MBB 267 Week 8: Mitchel 1

Question 1

What are the types of eukaryotic gene regulations?

Answer 1

Include

• Transcription regulation
• Translational regulation
• Pre-mRNA splicing
• RNA localisation

Question 2

When are genes expressed in eukaryotic organisms?

Answer 2

Differentially expressed depending on the environment they are in

Question 3

What can gene expression drive in metazoan organisms?

Answer 3

also drive cell differentiation and organism development

Question 4

What happens to dysregulated gene expression?

Answer 4

Dysregulated gene expression will disrupt processes such as cell differentiation and organism development, causing e.g. uncontrolled cell growth.

Question 5

What does transcriptional control involve/

Answer 5

involves cis regulatory elements and DNA-binding proteins. Transcription can be regulated by different factors at distinct sites in response to different signals.

Question 6

What is a TATA box?

Answer 6

A promoter sequence element with an A/T rich sequence made up of ~ 30 nucleotides long, on the 5’ of the transcription start site

Question 7

WHat are CpG islands?

Answer 7

A promoter region for constitutively expressed genes, which typically have clusters of dinucleotide “CG” (CpG islands) within their promoter regions. Methylation of CpG sequences (m5C) at the edge of CpG islands (CpG shores) correlates with transcriptional repression. CpG islands are aberrantly methylated in tumour cells.

Question 8

What is the general Organisation of Transcription Regulatory Elements?

Answer 8

So

• In mammalian genes;
• -contain both promoter-proximal elements (a regulatory sequence in eukaryotic DNA that is located close to (within 200 base pairs) a promoter and binds a specific protein thereby modulating transcription) and distal enhancers (further away).
• -Enhancers/ silencers can be up- or downstream of the promoter.
• Yeast genes;
• -contain an upstream activating or repression sequence (UAS, URS) and a TATA box promoter element.

Question 9

Why do we map transcriptional start sites of genes?

Answer 9

In order to analyse the promoter sequence of a gene

Question 10

How does gene mapping work?

Answer 10

The transcriptional start site of a gene can be mapped using a primer extension assay. Reverse transcriptase (RT) enzymes can produce a single-stranded complementary DNA (cDNA) strand using an RNA template. RT will extend the cDNA which will be measured by high resolution PAGE or by gel electrophoresis, and the position of the 5’ end of the RNA can be inferred.

Question 11

What is a common method used to analyse promoter regions of genes?

Answer 11

Regulatory regions within promoter regions can be defined by deletion analysis, using a reporter gene, e.g. lacZ, luciferase. Cell extracts from transformed cells are assayed for lacZ or luciferase activity.

Question 12

what is Linker Scanning Mutagenesis of Promoters

Answer 12

A method that uses many mutagenised sequences to identify sequences that are important for gene expression

Question 13

Explain how enhancer trapping works

Answer 13

Since Enhancer and silencer elements can be positioned kilobases away from the RNA polymerase binding site and so are not so amenable to analysis by the mutational approach. Enhancer trapping involves the random integration of a reporter gene such as lacZ into the genome using a transposon. A reporter gene with a weak promoter is randomly integrated into the genome using a transposon.Clones showing upregulated expression are isolated and the integration sites analysed by cloning and sequencing.