Chapter 1: Levels Of Gene Control

Question 1

[Protein content]

Methods used to study the expression of individual proteins in tissues and cells?

Answer 1

• Western blotting
• PAGE
• Mass spectrometry

Question 2

[Protein composition]

Methods to study overall protein composition of tissues and cells

Answer 2

-2D PAGE

Question 3

Principle of Western Blotting and the expected results

Answer 3

■Proteins are isolated from various tissue cells
■They are separated based on size by using gel electrophoresis (SDS-PAGE)
■ They are then transferred to a membrane
■ A protein-specific antibody is used to detect the protein of interest
■Results are observed by flouscerance

♤ Expected results
♡A band will be observed in tissue types where the antibody has bound.

Question 4

How to read western blotting and what the results mean

Answer 4

■If a band is shown in all tissues— it indicates that a particular gene is expressed in various tissue types

■If a band is shown on a particular tissue type— it means the gene is differentially expressed

Question 5

Gel electrophoresis (PAGE)

Answer 5

A technique whereby proteins are separated according to their size

Question 6

Principle of 2D PAGE Analysis & the expected results

Answer 6

■isolate proteins from tissue cells
■Separate proteins by their charge (isometric focusing)
■ then separate them based on size by using gel electrophoresis (SDS PAGE)
■has better resolution than SDS PAGE

Expected results
♤ protein spots observers with different intensities

Question 7

How to interpret 2D PAGE Analysis and What it means

Answer 7

■You check the size and it’s intensity
■ big spot/ high intensity —means the there is high level of the protein
■small/low intensity— means there is a low level of the protein

Question 8

[mRna content]

Methods to study the expression of individual mRNAs

Answer 8

■Northern Blot

■RT(q)- PCR

Question 9

[mRNA content]

Method to study mRNA population (many genes)

Answer 9

Microarray

Question 10

[Expression of mRNA/transcriptional level]

Principle of Northern Blot

Answer 10

■Isolated proteins 4rm tissues are separated by size
■transfered to a membrane and hybridized to a radioactive probe from gene encoding the mRNA of interest
○mRNA will anneal to single
Stranded DNA probe with
Complementary sequence
○Hybridize/anneal to agene specific probe
■visualize by xray

Question 11

[mRNA expression]” northern blot

Expected results and how to interpret the blot

Answer 11

■when a band is observed the mRNa of the gene is transcribed
■ no band= mRNA of gene is not transcribed

Interpretation
□when a band with similar intensity is observed in various tissue types= the gene is not differentially expressed

□when a band with different intensities or absent 4rm other tissue types= differentially expressed

□absence of band= gene not transcribed or transcribed at low levels
□ A band shows the gene is transcribed in the cell

Question 12

Method to detect low levels of mRNA

Answer 12

RT-PCR

Question 13

Why and when to use RT-(q)PCR

Answer 13

When we want 2 compare transcripts between samples with high accuracy n high sensitivity

■method used 2 detect low concentration of cDNA

Question 14

[mRNA content at low level]

Principle of RT-qPCR and expected results

Answer 14

■isolate RNA and change it to double stranded cDNA
■DNA is amplified using gene specific primers
■primers are Labelled with flouscerant markers
■The quantity of amplified DNA is related to the level of mRNA in the cell

□amplified DNA observed when mRNA is transcribed
□amplified DNA not observed when mrna is inactive

Question 15

[mRNA at low level]

How 2 interpret RT-qPCR

Answer 15

■high quantities of amplified gene —shows that more mRna are present in that tissue
■comparing results 4rm diff tissue types allows me to know in which cell type is a gene most abandantly transcribed

Question 16

2 Principles that:
1- measure how well a gene transcription is initiated (whether it is regulated at the level of transcription initiation or at a downstream level, such as RNA stability)

2- can compare the rate of transcription of different genes in different tissues

Answer 16

●Pulse-labelling (in vivo- in cells)

●Nuclear run-oon assay(In vitro-test tube(

Question 17

Pulse labeling principle

Answer 17

■The transcription rate of a gene in cells is monitored during RNA synthesis by adding a radioactive labelled UTP for Short period of time

■ Radioactive UTP is incorporated into newly synthesized mRNA during transcription by RNA Pol

■specific transcript of interest can be analyzed by hybridization using gene-specific probe

■visualize by auto radiography

Question 18

How 2 interpret pulse labelling[rate]

Answer 18

■high intensity means more mRNA is present–meaning there is a higher transcription rate happening= transcription activation

■no signal means no transcription rate and no transcriptional activation

Question 19

Principle of Nuclear run-on Assay[vitro-glass]

Answer 19

■Transcription rate is monitored by measuring the amount of radioactively labelled 3H-UTP

■labeling occurs for short period of time
■genes that are being transcribed in the labeling period will be radiolabelled
■specific transcripts of interest detected by hybridization to unlabeled genes

Question 20

Exceptional cases where changes to DNA occurs

Answer 20

1. DNA loss
2. DNA Amplification
3. DNA Rearrangement

Question 21

DNA Loss example of mammalian red blood cell

Answer 21

During differentiation of red blood cells the nucleus is removed and degraded in the erythroblast cells, resulting in a anucleated cell(no nuclear & DNA)

It sysnthesiz3s small amounts of other proteins by translation of mRNA produced in red blood cells before loss of nucleus

Question 22

DNA Amplification example

Answer 22

Case wherw high level of mRNA is required in short period of time such that a gene could not produce enough mrna/Protein

Chorion genes are amplified in DNA, allowing synthesis in these cells of the large amounts of chorion mRNA at short period of timr

Question 23

How to know if gene is regulated at Transcriptional or post-transcriptipmal control

Answer 23

• Nuclear run on assay

- pulse labeling

Question 24

When to know if it’s regulated at post-transcriptional level

Answer 24

■when genes are transcribed/expressed in all tissue types

Question 25

Name the main Regulatory RNA

Answer 25

• microRNA (miRNA)

- small interfering RNA (siRNA)

Question 26

miRNA are ____stranded, and folds to become a double stranded hairpin loop

Answer 26

Single

Question 27

Regulatory ncRNAs (miRNA n siRNA) inhibits what?

Answer 27

■inhibits transcription
■inhibits Translation
■can degrade transcripts

Question 28

MicroRNA (miRNA) example of embryonic development: lin-4 & lin-14

Answer 28

☆ lin-4 gene encodes a miRNA whilst lin-14 encodes a mRNA

☆lin-4 via same complementary sequence binds to lin-14, which inhibits lin-14 protein expression

Question 29

miRNA can target diff mRNAs and inhibit the gene expression how?

Answer 29

》miRNA has partial complementary sequence, thus, miRNA can bind to different mRNAs at different levels to inhibit the expression of a gene

Question 30

miRNA mechanism
Drosha(nucleas)
Dicer(cytoplasm)

Answer 30

》 miRNA are ssRNA which folds to form a ds hairpin loop called pri-miRNA
》Drosha will bind to the loop and cleave the legs of the pri-miRNA, to convert it to pre-miRNA it is then transported to the cytoplasm by exportin-5.
》Dicer will bind to the pre-miRNA and cleave the head of the dsRNA, and it will remove 1 ss RNA n degrade it, the other one will be the mature miRNA

Question 31

How do miRNA inhibit gene expression of target gene (3)

Answer 31

1. induces mRNA degradation
2. blocks translation
3. Induces closed chromatic structure

Question 32

siRNA mechanism

Answer 32

》processed from a dsRNA
》Dicer will bind to the dsRNA ,and cleaves it to smaller dsRNAs ,1 strand forms the siRNA
》can then bind to original RNA via complementary sequence to promote degradation