Cycle 8: Control of Gene Expression

Question 1

What is the difference between differential, spatial, and temporal gene expression?

Answer 1

differential gene expression - tissue specific transcription factors for different organisms

temporal gene expression - when are genes expressed (different lifetime, depending on environment)

spatial gene expression - where is the gene being expressed

Question 2

What are the levels of regulation? Which organisms?

Answer 2

• eukaryotes -
• transcriptional: will transcription occur or not and which genes transcribed

post transcriptional: RNA or premRNA already made, control if translation will occur or not

translational: target 3’ and 5’ UTR determines rate at which proteins are made

• post translational: protein already made, regulate amt of protein

Question 3

How does the E. coli lac operon work? Structure?

Answer 3

• ex. lactose metabolization gene expression in prokaryote –

structure:
- promotor
- operator
- regulatory gene lac I that codes for Lac repressor protein
- transcription unit of 3 structural genes: lacZ, lacY, & lacA
- termination sequence
- 3’UTR and 5’UTR

• lac repressor binds to operator and stops transcription (no RNA poly)
• when lactose present:
  1. lactose coverted to allolactose
  2. allolactose bind to repressor causing repressor conformation change to inactive shape
  3. RNA poly can transcribe

Question 4

What is the function of the products of lacZ and lacY gene?

Answer 4

lacZ codes for beta-galactosidase enzyme which metabolizes lactose to galactose and glucose

lacY codes permease transmembrane enzyme which is on the membrane and allows lactose in the cell

Question 5

What happens when repressor is mutated? lacZ? operator?

Answer 5

repressor - continuous transcript bc RNA poly can bind if inhibition without lactose present or over transcription

lacZ - lactose not broken down

operator - lac repressor unable to bind

Question 6

How are genes differentially expressed in different cells types (ex. liver vs lens cells)

Answer 6

tissue-specific transcription factors

Question 7

What is the role of tissue-specific transcription factors in the cell? How do they lead to spatial and temporal gene expression?

Answer 7

tissue-specific transcription factors bind to promoters only when needed
- positive or negative regulators

Question 8

Where can transcription factors be found?

Answer 8

transcription factors are PROTEINS!

nucleus – when they are transcribed and when functioning to bind to promotor

cytoplasm – when they are translated

across nuclear membrane – when they perform their function in the nucleus

Question 9

What is the mechanism of pre-mRNA splicing?

Answer 9

• SnRNPs 5’ (donor) and 3’ (acceptor splice sites go together
• 5’ looks for downstream 3’
• constitutive or alternative

Question 10

What is the 3’ splice site recognition sequence at the intron/exon border?

Answer 10

NCAGG

Question 11

How does mutations in the 5’ and 3’ splice sties lead to formation of aberrant (deviant) mRNA and proteins?

Answer 11

NCAGG mutated in 3’ :
- 5’ to next 3’ spliced out bc cell thinks the whole thing is an intron
- loss of exonic sequence

5’ splice site mutation:
- original intron is interpreted as exonic sequence
- changed reading frame
- adding intronic sequence

• abnormal when it is not evolutionary predetermined *

Question 12

What is the splicing mechanism behind the abnormal production of the beta-globin protein in beta-thallasemia?

Answer 12

blood disorder resulting in abnormal hemoglobin (oxygen carrier) made of two B-globin subunits and gamma-globin subunits

• b-globin subunit has mutation that results in it looking like NCAGG (TCAGG) which is 3’ splice site so cell thinks part of intron is exon and in the exon (which was an intron) now has TAG –> UAG –> premature stop codon!
• TAG in intron doesn’t mean anything
• shorter beta globin protein –> less functional heloglobin

** longer mRNA causes shorter protein

Question 13

How does ApoB100 and ApoB48 form during RNA-editing?

Answer 13

ApoB100 in liver, ApoB48 in small intestine

ApoB1000 - translation <– unedited mRNA –> gets deaminated causing mRNA to have stop codon so ApoB48 is a truncated version
- type of post transcriptional regulation

Question 14

What is the role of telomerase in telomere length? Why is telomerase activated in cancer cells and why is it present stem cells?

Answer 14

telomerase - enzyme that restores telomeres in some cells (cancer, germ cells, stem cells, early embryogenesis
telomere - repeated sequence at end of chromosome that protects chromosome that decreases in length with each replication

Question 15

What is the “hayflick limit” and “cell senescence”?

Answer 15

hay flick limit: number of replications that is safe for telomere length

cell senescence: stage where cell will no longer divide but will still function

Question 16

How do we develop from a one cell zygote?

Answer 16

The zygote is a stem cell that can differentiate into various different types of tissues to produce an entire organism

Question 17

What are the 3 factors that ensure temporal and spatial regulation?

Answer 17

1. every cell contains complete genome established in fertilized egg. DNA of all differentiated cells are “identical”
2. only a small percentage of the genome is expressed in each cell, and the transcripts are specific for that cell type
3. unused genes in differentiated cells are not destroyed or mutated. they retain the potential for being expressed

Question 18

What are the 3 factors that ensure temporal and spatial regulation?

Answer 18

1. every cell contains complete genome established in fertilized egg. DNA of all differentiated cells are “identical”
2. only a small percentage of the genome is expressed in each cell, and the transcripts are specific for that cell type
3. unused genes in differentiated cells are not destroyed or mutated. they retain the potential for being expressed (ex. telomerase can be turned on by cancer cells)

Question 19

What are stem cells? What are its properties?

Answer 19

cells that have the potential to develop into different cell types in the body during embryogenesis and adult life

properties:
- telomerase expressed
- potency

Question 20

What is potency?

Answer 20

differentiation potential
- potency for different types of stem cells are different –> not all stem cells have the ability to differentiate into any cell type

Question 21

What are the types of stem cells?

Answer 21

1. embryonic stem cells
2. somatic (adult) stem cells
3. induced pluripotent stem cells (iPS cells)
4. umbilical cord stem cells

Question 22

What are totipotent stem cells?

Answer 22

differential into embryonic and extraembryonic (umbilical cord) cell types creating a complete viable organisms (only zygote)

Question 23

What are pluripotent stem cells?

Answer 23

descendants of totipotent cells and cal differentiate into nearly all cells of the human body (except extraembryonic) so it can’t make an organism

Question 24

What are multipoint stem cells?

Answer 24

can differentiate into a number of cells but only those of a closely related family of cells
- ex. bone marrow contains multipotent stem cells that give rise to all cells of the blood, but not other cell types)

Question 25

What are unipotent stem cells?

Answer 25

can produce only one cell type but have the property of self-renewal, which distinguishing them from non-stem cells
- ex. skin stem cells
- difference between unipotent stem cell and regular somatic cell is telomerase activation

Question 26

What are the definition, properties and examples of adult stem cells (example: hematopoetic cells are adult stem cells)?

Answer 26

definition: cells of variable potency that can self renew
- niche (in tissue) houses stem cells which are in G0
- when stem cell signalled that an area needs regeneration they are stimulated to divide
- potency decreases as you go down differentiation path

Question 27

How do stem cells divide - 3 ways?

Answer 27

symmetric self-renewal: 1 stem cell to 2 stem cells

asymmetric self-renewal: 1 stem cell to 1 stem cell and 1 progenitor
– progenitor goes to which ever cell needed, stem cell goes back to niche (so that stem cell count doesn’t decrease)

symmetric differentiation: 1 stem cell to 2 progenitors
– neighbouring cell undergoes symmetric self-renewal to maintain stem cell count

Question 28

What is the difference between stem cells and progenitor cells?

Answer 28

stem cells can go back into the niche and stay but progenitors must leave to perform function (can’t go back to niche)

Question 29

What is the role of epidermal stem cells in the skin?

Answer 29

• in basal layer is niche
• epidermal stem cell moves up from niche
• can differentiate on its way up or move to top and then differentiate based on environmental signals

Question 30

how are cells regenerated in the small intestine?

Answer 30

• villa in SI has stem cell niche at the bottom groove
• groove has stem cells and other cells which stimulate stem cells when needed with factors (Int, EGF, and Notch)
  via stem cells present in the niche

Question 31

When of stem cells stay in Go? What do they re-enter the cell cycle?

Answer 31

stem cells sit in Go phase of the cell cycle (quiescent) and then re-enter cell cycle when activated by factors released from neighbouring cells

Question 32

How are iPS cells made and used in therapy?

Answer 32

• adult cells converted to stem cells
• embryo and adult has same genome
• discovered 4 transcription factors needed to drive backward to iPS cells (high potency - pluripotent)
• tissue specific transcription factors added to

Question 33

How were iPS cells used to create viable organism>

Answer 33

• took blastocyst that has embryonic stem cells which was removed and kept extra embryonic stem cell and added iPSC –> mouse!

Question 34

How were iPS cells used for treating burn victims?

Answer 34

take skin sample of burn victim covert to iPS and make more skin
- iPS has all properties: all three skin layers, hair folicles, sweat glands, oil-producing sebaceous glands and fat tissue

Question 35

How were iPS cells used for treating macular degeneration?

Answer 35

macula cells are retinal pigment epithelial cells (outermost layer in retina) –> gets degenerated

• took skin cell, converted to iPS and drove it to make retinal pigment epithelial cells
• result of patient: stoped macular degeneration and brightened vision
• …but eye tumour developed (probably bc of procedure)

Question 36

How are iPS cells used to regenerate injured cardiac cells after myocardial infection? How is this process connected to personalized medicine and application of pharmacogenetics?

Answer 36

regenerative medicine: reprogramming somatic cells

Newt - when limb is cut it forms blastema - undifferentiated cells
-> send out muscle progenitor cells
->migration of muscle fibre cells that dedifferentiate into ‘progenitor-like’ cells

Question 37

What are the important points of the “New England Journal of Medicine” study published in Jan 2021? CRISPR-CasP Gene Editing for Sickle Cell Disease and B-Thalassemia

Answer 37

• B-thamassemia from mis-sense mutation in DNA of B-globin gene causing sickle-cell shape of hemoglobin
• treatment with CTX001 drug
• fetal hemoglobin (alpha-alpha gamma-gamma) is different from adult hemoglobin (alpha-alpha beta-beta) (fetal is converted to adult)
• adult hemoglobin is the issue - treatment by turning on fetal hemoglobin bc entire genome is always present
• fetal has higher oxygen affinity bc oxygen comes from mother at a lower partial pressure
• 3-6 months of age shows sick-cell bc fetal hemoglobin completely depleted
• BCL11A - factor that represses fetal hemoglobin/converts it to adult by inhibiting gamma globlin gene
• KLF-1 transcription factor controls BCL11A
• target KLF-1 so inhibition of fetal hemoglobin doesn’t occur by mutating erythroid (RBC) enhancer region of BCL11A gene
• BCL11A not made so not restriction of HbF
• took healthy adult donor’s hematopoietic stem progenitor cells (who will not express HbF) –> CRISPR-Cas 9 to turn on HbF by targeting KLF-1
• grew edited stems cell in a Petri dish and transplanted it into bone marrow of B-thalassemia patients
• still makes sickle-cell hemoglobin, but transplanted stem cells will take over bc it can self-renew