LECTURE 8 (Analysis of Eukaryotic genes)

Question 1

What are the 3 techniques to determine the start and end of transcription sites?

Answer 1

HTS
Nuclease Protection Assays (RNA/DNA)
Primer extension

Question 2

Describe the primer extension method for determining the start and end of transcription.

Answer 2

you design a primer complementary to the sequence of RNA of interest. It’ll anneal and RT + dNTP will carry the formation of the DNA strand. By measuring the length on a gel and comparing with a seq of know size e.g a sanger seq for the same DNA, we can determine how long the transcript was and therefore where the start and end of transcription were. Remember that RT reads only 3’ to 5’.

Question 3

How are Protection assays used for determining the start or end of transcription?

Answer 3

This is used for DNA and RNA. You make a radiolabelled probe that will anneal with start/end of transcription and will have the 5’/3’ end radiolabelled. Then you treat the sample with a nuclease (DNase or RNase) that chops off the ss strand leaving the small seq that will be either the start or the end. Run on a gel to see the length of the fragment thats the end or start of transcription. Remember that the band on the gel is for the probe only!

Question 4

Describe HTS for mapping start and end of transcription.

Answer 4

It’s simply done by bioinformatics. Take into account that polymerases make way more mistakes at the beginning rather than at the end. The end is determined by reading the seq prior the polyA tail in mRNA

Question 5

Why is nuclease S1 used in PA for determining the length of start or end of transcription?

Answer 5

because it cuts everything that’s not double stranded

Question 6

What are ORF? How do they start?

Answer 6

Open reading frames are seq that code for a protein and do not have inter STOP codons, only one at the end however introns may intersperse the ORF in euk. They start with ATG (met)

Question 7

How can we determine whether there’s an intron interspersing a ORF?

Answer 7

Because most introns start with GU/AU and finish with AG this can be detected with a program

Question 8

What are pseudogenes?

Answer 8

DNA seq with similatitird with real gene but they have something that make them unfunctional, can be converted into RNA but not into proteins.

Question 9

Can we use primer extension to determine how long a transcript was?

Answer 9

no, it’s only used to know the length of the primer at the 3’ or 5’ end.

Question 10

How can we analyze enhancers and promoters? (3)

Answer 10

• repoter genes
• footprinting
• gel shift assays

Question 11

What are reporter genes?

Answer 11

they’re genes that serve as markers and are attached to a regulatory seq of other gene to make sure that gene is working.

Question 12

Give examples of reporter genes.

Answer 12

• Fluorescent proteins e.g GFP. it responds to blue light, there’s a conformational change and when it folds back it releases light.
• luminescent proteins: responds to the presence of ATP, theres a conformational change and make ADP, it folds back to the original state when ATP is released, when it does that, certain wavelength of light is released
• chromogen e.g Xgal. B galactosidase consumes X gal and produces glucose and blue pigments
• Chromoprotein is a protein with color, we attach to the gene of interest and it releases color when the gene is expressed.

Question 13

How can we use reporter genes to analyze enhancers and promoters?

Answer 13

• we attach a reporter gene to the DNA seq of interest. you introduce this into an organism, if the gene is expressed the reporter gene will give a signal showing that certain gene works

Question 14

When does a promoter end?

Answer 14

when the bases have no more influence in the transcriptional activity of the complex

Question 15

What are enhancers?

Answer 15

sequence that increases the utilization of a promoter.

Question 16

What are promoters?

Answer 16

region of DNA to which RNA polymerase binds in order to initiate transcription.

Question 17

What is the technique ‘footprinting’ for?

Answer 17

It’s in order to analyze which part of the promoter binds to the TF.

• take a piece of DNA that has enhancers and promoters and you label it at the 3’ end, treat with DNA binding proteins and treat with DNase which cuts DNA at random places but not at the places where the DNA binding protein was bound.
• digested pop is purified and denatured
• you run a gel and you can see that samples w/o binding proteins are the control

Question 18

What are gel-shift assays?

Answer 18

A technique that identifies DNA binding proteins in a complex mix of proteins isolated from nuclei Referred as EMSA. Electrophoresis, mobility shift assays.
You do two electrophoresis
1 . your DNA radiolabelled
2. your radiolabelled DNA + cell extract of different sizes
In the first one there’s only one band in the second one you see 6 bands and the smaller the cell extract or with no cell extract the DNA migrates to the +ve cathode, this suggests that there are 6 different specific binding proteins

Question 19

How do we analyse ORF?

Answer 19

using bioinformatics find start and stop codons

Question 20

Is splicing random?

Answer 20

No, it’s a sequential process

Question 21

What’s better bioinformatics or experiments?

Answer 21

bioinformatics give much more data than we could achieve experimentally and it’s cheaper but experiments confirm predictions, the best thing is a combination bt the two

Question 22

DNA footprint identifies 3 things;

Answer 22

• Areas of the promoters that interact with transcription factors
• Sequences in DNA that become particularly exposed by the interaction with DNA-binding proteins
• Single base pairs that interact with proteins

Question 23

DNA footprinting complement gel shift assay, true or false.

Answer 23

true.