Measuring Gene Expression

Question 1

H3K4me

Answer 1

active chromatin

Question 2

H3K27me

Answer 2

repressive chromatin

Question 3

H3K4 - monomethylation

Answer 3

found associated with enhancer DNA (can be many thousands of BP away

Question 4

H3K4 trimethylation

Answer 4

found associated with transcriptional start sites (activation)

Question 5

H3K27 trimethylation

Answer 5

found at promoters associated with early embryological development (therefore stem cell identity), associated with repression, has to be turned off to make progenitor cells- cell fate)

Question 6

H3K27 acetylation

Answer 6

associated with active gene transcription

Question 7

acetylation

Answer 7

almost always associated with activation of genes

Question 8

Methylation (1, 2, or 3)

Answer 8

can result in either activation or repression

Question 9

DNA binding proteins

Answer 9

TFs, repressors, enhancers, do not change genetic code

Question 10

chromatin binding proteins

Answer 10

can change the surrounding chromatin microenvironment in response to the microenvironment, important for cell fate (all cells have the same genome)

Question 11

Oct4

Answer 11

maintains pluripotentcy in embryonic stem cells, represses MyoD activity

Question 12

MyoD

Answer 12

responsible to designating muscle cell differentiation, once active (Oct4 repression relieved) MyoD can modify chromatin around itself, encouraging more binding of MyoD (self reinforcing)

Question 13

Pbx

Answer 13

In the first state - recruits histone deacetylases, associated with non-canonical E Box

Question 14

MyoD targeting chromatin remodeling complexes to myogenin locus prior to forming a stable DNA-Bound Complex

Answer 14

Pbx- in the first state recruits histone deacetylases,
MyoD- Pbx associated with non canonical E box)
recruits HATs
transfers acetyl group to surrounding lysines
opend up canonical E boxes as well as Mef2 binding sites
transcription of active genes occurs

Question 15

HATs

Answer 15

histone acetyl transferases

Question 16

Any particular nucleotide is only meaningful in context of its

Answer 16

flanking nucleotides

Question 17

epigenetics influenced by environment

Answer 17

genetically identical mice, one has a higher chance of developing yellow/obese phenotype because it’s mother had a diet rich in methyl donors

Question 18

How do we know that DNA methylation that controls Asia expression

Answer 18

when it is methylated (repressed) the mRNA is expressed at specific points in development and repressed at other times, when it’s not methylated, the gene is continually active and producing mRNA across the lifespan, the mouse develops yellow fur, obesity and diabetes

Question 19

directional flow of genetic information

Answer 19

all genes are written in DNA and encode an RNA product

Question 20

RNAs that encode proteins

Answer 20

mRNA (messenger)

Question 21

to measure gene expression

Answer 21

measure the RNA that has been transcribed from a specific from a specific gene

Question 22

RNA gels

Answer 22

isolate RNA from cells, resolve by size on a gel

Question 23

Problems of RNA gels

Answer 23

most mammalian cells express thousands of genes
-95% of the RNA in our cells is ribosomal, therefore all we see on the gel is the ribosomal RNA

Question 24

how to measure specific RNA

Answer 24

chemical affinity- probe that will bind our target RNA with high affinity, if we label the probe we can detect our RNA.
Measure the probe to measure the transcript

Question 25

Northern Blot

Answer 25

detecting RNA- detect a target mRNA using a complementary probe that is labelled

Question 26

Northern Blot steps

Answer 26

1) electrophorresis of RNA from cells 2) Transfer RNA to membrane 3) Allow labelled probes to base-pair with RNA 4)visualize probes (X-ray, fluorescence)

Question 27

cDNA

Answer 27

RNA chemically unstable and hard to work with, make a DNA copy of RNA (complimentary DNA)

Question 28

RT-PCR

Answer 28

reverse transcriptase coupled PCR (q RT-PCR if analyzed quantitatively)

Question 29

Reverse transcriptase steps

Answer 29

RNA 2) First stran cDNA synthesis 3) cDNA: RNA hybrid formation 4) DNA amplification

Question 30

problem with RT-PCR

Answer 30

by the time we have enough DNA to see on a gel, the PCR amplification has become inefficient

Question 31

How can we moniter PCR in real time

Answer 31

SYBR green emits a flurescent signal when it binds double stranded DNA (use a PCR machine that can check flurescence after every cycle)