Outcome 2 Flashcards
Describe genome strucutre in a prokaryote
Prokaryortic cells are haploid (one copy of every gene)
The prokaryotic genome is composed of a circular chromosome and often contiains a plasmid. The chromosome contains all of the genes required for growth and reproduction of the bacterium while the plasmid carries genes that provide a survival advantage i.e. resistance to an antibiotic. The chromosome does not contain much non-coding DNA and genes are located close to each other. Genes with related functions tend to be located close to each other on the chromosome and are under the control of a single promoter in arrangements called operons.
Descrime the organisation of the eukaryotic genome
In Eukaryotes, genes are divided between a number of separate, linear chromosomes. Each structural gene has its own set of response elements to which transcription factors bind. The chromosome consists of telomeres, centromemeres and DNA wrapped around histone proteins into nucleosomes. The chromatin can be loosely or tightly packed. There is an excess of junk DNA interspersed amongst the genes.
What is an operon
A set of adjacent structural genes which are functionally related as well as a shared promoter and regulator genes.
How is gene expression regulated in prokaryotes
At the transcriptional level by regulatory proteinds which bind to the sequences which control the transcription of structural genes.
Describe expression regulation in the Lac operon
The lac operon is an inducible operon. When lacose is absent the constitutively produced repressor protein binds to the operator preventing RNA polymerase from binding to the promoter. When lactose or IPTG are present they bind to the repressor protein, preventing the repressor protein from binding to the operator and allowing RNA polymerase to bind to the promoter.
Give an example of a repressible operon
The trp operon is a classic example of a repressible operon. When tryptophan accumulates, tryptophan binds to a repressor, which then binds to the operator, preventing further transcription.
At what three levels is gene expression regulated in eukaryotes
Transcpitional, post-transcriptional and post-translational
How are eukaryotic gene expression regulated at transcriptional level
By chromatin packing or by regulatory sequences. Regulatory sequences are dsDNA sequences that control transcription by bonding to regulator proteins.Contain promoter, enhancer, silencer operator and 5’utr,
What are introns and exons
In eukaryotes after transcription a primary mRNA transcript is formed in the nucleus that contains introns and exons.
Introns are intervening non-protein coding sequences
Exons code for protein expression.
What is a spliceosome
A large strucuture which contains proteins which are responsible for splicing.
How does a spliceosome know where to splice
It recognises splice sites - GU and AU which are cut.
What is alternative splicing
Not all exons in primary mRNA are necessarily incorporated into the final mRNA transcript. By altering which exons are spliced together, its possible to make different proteins. So alternative splicing allows for the production of different proteins from one gene.
Describe the poly(A) tail and its purpose
The poly(A) tail is a large sequence of adenosine around 200 bases long added to the 3' end of the primary mRNA transcript in the nucleus. It protects the mRNA from degradation in the cytoplasm and allows mRNA to enter ribosomes.
Describe the 5’caps purpose
Protects the mRNA from degradation and allows entry into the ribosomes for translation
What does post-translational regualtion consist of
The control of levels of active protein in the cell proteolysis and post-translational modifications are type of post-translational regualtion.
3 examples of post-translational modification
Glycosylation, phosphorylation and acetylation- adding sugar groups, phosphate groups and acetyl groups respectively
How can gene expression in a cell be measured
By analysing the type of and number of mRNA transcripts per cell.
Describe the process of extracting mRNA from cells
Cells are lysed into buffer containing agents that disrupt cell structure, disassociate proteins and inhibit RNases.
Cell lysate is loaded onto an affinity column containing immobilised oligo(dT). The column is washed through with a high salt buffer. The mRNA is able to bind to the oligo (dT). The cell components which are not bound to the oligo(dT) are washed out and removed.
The mRNA is eluted from the column by lowering the salt concentration of the buffer disassociating the mRNA from the oligo(dT)
The mRNA can be analysed by agarose gel electrophoresis.
How is RNA purity and concentration determined
By spectrophotometry - nucleic acid absorbs light strongly between 259nm and 271nm. By dissolving RNA in water and measuring absorbancy at 260nm
Why is RNA difficult to work with
RNA is unstable and degrades easily. RNases are found in all cel types, in the air and on surfaces in the lab. RNases absorb strongly to glass
What steps should be taken when working with RNA
Gloves worn at all times, DIsposable plastics used. All RNA solutions should be treated with diethylpyrocarbonate (DEPC). Work carried out at 4C if possible.
What is DEPC
A RNase inhibitor that can be added to solutions and left shaken for 2 hours. It can then be removed by breaking down at 70C for an hour.
How should RNA be stored
At -70C if a highly pure RNA sample
At -20C if in ethanol solution
What is cDNA
Complementary DNA - DNA that is originally synthesised from mRNA as a single strand of DNA complementary to the mRNA sequence.
