Final Exam Review

Describe how replica plating can be used to identify certain lac operon mutants

Start with an organism that exists on a medium where it does not need a particular gene, in this case, the lac gene

Mutate organisms using x-rays, some of the mutants will mutate in such a way that the lac operon is inactivated and the organism can no longer break down lactose

Press a velvet covered surface onto the plate and press it onto a new plate that contains only lactose

Monitor colony growth to see which colonies do not grow compared to the colony, these contain the mutants

Explain what a "constitutive" mutant is

A constitutive mutant is expressing a gene in an unregulated manner

Ex. Expressing the gene to process lactose even in the absence of lactose

Explain how "partial diploid" mutants provide evidence for negative control acting through a combination of a protein (repressor) and a DNA sequence (operator)

Partial diploids modify mutants by adding a functioning version of the gene

In this example, adding functioning version of lac gene to constitutive mutant

Bacteria are normally haploid but added another gene to make the organism partially diploid

Do the constitutive mutants regain control?

Some yes, some no, implies that there are two different components of negative control

One of the components was a diffusible factor, the other component was not diffusible

When the protein associates with the operator, the control is activated

Some of the constitutive mutants had a defect in lacI which encodes the protein, when adding the wild type, the functional gene can compensate

Some mutants had mutated operators (DNA sequence), adding the wild type would not restore control because the DNA sequence is not fixed

Define "operon"

Collection of genes that are under common control

Explain how, and why, glucose levels regulate expression of the lac operon

Glucose levels regulate expression to increase the efficiency of the bacteria

Glucose inhibits adenylyl cyclase

When adenylyl cyclase is not inhibited, it makes cAMP, which binds to CAP, which augments the promoter for the lac operon

Distinguish positive and negative control, and explain the value of the combination

Positive control: acts as a accelerator (glucose mechanism)

Negative control: acts as a brake (lactose mechanism)

Value of combination: the bacterium is able to conserve energy by only expressing the gene when the appropriate molecules are present or not present

Relate environmental conditions to the state of the lac operon

If glucose is high and lactose is high, lac operon is not expressed (adenylyl cyclase inhibited)

If glucose is high and lactose is low, lac operon is not expressed (adenylyl cyclase inhibited, operon is repressed)

If glucose is low and lactose is high, lac operon is expressed (adenylyl cyclase not inhibited, operon is not repressed)

If glucose is low and lactose is low, lac operon is not expressed (adenylyl cyclase not inhibited, operon is repressed)

LacX: codes for beta galactosidase

LacY: codes for permease

LacI: codes for the repressor protein

Explain the fundamental similarities and differences between the lac and trp operons

When trp is not bound to a small molecule, nothing blocks the synthesis of tryptophan

When tryptophan is adequately produced, it will bind to the repressor and allow the repressor to bind to the operon

For trp, the small molecule is referred to as a co-repressor

The small molecule for the lac operon is referred to as an inducer because it induces synthesis

Trp operon is referred to as repressible operon

Lac operon is referred to as inducible operon

****FUNDAMENTAL DIFFERENCE: the enzymes coded in the inducible operon are in a catabolic pathway, the enzymes coded in the repressible operon are in an anabolic pathway

Explain, with an example, how gene expression can be regulated at the level of translation

RNA thermometer

The mRNA for the sigma factor of heat shock proteins is not made at a normal temp

When temp raises, the mRNA dissociates from itself allowing the sigma factor to be transcribed and translated

The sigma factor can then go on and transcribed the heat shock proteins

Explain how DNAase-susceptibility experiments provide evidence of chromatin structure at particular loci, interpret such experimental data, and predict chromatin structure in relation to gene expression

If we add DNAase in vitro, regions of DNA that are open and easily transcribed should be readily attached, the regions of DNA that are more compact and less likely to be transcribed should be less eaten up

DNAase is NOT naturally present in cells

To determine if the gene is destroyed or not, can do PCR amplification to see if DNA sequence is present

Describe what information the Southern, Northern, and Western blots provide

Southern blot: is a particular DNA sequence present in a particular DNA sample, can be used to identify if dna samples are from different individuals

Bands that are at different locations indicate that the DNA is not identical

Start with DNA source, add restriction enzymes

Restriction enzymes cleave DNA at specific sequences

Fragments are separated by gel electrophoresis

After electrophoresis, dna is denatured and blotted onto filter paper

Molecule that is like a primer (allago nucleotide), referred to as a probe is added to the dna

Used to find complementary DNA

Wash away any probe that didn't bind

Visualize by exposing to xrays

Northern blot: is a particular RNA sequence present in a sample, this allows you to determine if a gene is being transcribed

Western blot: is a particular protein present in the a sample

Describe the typical effect of methylation of promotor CpG islands

CpG refers to C and G linked on the same DNA strand

General effect is to block transcription

Sustained through cell division (epigenetic effect)

Explain what genomic imprinting is and its effect on somatic cell gene expression

Particular genes get silenced based on if it is from the egg or the sperm

Crucial for gene expression because only one copy of each gene will be expressed

Define distal and proximal; explain the role of enhancers and promoter proximal elements in tissue-specific and coordinated gene expression

Enhancers are sequences in the DNA and they bind to activator proteins if they are available

If these are not available, they will not bind to mediator proteins that will efficiently transcribe genes

Are located in the introns

Distal control: enhancers that are far from from the gene being expressed

These allow for tissue specific gene expression where genes are only expressed in certain tissues

All cells have these enhancers, expression depends on what activator proteins are available

Promoter proximal: bind regulatory proteins in some cells, example is RNA polymerase binding site, close to promoter

Explain how the iron response element example illustrates UTR regulation of gene expression at the levels of translation and mRNA stability

Sequence in the mRNA called an iron response element

Element refers to the sequence

Found in both ferritin and transferrin receptor sequences

Protein that can bind to response element or to iron, called iron response element binding protein

When iron is low, the protein will bind to both sequences

For transferrin, the AU-rich region will break down the synthesized mRNA unless masked

For ferritin, the sequence is not blocked and will be read unless low iron levels are present and its translation will be blocked

Describe what RNA-interference is, ways in which microRNAs (miRNA) can silence gene expression, and a natural role of miRNA

RNA interference: RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules

miRNA can bind to a target mRNA and the mRNA will be degraded or by blocking translation

mRNA degradation is a catalytic process

This is also the natural role of miRNA

Crucial for normal embryonic development

miRNA can also be used to stop the overexpression of the onca gene which is expressed in cancer cells

Describe characteristics common to all viruses & common variations in structure and genome

Viral genome: instructions for producing new virus particles

Can consist of RNA or DNA, can be single stranded or double stranded

Plus sense: the RNA genome can also be used as the mRNA

Negative sense: the RNA genome is complementary to mRNA

RNA can serve as template for DNA synthesis

Segmented genome: Allows for the formation of recombinants

Protein wrapper: refers to capsid, which encases the nucleic acid

Purpose is to organize the viral genome, protect the nucleic acid of the virus

For non enveloped viruses, the capsid is what is exposed

Animal viruses also have a lipid bilayer surrounding the capsid which is derived from host cell membranes, have glycoproteins on envelope

Explain why antibiotics don't affect viruses, but how antiviral agents can

Antibiotics don't work because they typically target cell walls or ribosome which viruses do not have

Viruses depend on unique polymerases such as RdRp and proteases which can serve as potential targets for the antiviral

Describe the general roles and timing of our defenses against viruses, including interferons, antibodies and cytotoxic (killer) T cells—noting what vaccines do/don't promote

Interferons: innate mechanism, proteins made in response to viral infection by the cells, signalling molecules that induce interferon response which makes cells less susceptible to infection such as by reducing transcription, not enhanced by vaccines, take short time to develop

Antibodies: adaptive response, enhanced by vaccines, act to neutralize the virus by coating it to prevent it from binding, take 1 week to develop

Cytotoxic (killer) T cells: adaptive response, enhanced by vaccines, recognizes virus infected cells and activates programmed cell death in those cell, take 1 week to develop

Describe what CRISPR and Cas9 are, their natural function and location, and how CRISPR targets Cas9

Serves as an adaptive immune system in bacteria in the wild

CRIPSR: small regions of viral DNA incorporated into the bacterial chromosome

Cas9: Endonuclease that is a protein

When subsequent infection occurs, RNA is transcribed from CRISPR

The RNA associates with Cas9