Module 8: Genes Flashcards
stem cell definition
unspecialised/undifferentiated cell
potential to form different types of cells
how can a stem cell become specialised
differentiation
3 changes: cell shape, number of organelles, new content
occurs by controlling gene expression (some genes are activated and some are inhibited)
3 changes a stem cells needs to make to become specialised
cell shape
number of organelles
new content
stem cells in animals/mammals/humans
totipotent
pluripotent
multipotent
unipotent
totipotent
zygote
pluripotent
embryonic stem cells
multipotent
bone marrow stem cell
unipotent
tissues
what does iPS stand for
Induced Pluripotent Stem cells
what are induced pluripotent stem cells?
turning unipotent body cells into pluripotent cells (like embryonic stem cells), involves activating certain deactivated genes using transcription factors
What are the uses of stem cell therapy in humans
Use stem cells to produce tissues/organs for transplant
Use stem cells to treat irreversible diseases (inject stem cells at the site of disorder- will differentiate to become local specialised cells)
Stem cells in plants
In embryo= zygote/embryonic stem cells
In adult= meristem cells in stem/shoot/root
Uses of stem cells from plants
Traditionally cuttings were taken from plants (stem/shoot/root) and used to grow genetically identical plants, possible due to the presence of meristem cells Tissue culture (micro propagation)= large scale application of cuttings
Process of taking cuttings from a plant
Take cutting from shoot/stem/root (explant)
Place explant in the nutrient rich medium so meristem cells divide by mitosis
Produces a mass of meristem cells (callus)
Take each meristem cell and grow in a plant growth factor medium to promote differentiation and formation of shoot/root
Transfer plant to soil and greenhouse
Then transfer to field
What is controlling gene expression
Either activating a gene
Activating= protein made
Inhibiting= protein not made
Example of activating gene
Using oestrogen
Can enter a cell by simple diffusion and bind to receptors on the transcriptional factor
Causes transcriptional factor to change shape
Transcriptional factor can now enter nucleus and bind to promoters on the DNA to activate transcription= activated genes (protein to be made)
Example of inhibiting genes
Using siRNA (small interfering RNA) Making siRNA= double stranded RNA cut down into small sections, made single stranded, then attaches to an enzymes Will then bind to complementary sections on mRNA= the enzyme will cut the mRNA so translation can’t occur- gene inhibited (protein not made)
What is epigenetics
Heritable changes in gene function without changes to the base sequence of DNA
Changes may occur due to stress, lifestyle or diet
Chromatin and epigenome
What is chromatin
DNA- histone complex
What is an epigenome
Chemical layer
Chromatin and epigenome
Chromatin is surrounded by an epigenome
The epigenome can either cause the chromatin to become more condensed or more loose
More condensed= transcription factors can’t reach the DNA and the gene will become inactivated
More loose= transcription factors can reach the DNA and the genes will be activated
Changes may be brought about by acetylation or methylation
How does methylation affect the genome
Increased methylation= Adding methyl groups This attracts proteins Condensed the DNA-Histone complex Transcription factors can’t gain access Gene inhibited
How does acetylation affect the genome
Decreases acetylation=
Removing acetyl groups
Increases positive charges on the histone
Increases the attraction to the phosphate group on DNA
Which condense the DNA-Histone complex
Transcription factors can’t gain access
Gene inhibited
Gene mutation definition
A change in the base sequence of DNA
2 types of gene mutations
Substitution
Insertion/deletion
Substitution
Gene mutation
Replaces one base for another, changes one triplet code
(Can be silent, mis-sense or non-sense)
Silent
New triplet code codes for same AA
Mis-sense
Codes for a different AA so protein shape changes slightly
Non-sense
Codes for a stop cmon so polypeptide chain not produced
Insertion and deletion
Adding/removing a base
Cause a frame shift
All the triplet codes after the mutation will change so a normal polypeptide chain is not produced
Frameshift
Where all of the triplet codes after a mutation are changed
What is cancer
Formation of a malignant tumour
Due to uncontrolled cell division (mitosis)
2 types of tumours
Malignant
Benign
Malignant tumours
Rapid growth (rapidly dividing cells) Cells are unspecialised Cells can spread (metastasis) Systemic effects Requires surgery/chemotherapy/radiotherapy
What normally controls cell division
2 genes: proto-oncogene and tumour-supressor gene
Proto-oncogene- stimulates cell division, produces a growth factor and receptor protein, when growth factor binds to receptor protein on cells it stimulates DNA replication leading to cell division
Tumour-supressor- inhibits cell division, produces a protein that inhibits cell division
Explanation of cancer
Caused by mutation of genes that control cell division
Mutation of proto-oncogene leads to formation of an oncogene= over production of growth factor or receptor proteins permanently active= over stimulation of cell division (uncontrolled cell division)
Mutation of tumour-supressor gene= loss of protein to inhibit cell division (uncontrolled cell division)
Oestrogen and cancer
Oestrogen leads to activation of genes
High levels of oestrogen can lead to over activation of proto-oncogen forming an oncogene= cancer (uncontrolled cell division)
Epigenetics and cancer
Increased methylation of tumour suppressor genes leads to inhibition of tumour suppressor genes leading to cancer (uncontrolled cell division)
What is genetic engineering
Changing the genetic make-up of an organism’s DNA by adding or removing a gene
DNA becomes recombinant
Organism becomes genetically modified (transgenic)
Why do we genetically engineer animals
To give them additional characteristics
So they can make useful products (proteins)
Examples of genetic engineering in animals
Additional characteristics Add gene for disease resistance Add gene for growth hormone for growth Making useful products Use to produce anti-thrombin: protein used to make blood clot, use milk producing animal to produce, add gene for anti-thrombin next to milk producing gene in animal, therefore anti-thrombin protein ill be made in the milk (easily extracted)
Why do we genetically engineer plants
To give them additional characteristics
So they can make useful products
Examples of genetic engineering in plants
Add gene for:
- disease resistance
- pest resistance
- pesticide resistance
- promote growth for high yield
- produce GM fruits
Make useful products
Make golden rice
Make protein raw material for polymers
Why do we genetically engineer tomatoes
Prevent them from softening
Prevent formation of softening enzyme
Gene is added complementary to softening enzyme gene
It’s mRNA will bind to mRNA if the softening enzyme preventing translation of softening enzyme
Golden rice
Rice that contains beta-carotene
Pre-cursor to vitamin A to treat malnutrition deficiency
Why do we genetically engineer bacteria
So they can make useful products
Genetically engineering bacteria
To make useful products- insulin
Normally used animal sources (problems= limited supply, infection risk, immunorejection)
Involves adding human insulin gene to a plasmid then inserting this into a bacteria, bacteria now has gene/code to produce insulin protein
What are the 5 steps of genetically engineering bacteria
- Isolation
- Insertion
- Transformation
- Identification
- Growth/cloning
- Isolation
Either by reverse transcriptase, restriction enzyme, gene machine
End result= isolated human insulin gene
Use of reverse transcriptase in isolation
RT: enzyme found in virus, RNA -> DNA, obtained mRNA for insulin, RT convert it into single stranded complementary DNA (cDNA), DNA nucleotides and DNA polymerase added to make it double stranded
Use of restriction enzyme in isolation
RE: enzyme found in bacteria, cuts DNA at certain base sequences (recognition sites) by breaking bond between sugar and phosphate, cut staggered for GE as it leaves exposed bases “sticky ends” [cuts staggered at 6 base pair palindromes, where the 6 bases read forward are identical to 6 bases read backwards on both strands]
Use of gene machine in isolation
GM: build DNA sequence from known amino acid sequence of the protein (uses oligosaccharides)
- Insertion
Cut plasmid using the same RE from isolation stage
Leaves complementary sticky ends
Join human insulin gene with plasmid via the sticky ends
Use DNA Ligase to join the sugar-phosphate backbone
=recombinant plasmid (carrying human insulin gene)
- Transformation
Mix recombinant plasmid with bacteria
Add calcium 2+ ions and heat shock
Bacteria will become permeable and take up the recombinant plasmid
=genetically modified bacteria (carrying recombinant plasmid with human insulin gene)
- Identification
Identify which o the bacteria have taken up the recombinant plasmid and o these which ones have accepted the new human insulin gene
Has 2 stages
Stage one of identification
Choose a plasmid tat carries ampicillin resistance gene, when ampicillin is added only the bacteria that have taken up the recombinant plasmid will survive (will have obtained the resistance gene)
Stage 2 of identification
Use gene markers to identify which of the remaining bacteria have accepted the human insulin gene
Human insulin gene will be placed in the middle of these gene markers, if the bacteria accepts the human insulin gene they will reject the marker , rejects gene then will accept the marker
-antibiotic resistance= tetracycline resistance gene is lost if human insulin gene is accepted so bacteria is no longer resistant to tetracycline, add by replica plating, ones that die are ones we want
-fluorescent= gene lost if human insulin is accepted, identify bacteria showing no fluorescence
-enzyme= enzyme gene lost if human insulin gene is accepted, add colour less substrate, where there is no colour change select those bacteria (enzyme not made to breakdown colourless substrate for colour change)
End result= genetically modified bacteria
- Growth/cloning
Grow genetically modified bacteria (carrying human insulin gene)
They will produce the protein (human insulin)
What is PCR?
Polymerase chain reaction
Used to replicate DNA artificially
1. Heat to 95 degrees, hydrogen bonds break, double strands separate, left with 2 template strands
2. Cool to 55 degrees, primers bind (short single stranded sections of DNA) to start of each template strand, prevents template from rejoining and allows DNA polymerase to bind to build the new strand
3. Heat to 72 degrees, DNA nucleotides attach to complementary bases, DNA polymerase joins sugar-phosphate backbone of the new strands= 2 copies of DNA (1 original and 1 new)
Polymerase chain reaction VS semi-conservative replication
PCR only replicate short DNA fragments, SCR can replicate whole DNA
PCR uses 95 degrees, SCR uses DNA helicase
PCR uses primers, SCR doesn’t
In-vitro replication
PCR
More rapid, less complex
In-Vivo replication
Using bacteria to replicate DNA (add DNA fragment to the plasmid, then replicate the bacteria to make many copies of DNA fragment) More accurate (less mutations), less chance of contamination
What is a DNA probe
Short single stranded section of DNA
Has a specific base sequence so it binds to complementary genes
Is radioactively/fluorescently labeled
If gene is present in DNA, DNA probe will bind to it and show up be radioactive/fluorescent
What is genetic screening
Analyse an individuals DNA for the presence of a particular gene (mutated allele)
Use DNA probes (single stranded section of DNA, complementary to a particular gene, is radioactively labeled)
Obtain individuals DNA, make it single stranded, add the specific DNA probe for. The gene to be screened for, if the gene is present the DNA probe will bind, will show up as radioactivity on an X-ray film
What is genetic fingerprinting
Used to produce a unique fingerprinting of an individuals DNA (produces a specific banding pattern)
Used in forensics and paternity testing
Involves analyzing the individuals introns (non-coding DNA)
Introns contain repetitive sequences called variable number of tandem repeats (VNTR)
Number and the length of the VNTR are unique for each individual organism
What are the 5 steps of genetic finger printing
- Extraction
- Digestion
- Separation
- Hybridisation
- Development
- Extraction (genetic fingerprinting)
Extracting the individuals DNA
- Digestion (genetic fingerprinting)
Cutting the DNA down into fragments
Use restriction enzymes that cut just outside the VNTR (leaves the VNTR of the introns)
- Separation (genetic fingerprinting)
Separate out the DNA fragments by gel electrophoresis
Add alkali to make the separated fragments single stranded
Transfer the fragments to a nylon membrane by Southern Blotting
Add UV light so the DNA fragments set
- Hybridisation (genetic fingerprinting)
Add radioactively labeled DNA probes complementary to the DNA fragments
- Development (genetic fingerprinting)
Add photographic film and take an x-ray to produce the banding pattern picture
What is genome sequencing
Determining base sequence of a genome (full set of DNA)
Uses whole-genome shotgun (WGS) to cut DNA into smaller sections to be sequenced
Bioinformatics is the science by which the information is collected and analysed
Uses= supports phylogenetic classification, identify genes related to diseases
What is a proteome
Full set of proteins produced by a certain genome
