Molecular Genetics (Katherine Ford 25-30) Flashcards

1
Q

Give 5 reasons why we use model organisms?

A

1) Easy to grow and maintain in the lab.
2) easy to manipulate.
3) short generation times.
4) small, sequenced genomes.
5) can extrapolate from simple organisms to complex ones.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are a typical gram-negative bacteria used as a model organism?

A

E.coli

  • 20 min generation time!
  • can be frozen/stored
  • used to study conjugation 1946
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are a typical gram-positive bacteria used as a model organism?

A

Bacillus subtillis

  • found in soil & digestive systems of ruminants.
  • spores are tough
  • easily secretes proteins.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Why is Saccharomyces cerevisiae a good model organism?

A
  • doubles every 90 mins
  • full genome available
  • several plasmids to work with, including integrating plasmids.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Why are modified plasmids good cloning vectors?

A
  • easy to transfer between cells
  • easy to miniprep/ isolate DNA
  • abundant in cell
  • easy to screen for recombinants

(Note: PCRII-TOPO)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are the three types of artificial chromosomes that can be used as vectors?

A
  • BACs (circular)
  • YACs (Linear/ can take the largest fragments)
  • PACs

-used for genomic libraries

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Name an advantage and a disadvantage of using YIPs as vectors.

A
  • Yeast integrating plasmid = integrates into the chromosome.
  • produces stable transformants
  • but, transformation rate is slow.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

How do Lambda bacteriophages help with producing transformants?

A

Lambda replacement vectors are from a bacteriophage and can complete their lifecycle even with foreign DNA inserted into them.

  • The ds linear DNA has two cohesive ends (Cos sequences), once in the cell, these are cleaved off and the DNA circularises.
  • The middle of the vector is replaced with a multiple cloning site.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What are Cosmid vectors and how do the work?

A

They have highly modified Lambda vectors but everything between the cos sites is replaced instead.
- DNA up to 45Kb can be inserted

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What is Gateway cloning?

A

Exploits Cosmid vectors and can clone up to 4 DNA fragments.
- ccdB region is replaced with the gene of interest.
PCR product is taken up by the vector and clonases swaps the gene of interest into lots of destination vectors.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What method is useful if we want to clone more than 4 fragments?

A

Using Shuttle vectors as they have more selection markers.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

List the steps required for transforming vectors into yeast.

A
  • LiAOc makes yeast competent.
  • Add PEG, the plasmids and PCR product.
  • culture and heat shock
  • plate out on nutritional medium (auxotrophic selection) and recover plasmids via miniprep.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

List the steps required for transforming vectors into E.coli.

A
  • permeablise the cell wall by calcium chloride and heatshock & ice or by electroshock.
  • Blue-white screening
  • miniprep.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How can you transform fungi?

A

Electroshock, PEG-mediation or via Agrobacterium.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Which fungus is used for citric acid production?

A

Aspergillus niger produces citric acid with the same chemical composition.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Which microbe is often used to produce amino acids?

A

Corynebacterium glutamicum

  • accumulates glutamate under limiting conditions.
  • also, mutant forms which could withstand high lysine conditions (limiting) have been used to produce Lysine, an essential amino acid which is added to animal feed.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Describe how vitamin C is produced by biotechnology.

A
  • initially, Gluconobacter Oxydans was used alongside chemical synthesis.
  • Using Ewrwinia & Corynebacterium together is found to be more useful but they require different growth conditions.
  • Can GM the Corynebacterial gene into Erwinia so that all is required is acid treatment.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What are three disadvantages of using E.coli in recombinant technology and what can be done to overcome them?

A

1) Codon bias (AAA codes for lysine 75% of the time)
- Overcome by site-directed mutagenesis/ synthesising the DNA/ adding the missing tRNAs/ integrating relevant genes into E.coli.

2) Poor Secretion
- can be overcome by fusing protein to a bacterial protein that is usually secreted
- adding additional secretory sequences.
- expressing the protein in a gram-positive bacterium instead.
- modification of E.coli to give it better extracellular secretion.

3) Post-translational modifications are different to eukaryotes. (70% of human genes are glycosylated and add glycosyl groups to N-terminal groups; whereas prokaryotes at them to side chains.
- overcome by adding glycosylation genes to E.coli.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What model plant species is often used when investigating crops?

A

Arabidopsis thaliana

  • has a similar genetic response to stress and diseases.
  • crops are complex
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What are the three types of tissue culture used when producing transgenic plants?

A

1) Plant leaf discs
2) callus culture (immature embryos or meristems-totipotent)
3) suspension culture (immature protoplasts/ pollen.)

21
Q

What pathogen is exploited as a tool in plant transgenics?

A

Agrobacterium tumefaciens

  • gram-negative
  • attracted to plant wounds
  • induces the plant to produce excess auxin and cytokinin.
  • galls contain opines synthesised by a tumour inducing (Ti) plasmid.
22
Q

How does A.tumerfaciens act as a pathogen and how is this exploited by transgenics?

A
  • wounded plant cells produce signal molecules which are recognised by receptors.
  • Agrobacterium attaches to plant cell and activates VIR proteins which produce T-DNA.
  • T-DNA complex forms and transfers into cells, where it moves into the nucleus.
  • T-DNA is then expressed by bacterial proteins.

-we utilise this by inserting a selectable marker and gene of interest into the T-DNA.

23
Q

How does Agrobacterium tumefaciens regulate VIR proteins to produce T-DNA?

A
  • VirA detects wound and autophosphorylates.
  • VirA then phosphorylates VirG (a transcription factor) which initiates transcription of genes Vir B-E.
  • VirD1 and VirD2 are DNA nickases which cut and releases ssT-DNA.
  • VirD2 attaches to the 5’ end of the T-DNA and VirE2 coast it.
  • T-DNA exits the bacterium, into the plant and is moved to the nucleus.
24
Q

How can plant transgenics be used for herbicide tolerance?

A
  • make a crop resistant to glyphosate (roundup)
  • glyphosate inhibits synthesis of some amino acids by binding to EPSPS
  • by making mutant ESPS, glyphosate can’t bind to it.
25
Q

How can plant transgenics be used for insect tolerance?

A

Bt toxin:

  • cry genes from Bacillus thuringiensis produce a toxic crystal protein which binds to the epithelial cells of the digestive tract and creates holes.
  • CRY1 proteins are effective against Lepidopteran pests.
  • CRY3 proteins are effective against Coleoptera.
  • can choose Bt toxin specific to a pest.
  • expressed Bt in Maize and reduce problems caused by corn borers. (pesticide use was able to decline 11%)
26
Q

Why is Drosophila melanogaster a good model organism?

A
  • 2-week life cycle
  • easy to maintain the lab
  • well studied (only 4 chromosomes)
  • polytene chromosomes with characteristic banding patterns - good for mar marking.
27
Q

How can insects be used as vectors?

A
  • exploitation of baculoviruses
  • insect eats contaminated leaf and the intestinal epithelial cells release single virus particles which infect adjacent cells
  • Packages of virus particles are formed called polyhedrons.
  • Polyhedron promoters are v strong
  • a sequence can be placed in AcMNPV vector.

(could also use Bacmids from E.coli.)

28
Q

How may insect vectors be able to prevent the spread of human diseases?

A

Malaria is caused by Plasmodium sp.

- can engineer mosquitoes so they are less susceptible to Plasmodium by increasing levels of defensin A.

29
Q

Why are Nematodes good model organisms?

A
  • 2 sexes
  • the generation time of 3 days (4 larval stages before adult)
  • easy to maintain in the lab
  • transparency can be useful for reporter such as GFP.
30
Q

What is special about Flavr Savr tomatoes?

A

They were modified using antisense version of a gene.
- Agrobacterium tumefaciens was used to introduce an antisense copy of the polygalacturonase gene = delayed ripening and prolonged shelf life.

31
Q

Three ways in which RNAi can silence genes.

A

1) introducing antisense versions of genes
2) include a dual promoter for positive and antisense RNA
3) hairpins can produce dsRNA and form a loop, preventing translation.

32
Q

Why do extra RNAs silence genes?

A
  • may block translation by preventing ribosomal binding

- block post-transcriptional modifications by preventing binding of spliceosomes

33
Q

How can RNAi permanently repress gene expression?

A

RISC and associated siRNAs can bind complementary DNA.

  • siRNA directs heterochromatin- forming enzymes to the target loci.
  • when ‘open’ DNA is converted to heterochromatin, no more mRNA is produced.
34
Q

Advantages and disadvantages of using mammalian cells for recombination.

A
  • effective post-translation modifications
  • accurate folding
  • codon usage
  • accurate splicing
  • little regulation needed
  • difficult to culture cells
  • expensive and slow
  • limited large-scale production
  • low yield
  • susceptible to human viruses.
35
Q

What is the difference between cloning and transgenesis?

A

Cloning = generating progeny that are identical to the parent.

Transgenesis = introducing foreign DNA

36
Q

What are pluripotent cells?

A

Cells that can differentiate into all cells inside the embryo (not extraembryonic ones)

37
Q

What are the 3 methods to create transgenic mammals?

A

1) Microinjection of DNA into fertilised eggs and then implanted into a foster mother.
- need to cross heterozygous transformants to get 25 % homozygous progeny.

2) Transforming embryonic stem cells/ iPSCs (induced pluripotent cells from adults)
- insert vector with the gene of interest and the cell will express it.

3) Retroviruses
- ‘Infect’ the developing embryo with no need for microinjection
- viral DNA is also sometimes incorporated and sequences are limited to 8kb.

38
Q

4 Examples of transgenic animals.

A

1) Rat somatotrophin gene injected into mice was the first case of stable gene inheritance and normal function
- produced larger mice
- Note: mighty mouse/ smart mouse etc

2) Bovine growth hormone injected into cows for increased milk production.
3) Glofish on sale in the US
4) AquAdvantage Atlantic salmon has a gene from Pacific chinook and promotor from ocean pout = can grow all year round and reaches market size in half the time.

39
Q

What three methods may be used in gene therapy to deliver a gene to cells?

A
  • Direct injection
  • Aerosol inhalation
  • cell removal, treatment and implantation
40
Q

How do Adenoviruses cause infection?

A
  • penton fibre tip binds to CAR receptor found in many tissues.
  • penton base binds to integrins on host cell wall/ membrane
  • the membrane forms a vesicle and the virus is released into the cytoplasm
  • Virus disassembles and viral DNA enters the nucleus.
41
Q

Advantages and disadvantages of using Adenoviruses as vector.s

A

Advantages:

  • relatively harmless
  • easy to culture in large amounts
  • the life cycle is well understood
  • we know the function of its viral genes
  • the genome is sequenced.

Disadvantages:

  • limited by size (up to 5kb)
  • short-lived (2 weeks)
  • immunity can develop = can’t reinfect
42
Q

How did Adenoviruses help Cystic fibrosis sufferers, and why are they not used anymore?

A
  • In healthy cells, the CFTR protein forms an ion channel that opens and closes as a phosphate group is attached and detached.
  • In CF sufferers, this channel is the wrong shape = thickening of mucus coating the lungs. Leads to obstruction of airways, increased pathogen growth, scar tissue and respiratory failure.
  • Gene therapy used to clone a healthy CFTR gene into an adenovirus vector and inhaled through the nose.
  • Normal channel function was restored.
  • However, effect was only temporary as immunity developed and a severe immune response caused a death in 1999,
43
Q

How may Adenoviruses help with cancer treatments?`

A
  • Direct replacement of non-functional gene with a functional one.
  • Direct attack introduces a tumour necrosis factor.
  • Suicide genes may also be used.
  • Can provoke the immune system to attack also.
44
Q

How may Retroviruses infect a host?

A

(they’re made of +ve sense ssRNA)

  • enters the cell
  • RNA is copied into DNA by reverse transcriptase
  • DNA circularises and is integrated into the host genome
  • A strong promoter in the LTR region enables transcription of viral genes
  • New virus particles assemble and are packaged (the envelope contains glycoproteins acquired from the host cell membrane.
45
Q

How may Retroviruses be used to treat Murine Leukaemia Virus (MuLV)?

A
  • vectors have all viral genes removed except for the LTR promoter and the packaging signal.
  • The virus only contains the vector with the cloned gene insert and reverse transcriptase.
  • RNA is converted to DNA and the DNA is integrated into the host genome
  • No immune response and the effect is permanent.
46
Q

Describe the success of using Retroviruses to treat SCID (defective B&T cells)?

A
  • Add a functional ADA gene into a vector with a selectable marker.
  • Isolate bone marrow cells and culture them in vitro.
  • Infect the cells with the retrovirus and transfer them back to the body.
  • Produces functional lymphocytes
  • 100% of children survived and 75% needed no further treatment!
47
Q

What are the advantages of Adeno-associated viruses (AAVs) as vectors?

A
  • No immune response
  • No inflammation
  • Affects vertebrates (multiple cell lines)
  • Works in non-dividing cells.
  • Integrates into a specific site.

-But can only hold sequences up to 5kb.
(require another viral vector for replication and capsid production.

48
Q

What are two nonviral methods of gene therapy and how do they work?

A

Liposomes:

  • add DNA into a hollow liposome, which will fuse to plasma membranes deliver DNA by lipofection.
  • non-specific delivery but can inject into target cells.

RNA:
- can use antisense RNAi (delivered by liposomes also) to prevent transcription.
(antisense IGF1 stopped tumour growth)
(Can inject siRNA directly into the eye to target age-related macular degeneration.