Topic 7 - Modern genetics Flashcards
define ‘genome’
all of an organisms DNA includinfg mitochondrial/chloroplasts DNA.
these genes carry information for making proteins
What is genome sequencing?
identifying the DNA base sequence of an individual
What is dna sequencing?
the process of determining the order of the nucleotide bases (A,T,G and C) in a molecule of DNA
What needs to happen before DNA can be sequenced?
its important to know the location of the target piece on the chromosome
process used to identify the position is known as resirtiction mapping
**PCR **to apmplify sample
What are the ‘ingredients’ for PCR?
- original strand
- free nucleotides
- primers (x2)
- taq polymerase
What are the primers in PCR and what are they for?
- they are pieces of single stranded DNA which is complementary to the specific target sequence at the 3’ end of each DNA replicated strand.
- this specificity allows you to amplify any specific piece
What is taq polymerase?
the dna polymerase of thermus aquaticus (bacteria) is used to replicate DNA but is stable in high temperatures
Describe the method of PCR
- DNA heated to 95°C : hydrogen binds between chains break and DNA seperates into 2 strands
- Mixture cooled to 50-65°C : allows primers to anneal to each 3’ end of each strand
- Heated to 72°C for DNA polymerase (taq) to attatch nucleotides
- repeated cycles of heating and cooling amflify this region of DNA by thermal cycler (x30)
What can amplified samples produced by PCR be used for?
- to predict the amino acid sequence of proteins and possible links to genetically determined conditions, using gene sequencing. (also identify antigens and develop vaccines)
- in forensic science, to identify criminals and to test paternity, using DNA profiling.
How are genes located
- using a DNA probe (a short section iof DNA that has been labelled eg with a flourescent marker)
- the DNA probe DNA is complementary to the target gene
- sample has to be heated first to seperate original molecule (breaks H bonds)
- temp lowered to allow probe to anneal
- depedning on what kind of marker is used gene is identified
- in the case of a flurorescent maker a UV light and flourescent microscope is used.
What are the ways in whiuch transcription or trannslation of genes can be regulated in eukaryotes
- DNA regulatory sequences and transcription factors
- post-transcriptional modification of mRNA
- descruction of mRNA
- DNA methylation
- histone acetylation
what are the 5 factors affecting gene expression?
- regulatory sequences and transcritpion factors
- post-transcriptional modification of mRNA
- destruction of mRNA
- DNA methylation
- histone acetylation
what are the epigenetic factors that affect gene expression?
-destruction of mRNA
-DNA methylation
-histone acetylation
regulatory sequence
a sequence of DNA nucelotides that controls whether its target gene is transcribed or not
what are the 2 types of regulatory sequence?
- promoters
- enhancers
promoters
short base sequences that lie close to their target genes.
they initiate transcription by enabling RNA polymerase to bind to the gene they regulate
enhancers
short base sequences that lie some distance from tehir target genes they regulate.
they stimulate promoters causing an increase in the rate of transcription of the genes they regulate
what is ‘upstream’ of a target gene
going in the other direction (found in the other direction) to the direction of transcription
eg:
x when transcription —>
define transcription factor
(+how they work)
A protein or assembly of several proteins that regulate the production of mRNA.
(A specific transcription factor binds to a promoter region upstream of its target gene and either promotes or inhibits the binding of RNA polymerase to the target gene. )
what is necessary for transcription to happen
- in order for a gene to be transcribed the enzyme RNA polymerase must be able to attatch to it
- if this enzyme cannot, the gene will not be transcribed
- in eukaryotes RNA polymerase cannot initate transcription itself
- (attatchment of RNA polymerase is regulated by the genes promoter)
how do transcription factors work?
- specific proteins: transcription factors must bind to the promoter
- once done so an RNA polynmerase bind to the transcription factor complex and becomes activated to begin the synethsis of mRNA from a unique point on the target gene
how can post transcriptional modification of mRNA affect gene expression?
post–transcription modification of mRNA in eukaryotic cells
can result in different products from a single gene.
define exons and introns
exons= expressed regions of DNA
introns= intragenic regions that are not expressed
describe the post trabscriptional modification of mRNA (splicing) and how this leads to different products being formed from a single gene
- during transcription all the exons and introns all copied into the base sequence of an RNA molecule called pre-mRNA
- This molecule is then spliced; the introns are removed and the exons are rejoined (by spliceosomes) to form mature mRNA
- aliternative splicing of pre-mrna can produce mature RNA with different combinations of exons (isoforms)
(-this mRNA migrates to the cytoplasm where is base sequence is translated by ribsomes. )
what is an epigenetic modification
stable, long-term changes in the ability of a cell to transcribe its genes.
though these changes are inherited, they are not caused by changes to the base sequence of the DNA
how does the destruction of mRNA work?
incolves short regulatory RNA molecules binding to a protein to form a compex called an RNA induced silencing complex (RISC)
depending on the type of short regulatory RNA molecule (miRNA or siRNA) the compelx formed formed between the RISC and the regulatory RNA will bind with RNA and form bulges so it cannot be translated (miRNA)
siRNA forms a compelx with RISC that destroys mRNA
*check whether translated or trasncribed *
How can histone modification affect gene expression?
Addition of an acetyl group exposes promoters and target gene to transcription factors and RNA polymers
Addition of a methyl group can prevent the activation of RNA polymerase by effectively blocking the promoter
DNA methylation
involves addition of a CH3 group to cystosine bases which prevents trancription factors from binding therefore gene transcription is supressed.
Histone acetylation
the ‘tails’ of histone moelcules contain amino acid leucine. This can be acetylated, this is when an acetyl group (COCH3) is transferred onto it from a acetylene coenzyme A. When this happens the binding of the histines changes and they becomes more lossesly packed. This makes the promoter and target gene accessible to transcription factors and RNA polymerase
How are epigenetics incolved in cell differentiation?
Epigenetics control the amount of the cell’s DNA that is transcribed and therefore ensures that only the proteins needed for that specific cells function are produced.
What is a stem cell?
A stem cell is an undifferentiated cell which has the ability to differnetiate into other specific cell types
What is genetic engineering?
Involves inserting a foreign gene into an organisms genome resulting in the expression of the new gene.
How does recombinant technology work?
- The desired gene is located in the original organism and isolated
- Enzymes called restriction endonucleaseisolate the gene by cutting at specific sites
a) restriction endonuclease create staggered cuts referred to as sticky ends
b) a vector is cut with the same restriction enzyme
c) cutting the vector with the same restriction enzyme will create complementary sticky ends - The gene and plasmid are joined together by an enzyme called DNA Ligase
a. This creates a phosphodiester bond between the DNA of the gene and the vector
b. The vector is now referee to a recombinant vector - The recombinant vector is inserted into the target organism
- GM fungi is identified/cloned/cultured
What is a transgenic organism
+ example
contain genetic information from a different species
Eg when the human gene for insulin is transferred into E. coli the E. coli is transgenic
Vector
A vehicle used to artificially carry foreign DNA into another cell.
What are the two examples that are commonly used as vectors?
- bacterial plasmids
- viruses
How do bacterial plasmids work as vectors
Plasmids are rings of DNA found in bacteria
When bacteria reproduce the plasmids are copied
A recombinant plasmid will express the gene in a genetically modified bacterium
How do viruses work as vectors.
Viruses will infect the host organism
Viruses will force the host to express the recombinant DNA
How do gene guns work and what can they be used on
Technique that fires microscopic gold particles coated with the foreign DNA at the cells using a compressed air gun.
Designed to overcome the problem of the string cell wall in plant tissue since the particles can penetrate the cell wall and the cell and nuclear membranes and deliver the DNA to the nucleus where it is sometimes expressed
Name and define the threee types of stem cell
- Totipotent= can develop into any cell type including the placenta and embryo
- Pluriopotent = can develop into any cell type excluding totipotent stem cells
- Multipotent = can only develop into a few different types of cell
How can emmbryonic stemm cells be used in medicine?
To treat a variety of dieases eg: Parkinson’s and replace damaged tissue eg nerve cells
What is the main ethical issue around embryonic stem cell use?
+ two otyhers
Embryos are killed in the process of extracting stem cells
risk of infenction when transplanted and also could becomes cancerous
How do totipotent cells dvelop into other types of stem cell?
Epigenetic modifications result in selective translation of the relevant parts of the DNA. Totipotent cells devlop into pluripotent cells in the early stages of embryonic development and then later into fully differentiated somatic cells.
What is an induced pluripotent stem cell?
iPS cells are adult stem cells that have been reprogrammed to become pluripotenet again
Explain why totipotent stem cells have the capacity to divide and develop into all cells
becuase all the genes are activated/no genes deactivated.
What kind of cells can totipotent cells develop into making them special
3 pro,ary germ cell layers of the zygote and into extra embryonic tissue bsuch as placenta
where are the two places adult stem cells could come from?
- bone marrow (adult stem cells)
- human embryos (embryonic stem cells)
what are embryonic stem cells?
stem cells from ebryos. can grow into any typoe of cell found in the body., non specialised and can be removed from human embryos that are 4-5 days old e.g. from fertility treatment
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What is the type of stem cell created after fertilisation?
totipotent cell
How do cells specialise starting from a totipotent cell in zygote
- zygote created from fertilisation is a totipotent cell
- this divides into multiple totipotent cells
- due to epigenetic modifications after 4-5 days the totipotent cells begin to differentiate forming hollow ball of cells: blastocyst
- inner cell mass will form the animal (all cells in ICM are pluripotent)
- pluripotent cells soon undergo further specialisation into multipotent adult stem cells these can give rise to limited number of other types of somatic cells
list 3 conditions that could be treated with stem cell trasnplants
- type 1 diabetes
multiple scelerosis
spinal/brain injury
how are iPS cells made
- produced from adult somatic cells usinf appropriate transcription factors
- these transcription factors cause specific genes to be expressed which dedifferentiates a cell back to its pluriopotent state
- the genes they may add are: Oct4. Sox2, Klg4 and c-Myc usinf viral/vector gene transfer
- each individual can have their own pluripotent stem cell line produced from their body cells and these could potentially be used to generate transcplats without risk of immune rejection
how can liposomes be used for trasporting vectors?
Liposomes (small membrane vesicles) can encase vectors and fuse with the cell membrane (and sometimes the nuclear membrane too) delivering the DNA into the cell.
What are genetic markers and how do they work?
If the desired gene does not create an identiofyable change in phenotype or something obeservable, markers may be used .
Genetic markers are needed to identify cells that have successfully taken up a vector and so,
become transformed (into genetic recombinants).
Antibiotic resistance or flouresecent genes are good markers. When the desried gene is inserted into the middle of the marker gene, the marker gene is disrupted and won’t work. Then, in the case of an antibiotic resistance maker, replica plating where the second plate contains antibiotcs can be used. The colonies that do not grow do contain the desired gene now (as marker was destructed). In the case of UV, the colonies that took up the gene would not flouresce.
How does replica plating work?
Replica plating is a simple technique for making an exact copy of an agar plate. A pad of sterile cloth the same size as the plate is pressed on the surface of an agar plate with bacteria growing on it. Some cells from each colony will stick to the cloth. If the cloth is then pressed onto a new agar plate some cells will be deposited and colonies will grow in exaclty the same positions on the new plate.
How would youy use replica plating to test for bacteria taking up antibiotic resiance (desired gene)
In this case, the desired gene has an easily identifiable phenotype, namely they will not die in that antibiotic. No marker is needed here as the gene alone can be easily identified.
So replica plating is used where the master plate contains all the bacteria (some of whom have successfully taken up the vector, some havent). A sterile pad of cloth is then pressed against the culture, some of the bacteriua will stick to the cloth, and pressed onto a new agar plate, some cells will be deposited, however, only the successful recombinants will grow as the new agar should contain said antibiotic.
What are knockout mice?
A knowckout mouse in one where genes have been turned off or “knocked out”
What is the purpose of a knockout mouse?
Knockout mice are used to study what happens in an organism when a particular gene is absent. Studying knockout mice can provide information about how the knocked-out gene normally function, including the gene’s biochemical, developmental, phyiscal and behavioural roles.
How is a knock-out mouse made?
To create one, scientists genetically engineer the animal by disrupting a gene of interest. This may be done by deleting portions of the gene’s DNA sequence to make it non-functional or by replacing the gene with an altered sequence.
What is a GMO?
genetically modified origanism
have foreign DNA inserted into their genomes
Why is genetic modification done to plants? and how so
to increase crop yield or crop quality
- for resistance to prests
- to produce a vitamin (beta-carotene)
- increased nutiritional value
- to make bigger fruits
- can be grown in harsher conditions
- can be grown out of season
What are the downsides to GM crops
- the seeds are very expensive so many farmers may not be able to afford GM crops so will be outcompeted by ones who can as their crops will then produce higher yield and quality
- GM crops could breen with wild crops which would contaminate; the consequences are unknown
- GM crops could lead to a reduction in biodiversity
(fewer types of weeds- outcompeted by GM crop, fewer habitats and food sources for animals)
How have soya beans been GMed
Linoelic acid (polyunsaturated) is replaced by oelic acid (monounsaturated) which oxidise less easily and therefore don’t go off as fast and are also healthier.
Explain why an approach to protect mosquitos from malaria instead of killy them using GM plants containing a toxin (5)
- not unethical mosquitos not killed
- risk of other organisms being affacted by the GM fungus is avoided
- organisms that feed on mosquitos will not lose their food supply
- some people beleive GM is wrong unethical
- unkown risks
How do you use replica plating and antibiotic restistance marker to see whether bacteria has taken up the desired gene.
The desired gene will disrupt the gene for resistance, so when you replica plate, the spaces where the bacteria have failed to grow on the agar plate with the antibiotic are those who have successfully taken up the gene.
