Lecture 5

Question 1

In situ hybridization is an __________ method to visualize gene expression in ______ through ____ (spatiotemporal).

Answer 1

Immunohistochemical; space; time

Question 2

Histone octamers are made of two copies of _____, ______, _____ and _____.

Answer 2

H2a, H2b, H3 and H4

Question 3

Histone _____ can draw nucleosomes together into compact forms.

Answer 3

H1

Question 4

Histone tails that extend from the core are the sites for acetylation and methylation. What does acetylation lead to vs methylation?

Answer 4

Acetylation: DISRUPTS the formation of nucleosome assemblages

Methylation: STABILIZES the formation of nucleosome assemblages

Question 5

__________ of the β-globin gene creates a pre-mRNA containing exons and introns as well as the cap, tail, and 3’ and 5’ untranslated regions.

_________ the pre-mRNA into mRNA removes the introns.

__________ on the ribosomes uses the mRNA to produce a protein.

The β-globin protein is ______ until is modified and complexed with ________ and _____ to form an active hemoglobin.

Answer 5

Transcription

Processing

Translation

Inactive; α-globin; heme

Question 6

(T/F) Each of the steps where a gene turns into a functional protein can be regulated in various ways. It is this regulation during the development process that can yield different cell types and tissues. This can also generate novel organs and tissues in an evolutionary context.

Answer 6

True!

Question 7

The bridge between ______ and ______ can be made by transcription factors.

TFs assemble on the enhancer of the β-globin gene but the promoter is not used until the ______, TF binds to the promoter.

This TF can recruit several other factors including ______, which forms a ____ uniting the enhancer-bound factors to the promoter-bound factors, allowing transcription.

Answer 7

Enhancer; Promoter

GATA1

Ldb1; link

*multiple TFs and transcription machinery interact with each other to facilitate transcription.

Question 8

How does a gene with brain-specific and limb-specific enhancers only be expressed in those tissues?

Answer 8

In the brain cells, BRAIN SPECIFIC TRANSCRIPTION FACTORS bind to the enhancer, causing it to bind to the Mediator, stabilize RNA polymerase II at the promoter and modify the nucleosomes in the region of the promoter. The gene is TRANSCRIBED IN THE BRAIN ONLY; the limb enhancer does not function.

An analogous process allows for transcription of the same gene in the cells of the limbs. The GENE IS NOT TRANSCRIBED IN ANY CELL TYPE WHOSE TFS the ENHANCERS can not bind!

Question 9

For the gene with two enhancers (limb-specific + a brain specific enhancer), why could there not just be one enhancer that only binds limbs and brain TFS?

Answer 9

1) Having two different “control switches” allows for different timings of the gene to be expressed in the different tissues. Maybe in the brain it is expressed earlier, etc.

2) If there was a mutation in the limb-specific enhancer, the gene is still expressed in the brain, or vice versa.

Question 10

The Pax6 protein is critical in the development of several widely different tissues.

Enhancers direct Pax6 gene expression differentially in the ______, the _____ and ______ of the eye, the _______ and the ______ ____ (all tissues have their own specific enhancers).

The pancreas-specific enhancer’s sequence contains binding sites for _____ and ____ transcription factors; both must be present to activate Pax6 in the pancreas.

Answer 10

Pancreas; lens; cornea; retina; neural tube

Pbx1; Meis

Question 11

(T/F) Enhancers are located only upstream of the gene they control.

Answer 11

False! They can be anywhere on the gene!

Question 12

The LacZ gene codes for ___________ which breaks down synthetic sugars, resulting in a blueish/purplish colour.

Answer 12

β-galactosidase

Question 13

What happens when the LacZ gene is fused to the Pax6 enhancers for expression in the pancreas and lens/cornea?

Answer 13

β-galactosidase activity is seen in those tissues as they become blue.

This shows that the Pax6 enhancers are expressed in those tissues.

Question 14

How can the genetic elements regulating tissue-specific transcription be identified?

Answer 14

By fusing reporter genes to suspected enhancer regions of the genes expressed in particular cell types.

Question 15

What is the result of fusing the Green Fluorescent Protein (GFP) gene to a zebrafish gene that is active only in certain cells of the retina?

Answer 15

The result is the EXPRESSION OF GREEN FLUORESCENT PROTEIN in the larval retina, specifically in the cone cells.

Question 16

We can use genes as markers and visualize the tissues of interest.

For example, the enhancer region of the gene for the muscle specific protein _____ is fused to a lacZ reporter gene.

When stained for β-galactosidase activity, the 13.5 day mouse embryo shows that the reporter gene is expressed in the _______ of the eye, face, neck, and forelimb and in the segmented myotomes.

Answer 16

Myf5

Muscles

*Fusing a reporter gene to a gene of interest, we can see where that gene of interest is expressed.

Question 17

What happens when a mouse embryo that normally contains a transgene composed of the L1 promoter, which is a portion of the neuron specific L1 gene, a lacZ gene and a NRSE (neuro-restrictive silencing element) is lacking the NRSE?

Answer 17

Normal: cells are blue in the neural regions only.

No NRSE: whole embryo is blue; β-galactosidase is active everywhere, showing the L1 gene is expressed everywhere. Thus, we can conclude that NRSE is bound in non-neural cells to prevent transcription of the neural gene in those cells.

*reporter genes can help us study repressors!

Question 18

When histone tails are largely methylated, transcription is __________, but when they are largely acetylated, transcription is _______. Why?

Answer 18

Inactive; Active

Methyl groups condense nucleosomes more tightly, preventing access to promoter sites and thus preventing gene transcription.

Acetylation loosens nucleosome packaging, exposing the DNA and RNA to polymerase II and TFs activating the genes.

Question 19

What is MeCP2? What does it do?

Answer 19

MeCP2 is an enzyme that recognizes the METHYLATED CYTOSINES of DNA.

It binds to the DNA is thereby able to recruit:
a) Histone Deacetylases
b) Histone Methyltransferases

Both modifications promote the stability of the nucleosome and the tight packing of DNA, REPRESSING gene expression in these regions of DNA methylation.

Question 20

While histone deacetylases/demethylases are ________, histone methyl/acetyltransferases are _______.

Answer 20

Erasers; Writers

Question 21

Two DNA methyltransferases are critically important in modifying DNA.

What are they?

What do they do?

Answer 21

Dnmt3 and Dnmt1

The “de novo” methyltransferase, Dnmt3, can place a methyl group on unmethylated cytosines.

The “perpetuating” methyltransferase, Dnmt1, recognizes methylates cytosines on one strand and methylates the cytosine of CG pair on the opposite strand.

Question 22

Which one of the statements is true regarding MITF?

1) Transcription factors (MITF) have one main domain; a DNA binding domain.

2) The carboxyl termini forms the DNA binding domain that recognizes an 11-base-pair sequence of DNA having the core sequence CATGTG.

3) The protein protein interaction domain is located below the DNA binding domain.

4) MITF has the basic helix-loop-helix structure found in many TFs. The carboxyl-terminus of the molecule is thought to be trans-activating domains that bind the p300/CBP transcription co-regulator.

Answer 22

Statement 4 is true!

For 1) Transcription factors (MITF) have two main domains; a DNA binding domain and a Protein-protein interaction domain.

For 2) The amino termini forms the DNA binding domain that recognizes an 11-base-pair sequence of DNA having the core sequence CATGTG.

For 3) The protein protein interaction domain is located above the DNA binding domain.

Question 23

What are the 5 yamanaka factors?

Answer 23

Oct3/4, c-Myc, Sox2, Klf4, and Nanog

Question 24

What happens when the yamanaka factors are virally inserted into differentiated fibroblasts?

Answer 24

These cells will dedifferentiate into induced pulripotent stem cells (iPSCs), which are like stem cells as they give rise to progeny of all three germ layers.

Question 25

What happens when the yamanaka factors are virally inserted into differentiated fibroblasts?

Answer 25

These cells will DEDIFFERENTIATE into induced pluripotent stem cells (iPSCs).

Like embryonic stem cells, iPSCs can give rise to progeny of all three germ cells (mesoderm, ectoderm, endoderm)

Question 26

Briefly describe gene regulatory networks.

Answer 26

Transcription factors regulate their genes in a co-ordinate fashion through SPACE and TIME in the developing embryo, generating gene regulatory networks.

Question 27

__________ _______ can process the same pre-mRNA into different mRNAs by selectively using different exons.

Answer 27

Alternative/differential splicing

Question 28

Which one of the statements is false?

1) The Dscam gene of Drosophila is used for self-avoidance between dendrites that foster a dispersed pattern of dendrites.

2) Loss of the Dscam gene causes crossing and disrupted growth of dendrites from the same neuron.

3) It can produce only a few different types of proteins by alternative pre-mRNA splicing.

Answer 28

3!

It can produce 38,016 different types of proteins by alternative pre-mRNA splicing!