HP-1 binds this methyl group.
-Absence of HP-1 interferes with Heterochromatin formation and suppresses variegation (no way for heterochromatin to spread).