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Flashcards in Mutations and Gene Expression Deck (55):

What is a mutation?

Any change to a base (nucleotide) sequence of DNA
Can be errors during DNA replication
Rate of mutation can be increased by mutagenic agents


Types of mutation?

Substitution - swapping bases
Deletion - removing bases
Addition - one or more bases added
Duplication - one or more bases repeated
Inversion - a sequence of bases is reversed
Translocation - sequence of bases is moved from one location in the genome to another


Effects of mutations?

Sequence of amino acids changed so the polypeptide changes
Change active site of enzymes - will not bind to substrate
Genetic disorders by abnormal genes or chromosomes
Mutations in gametes can lead to hereditary mutations


Why will some mutations not effect the order of amino acids?

Code = degenerate, sequence of amino acid won't change
Substitution and some inversion mutations


Mutations causing change in amino acid sequence?

Additions, duplications and deletion will almost always cause this
Change the number of bases in the DNA code
Causes a frame shift in the base triplets
Triplet code is read differently


What increase the rate of mutation?

Mutagenic agents
UV radiation
ionising radiation
They can act as a base - base analogs - substitute as a base and change the sequence
Altering bases - delete or alter bases
Changing the structure of DNA - radiation causes problems in DNA replication


What are stem cells?

Unspecialised cells that develop into other cells


Where are stem cells found?

Some adult tissues


Totipotent stem cells?

Can mature into any type of body cell
only present in first few divisions of an embryo


Pluripotent stem cells?

After few divisions in an embryo
Can still develop into any cell in the body but lose the ability to become cells that make up the placenta


Multipotent stem cells?

Found in adults bone marrow
Able to differentiate into a few different types of cells - red and white blood cells


Unipotent stem cells?

Differentiate into one type of cell


How stem cells become specialised?

All contain the same genes but during development not all are transcribed and translated
Under the right conditions, some genes are expressed but some are switched off



Heart muscle cells - in mature mammals they can't divide to replicate
Heart cells couldn't be regenerated - problem if a heart attack occurs or becomes worn out by age
Scientists think that old or damaged cardiomyocytes can be derived from unipotent stem cells
Some think that this is constantly happening
- some think slowly and some are never replaced
- some think fast and all are replaced several times in a lifetime


Current stem cell therapies?

Bone marrow transplants to replace faulty bone marrow that is producing abnormal blood cells so that they can specialise to produce healthy blood cells
Used to treat leukaemia (blood cancer) and lymphoma (lymphatic system cancer)
Also used to treat genetic disorders such as sickle-cell anaemia and SCID


Potential stem cell treatments?

Spinal cord injuries - replace damaged nerve tissue
Heart disease - replace heart tissue
Organ transplants - organs can be grown


Medical benefits of stem cells?

save lives of people waiting for organ donor
Improve quality of life - replace damaged cells of those who are blind


Sourcing adult stem cells?

Body tissues - bone marrow
Simple operation - little risk
Arnt as flexible as embryonic stem cells - limited range of cells - multipotent


Sourcing Embroynic stem cells?

Embroys in early stages of development
embryo created in laboratory by IVF - egg cells fertilised outside of womb
4 to 5 days old - stem cells removed and embryo is destroyed
Pluripotent - unlimited


Sourcing Induced Pluripotent stem cells?

In the lab - 'reprogramming' specialised adult body cells to be pluripotent
Made to express transcription factors that cause the body cells to express genes associated with pluripotency
Can be introduce adult cells to a specially modified virus which has genes coding for the transcription factors within its DNA.
Virus infects DNA and these genes are incorporated into the cell's DNA so it produced the transcription factors


Ethical issues with embroynic stem cell usage?

People believe that
Destruction of embryo could of been used to make a foetus in a womb
Moment that egg is fertilised an individual is formed that has the right to life
People are less against egg cells that are artificially activated to start dividing
This is why iPS cells are important as they come from adult cells - which people are not against - and have the potential to be as flexible as embroynic cells and can be made from patient cells which will be genetically identical and won't be rejected by the immune system


Transcription factors?

Control the transcription of genes
Move from the cytoplasm to the nucleus
Bind to specific DNA sites near the start of their target genes - the genes they control their expression of
Control the rate of transcription
Activators (TFs) stimulate or increase the rate of transcription - e.g. help RNA polymerase bind to the start of the target gene and activate transcription
Repressors, inhibit or decrease the rate of transcription - e.g. they bind to the start of the target gene, preventing RNA polymerase binding


Role of oestrogen?

Steroid hormone
Binds to a transcription factor called oestrogen receptor, forming an oestrogen-oestrogen receptor complex
Complex moves from cytoplasm to nucleus where it binds to specific DNA sites near the start of target gene
Complex can act as an activator e.g. helping RNA polymerase bind to the start of the target gene


Role of RNA interference with siRNA

Are small doubled stranded RNA molecules
mRNA is transcribed - leaves nucleus for cytoplasm
siRNA associates with several proteins, unwinds to become a single strand
Binds to target mRNA of which it is complementary to
The proteins associated with siren cut the mRNA into fragments - can no longer be translated - moves to a processing body where it is degraded
Same thing with miRNA in plants


Role of RNA interference with miRNA

In mammals, miRNA isn't fully complementary to the target mRNA so it is less specific and can target many mRNA molecules
associates with proteins and binds to target mRNA in the cytoplasm
miRNA-protein complex blocks the translation of the target mRNA
mRNA then moved into a processing body where its stored or degraded
When stored it can be returned and translated later


Mutations leading to tumours?

Acquired mutations
Occur in the genes that control the rate of cell division by mitosis
Causes uncontrolled cell division
Results in tumour that invade and destroy surrounding tissue
Two types of gene that control cell division:
Tumour suppression gene can be inactivated by a mutation - normally slow cell division by stopping cells dividing or slef-destroying them (apoptosis) - mutation will mean protein is not produced so the cells divide uncontrollably
Proto-oncogene can be increased by mutation - called an oncogene - proto-oncogenes stimulate cell division by making proteins that make cells divide - oncogenes cause overactive cell division


Benign tumours?

Not cancerous
Grow slower
Covered in fibrous tissues that stops them invading other cells
Often harmless
Can cause blockages or put pressure on organs


Malignant tumours?

Grow rapidly
Invade and destroy surrounding tissue
Break off and spread to other parts of body in the bloodstream or lymphatic system


Differences in tumour cells to normal cells?

Have irregular shape
Nucleus is darker and larger
Don't produce all the proteins to function correctly
Different antigens on their surface
Dont respond to growth regulating processes
Divide by mitosis more frequently


What is methylation?

Adding a methyl group (-CH3)
Is important in regulating gene expression - can control if a gene is transcribed and translated


Cancer from abnormal methylation?

Hypermethylation - Tumour suppressor genes become hypermethylated, genes not transcribed, proteins that slow cell division not made, cells divide uncontrollably
Hypomethylation - porto-oncogenes act as oncogenes - increasing procession of protein that encourage cell division - cells divide uncontrollably


Oestrogen causing cancer?

Increased exposure to oestrogen over a long period of time increases chance of breast cancer
Theories why:
- Oestrogen stimulate breast cells to divide and replicated - more cell divisions increases chance of mutation - increased chance of malignant tumour
- If cells become cancerous, oestrogen helps their rapid replication
- Oestrogen can introduce mutations directly to DNA of breast cells, increasing chance of cancer


Genetic and environmental causes of cancer?

Genetic - specific inherited allies
Environmental - radiation, lifestyle choice (smoking, alcohol), high-fat diet


How do epigenetics work?

In eurkayotes
Controls whether a gene is switched on or off
Works through attachment or removal of chemical groups (epigenetic marks) to or from DNA or histone proteins
Dont alter base sequence
Alter how easy it is for enzymes needed for transcription to interact with and transcribe the DNA


Inheritance of epigenetics?

Most epiginetic marks on the DNA are removed between generations, some escape the removal process and are passed on to offspring.
Offspring can therefore be affected by environmental changes which affected their parents


Increased Methylation and gene expression?

Methylation of DNA is when a methyl group (an epigenetic mark) is attached to DNA coding for a gene
Always attaches at the CpG site, which is where cytosine and guanine bases are next to each other
Increased methylation changes the DNA structure so that the transcriptional machinery can't interact with the gene, gene is not expressed


Decreased acetylation of Histones and gene expression?

Histones are supercoiled around DNA to form chromatin, makes up chromosomes - can be highly or less condensed
Histones can be epigenetically modified by the addition or removal of acetyl groups (epigenetic mark)
When acetylated, the chromatin is less condensed. Transcription machinery can access the DNA, gene can be transcribed
When Acetyl groups are removed, chromatin becomes highly condensed and DNA can't be transcribed - machinery can't access them
Histone deacetylase is the enzyme responsible for removing the acetyl groups


Treating diseases caused by epigenetic disease?

Drugs designed to counteract the changes
Increased methylation leads to a gene being switched off, Drugs that stop DNA methylation can be used to treat these diseases - azacitidine in chemotherapy
Decreased acetylation of histones leads to genes being switched off. HDAC inhibitor drugs, stops HDAC enzymes so proteins remain acetylated and can be transcribed


Advantages of sequencing DNA of simple organisms?

Don't have much non-coding DNA
Easy to determine proteome and therefore identify antigens which can be used in vaccines


What is recombinant DNA technology?

transferring a fragment of DNA from one organism to another
Possible as genetic code is universal
Organisms that contain transferred DNA are known as transgenic organisms


Making a DNA fragment from Reverse Transcriptase?

mRNA molecules used as templates to make lots of DNA
Reverse transcriptase makes DNA from an RNA template
cDNA is produced (complimentary DNA)
For example, only two copies of insulin gene, but many mRNA copies, possible to make cDNA from insulin mRNA.
mRNA is isolated from cells, mixed from free nucleotides and reverse transcriptase


Making a DNA fragment from Restriction Endonuclease?

DNA nucleotide strand are palindromic and are antiparallel
Restriction endonucleases cut DNA at palindromic sequences
Different Restriction endonucleases cut at different sections due to its active site being complimentary
DNA is incubated with specific endonuclease and the DNA fragment is cut via a hydrolysis reaction
The cut either leaves sticky ends - can be used to bind (anneal) the DNA fragment to another sticky end DNA fragment with complimentary sequences - or blunt ends


Making a DNA fragment from a gene machine?

Database includes the information needed to produce the DNA fragment
Any sequence can be made
Sequence required is designed
First nucleotide sequence is fixed to some sort of support
Nucleotides added step by step in correct order, adding protecting groups to stop branching
Short sections of DNA called oligonucleotides are made and are added up to make longer DNA fragments


In vivo: Inserting into vector?

Vector: organism used to transfer DNA into a cell
Vector DNA cut open using same restriction endonulease that was used to isolate DNA - sticky ends are complimentary
DNA and DNA fragment are mixed together with DNA ligase - joins the sticky ends together - ligation
Forms recombinant DNA


In vivo: Transferring DNA into Host Cells?

Vector with recombinant DNA transfer the gene into cells
Plasmid vectors: heat shock
Bacteriophages: infect the cells
Host cell take up the gene and are transformed


In vivo: Identifying Transformed cells?

Only 5% are transformed - use marker genes
Inserted at same time
Host cells grown on agar plates. Transformed cells will form a colony.
Marker gene can code for antibiotic resistance, so only transformed cells will survive and grow when exposed to antibiotic
Marker gene can be fluorescent and visible under UV


Producing proteins with transformed cells?

To produce protein from transformed cells which are coded for by the DNA fragment, the vector needs a specific promoter and terminator region
Promoter regions are DNA base sequences that start RNA polymerase producing mRNA. Terminators stop it.


In vitro amplification?

Reaction mixture: DNA sample, free nucleotides, primers and DNA polymerase
Primers are short pieces of DNA that are complementary to the bases at the start of the fragment desired
DNA mixture heated to 95C to break hydrogen bonds between strands of DNA
Mixture then cooled to 55C so primers can bind/anneal to the strands
Reaction mixture heated to 72C so DNA polymerase can work
DNA polymerase lines up free DNA nucleotides alongside each template strand
Base pairing forms new complementary strands
Two new copies of the fragments of DNA are formed every one cycle
Cycle starts again at 95C and all four DNA strands are used


Gene therapy?

Altering the defective genes inside cells that cause disorders
If caused by 2 mutated recessive alleles you can add a working dominant allele
If caused by a mutated allele you can silence the allele by putting DNA in the middle of the allele so it does not function
Both involve inserting a DNA fragment into the person's original DNA
Uses vectors to do this
Somatic therapy - involves altering alleles in body cells - most affected by disorder - doesn't affect sex cells so offspring can still inherit
Germ line therapy - involves altering alleles in the sex cells - offspring will not be affected by disorder - currently illegal


DNA probes?

Short strands of DNA.
Specific base sequence
Will bind (hybridise) to target allele if present in sample
Has a label attached so it can be detected - radioactive X-ray or fluorescent


How are DNA probes used?

Sample of DNA turned into fragments using restriction endonucleases and separated using electrophoresis
DNA fragments are transferred to a nylon membrane and incubated with the fluorescently labelled DNA probe
If allele present the DNA probe will hybridise(bind) to it
Membrane is then exposed to to UV light and if the gene is present there will be a fluorescent band



Variable Number Tandem Repeats
Base sequences that don't code for anything and repeat
The number of repeats varies in everyone and can be used for genetic fingerprinting



Sample of DNA from blood, saliva
PCR to make many copies that contain VNTRs - primers used to to either side of repeats so the whole repeat is amplified
DNA fragments produced have lengths that correspond to the number of repeats
Fluorescent tag added so the DNA fragments can viewed under UV light
DNA mixture placed into well and covered in buffer solution that conducts electricity
Electrical current added, DNA is negatively charged so they move toward positive electrode at other end of gel
Small DNA fragments move faster and travel further - DNA fragments separate according to size
DNA fragments viewed as bands under UV light


DNA fingerprinting process?

DNA cut
Using restriction enzyme
Separates according to mass
Transfer to nylon membrane
Make single stranded - DNA helices
Apply probe
Radioactive or fluorescent
Reference to VNTRS


Definition of epigenetics?

Heritable changes in gene function
Without changes to the base sequence of DNA