Molecular Genetics 19-24 Flashcards
Where can variation be found (3 places)?
- In the surrounding population
- In distant populations
- In related species
Why might variation in the local population be limited?
Due to previous selection
What is the A. E. Watkins collection?
A collection of different breeds of bread wheat collected in the 1920s/30s from 32 countries by A. E. Watkins to capture diversity
What is the germplasm development programme?
The transfer of an entire distant relative genome to wheat in overlapping segments
What grass plant that is distantly related to wheat can provide salt resistance to wheat?
Thinopyrum bessarabicum
What does a chromosome from rye do to wheat?
Makes the wheat more resistant to drought, heat resistant and disease resistant, so it is suitable for animal food
What is the solution when the available variation is not enough?
Carry out a mutagenesis programme
What are examples of physical mutagens?
Ionising radiation (X-rays, gamma rays etc.). These form radicals that break DNA strands Non-ionising radiation (UV). Absorbed by pyrimidines in DNA, causing adjacent bases on the same DNA stand to bond covalently and form pyrimidine dimers
What are examples of chemical mutagens?
- Base analogues such as 2-amino purine (which resembles adenine) and 5-bromouracil (which resembles thymine)
- Intercalating agents such as ethidium bromide, proclaim and acridine orange are compounds that slip between adjacent base pairs in DNA
- Base-modifying agents include alkylating agents (eg ethyl methane sulphonate (EMS)), deaminating agents (eg nitrous acid) and hydroxylating agents (eg hydroxylamine)
What does EMS (ethyl methane sulphate) do?
- Adds an ethyl group to guanine and produces 6-ethylguanine, which pairs with thymine and leads to CG:TA mutations
- Also adds an ethyl group to thymine to produce 4-ethylthymine, which then pairs with guanine leading to a TA:CG mutation
What are the two types of genetic screening using mutagenesis?
- Forward genetics
2. Reverse genetics
What is forward genetics?
A forward genetic screen is an approach used to identify genes (or a set of genes) responsible for a particular phenotype of an organism
What is reverse genetics?
A reverse genetic screen analyses the phenotype of an organism following the disruption of a known gene
What is the method used to make forward genetics cheaper and save sequencing the whole genome including non-coding DNA?
Use an Exome capture array
How much larger is the wheat genome compared to the human genome?
6 times larger
Outline the steps of carrying out an Exome capture array:
- Sonicate DNA
- Expose fragments to a support - only coding sequences hybridise to exome baits on support as the probes on the support are each specific to small parts of the coding DNA
- Only costs £300-£600 as only exome DNA is sequenced
What does sonicate mean?
Subject a sample to an ultrasonic vibration so as to fragment the sample
What is Exome capture array useful for?
- Used to sequence coding regions from wild type and mutant lines
- Differences enable you to narrow down the gene underpinning the observed phenotype
- Use sequence to design molecular markers to track the differences and hence find out which are linked to the phenotype
When was targeting genome editing first discovered?
2005-15
What were the old methods of targeted genome editing?
Zinc Finger Nuclease
and
TALENs and TAL effectors
What does TAL stand for?
Transcription Activator-Like
What does TALEN stand for?
Transcription Activator-Like Effector Nuclease
What were Zinc Fingers / TALEN nucleases?
They produced protein subunits that recognised specific DNA sequences in the genome. The protein subunits could each recognise around 3 based, but several subunits could be used in combination to recognise and bind to a sequence of 9-18 base pairs. Each protein had a restriction endonuclease attached to them (e.g. fok1) to cut the sequence between the proteins
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
What is CRISPR?
Functions as the prokaryotic ‘immune system’ to fight phage infections
When was CRISPR-Cas9 first discovered and in what species?
In 1987 in E. coli
How many types of CRISPR systems have been identified so far?
3 types, Type I, II and III
Which type of CRISPR system is the basis for current genome engineering applications?
Type II
What species is the Type II system of CRISPR taken from?
Streptococcus pyogenes
Outline the steps of a bacteriophage infection and the use of the CRISPR system to fight it
- Phage infection
- Spaced acquisition: certain CRISPR-associated (Cas) enzymes acquire spacers from the exogenous protospacer sequences and install them into the CRISPR locus within the prokaryotic genome
- crRNA biogenesis and processing: these spacers are segregated between direct repeats that allow the CRISPR system to mediate self and non-self recognition. The CRISPR array is a noncoding RNA transcript that is anzymatically maturated through distinct pathways that are unique to each type of CRISPR system
What does the Cas9 protein do?
Cut the phage DNA and remove it from the bacterial genome
What does genomic manipulation using CRISPR systems require?
- The Cas9 protein
- An engineered small guide RNA (sgRNA) with a PAM sequence upstream of complementary target sequence
What happens when the sgRNA meets the target DNA?
There is base-pairing between the sgRNA and the target DNA, which causes double-strand breaks due to the endonuclease activity of Cas9
Is CRISPR-Cas9 considered GM?
It was initially, as you were introducing foreign DNA into the cell.
Nowadays you can simply introduce the Cas9 protein into the cell and RNA rather than DNA - but is this still GM technology?
With any guide RNA, there must be a PAM sequence, what is a PAM sequence?
NGG
N = any nucleotide
How do cells repair double strand DNA breaks?
- Non-homologous end joining (error-prone): Joins the two cleaved ends back together, even though DNA has been removed - many insertions/deletions
- Homologous recombination (faithful): brings in one strand of other chromosome and carry out the repair based on the sequence of a template strand of the other chromosome - can make individuals homozygous when they were previously heterozygous
What is the strand cleaved (cut) by?
RGEN
RNA-Guided Endonuclease
What is Spo11’s role in meiosis?
Its a protein which influences whether recombination occurs
Ways to knock out Spo11 in hexaploid wheat lines
- Gamma deletion line
- EMS tilling lines
- Gene editing
What are the problems with using gamma deletion lines and EMS tilling lines to knock out Spo11 in hexaploid wheat lines?
Both methods imprecise and require screens to identify desired lines
How is gene editing used to knock out the Spo11 gene?
All 6 copies of Spo11 need to be knocked out (because it is hexaploid). Protoplasts were prepared from leaf tissue of plants transformed with Cas9-P2A-GFP to test the construct. Once it was obvious the process worked, it was applied to wheat. After one attempt, 5 out of 6 of the Spo11 gene had been altered
What happens when a wheat plant has had all 6 copies of the Spo11 gene edited out?
It is sterile
How can you modify the Cas9 nuclease to target any protein/structure in the genome and stay on the DNA rather than cutting it?
You can modify it so that it binds to the RNA but does not cut, instead it stays bound to the guild RNA
CRISPR-Cas9 applications
- Targeted mutagenesis
- Insertions
- Deletions
- Precise insertions or gene replacements
- In vivo imaging of specific genome loci (attach GFP)
- Up-regulation (activators attached which switch genes off in that region)
- Silencing (CRISPRi) (repressors attached which switch genes off in that region)
How is CRISPR-Cas9 used to activate certain genes?
Put activation gene VP64 to express gene and cause protein to be produced
Can the method be detected after genome editing?
No, impossible to detect his modification was made. Once you have created the mutant you simply cross out the transgene (so no longer GMO)
What is PHS?
Pre-harvest sprouting
The germination of grains in the ear before harvest
What causes PHS?
Heavy rainfalls and high humidity are factors
What are the main effects of PHS?
Lower yield and diminished quality of grain
Flours obtained from sprouted grains have less thickening power and the bread baked with these flours have smaller volume and a sticky structure
What is the name of the quality control test used to evaluate the amount of ‘sprout damage’ in wheat?
The Hagberg falling number test (HFN)
How does the HFN test work!
If germination occurs there is a dramatic increase in the amount of alpha amylase.
The falling number is the time in seconds for a stirrer to fall through a hot slurry of ground wheat.
The greater the amount of alpha amylase in the wheat, the thinner the gelatinised starch paste and the faster the plunger will fall through the slurry.
A high falling number indicates the wheat is sound and satisfactory for most baking purposes
What is the falling number of a batch of No.1 wheat?
Over 300 seconds
What is the gene underpinning preharvest sprouting?
Viviparous 1 (vp1)
What does the vp1 gene do?
It encodes a transcription factor which represses genes encoding hydrolytic enzymes such as a-amylase
What class of transcription factors does Vp1 belong to?
B3 domain transcription factors
What is the problem associated with vp1 and PHS?
No mutation in vp1 has been detected which might lead to pre-harvest sprouting - it appears to be highly dependent on the environment
What is alternative splicing?
The different ways the exons of a gene may be combined, producing different forms of proteins within the same gene-coding regions
What are the types of alternative splicing?
- Primary isoform: using every exon
- Cryptic exon: a small bit of intron becomes an extra exon
- Exon extension: certain exons are extended beyond their regular length (5’ or 3’)
- Exon skipping: one exon left out
- Exon truncation: shortening an exon
What is RT-PCR?
Using reverse transcriptase to convert an mRNA template with oligo dT primer into cDNA. The gene activity is seen on an agarose with ethidium bromide staining
What base pairings are more common in exons?
C-G
What bass pairings are more common in introns?
A-T
What is the consequence of introns having more A-T base pairings?
Introns are more likely to contain a stop codon (TAG, TAA or TGA). If these introns are included in the protein due to alternative splicing the stop codon will truncate the protein
When McKibbin ran products of RT-PCR from mRNA of Vp1 gene, why was there multiple bands on the gel as opposed to one band for the vp1 gene?
Evidence suggests that bands are due to alternative splicing of Vp1 gene product leading to a number of different proteins but with insufficient functional vp1 protein to ensure a high degree of embryo dormancy
What is the link between the wetness of summer and the amount of alternative splicing
The wetter the summer the greater the amount of alternative splicing, therefore the higher the likelihood of alternative splicing
What plant often grows alongside the wheat, which germinates much later than wheat?
Wild oat
How could GM technology use the wild oat late germination to delay the germination of wheat? What is the problem with doing this?
The wild oat vp1 gene could be put into wheat, producing a transgenic wheat plant with much less pre-harvest sprouting. The problem is that the wheat will not germinate at all with the wild oat gene
How could scientists possible control when the wheat germinates?
Remove the promoter of the vp1 gene and replace it with a promoter that could be controlled
What is epigenetics?
The study, in the field of genetics, of cellular and physiological phenotypic trait variations that are caused by external or environmental factors that switch genes on and off and affect how cells read genes instead of being caused by changes in the DNA sequence
Who coined the term epigenetics?
C. H. Waddington
What does ‘epi’ mean in epigenetics?
Above or in addition to
What is mosaicism?
When an individual has two different eye colours or an eye with two colours
Does DNA usually exist in ‘standard form’ (a naked DNA molecule)?
Rarely, the DNA bases are chemically modified and the histones associated with the DNA in nucleosides are often also chemically modified
What are the two forms cytosine can appear in?
Methylated and non-methylated - there are technically more than 4 bases if you also think about form
What differentiates totipotent embryonic stem cells from unipotent adult stem cells?
DNA methylation - more CGs are methylated in a unipotent adult stem cell, which reduces differentiation potential by reducing or stopping gene expression
Where is the site of DNA methylation along the DNA strand?
CG
What is the result of cytosine methylation?
It switches off gene expression
What is the epigenetic code?
The specific epigenetic modification in each cell, consisting of histone modifications and DNA methylation. It is cell and tissue specific
What is histone modification?
A combination of different molecules can attach to the “tails” of proteins called histones, which alter the activity of the DNA wrapped around them
What is euchromatin?
The transcriptionally active form of chromatin, which is found in decondensed form. 92% of the human genome is euchromatic
What is heterochromatin?
The more tightly condensed form of chromatin, in which the activity of the gene is modified or repressed
What epigenetic features does euchromatin have?
- High histone acetylation
- Low DNA methylation
- H3-K4 methylation
What epigenetic features does heterochromatin have?
- Low histone acetylation
- Dense DNA methylation
- H3-K9 methylation
What was Lamarcke’s theory of Inheritance?
Adaptation created new variations within an organism’s lifetime
These characteristics were inherited by offspring
What was Darwin’s theory of evolution?
Random hereditary variation occurred first, followed by selection of the variations
What was Lamarcke partially correct about?
Traits due to epigenetics CAN be inherited to offspring
Outline the agouti mouse example
The agouti allele Y is lethal when homozygous and heterozygous individuals have a tendency towards life-threatening diseases and a shorter life expectancy.
When researchers crossed heterozygous agouti mice, their offspring were expected to be the same: overweight and yellow.
However, they changed the mother’s diets and fed them a diet rich in methyl donors. These made their way to the embryos’ chromosomes and reduced the deleterious effect of the Y allele
Outline the Hongerwinter example of epigenetics
The German army blocked food to the Dutch in 1944. There was a famine
Children born or raised in this time were small, short in stature and had many diseases.
It was found that women living during this time had children 20-30 years later with the same problems despite being conceived and born during a normal dietary state
What high blood pressure medication is produced from fungi?
Lovastatin
What are non-ribosomal peptides?
Peptides produced by nonribosomal peptide synthetases rather than ribosomes, and independently of mRNA
What are polyketides?
Chains of acetate-derived units. They are secondary metabolites
What is a secondary metabolite?
It is an organic compound produced by bacteria, fungi or plants which is not directly involved in the normal growth, development of reproduction of the organism. Their absence does not result in immediate death, but rather in long-term impairment of the organism’s survivability, fecundity or aesthetics
What is a hybrid system when talking about classes of compound produced by fungi?
Polyketide/Non-ribosomal Peptide hybrid
Polyketide chain joined to one or more amino acids, made by a large hybrid enzyme
What was the first immunosuppressant (produced by fungi) which allowed organ transplants?
Cyclosporin A
What class are most antibiotics produced by bacteria or fungi in?
Polyketides
Outline the old method of finding the genes responsible for producing polyketides and nonribosomal peptides
- Polyketide syntheses (PKS) are conserved in structure
- Design degenerate primers for PCR
- Amplify from genome
- Clone and sequence PCR product - you will have many polyketide synthase gene products as they will all have the same conserved region
What is useful about an inosine base?
It can be used to synthesise primers where there is degeneracy in code as it will bind to any nucleotide
How to then find the whole gene encoding the polyketide using just the small piece amplified through PCR?
Use a lambda library made from the genomic DNA of your organism
What is a lambda library and how is it used to identify the genes responsible for producing polyketides?
Lambda libraries are where you’ve taken the genome of your fungus, extracted the DNA, randomly broken it up into big pieces and cloned each piece into a different vector. This is all done at once. The vector is a bacteriophage lambda. You can chop out a piece of the bacterial genome and clone in DNA from the fungus (15-20kb per virus particle). This must happen millions of times to cover the whole fungal genome. You then let the bacteriophage grow on a petri dish, and place some of these on a membrane. Then probe the phages on the membrane with your PCR product, and whichever it binds to is your gene of interest, so you can select that phage, grow it and sequence it. If the whole gene is not there, you take the end of your sequence, design a new primer, and find the corresponding piece of phage containing that sequence
What is a bacteriophage lambda?
It is a double-stranded DNA virus that infects E. coli
What is the advantage of gene layout in fungi that allows you to find genes with a similar function easily?
Genes with similar functions are found in clusters together in the genome, all you have to do is find the conserved synthase and work outwards
How do you prove you have found the right set of genes that code for polyketides?
Gene disruption
How does gene disruption work?
- Target gene removed and replace with selectable marker
- Fungi are haploid, so get an immediate phenotype
What is the other name for gene disruption?
Gene knockout
How to work out which gene does which step of producing polyketides?
Create a series of expression plasmids and build up the pathway step by step in a different fungus (usually an aspergillosis species) which doesn’t produce the polyketide.
Each gene expressed by amyB promoter and terminator - strong promoter which expressed gene at high amounts, so this fungus produces much more of the proteins than the original species which produced it
What is tennelin?
Tennelin is a polyketide-amino acid hybrid produced by the enzyme TenS
What is heterogous expression?
Expression of a gene in a host of a different species
What is DMB?
A polyketide produced by the polyketide synthase DMBS. It is produced in a related species of fungi to the species that produces tennelin
What is the strategy of domain swaps?
Swapping sequential domains of enzymes to make hybrid enzymes, which helps to understand what parts of the enzyme programme the substrate specificity
What are focussed domain swaps?
Creating hybrid enzymes that will produce polyketides with specific methylation and extensions, which can be controlled and altered
Outline the identification of the tropolone pathway
- It is extremely hard to convert a 6-carbon aromatic ring to a 7-carbon aromatic ring
- Bacteria and fungi are able to do this
- Two fungi species were identified which could carry out this reaction
- Sequenced the genome of both fungi and annotated the gene assembly
- Searched for polyketide-producing gene clusters in genome
- Narrowed down to 8 possible genes that are non-reducing
- Narrowed down to 2 possible genes that are non-reducing and methylating
- Narrowed down to 1 gene through comparison of the two genomes to find one gene they shared in common
- Gene knockout of tspks4 showed that the mutant stopped making the 7-ring compound, so they had found the correct gene
What are the two fungi species which possess the tropolone pathway?
Acremonium and Talaromyces
How many polyketide-producing gene clusters were found in Talaromyces stipitatus?
39 gene families
Isolating the gene involved in the tropolone pathway - TsL1
- Cloned the gene into an E. coli expression vector with 6 histidines tagged on the front of the protein
- Under the control of a T7 promoter under the control of lacZ
- Add IPTG to switch on expression
- The protein produced is impure as it has been produced alongside many other E. coli proteins
- Take mixture and run mixture down column with nickel bound all the way through the column which catches all the proteins with the his tag
- Put proteins in a column that is based on size exclusion and purifies proteins based on size
Testing if the correct protein has been isolated which is involved in the tropolone pathway
Chemists synthesise substrate with 6-carbon ring, purified substrate is added to see if it will convert to 7-carbon ring.
Result is that 6-carbon compound has been changed - this is the first enzyme involved in the reaction which produces a 7-carbon compound
Finding the pathway that produces cornexistin
- Whole pathway needed to be found
- Cornexistin and byssochlamic acid are very similar compounds produced by different fungi
- Sequence genomes of fungi that produce each of these compounds
- Look for similar gene clusters with the right sorts of activity
- Similar process to finding tropolone pathway
What is cornexistin?
Potential selective herbicide, made by a fungus
Kills grass but not maize
What is byssochlamic acid?
Used to be a problem in food processing - a toxin that would be produced when bottling food for storage as the fungus is thermotolerant
What is the modern way to express the genes involved in polyketide synthesis?
Express up to 4 genes per plasmid
5 selectable markers (plasmids) can be introduced into one cell, so 20 genes can be coordinately expressed
What is the feature of yeast that allows it to be used to express gene clusters?
It easily becomes recombinant - has efficient recombination system.
What is a bipartite system?
An improved form of gene disruption: Selectable marker split into two separate pieces of DNA. Only homologous recombination will give the full market and increases targeting efficiency, as if selectable marker is not split in two it could go into the wrong place within the vector, so the transformed cell will be resistant to antibiotics, but the targeted gene would not have been knocked out.
What is genome mining?
-Searching for new clusters with unknown products which may be useful
Why is RNA more challenging to work with than DNA?
- It degrades easily - enzymes within many organisms which break down RNA due to viruses having RNA genomes
- You have to get it from the right tissue
- You can’t directly clone it
- You can’t directly sequence it - have to convert it to cDNA first
- Can’t directly PCR it
Why is working with plant viruses difficult?
- Most have RNA genomes
- They are viruses so only replicate in a living host
- No effective chemicals for their control
- No drugs can be used in agriculture to slow the virus
What are the best ways to prevent virus spreading between plants?
- Good sanitation in the lab
2. Plant breeding for resistance
Example of a plant affected by virus
Pepino (small evergreen shrub) Cultivated in South America Produces sweet fruit Susceptible to a viral disease: pepino mosaic virus (PepMV) Only a small problem affecting one plant
The bigger problem with PepMV
It JUMPED HOST
In the 1980s tomatoes became susceptible to PepMV
It spread to Europe and began threatening commercial tomato production
What is the structure of PepMV?
- Single-stranded positive sense RNA genome
- 6.4kb in length
- 3’ polyA tail
- 5’ cap
- Translated into 5 peptides
- Structured similarly to a bacterial operon - 5 different ribosome binding sites
How is it confirmed that PepMV is a member of the Potex family?
- RNA removed from diseased plant
- Degeneratr Potex primer which binds to a conserved region found in all potex viruses is used to prime the RNA and reverse transcription is carried out to convert the RNA to cDNA
- PCR amplifies the cDNA product
- Product put in a PCR cloning vector and sequences to find the internal part of the virus’s sequence
How to work out the rest of PepMV’s sequence using the result of PCR and primers?
- Grind up sample and leave out, ssRNA will be degraded but dsRNA is much more stable so is not
- Run it on a gel to separate out the dsRNA
- Add a polyA tail to the ends of the double strand using the enzyme terminal transferase
- Create primers for the middle sequence you know and the polyA tails
- They will ligate with the known sequences
What is an infectious clone?
An artificial version of a virus.
RNA genome made in lab which can infect plants and become a full working virus
What are the two methods of producing an infectious clone?
- Create the RNA genome in vitro through transcription of the cDNA
- Drive the expression of the cDNA in a plasmid vector WITHIN the plant with a strong 35S promoter
What is homologous recombination?
A type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical pieces of DNA
Outline the process of making an infectious clone in vitro
- Amplify up the virus genome in 5 pieces
- Put them together in a yeast vector URA3 using a crossover system
- The 5 pieces overlap slightly by about 50 bases
- The beginning and end of the viral code has a small part of the vector sequence added on
- Transform all pieces at once into the vector and select for transformants
- Produces a circular plasmid with full viral cDNA
- Purify plasmid and linearise it with Kpn1 so polerase will stop transcribing at that point
- Add T7 RNA polymerase and NTPs to carry out transcription
- RNA produced is the virus and will infect a plant if rubbed onto it
How were the different parts of a virus that cause virulence and specific symptoms found?
Chimeras were created between a more and less virulent strain of the virus, where specific parts of the RNA were swapped around
What gene can confer resistance to PepMV, but what is the problem with this?
Rx from potatoes
However this makes the plant GM, and the virus will mutate to overcome the resistance quickly
Challenge with unstable DNA when creating an infectious clone and how to get around this
- Some viral cDNA is very unstable in E. coli. The plasmids will fall apart because the DNA sequences have hidden ORFs which E. coli recognises and transcribes, but they produce toxins which kill E. coli
- The solution to stabilise the DNA is to put plant introns in the unstable regions of the virus. E. coli cannot splice out introns so the introns will not be removed and the toxic genes will not be transcribed
What is the problem with putting introns in unstable viral sequences within E. coli and what is the solution?
Only eukaryotes carry out splicing so the introns cannot be removed by E. coli
The solution is to put a 35S plant promoter before the genes and transform agrobacterium to contain the vector containing the viral sequence. This will transport the vector to the plant nucleus where it can undergo splicing and transcription
What does Agrobacterium normally do to a plant when infecting it?
Transfers cytokinine genes into the nucleus of the plant causing uncontrolled growth and opine genes to feed the bacteria. It is tumour-inducing
What is transient expression?
The agrobacterium doesn’t need the DNA to be integrated into the chromosome, it just needs to reach the nucleus to undergo transcription
Outline the steps of agroinfiltration
- Syringe-inject Acetosyringone-induced Agrobacterium cultures through the plant’s stomata
- T-DNA moves into the plant cells
- Migrates into the nucleus
- Transcription from p35S
- RNA is spliced, removing introns
- mRNA is exported into the cytoplasm
- Gets translated to give proteins
- These replicate the RNA
How can viral proteins be tagged to track the action of a virus?
By fusing GFP to the viral proteins
How do scientists work out how plant viruses are transmitted via aphids?
The GFP-tag all the viral proteins
See where the proteins stick on/within aphid e.g. the end of the stylet, through the salivary glands etc.
How to exploit plant viruses to use in human healthcare
- If you can use infectious clones to make GFP, you can use them to make any protein
- The plant isn’t GM, only the virus has been genetically modified
- Infect the plant, grow and harvest the leaves, then extract your protein
How can viruses be modified to be vaccinations by changing their coat protein?
- Modify the viral DNA so it no longer has the viral coat protein and replace this with the antigen to an infectious disease as its coat protein
- The virus will replicate within the plant but cannot move from plant to plant
- Extract these viral proteins - an animal disease coat protein with a plant virus genome inside cannot infect and human and make them sick, so would be safe for vaccinations
