Gene Expression and Cell Decisions Flashcards
Identical twins have the same DNA and different people have different DNA, is this the same for cells?
No, different cells all have the same DNA
Who did the experiment and what was it that showed cells from the same organism possess the same DNA and hence changes in cells must be due to the way the DNA is expressed?
John Gurdon took a nucleus from somatic cells of a Xenopus tadpole. And placed in enucleated oocyte. Modified egg grew into a frog - differentiated cells still contain full set of genetic info required for development
What is an experimental method to analyse the cause of a change in phenotype?
Complementation tests: look for mutants in which the process has gone wrong, gather the mutants and organise into complementation group. Will only complement if mutations in different gene so can tell you if they map to one gene or more and about dominance relationships without actually knowing what gene product is doing
What is a conditional mutation?
One that only mutated under certain conditions edge temperature sensitive mutations
What is the difference between cis and trans mutations?
Cis act only on the chromosome in which they reside, trans act on a different chromosome
Why does E.coli prefer to metabolise glucose?
Lactose is a disaccharide that must first be cleaved to glucose and galactose
Who uncovered the notion of the lac operon and how?
What was he basis to suggest co-linearity of the genes lac Z, Y and A?
What was the dominance relationship of each of the genes lacZ, Y, A and I?
Jack Monod and F. Jacob by isolating mutations that would affect the growth of e.coli on lactose and analysing mutations (complementation, cis/trans, dominance)
Polarity - the mutations of Z affect Y and A and the mutations of Y affect A (a polar mutation affects the expression of upstream or downstream genes) however, in general most of the mutations in these three genes affects only the gene they are in, making the cis mutations
Mutations in any of these genes are recessive as presence of the other functioning allele will still result in functioning gene product
How are the lac operon genes transcribed?
As part of the same unique mRNA so they will also be translated in series therefore
What helps to confer specificity of the operon?
The binding site of the repressor molecule is composed of a duplication of the 5bp binding site of the recognition alpha helix of the repressor, so requires a dimer of the repressor.
being made up of two parts reduces the chances of a binding site being elsewhere in the genome as the 5bp binding sequence of the helix alone would be to likely to appear elsewhere
How do mutations in the I gene differ to those in the lacZ, Y or A genes?
Mutations in the lac I gene are trans rather than cis as they alter the expression on the other three genes
How do mutations in the O gene (operator) differ?
Where is P in relation to O?
Not affecting a protein product but a binding site
Two types of mutation:
Constitutive (cause X,Y and A to be always expressed even in absence of lactose)
Superrepressor - no expression under any conditions
They are cis acting and dominant
p is just upstream of O suggesting mutations in O operate by affecting activity of RNA polymerase
How does the superrepressor O mutation prevent expression?
The repressor molecule binds so tight it won’t come off
What ensures glucose will always be metabolised first even though both may be present?
CAP = catabolise activating protein which binds cAMP causing the DNA binding site on CAP to rotate 60 degrees, increasing affinity for DNA by allowing CAP to fit into the major groove without steric clashes. By binding DNA CAP bends DNA, stabilising the binding of RNA polymerase.
cAMP levels are kept low in the presence of glucose, when glucose levels low cAMP rises to promote operon transcription, aided by the fact that the inducer molecule prevents repression
How does the repressor molecules binding in dimers aid the system?
Through cooperativity: when one repressor on its own bind it can unbind quite easily but when another binds nearby (on the repeat) binding is stabilised
By stabilising binding fewer repressor molecules are required to regulate the system, increasing specificity and sensitivity of the system
What further aids this cooperativity?
What is the result?
The are an additional two operator regions, one either side of O1 but far away. They increase cooperativity by binding repressor and through looping of DNA to fix repression in place .
Results in one functional molecule of repressor per cell which has consequences
What do we call the use of proteins like CAP to regulate binding of proteins like RNA pol?
Regulated recruitment
What is the significance of having only one functional repressor per cell?
Gene leakage - the repressor continually binds and unbinds, when unbound there is no other molecule to do its job so some expression is allowed (leakage) this allows some molecules of permeate to be made so that when lactose eventually is present it can get into the cell to induce the system (via allolactose)
Allows positive feedback loop
How are feedback loops used in maintenance of the genetic system?
Permease allows some lactose in, which induces the system so you get more permeate and more lactose in…positive feedback that supports economy of system
What is a bacteriophage?
What plaques can form on infection?
What is the result of a bacteriophage persisting within the cell?
Can lytic plaques give rise to a lysogenic plaque?
A bacterial parasite that, when it infects a cell, will either kill the cell in the process of making many replicates of itself, or persist in the cell by integrating its DNA into the bacterial chromosome
If the bacteriophage just kills in a lytic cycle a clear plaque called a lytic plaque forms
If is sometimes kills and sometimes persists a turbid plaque forms
The infected bacterium gives rise to a lysogen not which is resistant to further infection
No but you can induce lysis of a lysogenic plaque using UV light
What is a complementation group?
A set of mutations that don’t complement one another make up a complementation group
What are the four complementation groups that determine the plaques formed?
cI, cII, cIII and cro
