Topic 8- inheritence etc

What is a gene mutation

Alteration of a base in sequence of bases for one gene. This is likely to occur during DNA replication, which is during interphase of cell cycle.

What are mutagenic agents and types

High energy and ionising radiation

• these mutagenic agents include radiation such as alpha and beta particles and xray and gamma rays. Can damage and disrupt structure of dna

Carcinogens

• Chemicals which can alrer structure of dna and interfere with transcription. eg tobacco smoke

What is addition mutation

One extra base being added to the sequence

Means that subsequent codons altered, causing frame shift. The altered codons could potentially code for different amino acids and result in different sequence of amino acids & potentially non functioning protein.

Eg TAC TTC AGG

to TAC ATT CAG G

deletion mutation

deletion of a base in a sequence

causes a frame shift left

eg TAC TTC AGG

to TAC TCA GG

what is substitution mutation

one base changed/ swapped for a different base.

Base number same so no frame shift.

Genetic code is degenerate so may still code for same amino acid and so have no impact.

inversion mutation

a section of bases detach and rejoin inverted. Results in different amino acids coded for.

eg TAC TTC AGG TGG

to TAC GGA CTT TGG

What is translocation in terms of mutation

a section of bases on one chromosome detached and attaches onto a different chromosome.

This can cause significant impact on gene expression and phenotype

what are stem cells

Undifferentiated cells that can continually divide and become specialised. Differentiation is the process which stem cells become specialised

what are totipotent

stem cells that can divide and produce any type of body cells, they occur for a limited time in early mammalian embryos

What are pluripotent cells

These stem cells are found in embryos and become almost any type of cell.

Issues with pluripotent cells

• can continually divide to create tumours

• ethical debate of therapeutic clone and destroying embryo

what are multipotent and unipotent cells

Stem cells in mature mammals that can divide to form limited number of different cell types.

Multi-potent cells such as in bone marrow can differentiate into a limited number of cells. unipotent only differentiate into one type of cells

sources of stem cells in mammals

• embroys up to 16 days after fertilisation contain stem cells that are pluripotent

• umbilical cord blood contains multipotent stem cells

• placenta has multi-potent cells

• adult stem cells such as in bone marrow

What are IPS cells

Induced pluripotent cells

created using adult unipotent cells, get altered in lab to return to state of pluripotency.

To do this genes that were switched off to make cell specialised must be switched back on, using transcriptional factors. Very similar to embryonic pluripotent stem cells, but dont cause destruction of an embryo.

The ips have shown self renewal property, so they can divide indefinitely so could be used instead of embryonic

what is epigenetics

heritable change in gene function, without changing the dna base sequence. These changes are caused by changes in environment and can inhibit transcription

what js an epigenome

A single layer of chemical tags on the DNA and it impacts the shape of DNA histone complex and whether the DNA is tightly wound so wont be expressed, or unwound therefore expressed

what does methylation of DNA do

When methyl groups are added to DNA they attach to cytosine base. This prevents transcriptional factors from binding and attracts proteins that condense the DNA histone complex. Methylation prevents a section of DNA from being transcribed.

Increased methylation of dna inhibits transcription

what does acetylation of histone proteins do

decreased acetylation of associated histones proteins on DNA inhibits transcription.

if acetyl groups removed from dna then histones become more positive and are attracted mlre to the phosphate group on dna. this makes DNA and histones more strongly associated and hard for transcription factors to bind

what are tumour suppressor genes

genes produce proteins to slow down cell division and cause cell death if DNA copying errors are detected. If a mutation results in the tumour suppressor gene not producing proteins to carry out function the cell division could continue and mutated cells not identified and so not destroyed.

Abnormal methylation and tumour supressor genes

methylation can cause a gene to turn on or off.

if tumour suppressor genes become hyper methylated meaning an increased number of methyl groups attached to it so gene becomes inactivated and turns off.

Oncogenes and methylation

may be hypomethylated reducing the number of methyl groups attached causing the gene to be permanently switched on

Example of oestrogen and transcription factors

Oestrogen is a steroid hormone so is lipid soluble, and can initiate transcription.

Binds to receptor site of transcriptional factors, when it binds to transcriptional factor and causes ir to change shape slightly and makes it complementary to bind to DNA to initiate transcription.

how does sirna cut up mrna

An enzyme can cut the mRNA into siRNA

One strand of the siRNA then combined with another enzyme

This siRNA -enzyme complex will bind via complementary bade pairing to another mRNA molecule

Once bound the enzyme cannot cut up the mrna so cannot be translated, therefore polypeptide chain is not made

Benign tumours

Can grow large but at slow rate

Non cancerous because they produce adhesion molecules which causes them to stick to a particular tissue and they are often surrounded by a capsule so they remain compact and can be removed by surgery

Localised, do not break off and spread

Malignant tumours

cancerous and grow large rapidly

become large and can become unspecialised again

they do not produce the adhesive so instead metastasis occurs meaning tumour breaks off and spreads to other parts of body.

secondary tumours can develop

tumour not encapsulated and can instead can grown projections into surrounding tissues and develop own blood supply

what are oncogenes

Oncogenes are mutated version of proto oncogenes which profuce a protein involved in the initiation of dna replication and mitosis cell division when the body needs new cells. Oncogenes can result can be permanently activated to cause cells to constantly divide

what is the genome

the entire generic material of an organism in the nucleus of a cell (in eukaryotes)

why are simple organisms useful for vaccines

Simpler organisms (eg bacteria) do not contain introns in their DNA.

This means that the genome can be used directly to sequence the proteins that derive from the genetic code (proteome) of the organism

this is useful for identifying potential antigens to use in a vaccine

whats recombinant dna technology

the combining of different organisms dna, which would enable scientists to manipulate and alter genes to improve industrial processes and medical treatment.

How can reverse transcription be used to produce dna fragments

1. Reverse transcriptase makes dna copies fron mRNA

2. naturally occurs in viruses such as HIV

3. A cell that naturally produces the protein of interest is selected

4. These cells should have a large amount of mrna for the protein

5. reverse transcriptase enzyme joins dna nucleotides with complementary bases to mrna sequence

6. Single stranded dna is made (cDNA)

7. To make this dna fragment double stranded the enzyme DNA polymerase is used

advantage of reverse transcription for dna fragments production

The cDNA is intron free because its based on the mrna template

particularly helpful for prokaryotic cells which cant remove the introns

What are restriction endonucleases

enzymes that cut up dna, and naturally occurs in bacteria as defence mechanism

how do restriction enzymes / endonucleases work

Restriction enzymes have active site complementary in shape to a range of different dna base sequences, described as recognition sequence and therefore each enzyme cuts the dna at a specific location.

Some enzymes cut at the same location and create a blunt end whereas others cut to create staggered ends and exposed dna bases.

Exposed ends are palindromic and called “sticky ends” because they have ability to join to dna with complementary base pairs

Dna fragments can be created in a lab using a computerised machine.

scientists first examine the protein of interest to identify the amino acid sequence and from that work out the mrna and dna sequence

the dna sequence is entered into the computer which checks for biosafety and biosecurity that the dna being created is safe and ethical to produce.

The computer can create small sections of overlappinf single strands of nucleotides that make up the gene called oglionucleotides.

these oglionucleotides can then be joined to create dna for the entire gene

PCR can be used to amplify the quantity

advantage of gene machine

very quick and accurate process. makes intron free dna so can be transcribed in prokaryotic cells

draw a table showing advantages and disadvantages of each technique

• reverse transcriptase

• restriction endonuclease

• gene machines

what must happen to dna fragments to ensure transcription of these genes can occur

1. a promoter region must be added - added at the start of the dna fragment. this is a sequence of dna which is the binding site for RNA polymerase to enable transcription

2. A terminator region- at the end of gene. causes rna polymerase to detach and stop transcription, so only one gene at a time is copied into mrna.

What is a vector

something that can carry isolated dna fragments into host cell.

Most common vector is bacterial plasmid as they are circular dna which are separate from main bacterial genome and only contain few genes

How would you insert dna into a vector

plasmid cut open using same restriction endonuclease

causing sticky ends

therefore dna fragment sticky ends (exposed nucleotides) complementary to the sticky ends on plasmid

the dna fragment and cut plasmid are combined and then enzyme ligase sticks them together

ligase catalyses the condensation reaction to form phosphodiester bonds between nucleotides

Transformation : how to insert vector into host cell to get the gene expressed

to Increase permeability, host cells mixed with Ca2+ ions and are heat shocked, which enables the vector to enter host cells cytoplasm

issues with host cells taking up recombinant plasmid

1. the recombinant plasmid doesnt get inside cell

2. plasmid rehoins before dna fragment entered

3. The dna fragment sticks to itself rather than inserting into the plasmid

what are marker genes and Three different marker genes

marker genes can be used to identify which bacteria successfully took up the recombinant plasmid

1. antibiotic resistant genes

2. genes coding for fluorescent proteins

3. genes coding for enzymes

how do you use antibiotic resistant marker genes

eg resustance to tetracyline and ampicillin, inserting the dna fragment which disrupts the tetracycline resistance gene, causing it to no longer generate funcitonal protein.

grow colonies on agar and determine which is present to see if gene has been inserted

Fluorescent markers

GFP - green fluorescent proteins genes; which can be inserted into the bacteria plasmid

DNA fragments interrupts the GFP gene, so only non glowing colonies contain recombinant plasmid

Enzyme markers

Enzyme lactase can turn certain substance blue from colourless, the gene for this enzyme is inserted into the plasmid and dna fragment is inserted in middle to disrupt it. prevents lactase production

Then grow bacteria on agar plate with colourless substance

colonies which cannot turn substance blue contain recombinant plasmid

what is used to grown the host cell

fermenter grows multiple copies of host cells which have recombinant plasmid.

The host fell can then produce protein coded for by inserted dna

how to collect for dna for pcr

• blood body cells or hair cells. Pcr can be used to amplify amount of DNA by cloning

digestion in genetic fingerprinting

restriction endonucleases are added to cut up dna into smaller fragments, enzymes which cut close to target VNTRs are added

separation in genetic fingerprinting

The DNA samples are loaded into small wells in agar gel, the gel is placed in a buffer liquid with an electrical voltage applied.

DNA is negatively charged so the DNA samples move through the gel towards the positive end of gel.

Gel electrophoresis - agar gel created resistance for the moving DNA and smaller dna move faster and further along gel, to separate different lengths of dna/vntr.

alkaline added to separate double strands of dna

what are dna probes

DNA probes are short single stranded pieces of dna complementary in base sequences to the VNTRS the probes are radioactively or fluorescently labelled.

hybridisation

different dna probes are mixed with single stranded vntrs on the agar gel for them to bind (hybridise)

development

the agar gel will shrink and crack as it dries therefore vntrs and dna proves are transferred to a nylon sheet - so it will last longer

the nylon sheet can be exposed to xrays or uv

In vitro cloning in PCR method

1. Temperature is increased to 95° to break hydrogen bonds and split the dna into single strands (denaturing)

2. the temperature is then decreased to 55° so primers can attach (annealing)

3. the enzyme dna polymerase then attached complementary free nucleotides and makes a new strand to align next to each template (synthesiss). Temperature increases to 72 which is optimum

advantages of pcr

Automated - more efficient

rapid - lots of clones can be made within hours

Doesn’t require living cells - quicker and less complex techniques needed

what are dna probes used for

locating soecific alleles of genes and screening patients for heritable conditions etc.

process of dna hybridisation

1. patients dna sample heated to make it single stranded

2. heat causes hydrogen bonds between bases to break (denaturing)

3. the patients single stranded DNA sample is mixed with the DNA probe and cooled , and any complementary sequences can alogn and form hydrogen bonds (anneal)

4. some lf dna anneals with probe and some back together

how to locate specific alleles of genes

1. DNA base sequence must be known to create the DNA probe

2. This can be determined using DNA Sequencing techniques

3. The fragment of DNA can be produced using a gene machine

4. this fragment can be amplified using PCR

5. THE LABEL IS THEN ADDED EITHER A RADIOACTIVE NUCLEOTIDE CONTAINING THE IS 32P Or a fluorescent label which emits light under uv

6. After hybridisation the DNA is washed so that any unbound DNA probes are washed away

7. The presence of the radioactive or fluorescent labels Therefore, indicates that the allele of interest is present in the patient’s DNA

How can dna screening help personalised medicine

Can help determine best dose, increase effectiveness, safety and save money

Some painkillers are more or less effective depending on genotype.