intro to the control of gene expression

Question 1

what is the genotype

Answer 1

Genotype : human cells all contain the same genetic blueprint.
•Genetic estimates indicate that the human genome contains about 50,000 genes
•In any given cell type only about 10,000 genes are expressed
•Genome is effectively identical in every cell

Question 2

what is differential gene expression

Answer 2

•Genome must be differentially interpreted in different cells at different times. This is differential gene expression.

Question 3

when is differential gene expression tightly regulated ?

Answer 3

Time :
• Development (ie embryos vs adults)
• In response to hormones, infection, other signals

Space :
• Different tissues or cell types express different genes (ie brain versus muscle cells)

Question 4

why is it important for differential gene expression to be tightly regulated ?

Answer 4

• Dna is transcribed into rna, which is then translated to proteins. These proteins can be grouped into 4 groups: metabolism, cell shape/motility, cell differentiation, cell proliferation.
• If this flow of information is not tightly controlled it can lead to metabolic diseases if we are affecting the expression of proteins involved in metabolism, it can lead to metastasis because the proteins cause different motility. problems with cell differentiation of specific tissues or or organs, leading to congenital disorders. failure to regulate the expression of proteins involved in controlling cell proliferation/division, it can lead to cancer.

Question 5

What are the drosophila homeotic mutants

Answer 5

• Bithorax gene ( present in flies) : gene that controls the number of segments in an organism. A mutation in the bithorax gene can cause flies to have 2 thoraces, 2 pairs of wings in each thorax, and 2 stomachs. There is a duplication.
• Antennapedia gene : gene that is involved in the formation of the legs in flies. If the gene is expressed incorrectly, there is the formation of 2 legs in the place of the antenna.

Question 6

what are totipotent cells

Answer 6

• Totipotent cells are cells that can give rise to all tissues in the organism. They can only be found in the early stages of embryogenesis. They differentiate to form pluripotent stem cells.

Question 7

what is the goal of reprogramming committed stem cells?

Answer 7

• The ability to isolate adult stem cells from all developmental compartments
• The ability to drive these cells to develop either in vivo or in vitro in a completely controlled manner so that we can regenerate tissues

Question 8

what is beta-thalassaemia

Answer 8

• A group of genetic diseases caused by insufficient expression of beta-globin. B-globin is the protein that transport oxygen in our rbc.
• In most types of beta-thalassaemia the beta-globin protein is structurally normal (unlike sickle cell disease)
• mutations that cause this disease are usually single base changes

Question 9

outline the mutations that can cause beta -thalassaemia

Answer 9

1) the single base change from T to C results in no expression of this protein.
- The tata box region in the promotor that recruit rna polymerase to this promotor to copy the DNA into RNA, this mutation destroys the tata box of the beta globin gene. –The tata box is no longer recognised by the RNA polymerase II and cannot be expressed.

2) • This is a mutation at the exon intron boundary from A to G.
• This means that the end intron cannot be removed from the pre-mRNA molecule.
• Cells will degrade the RNA that is not processed.
• The single point mutation will result in this gene not being expressed.

3)• There can be mutations in other parts of the promotor region which will also result in the gene not being expressed

4)
• Mutations within introns will prevent the introns from being spliced.

5)• There can also be mutations in the 3’ UTR region which will destabilize the RNA for this gene.

Question 10

when can we regulate gene expression?

Answer 10

at the level of transcription and post transcription

Question 11

list the processes that are involved in post transcriptional regulation

Answer 11

• Polyadenylation
• Capping
• Splicing
• Translation – mature mRNA in cytoplasm
• RNA stability –same

Question 12

give examples of translational control

Answer 12

• Early embryogenesis – during first 4 – 8 cell divisions there is virtually no gene expression. At the end of blastocyst formation first genes to be expressed are due to up-regulation of translation from maternally-derived pre-formed mRNAs
• Environmental stress – exposure to heat shock or pathogens can cause global changes in translation. Some pathogens e.g viruses when they infect cells they inhibit translation of all mRNAs.

Question 13

why do viruses inhibit translation of all mRNAs when they infect cells?

Answer 13

By inhibiting all translation machinery, they are stopping the cells from producing proteins that can be part of an anti-viral response mechanism . the viruses can also take the translational machinery to express their genes. They can inhibit translational machinery by inactivating EIF3 and EIF4.

Question 14

explain how ribosomes finds the start sequence

Answer 14

• During translation, the ribosome will bind to the cap and start scanninig the mRNA molecule until it finds the kozak sequence.
• The kozaq sequence precedes the AUG codon, which is the first codon that will be translated into met , and it is at this stage the ribosome will start synthesising proteins.
• The first AUG codon that follows a kozak sequence will code for methionine. The kozak sequence must be present for initiation of protein translation.

Question 15

what must be present for initiation of protein translation ?

Answer 15

kosaq sequence.

Question 16

what is the function of 5’UTR

Answer 16

play a major role in determining how efficiently the ribosome initiates translation

Question 17

describe the mRNA of beta globin .

Answer 17

–Globin (very efficiently translated). The beta globin mRNA has an efficiently translated 5’ UTR regions. This is because Beta globin Is the protein that transports oxygen and is expressed in RBCs which have no nucleus. It does not transcribe DNA into RNA. Instead, the rbcs contain and express high levels of beta globin gene which is efficiently translated 5’UTR .

Question 18

why kind of 5’UTR does globin have ?

Answer 18

very efficently translated

Question 19

what kind of 5’UTR does ferritin have

Answer 19

–Ferritin (very inefficiently translated). Will only be translated efficiently in the absence of stimuli.

Intracellular Iron levels are translationally controlled

• Ferritin - binds Iron and retains in the cytoplasm as a store for excess
• Only need Ferritin at times of Iron excess

Question 20

how does ferritin work ?

Answer 20

• When iron is in excess the ferritin is expressed to bind to the excess iron.
• In the absence of iron the ferritin mRNA molecule, the 5’utr region can fall back by complementarity and form hairpin structures.
• The hairpin structure and the 5’UTR of the ferritin RNA is recognised by a protein called the ferritin starvation inhibitor.
• the ribosomal unit can bind to the 5’cap but it cant move along the mRNA because its movement is prevented by the ferritin inhibitor.
• Therefore this rna is expressed in cells but in the absence of iron it is not translated.

In the excess of iron:

• The iron binds to the inhibitor which makes the inhibitor dissociate from the 5’UTR region and now the ribosome is able to move along and translate the ferritin RNA to protein
• The ferritin protein will then be able to bind to the excess of iron.

Question 21

describe 3’UTRs and give examples

Answer 21

•3’ UTRs confer very different stabilities on mRNAs as the length of the polyadenylated tail will determine the time it takes for degradation to take place.

–Globin 3’UTRs confer stability e.g beta globin gene. It stops the rna from being degraded, and if the rna is present most of the time and its not degraded then it will give rise to high levels of Beta globin . this is because rbcs don’t have DNA so they cannot transcribe the beta globin gene. so a way for rbcs to make a lot of beta globin protein is by having a stable RNA by having a 3’utr and 5utr for translation.

–some 3’UTRs are unstable. The cells express the mRNA, which will be translated into proteins, and as soon as it is translated the mRNA will be degraded and can no longer produce proteins. Eg. immune stress hormones (very unstable mRNAs)

Question 22

what are miRNAs

Answer 22

• Mi RNA is small and non coding RNA
  • Regulate gene expression
• transcribed by RNA polymerase II
  •These RNAs are referred to collectively as microRNAs or miRNAs
  •These miRNAs act to control the post-transcriptional regulation
  •Any given miRNA can regulate several target genes

Question 23

what are microRNAs derived from ?

Answer 23

Micro RNAs are derived by processing from a larger precursor.

Question 24

deacribe the synthesis of miRNAs

Answer 24

• As a result of transcription , there is a polyadenylated RNA molecule , which will fall back together because of areas of complementarity, such that parts of the single stranded molecule binds to complementary regions further along the sequence.
• The precursor mRNA molecules are transported to the cytoplasm where the loop will be degraded. The single stranded RNA part of the molecule will also be degraded. As a result, the large precursor is cleaved to form a double stranded RNA.
• Double stranded RNA is recognised by RISC proteins and is unwound to form single stranded miRNA molecules, forming risc complexes.
• Once the complex is formed, RISC will search for complementary target mRNA.
• If the single stranded RNA molecule has an extensive match with another mRNA molecule, the risc will cleave and degrade the extensive match, ie the RNA molecule
• This allows miRNA to shut down he expression of genes by degrading mRNA molecules.
• Less extensive match will cause gradual degredation.
• Translation is reduced, mRNA is sequestered and eventually degraded.
• Risc is released.

Question 25

what are the applications of miRNAS

Answer 25

• This allows us to chemicaly synthesise double stranded molecules
• Allows us to dertemine function of genes.

Question 26

what is an example of a congenital disorder /

Answer 26

bithorax gene mutation in drosophilia

Question 27

what tissue can pluripotent cells not differentiate into ?

Answer 27

placental tissue

Question 28

how are iPS formed

Answer 28

introducing new genes and transcription factors to turn on genes that were off

Question 29

what does the primary transcript consist of

Answer 29

both exons and introns

Question 30

why dont zygotes need to transcribe genes

Answer 30

they have pre existing maternal RNAs

Question 31

how do cells respond to viral infections?

Answer 31

they transcribe genes that code for antiviral proteins

Question 32

what recruits the ribosome?

Answer 32

• The 5’cap is not enough to recruit the ribosome, there are eukaryotic initiation factors. E.g eif3 and 4 that promote the binding of the ribosome, recognition of the cap and initiation.

Question 33

what is the promotor region in eukaryotes and prokaryotes ?

Answer 33

tata box and pribnow

Question 34

How can committed stem cells, like skin cells, be reprogrammed into pluripotent stem cella?

Answer 34

• The way we reprogramme committed stem cells is by forcing them to change the expression of the genes. The pluripotent stem cells can then be forced to differentiate into the tissue that is required. This reprogramming of the pluripotent stem cells in the tissue of interest is by affecting the expression of genes.