Notesale: Turn your study into money

Document Preview

Extracts from the notes are below, to see the PDF you'll receive please use the links above

---

DNA Replication
When/ Why/ How does DNA need to Replicate?
 Replication occurs during Interphase before Cell Division
 Each daughter cell contains an exact copy of parents genetic material- cannot survive of
function without DNA
 Accuracy- Allows organisms to reproduce themselves to preserve a species





Helicase separates the 2 stands of DNA
DNA Polymerase picks up free nucleotides from the nucleoplasm, working from 5 prime to 3
prime
DNA polymerase creates phosphodiester backbone by complimentary base pairing onto the
two parent strands which act as templates

How?
 Replication begins at specific sequence known as replication origin
 Helicase enzyme unwinds and unzips the parental DNA, complimentary strands held by weak
Hydrogen Bonds
 Whole DNA molecule will have been unwound and replicated by end
 Parent strands act as template, new strands built up from free DNA nucleotides that are
abundant in nucleoplasm
 Free nucleotides attach (via Hydrogen Bonds) to exposed parental strands
 DNA Polymerase enzyme joins new nucleotides to each other by strong, covalent
phosphodiester bonds- forms sugar-phosphate backbone of new strand
 Each new identical molecule is made up of one new and one original chain from the parental
molecule- semi-conservative replication
 Meselon-Stahl 1958

Is There Evidence for Semi- Conservative Replication?
Meselon and Stahl grew successive generations of E
...
In time all the DNA in the E
...
The weight of the DNA molecule was considerably higher than that of DNA with
14
N as DNA contains so much Nitrogen
...
Coli containing heavy DNA was placed in medium containing normal nitrogen 14N
...

The E
...
The weight of the DNA was determined
using a centrifuge in Caesium Chloride (of same density)
...

Second generation DNA will have half intermediate and half light DNA
...


The Genetic Code
 Sequence of bases on DNA determines primary structure of protein (Number, Type and
Sequence)
 Segment of DNA which codes for protein is a gene
...
Genes
control the production of enzymes, which ones are produced can determine the organisms
characteristics
 The code is universal, same in all living organisms
Number of Letters
 Four bases,3 base code so 43 (64) different combinations to code for amino acids
 Vocabulary not large if each had just 2 bases in a code (16) as there are 20 amino acids
 Each amino acid codded for by triplet code in nucleus
 Some amino acids can be coded for by more than one triplet code so the code can be
described as degenerate
 Having 44 (256) combinations would involve too much DNA in the nucleus and be insufficient
Non-Overlapping Code
 The code must be read in non-overlapping triplets
 If the code was overlapping, one base could affect up to three amino acids- mutations would
have a more drastic effect
 The code has INITIATION triplets which are first encountered by the ribosome’s- AUG
 The code has TERMINATION triplets where protein synthesis stops- UAG, ATC

Protein Synthesis
RNA (Ribonucleic Acid)
RNA plays an important role in protein synthesis but its structure differs to DNA
...
A messenger RNA molecule is produced by a process known as transcription during protein
synthesis
...


Transcription
Definition; The process that comprises of using the base sequence of a gene upon a DNA
strand as a template to produce a molecule of mRNA with a complimentary base sequence
...
A-U, T-A, C-G, G-C
6) The RNA Polymerase then creates phosphodiester bonds between the RNA nucleotides to
generate a single strand of mRNA
7) When the enzyme reaches the end of the gene it releases the mRNA and the DNA rewinds to
form its double helix
8) The new mRNA molecule leaves the nucleus through a nuclear pore, enters the cytoplasm
and binds to a ribosome
A Codon is a sequence of three nucleotide on the mRNA molecule which codes for one amino acid
...

The sequence of mRNA is complimentary to that of DNA Sense Strand
...
If DNA entered the
cytoplasm for protein synthesis it is likely the DNA molecule will be damaged by the harmful
substances in the cytoplasm then a mutation would occur
...


Translation
Definition; Process allowing the assembly of amino acids into polypeptides based on the
original DNA code, occurs on the ribosomes
...

Transfer RNA
Plays an important role in translation
...

 At the top there is an amino acid
attachment site to which a specific amino acid is
bound
...





tRNAs with amino acids move towards the ribosome
At the bottom of each molecule is a specific sequence of three unpaired RNA bases which
are called the anticodon
The Specific amino acid carried by tRNA is determined by the anticodon on the tRNA
...
One site binds with the tRNA with the growing polypeptide
b
...
This process uses ATP (to perhaps cut the tRNA from the amino acid)
7) The empty tRNA leaves the ribosome and the ribosome moves along the mRNA so that the
second codon is in the first site
...
A
peptide bond forms
...
The
process of matching codons to anticodons repeats until STOP codon is reached
9) At STOP codon the mRNA and the polypeptide will be released
...


Blood Tests
In the case of cystic fibrosis, abnormal proteins can be detected in the blood
Amniocentesis
 Amniotic fluid removed from embryo during early stages of pregnancy
 Fluid contains foetal cells allowing the analysis of chromosomes, proteins, enzymic reations
and other biochemical properties
 Method detects defects such as haemophilia and Down’s syndrome
Chorionic Villus Sampling (CVS)
 Small sample of chorion (foetal tissue) is removed from the uturus via the vagina using a
long syringe, without the need for surgery
 Cells can be then quickly cultured and examined microscopically or using other biochemical
tests
 CVS can be performed earlier during pregnancy (8-10 weeks) than amniocentesis, which
must await for large enough volume of amniotic fluid

Genetic Counselling
 4000 diseases have genetic basis at present, affecting 1-2% of population
...

Cystic fibrosis sufferers produce very thick, sticky mucus that does not flow normally

Thick secretions;
 Congest the lungs, lead to recurring infections
 Block the pancreatic duct, stopping pancreatic enzymes from reaching the small intestine so
food digestion is not completed
 Cause the protective lining of the intestines to be defective
Symptoms;
 Distress with breathing (frequent daily massage needed to keep airways open)
 Impaired digestion
 Limited absorption of digestion products (Result is large appetite)
Someone with cystic fibrosis can live to be about 30, with daily physiotherapy and regular treatment
with antibiotics

Other Genetic Disease;
Huntington’s Disease
 Due to autosomal (non- sex chromosome) dominant allele on chromosome 4
 Rare disease causes nerve degeneration, accompanied with involuntary muscle contractions
 Most sufferers are heterozygous for this allele
Sickle Cell Anaemia
 Haemoglobin consists of two alpha and two beta chains, folded together
 In single cell anaemia there is a single base pair mutation on one allele controlling formation
of one beta chain
 Leads to abnormal folding of the haemoglobin
 Means red blood cells carry less oxygen and are fragile
 Sickle shaped cells ca not pass through capillaries very easily and so can cause blockages
 Disease is autosomal recessive, sufferers die at an early age
 Carriers have no symptoms and can be beneficial against malaria
Haemophilia
 Caused by recessive gene carried on X chromosome
 Sufferer unable to clot blood which occurs in a series of enzyme controlled steps
 Haemophiliacs do not produce enzyme facto VIII and so clotting process not completed
 Disease only affects XY males as Y chromosome is shorter than X chromosome and so does
not carry out factor VIII allele
...
Parents may have child with disorder and seek advice regarding chances
of second child with disorder
Advice given will be based on; History of the family disorder, Frequency of faulty gene in
general population, or whether parents are closely related
Parents are closely related, there is an increased chance of genetic disorders being passed
on by sharing similar genes
Given severity of genetic disorders, effect will have on quality of life of individual and family
creates difficult questions
Genetic counsellors can provide information, probabilities and genetic screening

Gene Therapy
Insertion of a new DNA sequence to replace function of a faulty gene that causes genetic disease
Gene therapy can be in two forms; germ- line therapy and somatic cell therapy
Germ- line Therapy
 Germ- line cells are those cells in a eukaryote, from which the gametes are derived i
...
the
tissue in the ovaries, which produce ova, or the tissue in the testes from which spermatozoa
are developed
 Research involves injecting the ova with replacement gene, or the early developing embryo
Advantages;
 Tissues of the new adult contain new gene, so genetic disorder is cured, and this can lead to
an increased chance of that adult producing progency without the defective gene
Difficulties;
 Chance of genome being disrupted: if this occurred the defective gene would still be present
and any additional disruption could be passed on to future generations
 There are ethical issues associated with germ- line therapy e
...
engineered babies etc
...
Either add directly within the body or
withdraw tissues and then replaced the treated cells
...
This fragment of DNA is replicated using
Polymerase Chain Reaction (PCR)
...
Special primers are added to a heated mixture of DNA
...
Primers attach to the
DNA at their target sequences, only a few bases long
...
New double helix sequences are then heated and
the process repeated until as large numbers of copies of the gene are made
...
Malfunction of the lungs is usually the cause of death and poor functioning affects the
sufferer most it has been under intense research
...


Replacement of Target Cells- Bone Marrow
-Affected tissue e
...
bone marrow, can be removed from the body and cultured
-DNA of the cells is treated with the new DNA and then the tissue is reintroduced to the patient

Problems With Gene Therapy
-Locating gene in human genome (to obtain copies of the unaffected DNA)
-Finding an efficient means to introduce the normal gene into target cells- solution for CF= aerosols,
liposomes, virus’
-When a new gene introduced into a cell, may not combine with the DNA and be degraded by the
cell

Dangers of Gene Therapy
-Introduction of new sequence of DNA in wrong area may trigger the activation of oncogenes which
are mutations of normal cellular genes, which contributes to development of cancer
-Possible introduction of other genes as well
-Unexpected disease caused e
...
leukaemia
-Allergic response
-Viral vectors used may have adverse effect

Benefits of Gene Therapy
-Symptoms of inherited diseases are alleviated more effectively than previous methods
-But condition not cured (Improvement only lasts a short time)

Ethical Considerations for Gene Therapy
-What is normal and what is disability?
-Are disabilities diseases? Do they need to be cured or prevented?
-Does searching for cures demeanthe lives of individuals presently affected by disabilities?

-Is somatic gene therapy (done in adult cells of a person known to have the disease) more or less
ethical than germ- line gene therapy (done to sperm and egg cells and prevents the trait from being
passed on to further generations)
...
expensive
...
g
...
g
...

Advantages;
-Large number of plants grown in sterile conditions so greater survival and quicker than if seeds
were planted outside
-Good/ quality healthy stock are selected with disease resistant or high yield, diseases can be
eliminated
-Crop is uniform as it is genetically identical- commercially important
-Large numbers can be stored in small areas so reduced heating and lighting costs
-Unique genotypes preserved
-Reduced space needed for transport
Disadvantages;
-Sterile conditions must be maintained (bacterial/ fungal contamination of culture medium could
result in loss of plants)
-Plants are genetically unstable with an increased mutation rate in medium- grown cells resulting in
abnormalities in plantlets- regular inspection is needed to remove defective individuals so higher
labour costs
1) A palnt is selected with desired characteristics
2) Scalpel used to remove meristem from the shoot
...
5cm3 blood which contains white blood cells, 0
...
HVRenzme cuts around to keep HVR intact
3) DNA has (-ve) charge so attracted to anode so is separated into bands
...

4) Denatured- gel immersed into alkali, which unzips double strand DNA
...
Single strands of DNA fragments are
drawn up into membrane where they stick
6) Radioactive/ UV gene probe added which binds to single stranded DNA fragments that
contain complimentary base sequences
...

7) Radioactive probe exposes film so patterns of dark and light bands are revealed producing a
DNA profile unique to each individual
Genetic fingerprinting is used for paternity tests, genetic screening and forensics
...
g
...
g
...
coli)
-Splicing= breaking open the plasmid DNA and inserting small fragment of donor DNA
(containging required gene) into it
-Plasmid treated with SAME restriction endonuclease as donor DNA so the foreign gene will
complimentary base pair with the plasmid so hydrogen bonds can form because of complimentary
sticky ends
-Recombinant DNA is formed via complimentary base pairing between sticky ends
-Ligase enzyme creates phophodiester bonds between unjoined bases on phosphodiester backbone
so joins plasmid and gene DNA permanently together
...
Overcome
strong cell wall
...
coli
-Only 1% of the bacteria will actually take up recombinant plasmid
-Those that do are transgenic organisms or described as transformed
Selection of Newly Transformed organism for Cultivation
-Antibiotic resistance sequences in plasmids are used as marker genes to identify and sort bacteria
containing recombinant DNA
-The transformed bacterium will not be killed by the antiobiotic and replicate while the nontransformed cells will die
Cloning
-Within each transformed bacterium the foreign DNA is replicated with each bacterial cell division
-Cloning of the recombinant containing bacteria results in multiple copies of the recombinant genes
-Repeated division produces a large population of bacterial cells all containing replicas of the foreign
DNA
Culturing

-Recombinant containing bacteria are used to make pure cultures
-Grown on industrial scale in fermenters
-Conditions ensure optimal growth of transgenic bacteria
-Human protein is purified from culture solution
-Large quantities made relatively quickly and cheaply



---