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