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How is DNA organised?

DNA is wrapped in an octamer of histones. There are 166bp per histone, stabilised by histone 1 and organised around chromatin.


How do you inactivate chromatin?



What are the 2 types of chromatin?

Euchromatin - lightly packed and under active transcription.

Heterochromatin - tightly packed and the genes are unexpressed.


What are the stages of mitosis and what occurs in them?

1). Prophase - nuclear membrane disappears, chromosomes condense and spindle appears.

2). Metaphase - the chromosomes align in the middle of the cell and spindle attaches.

3). Anaphase - spindle contract and pulls chromatids to poles of the cell.

4). Telophase - nuclear membrane reforms and chromosomes decondense.
- cell divides via cytokinesis.


What are the stages of meiosis and what occurs in them?

1). Prophase I - chromosomes condense and duplicate and nuclear membrane disappears.

2). Metaphase I - crossing over occurs at a chiasma = genetic variation
- chromosomes lie in pairs at the middle of the cell (a tetrad).

3). Anaphase I - homologous pairs of chromosomes are pulled apart by spindle.

4). Telophase I - the nuclear membrane reforms and chromosomes decondense giving 2 daughter cells.

5). Now stage II continues.


What a the stages of the cell cycle?

M = mitosis
G0 = cell is in arrest/not in the cell cycle
G1 = cell contents replicate
S phase = DNA replication
G2 = DNA is checked and repaired.

There are checkpoints between G1 and S, and G2 and M


How is DNA replication driven?

By the hydrolysis of PPi (pyrophosphate)


What are the stages of DNA replication in eukaryotes and what occurs in them?

1). Initiation - recognition of origins of replication and DNA polymerase recruited. DNA primate forms a small RNA primer = DNA can be extended from 3' end.

2). Elongation - DNA helicase unwinds the helix of DNA and DNA polymerase reads in a 3' to 5' direction and extends. The leading strand is formed on 3' to 5' and Okazaki fragments (the lagging strand) on 5' to 3'.
DNA ligase then cements these fragments.

3). Termination - the forks from multiple origins of replication meet and DNA ligase cements them together.


What are the stages of DNA replication in prokaryotes and what occurs in them?

Similar as in prokaryotes except:
1). There is one origin of replication
2). When the forks meet (lagging and leading strand) DNA ligase cements them together = 2 identical double stranded molecules.



What does meiosis provide in spermatogenesis and how long does this take?

4 sperm and it takes ~48 days.


What does meiosis provide in terms of oogenesis and how long does this take?

It gives us 1 oocyte and 3 polar bodies.
It takes 12-50 years.


How does meiosis maintain genetic diversity?

1). Random assortment of chromosomes during lining up in metaphase.

2). Crossing over between the homologous pairs of chromosomes swaps some alleles.


What is autosomal recessive?

CF and it can skip generations.


What is autosomal dominant?

Huntingdons and it cannot skip generations.
It is usually fatal if homozygous.


What is X-linked recessive?

Haemophilia A and it has a male preponderance.
Affected males will have a carrier mother and can't pass it on to their sons.


What is co dominance?

It is where out of two alleles, one is not dominant over the other so they are expressed equally in the phenotype e.g. Blood group.


What is complementation?

This is where more than 1 gene is involved in producing the phenotype e.g. Albinism.


What is linkage and recombination?

Genes close together on a chromosome are tightly linked, and therefore, have a lower recombination frequency during crossing over etc.


What are the types of mutation?

1). SNP's (single base substitution)
- nonsense - produces an early stop codon
- missense - changes one a/a for another
- silent - makes no change
- can change the frame shift = altered splice site

2). Insertions/deletions - A base is added or taken from the DNA.
- disrupts the reading frame if not x3
- causes premature termination codons (PTCs) but mRNA with PTCs are degraded by NMD.


What a the chemical causes of mutation?

1). Disruption of base stacking
Substances such as IQ can bind to DNA and push between bases = DNA polymerase misreads and skips the next base ( usually a GC bond).
IQ is found in cigarette condensate and cooked meats.

2). EMS (ethyl methane sulphonate) removes purine rings and apurinic sites can be paired with any base.

3). Nitrous acid changes the amino group of the base to a keto group
- C changes to U
- A changes to H and pairs with C
- G changes to X and pairs with C


How can sequence changes in replication cause a mutation?

1). Slippage
- A 'kink' occurs in the newly made or template strand = addition or omission of a base.

2). Tautometric shift
- proton in the bases changes place and causes altered base pairing properties
C (T seen as C) -->A
T (C seen as T) -->G


How can radiation cause mutations to occur?

1). UV light can form thymine dimers = adjacent T bases bond
- BUT these usually correct themselves via photoreactivation.

2). Ionising radiation produces ions which react with cellular molecules.


How can mutations be detected in a foetus?

1). Foetal DNA can be isolated and sequenced but this isn't often used.

2). Amniocentesis
- 0.5-1% chance of miscarriage
- occurs at around 15-20 weeks and takes 2 week to culture cells

3). Chorion villus biopsy
- 2-3% chance of miscarriage
- foetal villi taken from mothers tissue
- occurs at 10-13 weeks


What is MLPA?

It is a way of detecting exon deletions or duplications (counts exon no.)

1). 2 short pieces of DNA a arranged with a gap between them to place patients DNA in.

2). Both short pieces of DNA have primers on the end to allow for PCR.

3). If the patient is missing an exon, the DNA will not hybridise, so no ligation will occur and no PCR product will be formed.


What is SSCP?

It identifies mutated regions in DNA.

- If you are heterozygous for a disorder, then the normal and mutant sequences will both show.

1). Heat DNA to denature
2). Cool to allow annealing = ssDNA will curl up on itself.
3). Gel electrophoresis - different sequences will form different shapes.


What are the types of DNA repair?

1). Nucleotide mismatch - enzymes check new strand for mismatched bases and repair them with a patch of DNA.

2). Nucleotide excision base repair - bases damages by ROS or UV are removed and replaced.

3). Base excision repair - bases damaged by deamination or containing uracil are detected, removed and replaced by enzymes.


What are the numerical forms of chromosomal abnormality?

1). Polyploidy - chromosome number is a multiple of 23, usually as a result of polyspermy.

2). Aneuploidy - chromosome number is not a multiple of 23
--> trisomy = one more
--> monosomy = one less


How can aneuploidy occur?

1). Non disjunction during meiosis or mitosis
--> if in mitosis, can create mosaics.

2). Anaphase lag where a chromosome is left behind and lost.


What are the 6 structural chromosomal abnormalities?

1). Translocation - where one chromosome gives part of itself to another.

2). Reciprocal translocation - this is balanced, where 2 chromosomes exchange parts. Usually the breakage point is between genes.
--> can be passed onto gametes

3). Robertsonian translocation - the q arms of two chromosomes combine and the p arms are lost, to create one 'super chromosome'. This occurs when the satellite regions of the p arms combine inappropriately.

4). Ring chromosome - ends of the chromosome are lost and they fuse to form a ring.

5). Isochrome chromosome - 2 non identical chromosomes are formed, usually by the joining of 2 long arms and 2 short arms.

6). Interstitial/terminal deletion is unbalanced, where parts of the chromosome are lost.


How are chromosomes studied?

1). Add phymohemaglutinin to stimulate the division of lymphocytes.

2). Add colcemid to arrest cells in metaphase

3). Add KCL to swell the cells then place on a slide.

4). Digest using trypsin to produce fragments and stain using Giemsa.


What occurs during transcription?

1). Initiation - a transcription factor recognises and binds to the specific promoter sequence in a 3' to 5' direction, causing a complex to form in which RNA polymerase is recruited.
RNA polymerase binds at the TATA box (30 downstream) which is followed by a variety of upstream sequences.

In prokaryotes, it binds at the prinbow box, 70 downstream.

2). Elongation - RNA polymerase makes RNA in the 5' to 3' direction.

3). Termination - mRNA is processed.


What are the processing types in termination of transcription?

1). Capping at the 5' end
- protects against degradation
- made of methyl-guanine and bonded to a 5'-5' triphosphate.
- nuclear export is regulated by cap binding complex.

2). Tailing at the 3' end after stop codon
- protects against degradation

3). Splicing
- removes intron DNA using spliceosomes
- a change in the sequence can alter splicing


What is the coding strand?

The DNA strand that is not being copied in transcription.


What occurs during translation?

1). Initiation- Recognition of AUG start codon after cap = cap binding proteins (GTP) bind to cap and recruit tRNA. Energy moves tRNA along until it hits a start codon = recruits 60s ribosome subunit.

2). Elongation - Met-tRNA is bound to the P site and A site is free = energy moves complimentary charged aminoacyl tRNA in.
Peptide bond forms (via peptidyl transferase) and met 'jumps' = tRNA no longer charged = released from P site.
Translocation occurs using energy.

3). Termination - the last A site becomes occupied by a stop codon = a releasing factor like tRNA binds and pushes out the last tRNA.


What a the differences between eukaryotic and prokaryotic transcription/translation?

Eukaryotes have many RNA polymerases, prokaryotes don't.

Eukaryotes have 80s ribosomes, prokaryotes 70s.

Eukaryotes have complex promoters, prokaryotes are simple.

Eukaryotic mRNA is longer lived.

Eukaryotes have post transcriptional/translational processing, prokaryotes don't.


What are the 3 autosomal aneuploidy disorders?

Trisomy 13 - Pataus syndrome
= polydactyly and rarely live to a year.

Trisomy 18 - Edwards syndrome
= 5-15 day lifespan and female preponderance

Trisomy 21 - Down's syndrome
= greater risk when mother is older than 35
- intellectual disability
- congenital heart disorder
- hypothyroidism
- hearing and visual disorders


What are the 4 sex aneuploidy disorders?

1). 47, XXY (Klinefelters syndrome)
- gynocomastia
- less testosterone
- learning impairment

2). 47, XYY (XYY syndrome)
- normal phenotype
- increased growth
- slightly lower IQ

3). 47 XXX (triple X syndrome)
- microcephaly
- tall
- scoliosis
- learning disability

4). 45, X (turners syndrome)
- broad chest
- short stature
- webbed neck
- CV and renal issues


Why is not 1 X an issue?

Only 1 is ever active, so the others form Barrs bodies at nucleus periphery.

In XY, both chromosomes still have areas in common at the ends of the arms.


What is restriction analysis?

Restriction enzymes + gel electrophoresis.


How is DNA cloned?

1). Cut 2 DNA molecules with same restriction enzyme = forms complimentary sticky ends.

2). DNA ligase forms phosphodiester bond = recombinant DNA that is taken up by bacteria.

3). Bacteria is cultured in a Petri dish of antibiotic that the plasmid had resistance to = those who haven't taken it up die.


Why is DNA cloned?

Can make proteins
- remove useful mRNA
- use reverse transcriptase = ds cDNA
- form a plasmid and add to bacteria
Gene therapy
Screen for genetic disease


How does PCR work?

1). Heat to 95degrees = denature DNA = break H bonds.

2). Cool to 55degrees = primers can anneal

3). Heat to 72degrees so TAQ polymerase can form complimentary strand.


How is DNA profiling performed?

Small tandem repeats are individual in all people = can be replicated by PCR and examined.


What does SDS-PAGE electrophoresis do?

Adds SDS detergent = net -ve charge on protein and no secondary structure

Add betaME (reducing agent) to rid of disulphide bonds

Separate based on size only


What does isoelectric focusing do?

A gel with varying pH as you go down is made = proteins migrate until they reach their isoelectric point.

Separates on basis of charge only!


What is 2D PAGE?

Combines IEF and SDS-PAGE = separates proteins with the same charge and size.

Add to a mass spec, to compare gained values to known values to identify the proteins.


What is western blotting?

Use a nitrocellulose replica of a gel after electrophoresis.
- Add primary antibody = binds
- Add secondary antibody which is marked with fluorescence = binds to primary


Compare and contrast polyclonal and monoclonal antibodies.

Both are specific to one antigen.
Polyclonal antibodies are produced by many B lymphocytes, monoclonal by only 1.
Polyclonal antibodies have multiple epitopes (sequence it will recognise on the protein), whereas monoclonal have 1.


What is microarray?

DNA or mRNA is taken from the patient and a healthy person, and both are labelled in different colours.
Equal amount of each persons are added to wells so if one colour dominates, this gives an indication of gene expression.


What is reverse transcriptase PCR?

1). A 'TTTTTT' primer is made to bind to the polyA tail of mRNA.
2). Reverse transcriptase can bind and make cDNA.
3). RNAase breaks the mRNA down = ss cDNA left.
4). PCR primers added and amplification occurs.


What is FISH?

1). A labelled DNA probe is made.
2). DNA denatured in situ.
3). Add the probe = detection.


How does ELISA work?

Works out the conc or presence of a specific protein.

1). Sample taken and placed in a microtatum plate.
2). Specific antibody is added.
3). Add a secondary enzyme linked antibody.
4). Add substrate that changes colour in presence of that enzyme.


What do ELISA and RIA detect?

RIA detects cortisol, T3/4, TSH and insulin.

ELISA detects T3/4, TSH and insulin.


How is southern blotting linked to DNA fingerprinting?

You can analyse the no. of satellite regions made = fingerprinting.


What is high resolution banding?

Monochrome paints in FISH and banding can be limiting = in mFISH and mBAND, fluorescent bands of chromosome specific probes are hybridised and the images analysed.


What is a tumour cell defined as?

1). Ignores external antigrowth growth signals.
2). Avoids apoptosis.
3). Divides indefinitely.
4). Can invade other tissues to form secondary tumours.
5). Stimulation is sustained by angiogenesis (formation of new blood vessels).


What can result in cancer?

Failure of the repair enzymes involved with DNA mutation.


What do all cancer cells exhibit?

Chromosomal and microsatellite instability.


What are oncogenes and proto-oncogenes?

Oncogenes are retro virus genes that can transform cells into a cancerous phenotype.

Proto-oncogenes are similar but in humans which form cell control functions.


Give 2 examples of protooncogenes.

Gene Human function
SRC - Tyrosine kinase
MYB - Nuclear protein


What can cause an protooncogene to turn into a cancer causing oncogene?

Key a/a substitutions


What is native gel electrophoresis?

Gel electrophoresis of proteins