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BB10006 – Cell and Molecular Biology
DNA – Momna Hejmadi
Nucleic acids
o Timeline:
o 1869 – F
...

o 1828 – F
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o 1944 – Avery, MacLeod, and McCarty – Transforming principle is DNA – transferred substance
found to be DNA (when protein, RNA, or fats removed or destroyed, cells were transformed,
when DNA destroyed no transformation occurred)
...

o 1952 – Hershey-Chase – ‘Blender’ experiment - viral DNA enters cell during infection, viral
protein doesn’t, so DNA must be genetic material
...
Franklin and M
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o 1953 – J
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Crick – DNA structure solved - two antiparallel strands in double helix,
unwound and unzipped for replication and transcription
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o Carbons in sugar labelled “1’” etc
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o Purines – adenine and guanine
...

o 5’-3’ polynucleotide linkages – phosphodiester bonds (5’ and 3’ links to pentose sugar), Nglycosidic bonds (base to C1’ pentose)
...

o Purines pair best with pyrimidines as they form the right diameter for the helix
...

o RNA structure – single stranded (intramolecular base pairs), less pronounced major and minor grooves,
uracil instead of thymine, structural, adaptor, and transfer roles all involved in decoding information
carried by DNA
...

o DNA replication is the process of duplication of the entire genome prior to cell division, it preserves the
integrity of the genome in successive generations, replication rates vary (slower in eukaryotes)
...

o Elongation – proteins connect correct sequences of nucleotides into continuous new strand of DNA
...

o Basic rules of replication:
o Semi-conservative
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o Uni or bidirectional – replication forks move in one or opposite directions
...

Synthesis 5’-3’ – nucleotide recognition, enzyme catalysed polymerisation (DNA polymerase),
complementary base pair copied, substrate is dNTP, can’t go other way due to chemistry of DNA
where the incoming nucleotide has three phosphates attached so no other bases can join afterwards
(has to be in this direction as energy for the formation of the phosphodiester bond comes from the
dNTP, a nucleotide with two additional phosphates attached to its 5’ end, which has to be added, in
order to join the 3’OH group with the phosphate of the next nucleotide one oxygen has to be
removed and added to two extra phosphates when are attached to a Mg2+ which pulls up the
electrons of the oxygen weakening the bond, and the nucleophilic attack of the oxygen from the
3’OH succeeds, forming the phosphodiester bond, the other way no high-energy bond would be
cleaved)
...

Core proteins at replication fork:
o Topoisomerases – prevents torsion by DNA breaks
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o Primases – RNA primer synthesis
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o DNA polymerase – synthesis of new strand
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o DNA ligase – seals nick via phosphodiester linkage
...
2Gbp), 24 types of linear chromosomes, around
30,000 genes (which was seriously overestimated), less than 5% codes (is information that may affect how
genes are expressed), mitochondrial DNA is 16
...

o Why do we need the DNA blueprint?
o Helps us understand genes
...
g
...

o Risk of disease: e
...
functional variant in coronary disease causes changes to receptor so can’t take
up LDLs, can give advice once aware
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o Forensics: mitochondrial DNA in hair shaft, nuclear in root
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o Synthesis is always 5’ to 3’
...

o Gene expression allows selective expression of genes, regulation of transcription controls time, place and
level of protein expression
...


o

Mechanism of transcription
o Initiation – RNA polymerase binds to promoter and opens helix
...

o Termination – stops at termination signal, RNA polymerase dissociates
...


o

o

Post transcriptional modifications
o Capping – addition of 7-methylguanosine at 5’ end, mediated by guanylyltransferase, probably
protects against degradation, serves as recognition site for ribosomes, transports hnRNA (premRNA) from nucleus to cytoplasm
...

o Splicing – highly precise removal of intron sequences, performed by snRNPs and spliceosomes (large
RNA-protein complex made of small nuclear ribonucleoproteins), recognise exon-intron boundaries
and splice exons together by transesterification reactions
...


DNA Translation
o Translation requires mRNA, aminoacyl-transfer RNA (aatRNA), and ribosomes, ribosomes engage the
mRNAs and form a catalytic domain into which the tRNAs enter with their attached amino acids, the proteins
of the ribosomes catalyse all the functions of polypeptide synthesis
...

o Non-overlapping – only read in groups of three, frame shift can stop or truncate the protein
...


o

Structure of tRNAs – class of small RNAs which form covalent bonds to amino acids, allows correct insertion
of amino acids into the elongating polypeptide chain
...

o Correct aminoacylation – recognise specific tRNA identifiers present on acceptor stem (3’ end) and
anticodon loop, positions of identifiers are specific to tRNA types and are highly conserver,
aminoacyl-tRNA synthase specific to amino acid fits amino acid and tRNA into separate active sites
then forms a high-energy ester bond
...

Ribosomes – made of rRNA and ribosomal proteins, forming two subunits (one large and one small),
subunits are self-assembling and combine only in the presence of mRNA and a charged aatRNA
...

Initiation in eukaryotes:
o Formation of 43S pre-initiation complex (smaller sub-unit) – first charged tRNA with methionine as
its amino acid (tRNAimet) attached to eIF2 and GTP, then bound by eIF1 to 40S-eIF3, then eIF5
attached, to form 43S pre-initiation complex
...


o

o

Positioning at start codon – initiation complex moves down mRNA unwinding using eIF4 helicase,
stops at start site AUG (Kozak sequence ANNAUGG signals it’s the right AUG), allows irreversible GTP
hydrolysis of eIF2 preventing further unwinding of mRNA
...


Elongation in eukaryotes:
o aatRNA binding – aatRNA binds to a site on ribosome by base pairing with codon, elongation factors
(EF) mediates and GTP hydrolysis drives reaction
...


o

Transpeptidation – peptidyltransferase transfers peptide bond from tRNA to next amino acid
...


Termination in eukaryotes – release factors (eRFs) recognise and bind to stop codons, inducing peptidyl
transferase to transfer peptidyl group to water instead of aatRNA, uncharged tRNA released from ribosome,
inactive ribosome then releases mRNA
...

Post translational modifications
o Protein folding – nascent protein is folded and/or modified, mediated by molecular chaperones (e
...

Hsp70) or chaperonins (Hsp60 complexes)
...
g
...
),
dictate the activity, life span or location of proteins
...
g
...

Antibiotics that inhibit translation
o Prokaryotes: streptomycin prevents initiation-elongation, chloramphenicol blocks
peptidyltransferase (only in mitochondria)
...

o Both: puromycin causes premature release of polypeptide
...

o Isotopes with excess neutrons emit beta particles (negative electrons) from nucleus giving one less
neutron and one more proton, by converting a neutron into a proton
...

o Radionuclides of atomic number 60 or higher can emit alpha particles, consisting of two protons and
two neutrons
...

o Radiation properties
o Alpha – slow moving, range ~3cm in air, no external hazard, contamination is very serious
...

o Gamma – long range (most we’re exposed to from space), external radiation hazard
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o Gamma and X-rays stopped by lead and concrete
...

▪ 32P – 14
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o Legislation
o Radioactive Substances Act (1993) – concerned with record keeping (use, accumulation and
disposal), registration of users (establishment and people)
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o Transgressions are criminal offences
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o Shielding
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o Reduce exposure time to a minimum
...

o Never work alone
...
3 days
...

o Range – 790cm in air
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o Shielding – 1cm Perspex
...
g
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Sulphur-35 35S
o Half-life – 87
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o Radiation – beta (low energy electrons)
...

o Detection – Geiger-Muller
...

o Uses – labelling proteins (methionine), DNA sequencing (S substitutes O in phosphate – used
instead of radioactive phosphorus as it’s lower energy so tighter band on X-ray film, and safer)
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4 years
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o Range – 6mm in air
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o Shielding – none (can be absorbed through skin, danger of contamination
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Iodine-125 125I
o Half-life – 59
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o Radiation – gamma, X-rays
...

o Detection – scintillation counting, thyroid scans (gets concentrated there so check there for
contamination)
...

o Uses – labelling peptides (once labelled not dangerous)
...

o Basic cloning involves: insertion of DNA fragment into vector to form recombinant molecule; transport of
recombinant molecule into a bacterial cell; multiplication of cell and recombinant molecule; production of
bacterial colonies containing recombinant molecule, each colony is a clone of identical host cells containing
the recombinant molecule
...

o Requires: a thermostable DNA polymerase, Taq; short DNA primers that can anneal to sequences
that flank the gene of interest; a thermocycler, computer controlled heating block that can cycle
through precise temperature changes
...

o Why gene cloning?
o Provides a pure sample of the gene of interest – the raw material for the study of gene structure and
function
...

o Enables genes and genomes to be sequenced
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o
o

o

Problem of identification – cloning can produce hundreds, thousands or millions of clones so finding the of
the clone of interest requires strategies for selection and identification
...

Production of animal protein – can clone gene for important animal protein (like a hormone) in bacteria and
express it there, then purify the protein and use
...
g
...

o RNA – mRNA, rRNA (majority), tRNA, viral (single and double stranded)
...
g
...

o Removal of protein – makes up large proportion of material (e
...
phenol extraction, denatures
protein, or digestion of protein, e
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proteinase K
...
g
...

o Concentration of nucleic acid – usually by precipitation (e
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ethanol and salt precipitation)
...

▪ Media can be defined/minimal (known chemical components) or undefined/complex
(mixture of organic extracts, like tryptone or yeast extract)
...

▪ Harvested by centrifugation, cells form pellet and supernatant can be discarded
...

o Purify DNA from cell extract – degradation of or separation from contaminants
...

▪ Separation from contaminants – ion-exchange chromatography, NAs are negatively charged
and will bind to a positively charged resin, increasing salt concentration elutes (washes off)
bound components, DNA is tightly bound and is eluted at high salt concentrations
...
g
...

o Purity and concentration of DNA – measured in a UV spectrophotometer, requires a quartz cuvette
transparent to UV light, at a wavelength of 260 nm (A260) one absorbance unit is equivalent to 50 μg
of double stranded DNA per ml
...
8, ratios of less show contamination (by protein or phenol)
...
g
...

o Purification of plasmid DNA from bacterial cells
o Plasmids – autonomous, self-replicating circles of double stranded DNA found in many bacterial
cells, not essential to the bacteria, replication is coordinated and controlled with replication of
bacteria, they are the basis of many plasmid cloning vectors, smaller than chromosomal DNA
...


o

o

o

o

o

Plasmid DNA can be precipitated directly from cleared lysate but often further purification is
required, the classical method that produces extremely high-quality DNA is ethidium bromidecaesium chloride density gradient centrifugation (steps after preparation of cleared lysate are long
and complicated so only do them if you need ultra-pure DNA)
...

Ethidium bromide-caesium chloride centrifugation of DNA – extended centrifugation (over 24
hours) with caesium chloride and ethidium bromide at high rpm creates a density gradient in the
tube, molecules band according to their buoyant density, the position of the bands can be seen
with UV light, plasmid DNA is removed with a hypodermic syringe, EtBr is removed with butanol,
CsCl is removed by dialysis, pure plasmid DNA can be concentrated by ethanol precipitation
...
g
...
) – variations on methods depending on
the nature of the tissue or the sub-cellular fraction in which the DNA is found and the biology of the
organism concerned, for example bacteriophage particles are found in the intracellular medium of
an infected culture rather than in the E
...

o RNAs – use methods, reagents and buffers that favour the conservation of RNA and elimination of
DNA, start with a tissue in which the RNA of interest is abundantly expressed
...

o Oligo-dT chromatography
▪ Prepare a small column containing oligo-dT (primer, single stranded sequence of
deoxythymine used for priming reactions catalysed by reverse transcriptase) bound to a
cellulose matrix
...

▪ Add medium salt buffer – washes off rRNA and tRNA
...

▪ The RNA can then be concentrated with ethanol precipitation
...


Enzymes for manipulating nucleic acids
o Nucleases – cut, shorten or degrade NAs
...

▪ Bal31 – digests ends of DNA, degree of digestion is dependent on time and concentration,
can be used to shorten DNA molecules
...

o Endonucleases – able to break internal phosphodiester bonds within a NA molecule
...

▪ Deoxyribonuclease I (DNase I) – chops up all DNA, useful to get rid of DNA or to produce
small fragments
...

o Ligases – join NA molecules together, natural function is to repair single-stranded breaks, can be used to
join DNA molecules together for cloning, most have already been cloned so are produced rather than
purified
...

o Polymerases – make copies of NA molecules, synthesise new strands of DNA complementary to NA
template, require short primer sequence base-paired to a single-stranded template (provides doublestranded region for polymerisation) and a supply of nucleotides
...
coli, repairs nicks in DNA by synthesising new strand and replaces any
existing strand as it proceeds (proof-reading capability) so has dual activity (DNA polymerisation and
DNA degradation), can be modified by removing the amino acid segment involved in degradation to
form the Klenow fragment which lacks the proof-reading capability so will only synthesise the
complementary strand, this is used in DNA sequencing
...

o Reverse transcriptase – from retroviruses, uses an RNA template and a DNA primer, synthesises a
complementary copy of RNA, used for cDNA cloning (cloning mRNA)
...

o Natural function – host-controlled restriction, cleave bacteriophage (bacterial virus) DNA and prevent
infection, bacterial DNA is methylated at the RE sites so is protected against its own, each RE has its specific
methylase (in nature they come in pairs)
...

o Production of blunt and sticky ends – some Res cut straight across double-stranded DNA leaving blunt ends
(AluI), and some cut in a staggered fashion leaving sticky ends (EcoI) which can base-pair together with
hydrogen bonds so is helpful for re-joining ends
...
g
...

o Frequency of recognition sites – in a random sequence of DNA the average frequency of occurrence of a RE
recognition site can be calculated (although DNA is not always random, 4-cutters should cut around 16x
more frequently than 6-cutters)
...

o Hexanucleotide – once every 46 – 4096 nucleotides
...


o

Agarose gel electrophoresis – invaluable tool for analysing, mapping and purifying DNA molecules, agarose
is a highly pure form of agar, use to form a transparent gel matrix which can then be used to separate DNA
molecules (like the products of restriction digests)
...

o Preparing for DNA electrophoresis – the gel formers and comb are removed from the gel which is
then placed in the electrophoresis tank and covered with the electrophoresis buffer, the digested
DNA is mixed with a loading buffer which contains a dye (for visibility) and sucrose or glycerol (to
make the sample heavier than the buffer so it sinks to the bottom of the well)
...

o Visualising DNA fragments – DNA fragments are stained with ethidium bromide (EtBr) either during
or after electrophoresis, then gel is placed on UV transilluminator, DNA bands fluoresce and can be
photographed, can detect >10 ng DNA
...

Restriction mapping – perform single and double RE digests on DNA (with one and then two Res),
electrophorese them in parallel, accurately measure sizes of fragments, work out relative positions on DNA
molecule
...

DNA digest – agarose gels can also be used to isolate particular restriction fragments of DNA which can then
be used for cloning or other purposes
Ligation – construction of a recombinant molecule
o Gene is inserted into a cloning vector by the action of T4 DNA ligase which originally comes from
bacteriophage T4 where its function is to repair nicks in DNA
...

o Inter- and intra-molecular association – in ligation you have no control over which complementary
sticky ends join, so the insert and the vector may ligate with themselves rather than form the
desired recombinant molecule
...
coli
...
coli as
well as its plasmids and bacteriophage are the principle tools of the genetic manipulator (although other
bacteria and organisms can be used), other prokaryotes and eukaryotes can be used as well
...
coli cell containing a
recombinant DNA molecule, produce a bacterial colony on petri dish, use colony to inoculate a liquid culture,
grow overnight, purify recombinant DNA (note: the polymerase chain reaction can play a similar role)
...

Desirable characteristics
o Small size – easy manipulation and less vulnerable to damage, high copy number, more insert per g
of plasmid, easier transformation
...
g
...

o Range of unique restriction sites – permits cloning fragments produced by several enzymes
...

o High copy number – more plasmid DNA per cell
...

pBR322 cloning vector – was constructed by Bolivar and Rodriguez from pieces of different naturally
occurring E
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o About 4
...

o Two selectable markers - ampicillin and tetracycline resistance
...

o Selection for recombinants via insertional inactivation
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Cloning into the BamHI site of pBR322 – cut pBR322 with BamHI (+/- removal of 5’ phosphates with alkaline
phosphatase), DNA to be cloned also cut with BamHI, mix the DNAs in a buffer with T4 DNA ligase and ATP
then incubate for a few hours, ligated products will be linear and circular (all possible products produced)
...

Transformation – the uptake of DNA by bacterial cells
...
coli cells treated with cold CaCl2 – become competent to take up DNA
...

o Heat shock at 42C – DNA enters cell
...
01%)
...

All circular plasmid-containing molecules are cloned – all plasmid-containing cells will grow into colonies,
there is no differentiation between empty plasmids, recombinant plasmids and the clone, insertional
activation is used to differentiate between recombinants and non-recombinants:
o Insertional activation – inserting extra DNA into a gene inactivates it, the normal pBR322 provides
resistance to ampicillin and tetracycline (ampRtetR), the BamHI site is within the tetracycline
resistance gene so inserting DNA into that site inactivates the gene and recombinants are ampicillin
resistant but tetracycline sensitive (ampRtetS), this can be used like positive selection
...

Limitations of pBR322 – lack of cloning sites (especially located conveniently together), and although
insertional inactivation is useful some more positive selection of recombinants would be advantageous
...

o lacZ’ gene – part of -galactosidase from lac operon, complete pUC8 is ampR-gal+, new DNA
inactivates lacZ’ so recombinants are ampR-gal-, this is the basis of positive selection for
recombinants, a mutant E
...

o Identifying recombinants with pUC8 – the agar which the transformed cells are plated onto contains
ampicillin and also IPTG (an inducer for -galactosidase) and X-gal (an artificial substrate for galactosidase) which gives a blue colour when broken down, so non-recombinant clones grow into
blue colonies as the enzyme is produced and recombinant colonies are white, these can be picked
off for further analysis
...

o Small fragments are easier to clone than large ones – size limitation on clonable fragments is around
5000 bp
...


Bacteriophage cloning vectors
o Bacteriophage – viruses that infect bacteria, used in vector construction and use, consist of a nucleic acid
molecule surrounded by a protein coat, can have DNA or RNA but ones with DNA genomes are generally
used, head and tail structure is typical of bacteriophage lambda () which has a double stranded DNA
genome of nearly 50,000 bp, vectors derived from lambda are used for cloning large pieces of DNA,
filamentous structure is typical of M13 which has a single stranded DNA genome of about 10,000 bp and
cloning vectors derived from it are used for DNA sequencing
...
coli cells IF is converted to double stranded replicative form (RF) and replicates to
produce many copies, replication changes to rolling circle mechanism which produced single stranded linear
molecules which then circularise to IF and are packaged into the protein coat to be extruded from the cell, IF
can be purified from the medium and RF from the cells
...
coli where IF is produced, infective form (IF) is single
stranded so is an ideal template for DNA polymerases and can be used in DNA sequencing, lacZ’ and RE sites
from pUC vectors can be put into M13 which enables blue/white selection and enables shuttling DNA from
pUC to M13 vectors
...
coli with recombinant
plaques being white and non-recombinant being blue, also come in pairs enabling fragments to be
turned around (like pUC plasmids)
...

o Ligation products are transfected into E
...

o Cells are plated out onto an E
...

o E
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o Use white plaques to infect E
...

o Transformation – the introduction of plasmid DNA into a bacterial cell (inefficient)
...

o Transfection – the introduction of naked bacteriophage DNA into a bacterial cell by transformation (cold
CaCl2 and heat shock, once in the cell the phage’s genes are expressed and it behaves like a normal
bacteriophage, inefficient)
...
coli by injecting linear DNA molecule which then circularises by means of its 12 bp

o

o

o
o

o

o

complementary single stranded ends (called cohesive or cos sites), then replicates as a circular
molecule, after a while replication switches to the rolling circle and it produces long strings of
genome joined end to end and separated by cos sites, enzymes recognise the cos sites and cut the
DNA there, packaging individual lambda genomes into their respective protein coats, then the E
...

Lysogenic – of interest to geneticist, lambda infects E
...
coli genome and is passively replicated with that, under certain circumstances
the lysogenic lambda can de-integrate from the E
...


The importance of cos ends:
o Base pairing facilitates circularisation on infection – cohesive ends hydrogen bond and E
...

o In late replication  replicates by rolling circle and produces concatamers of  genomes, enzymes
recognise cos sites and cleave DNA there which is then packaged by other  proteins to form phage
particles
...
coli by lambda particles in highly efficient, so more clones
of DNA
...
coli, but protein coat imposes physical limitations on size of DNA molecule so can only be 52
kb, so only 3 kb of DNA can be inserted into wild-type lambda – solution: remove genes for
lysogenic cycle which is about 15 kb
...
g
...
coli strain that produces EcoRI, most  DNA molecules that invade are destroyed by
the restriction endonuclease, but a few survive and produce plaques, these are mutants which have
lost their EcoRI site spontaneously), or change sequences at sites using in vitro mutagenesis
...

o Replacement vectors – for cloning large fragments (15 – 22 kb) with very high efficiency, used for
producing genomic libraries (a collection of clones that together contain all the DNA of an
organism)
...


λgt10 – cl gives clear plaques for recombinants & turbid for non-recombinants
...

Used for high efficiency cloning of small DNA fragments; e
...
cDNA cloning
...
coli, plate out on
E
...

Replacement vectors - packaging constraints impose a limit to the size of insert that can be cloned in
insertion vectors, if all the non-essential region could be removed this would increase the clonable fragment
size but there is also a minimum size that can be packaged so use a ‘stuffer fragment’ (conveniently sized
chunk that can be cut out when needed)
...

o Method of cloning - vector is cut with RE, purify left & right arms from stuffer fragment, ligate to
digested DNA, package in vitro & infect E
...

coli lawn (determine its titre in plaque-forming-units per ml (pfu/ml)), arms alone are too small to be
packaged so all plaques should be recombinants
...

o
o
o
o

o

Genomic and cDNA libraries
o Cosmids - Hybrid between λ and plasmid (plasmid that carries a cos site, also needs a selectable marker, and
a plasmid origin of replication), enables large pieces of DNA (around 35 kb) to be cloned, use efficient
infection of E
...
coli behaves as plasmid, form colonies on selective media like plasmids,
cos sites are the key at the ends of linear λ molecule as enzymes that package λ DNA into the protein coat
only need these to function (package any DNA that carries cos sites separated by 37-52 kb of DNA)
...
coli cells where the cosmid recircularises, infected cells
placed on a selective medium and antibiotic resistant colonies are grown (non-recombinant linear
cosmids are too small to be packaged)
...

o Number of clones needed:
ln(1 – P)
N = ---------------ln(1 – a/b)
N – number of clones required
...

a – average size of DNA insert in vector
...

The larger the size of the average cloned insert, the smaller the total number of clones required to
contain the whole genome
...

Construction of genomic library
o Problem: 6-cutter enzymes cut on average every 4096 nucleotides so many fragments will be too
small and some will be too long, 4-cutter enzymes cut on average every 256 nucleotides so most
fragments will be too small to clone
...

▪ Cosmid genomic library – then packaged into λ particles, infect E
...

Cloning even larger fragments
o Bacterial artificial chromosomes (BACs) - based on F plasmid of E
...

o Bacteriophage P1 - clone up to 110 kb
o P1-derived artificial chromosomes (PACs) - cosmid-like vectors based on P1, clone up to 300 kb
...

o Differential gene expression - limited number of genes are switched on in any particular cell type or
during a particular stage of development so Isolate RNA from tissue where genes of interest are
expressed to maximise chances of cloning mRNA for the gene of interest
...

o cDNA cloning – to clone mRNA need a double-stranded DNA copy of it, reverse transcriptase will
synthesise a complementary DNA strand to a RNA template (requires single-stranded RNA template,
short DNA primer base-paired to template, supply of deoxyribonucleotides (dNTPs)),
o Annealing of oligo(dT) primer - oligo(dT)s can be made to anneal to the poly(A) tail of mRNA and
serve as a primer for reverse transcriptase
...

o Partial degradation of RNA – ribonuclease (RNase H) used to partially degrade the RNA half of this
hybrid molecule, experimental conditions are such that RNA fragments are left base-paired to the
DNA strand
...

o

o

o

o

Cloning cDNAs - many steps in cDNA synthesis means little product and cDNAs are blunt ended so inefficient
to clone, best approach is to add linkers to cDNA ends (short synthetic double-stranded oligonucleotides
that contain a restriction site, used in such high concentration that despite their blunt ends they are ligated
to the ends of the cDNA), cDNA with ligated linkers is digested with RE that cuts restriction site in linker to
end up with cDNAs with sticky ends, cDNAs can then be ligated to a λ insertion vector (e
...
λgt10) and
cloned with high efficiency, generates cDNA library
...


Cloning beyond E
...
coli is the principal workhorse of the molecular biologist, plasmid (and a few
phage) vectors exist for other bacteria (e
...
Streptomyces, Bacillus, Pseudomonas) including broad hostrange vectors which will work across several bacterial species and use horizontal gene transfer, similar in
mode of action to E
...

o Cloning in yeast (Saccharomyces cerevisiae) – model eukaryotic organism (know a lot about it, one of the
oldest organisms to be used by humanity, one of the first organisms to have its whole genome sequenced),
vectors based on 2 μm plasmid (one of the very few found in eukaryotes)
...

o LEU2 as a selectable marker - use an auxotrophic yeast host that has a mutant leu2 gene (cannot
synthesise leucine so must be grown on medium containing leucine), 2 μm plasmid vector contains
LEU2 gene so transformed cells plated on minimal medium, functional LEU2 in plasmid will
complement mutant leu2 gene in host enabling growth of transformed yeast cells
...
coli and yeast, can replicate as independent plasmid in yeast, can
integrate into yeast genome via homologous recombination between LEU2 and leu2, hence
episomal, do bulk of manipulaiton in E
...

o Cloning in plants - most vectors based on the Ti plasmid of Agrobacterium tumefaciens (soil bacteria that
causes crown gall disease, tumours), transgenic plants for research and crops for food, feed and products
...
tumefaciens, of no use to plant ), A
...

o Ti vectors - Ti plasmid and T-DNA are modified to make vectors, oncogenes (genes for biosynthesis
of plant hormones which causes tumours) removed from T-DNA and replaced by selectable markers
(bacteria and plant, including to T-DNA) as well as restrictions sites and useful promoters (control
expression of introduced genes), used to transform plant cells and regenerate whole plants,
transgenes inherited in Mendelian fashion
...
9 kb, contains
three genes flanked by short inverted repeat sequences at either end
...

o Cloning in animals – to generate gene “knock outs” inactivate specific gene and observe changes in
phenotype, for “pharming” to synthesise important protein in e
...
milk, for gene therapy to study or treat
human disease
...


Clone identification
o The problem - how to identify your clone from amongst the other colonies or plaques, particularly difficult
when dealing with nucleic acids from eukaryotes (with prokaryotes it is sometimes more straightforward as
their genomes are smaller and because the cloning is carried out in E
...

o Direct selection – set up so only the desired clone can survive, selection occurs at the plating out stage (only
possible in a few circumstances), works with some bacterial genes but not eukaryotic genes because the
genes can be expressed in E
...

o E
...
you want to clone kanR gene, clone all EcoRI fragments, plate out on kanamycin agar, only kanR
recombinants can grow
o Marker rescue (variant of direct selection): use a mutant deficient in the gene you want to clone
(auxotrophic mutant), idea is the gene you insert with substitute/complement the mutation –
rescuing the genetic marker, works best for bacterial genes in metabolic pathways as enzymes are
often essential for growth on particular substrates (so without they die)
...
g
...
coli has trpA- mutation can’t synthesise tryptophan and requires it in medium to grow, so
after marker rescue only trpA+ recombinants can grow on minimal medium – complementation
...

o ‘Magic pointers’ - search the literature to see if “your” gene has already been cloned from another organism
in another lab, due to descent from a common ancestor there is some degree of sequence similarity
between the same genes from different organisms, the more closely related the organisms the better as the
sequences will be more similar, ask for a clone of the heterologous gene, perform nucleic acid hybridisation
...

o Heterologous probing – use gene from organism A to identify the same gene in a library (cDNA or
genomic) from organism B: immobilise library B on a nylon membrane and denature DNA to
separate strands, make gene from A radioactive and denature gene probe, hybridise gene A (probe)
with library B, wash off non-specifically bound probe (less than 80% complementary), expose
membrane to X-ray film and hybridising clones give black spots, pick out correct clone from library B
...

o Library screening: hybridisation - probe gene is made radioactive (32P) and denatured, hybridised to
library, wash to remove non-specifically bound probe, control stringency through temperature &
salt concentration, exposed to X-ray film (autoradiograph), hybridise duplicate membranes to
eliminate false positives (usually find spot then repeat with small surrounding area at lower
concentration to get better separation between plaques)
...

o Radioactive labelling of DNA to make a probe - generally radio-labelled using a radioactive isotope of
phosphorus (32-P) as part of the phosphate part of the base that makes up the DNA (generally alpha
phosphate as beta and gamma are lost during formation of the phosphodiester bond)
...

o Identification of a ‘new’ clone - you cannot always use someone else’s gene (becoming rarer), usually
interested in a gene because you are also interested in the corresponding protein/enzyme so purify protein
to homogeneity, use protein to provide amino acid sequence data (synthesise an oligonucleotide) or raise
antibody, both can be used to identify “your” clone
...

Oligo probing of gene library – oligos are end-labelled with γ-32P-dNTP, hybridise to library (colonies
or plaques) immobilised on nylon membrane, check with second oligo if possible (eliminates false
positives)
...

Antibody identification of cDNA clones in λgt11 – λgt11 has a unique EcoRI site in lacZ’, cDNA
inserts create hybrid genes with LacZ’ , IPTG induces expression to give fusion proteins, cDNA can be
cloned in two possible orientations and three possible reading frames so 1/6 clones will express
fusion protein in correct frame, labelled antibody will recognise the corresponding protein and thus
“your” clone
...
g
...


Polymerase chain reaction and southern hybridisation
o Polymerase chain reaciton (PCR) – requires a minimum of sequence information on DNA molecule of
interest, gives the exponential amplification of a singe gene, enables identification, amplification and
isolation of DNA of interest from a minute amount (one or two copies) of a mixed DNA population
...

o Can withstand high temperatures (~95oC) required to denature double-stranded DNA - works best at
~75oC
...

o PCR:
o Mix DNA, forward and reverse primers, Taq polymerase, buffer, and water
...

o Cool (~55oC) to anneal primers to complementary sequences
...

o Repeat cycles, ‘short’ products accumulate exponentially
...

o Typically 17-20 bp – short primers will hybridise at random due to chance so aren’t specific enough
...

o Tm – melting temperature at which the correct base-paired hybrid dissociates, controls annealing
temperature of primers to template (both should be similar), Tm = (4 x [G + C]) + (2 x [A +T]) oC
...

o T/A cloning of PCR products – PCR products often have an extra 3’ A, prevents blunt-end cloning,
can only clone in a vector with an extra 3’ T, such T/A vectors can be made or bought
...

Taq error rate – all polymerases have an error rate but can correct via proof-reading, Taq lacks proofreading so has error rate of ~1/9000 nucleotides, with 30 cycles of replication this adds up to ~1/300 bp
which can be a problem if you clone individual fragments (each clone has different mutations), there are
high-fidelity polymerases that can be used in PCR, but these have there own complications (more expensive
and harder to use)
...

o If the PCR products are cloned it is an issue as each clone contains multiple copies of a single
amplified fragment and this may not be the same as the original sequence
...

o Transfer to nylon membrane by capillary action
...

o Denature DNA and hybridise with labelled probe for gene of interest
...

Southern hybridisation and whole genomes - used to detect genetic differences via restriction fragment
length polymorphisms (RFLPs) (changes in DNA sequence that change the length of restriction fragments),
gives information on differences and changes in genes or pieces of DNA between individuals and within
populations
...

o Due to: change to restriction site; insertion or deletion between restriction sites
...

o Agarose gel is Southern blotted and hybridised with probe, hybridises to region containing gene of
interest which contains several restriction fragments
...

Genetic diversity – RFLP data can be scored for presence or absence of bands, measure of similarity
calculated, trees plotted showing similarity (can compare diversity within and between populations of same
species, if comparing different species can be used for phylogenetics)
...

o DNA sequencing: chain termination (Sanger) – exploits M13, but can also be used with plasmids and PCR
products; chemical degradation (Maxam & Gilbert); there are automated methods based on chain
termination
...

o Dideoxynucleotides – dideoxy NTPs lack 3’ hydroxyl group, once incorporated into a growing chain the
ddNTP cannot form another phosphodiester bond because no 3’ hydroxyl so the chain terminates
...

o In sequencing, also a very small amount of ddNTPs (ddATP, ddCTP, ddGTP & ddTTP), each labelled
with a different fluorescent marker
...

o End up with four families of molecules that that stopped at a particular ddNTP
...

o Location of primer determines sequence read: universal primer to vector DNA sequence flanking DNA
insert is often used, can’t sequence long bits of DNA so use universal primer to get as far as possible with
confidence then use internal primers based on sequence obtained can be synthesised, total sequence is
built up from these partial sequences, best to sequence genes in both directions for added confidence
...

o DNA fragmented (sonicated), separated on agarose gel, fragments 1
...
0 kb collected, clone library
created, end sequences of clones obtained, overlapping sequenced identified to obtain contigs, gaps
closed between contigs through directed approach involving hybridisation to identify adjacent
clones followed by sequencing
...

o Clone contig (more expensive and time consuming but better for larger genomes) – overlapping clones
identified and map generated prior to sequencing (order them)
...
g
...

o Post genomics – after sequence is available genes and their funcitons can be identified, need sophisticated
software which can learn
...

o Identifying open reading frames – 6 possible, start with initiation codon and end with stop codon in
the same reading frame so search for these with more than 100 codons to find potential genes, in
prokaryotes genes are densely packed and longer ORFs are usually genuine
...

Homology searches - BLAST searches of data bases can pick out related sequences – use genes
already sequenced to search genome for sequences that look like that gene and search all possible
reading frames, translate into amino acids in the six different reading frames then compare to
already sequenced
...
g
...


Gene analysis and expression
o Transcription and translation: genes transcribed into mRNA which is translated into protein, mRNA is
processed (introns removed, capped, polyadenylated)
...

o Cloned genes & cDNAs can be used to analyse many of these processes involved in transcription,
translation & the regulation of gene expression – relationship between a gene and its transcript
(mRNA) studied by comparing gene and cDNA sequences (introns, poly(A) addition sites), but start of
transcription has to be determined experimentally
...

o Northern blots – band intensity gives measure of abundance of transcript (mRNA), can be used to
assay changes of gene expression (over a time course, indifferent tissues, during development, in
response to an environmental cue)
...

o Control of gene expression – gene is more than just the region that encodes the mRNA, upstream or 5' of
the gene are regulatory sequences which include the promoter to which the RNA polymerase binds, some
determine the level of expression of the gene while others may determine developmental state or tissue in
which the gene is expressed, if genomic clone includes a long 5' region it is probable some regulatory
sequences have been cloned, some (e
...
RNA polymerase binding site) are quite well characterized and can
be recognised from the sequence itself, others have to be identified experimentally
...

o Identification of regulatory motifs – deletion analysis: start deleting small bits in regulatory region and see
the effect it has, indicating the function of the deleted motif (sequence that binds regulatory protein, often
has a consensus sequence which is a common pattern of bases), assumes that deletion of a motif will change
regulation of expression of cloned gene
...

o Reporter gene – readily assayable, visible phenotype, not normally present in organism, replace
genetically modified gene with reporter gene and keep the regulatory sequences being tested the
same, e
...
GUS (β-glucuronidase, blue, most widely used), LUX (luciferase, light, non-destructive),
GFP (green fluorescent protein, green fluorescence, non-destructive)
...

o Promoter deletions can be made by – removal of restriction fragment, site-specific mutagenesis
(often via PCR) to change or delete specific nucleotides
...


Biotechnology
o Culture methods:
o Batch culture in larger vessels used to produce products from microorganisms, after growth they’re
concentrated by centrifugation and the product purified from the pellet or supernatant
...

o Industrial microorganisms – a wide range of organisms are used on an industrial scale to produce useful
product, most were produced in this manner before genetic modification which has allowed expression of
genes in hosts other than that the gene was isolated from and enhancing of expression in normal hosts
...

Cephalosporamins
CephalosporiumI spp
...

Invertase
Saccharomyces cerevisiae
Alcohol
Saccharomyces cerevisiae
Glycerol
Saccharomyces cerevisiae
Butyric acid
Butyric acid bacteria
Acetone, butanol
Clostridium spp
...

o Important gene control signals: promoter – where transcription of gene starts; terminator – where
transcription stops; ribosome binding site – where ribosome attaches to mRNA; these are all
different between eukaryotes and prokaryotes so eukaryotic genes won’t work in a prokaryotic
background
...

o However, E
...

Expression vector – E
...

Different promoters - different efficiencies (strong and weak), regulatable (controlled by chemical, induction
and repression possible, useful if product is toxic to host, can control timing of expression during bacterial
growth, e
...
lacZ promoter controlled by IPTG
...

o Trp promoter – induced by 3-β-indoleacrylic acid, repressed by tryptophan
...

Protein expression in eukaryotes: yeast (Saccharomyces cerevisiae) – well characterised system, range of
useful promoters, some regulatory differences from mammals (not a problem), problem of
“hyperglycosylation” of proteins, secretion of proteins not very efficient (need active secretory mechanism)
...

Pharming (proteins from transgenic organisms)
o Transgenic animals – gene injection into somatic nucleus, nuclear transfer to oocyte, implantation
into foster mother
...


Pharmed animals – proteins have been produced in the blood of transgenic animals and the eggs of
transgenic sheep but the most successful approach has been in farm animals where the clones’ gene
is driven by the animals β-lactoglobin gene which is active in the mammary gland so the protein can
be harvested from the milk (cows produce 8,000 litres per year – 40 – 80 kg protein)
...

Recombinant pharmaceuticals – many human disorders due to imbalance of proteins (absence or
malfunction) which can be corrected by administering human protein, e
...
insulin controls blood glucose
levels and pig insulin can be used but there are slight differences so human insulin would be best but it
needs post-transcriptional processing
...
coli, clone A and B chain gene fragments into separate
inducible vectors, synthesise fusion proteins and purify, cleave off β-galactosidase segment with cyanogen
bromide which cleaves at methionine, purify A and B chains, join by disulphide bridge formation (not very
efficient), alternatively the synthesis of proinsulin alone can help form disulphide bonds followed by
proteolytic cleavage of C chain
...

Live recombinant virus vaccines – virus coat protein ligated into vaccina virus under control of vaccina
promoter, virus replicates and expresses hepatitis B protein, elicits immune response to both smallpox and
hepatitis B
...

o Demonstrated with – hepatitis B HBsAg, measles virus, respiratory syncytial virus
...

Human genetic diseases:
Disease
Symptoms
Frequency
(Births per year)
Inherited breast cancer
Cancer
1 in 300 females
Cystic fibrosis
Lung disease
1 in 2000
Huntington’s chorea
Neurodegeneration
1 in 2000
Duschenne muscular dystropy
Progressive muscle weakness
1 in 3000 males
Haemophilia A
Blood disorder
1 in 4000 males
Sickle cell anaemia
Blood disorder
1 in 10,000
Phenylketonuria
Mental retardation
1 in 12,000
β-Thalassaemia
Blood disorder
1 in 20,000
Retinoblastoma
Cancer of the eye
1 in 20,000
Identifying genetic diseases: some genetic diseases appear early (cystic fibrosis), some late (Huntingdon’s,
Alzheimer’s), many infectious diseases are now controlled (vaccines, antibiotics), increases in life expectancy
so greater %age of population now die from diseases with genetic component, gene identification may
enable identification of biochemical basis and therapies to be designed, identification of mutations permits
screening programmes (identify carriers early, counselling about probability of children inheriting
gene/disease, early identification enables treatments and precautions to reduce risk), identification of gene
necessary for possible gene therapy
...

o E
...
Breast cancer gene – women with high incidence of breast cancer, all had the same RFLP
(D17S74) on long arm of chromosome 17 (~1000 genes in the 20 Mb region), fine scale mapping of
short tandem repeats reduced target region to 600 kb (~ 60 genes), strong candidate for BRCA1
eventually identified in this region, expression profiling – expect mutant gene to express in breast
tissue
...

o Germline therapy – introduction of the correct gene into a fertilised egg and reimplantation into the
mother, if successful the correct gene is expressed in all cells and can cure genetic disease
...
g
...

▪ Cystic fibrosis – use adenovirus vectors or liposomes, inhaled into lungs and gene is taken up
into epithelial cells, has to be repeated every few weeks to be effective
...

Gene cloning in plants – addition to conventional breeding (precision breeding), gene addition (introduce a
novel gene), gene subtraction (inactivate an existing gene), many agrochemical and seed companies have
invested heavily in this area
...

▪ Engineer expression only in those parts of the plant that need protection – reduces
exposure to insect
...

Herbicides – want them to kill weeds but not crops, be harmless to animals, and to break down rapidly in
soil to harmless products
...

o Roundup Ready crops – mutant EPSP synthase that is resistant to glyphosate isolated from
bacteria, modified into a eukaryotic gene (constitutive promoter, poly (A) addition signal),
transformed into crop plant and expressed in all of plant, provides resistance to glyphosate, so
glyphosate can be applied whenever
...

o However, selective pressure of herbicide leads to glyphosate resistance in weeds so need improved
strategies
...


Plant Development – Rod Scott
Why study plant development?
o Utility – all food is derived directly or indirectly from plants, increasing production requires knowledge of
development to help plant breeders and genetic engineers improve crop plants, need to increase crop yields
as population is increasing so fast, from 1950 to 1984 the green revolution increased grain production by
250%
...
6 billion so grain production must double, neo-Malthusians
believe people are the problem
...

o Wheat and rice – contained Rht genes which are green revolution genes (natural mutant alleles),
inhibits normal stem development (elongation), so more resources available for grain filling,
introduced in mid 1960s
...

o Barriers to progress (anti-science) – genetic engineering is controversial
...

o Life cycles are descriptions of development, plant and animal life cycles have a different emphasis
...

o Cell movement and planes of division: plant cells have cell walls which cement them in place so the
cells cannot move or migrate during embryonic development (pollen is moved by other organisms),
so development of plant form is dictated by division planes and expansion of immobilised cells –
anticlinal divisions are with cell plate at right angle to surface, periclinal divisions are with cell plate
parallel to surface; altering division planes allows building of depth and in three dimensions
...

Embryogenesis:
o Pollination/fertilisation – double fertilisation event
...

Fertilised egg – embryo
...

First zygotic division: asymmetric physically and a molecular level, and mutants which block
asymmetry develop differently; produces small apical cell (embryo) and large basal cell (suspensor)
which both have different fates (what the cell will normally become), cell fate is a component of
position
...


The ABC Model for the regulation of pattern in flower
o Fate of the floral meristem (making a flower): vegetative meristem has structures coming off the side which
are destined to become leaves, the shoot meristem then receives signals to switch from vegetative to
reproductive phases of development to become the inflorescence meristem, this goes on to produce the
floral meristem which makes a shoot which makes buds which make flowers; there are four organ types in
specific positions on the meristem and the flower has a specific pattern of organs (sepal, petal, stamen,
carpel)
...

Inducing mutants in the model plant Arabidopsis thaliana: its diploid with 10 chromosomes, has
hermaphrodite flowers which simplifies the genetics as they self-fertilise (homozygosity), and makes
thousands of shapes; chemical methods or ionising radiation are used to mutate the organism, many
mutants (phenotypes) can be identified
...


o

Core features: combinatorial – A sepals, A+B petals, B+C stamens, C
carpels; antagonism – A and C are mutually antagonistic maintaintains
A and C fields (cadastral)
...


o

o
o

Genes: Agamous was the first homeotic gene identified in Arabidopsis; DNA sequencing and database
comparisons revealed 3 significant matches (DEFA in plants, SRF in mammals, and MCM1 in yeast)
...


Green Revolution Genes
o Stem length reduction and day length genes, lead to greater yields (increased harvest index), technology has
also increased yields with significant increases in irrigation
o For grain crops harvest index (HI) is ratio of harvested grain to total shoot dry matter and this can be used as
a measure of reproductive efficiency
...

o Dwarfing genes in Arabidopsis thaliana: gibberellic acid insensitive (gai) mutants are dwarf and GA
treatment doesn’t restore normal height
...

o gai (mutant allele) encodes mutant protein that cannot bind GA but retains repressor function
resulting in dwarf plants
...

o Extending the Green Revolution: basmati rice varieties are tall, have weak stems and are damaged by wind
and rain (lodging); previous attempts to reduce height using breeding failed (lost superior cooking
characteristics), so the Arabidopsis gai (mutant) gene was introduced, achieved dwarfing without disrupting
genotype leading to acceptable dwarf basmati
...

o Genetic models: large array of mutants available, for phenotypic difference; large number of
offspring, for repeat; short generation time/gestation time
...
g
...

o Genomic models: relevance to human genome (gene conservation; disease models, allow good
comparison; drug testing
...

Genetics – lots of mutants
...

Xenopus laevis
African clawed frog
External development, experimental
embryology
...

Mus musculus
Mouse
Genomics, genetic modification
...

o Cleavage: fertilisation triggers cell division; three cleavages form eight cells, which aren’t even in size
as yolk is quite hard so it’s difficult for cells to divide; cell divisions continue to occur without an

o

increase in size of the blastula so the cells become progressively smaller; blastomeres (animal pole)
divide more rapidly than the yolk cells (vegetal pole) and therefore become more numerous; no
specification of cells yet; first three cleavages are perpendicular to one another, the following are in
a radial fashion
...

30

o

Dorsal lip
o

o
o

Gastrulation: cells move into the embryo and generate the three germ layers – the ectoderm,
mesoderm, and endoderm; the blastocoel (hole inside) allows space for cells to move and as they
move they pull other cells with them, forming three germ layers and displacing the blastocoel
completely; the animal pole cells at the dorsal lip of the blastopore begin to involute causing the
future mesodermal and endodermal cells to be moved interiorly; the archeneteron (primitive gut) is
formed, displacing the blastocoel, it will eventually surround and enclose the yolk cells; the animal
pole cells on the ventral side of the blastula meanwhile envelop the vegetal pole cells by epiboly,
followed by some involution through the ventral lip of the blastopore; the head end (anterior end)
will be formed at the leading edge of the mesoderm
...

Definitions:
Blastula stage
Ball of cells
Gastrulation
Cell movements which produce germ layers
Germ layers
Ectoderm, mesoderm and endoderm
Neural plate stage
Embryo after gastrulation
Neurulation
Cell movements which roll up the neural tube
Dorsal
Back
Ventral
Belly

Animal Cloning (somatic cell nuclear transplantation)
o Method: took unfertilised frog egg, exposed to UV to disrupt DNA; took nucleus from one cell and injected
into the unfertilised egg; a blastocyst and eventually a frog formed, e
...
Dolly the sheep (not identical, some
technical differences)
...

o Lessons: DNA can be reprogrammed to allow development following differentiation, DNA is not lost during
development, gene expression must be regulated
...

o The presence, absence and combination of
different transcription factors determines what cell
type a cell forms, mutations to transcription
factors cause abnormal levels of proteins in cells
so different cell types form, can end up with
tissues and structures becoming other types
...

o One pole of the embryo acts as a signalling centre, making high levels of inductive signals at that end
of the embryo
...

Understanding an inductive signal: find the signalling centre; find the gene responsible; check where the
gene is expressed
...

o Took fertilized eggs of newts, formed a ligature (with baby’s hair), stopping cells from forming on
one side of the ligature until pressure got too much, then one cell would pop over, the ligature was
kept tight enough to make them develop separately
...

o Spemann Organiser graft: cut out cells around dorsal lip – used very thin needle with hair in the
end; transplanted cells into ventral side, this formed a two-headed conjoined embryo, joined at the
belly region (Spemann organiser is group of cells which produces signals to produce second axis
including head, organiser graft induces dorsal fate in cells which would otherwise follow ventral
fate)
...

Finding the gene responsible: over-express gene products in embryos – inject mRNA from a plasmid library
and let it develop (can induce a mutant phenotype or rescue a mutant phenotype), will be translated into
protein, resulting embryos can be checked to see what changed and if they’re normal, shows whether the
protein altered development
...

Checking where the gene is expressed: in situ hybridisation shows where the mRNA for the gene of interest
is expressed; generate RNA probe, complementary to small portion of mRNA of interested, then track; add
markers - e
...
DIG as marker, recognised by antibodies which can have molecules attached (alkaline
phosphatase enzyme attached, forms coloured precipitate with substrate, colorimetric reaction occurs
which is used to visualise the location of mRNA of interest); probe can penetrate through tissues to cells
then wash away excess, can do whole-mount or slices of tissue mounted on microscope slides
...


Drosophilia melanogaster Development (Larval stage insect)
o Lifecycle: very rapid – two weeks from egg to adult; lay fertilised eggs – fertilised inside fly
...

Gastrulation: a head region forms at the anterior end, demarked by the cephalic fold; a ventral furrow
appears on the ventral surface (a single layer of epidermal cells) and cellular movements begin to occur;
future mesodermal cells are moved into the embryo by invagination (folded back on themselves to form a
cavity), this tube of mesodermal cells then dissociate to form a layer internally
...

Central nervous system and segmentation: following invagination of future mesodermal cells, the ventral
furrow closes and ectodermal cells either side of the closed ventral furrow dissociate and start moving as
individual cells into a position between the external ectoderm and the internal mesoderm; these cells
become neuroblasts which form the nerve cord on the ventral side; they form segments which acts as the
body plan for the embryo, with larvae and adult proteins in the right place for structures to form
...

o Generate the mutants:

Males produce large number of gametes, mutate male with radiation so germ cells are mutated,
breed for F1 population, lots of offspring with different gametes (always wild type from female), if
recessive mutation then can’t see straight away
...

o Interbreed two populations for F3 (get mutated from mixing to +/-)
...

Drosophila genetics: 7 homeotic mutations (convert one structure to another)
...

o

o
o

o

o Thorax: Sex combs reduced (SCR), Antennapedia (Antp), Ultrabithorax (Ubx)
...

Mapping of the homeotic genes – found near each other in the genome: homeotic mutants are expressed in
different anterior/posterior positions, the order of expression in the embryo matches the order of the genes
on the chromosome (collinearity)

Homeotic Genes and a Transcription Factor Code
o Homeotic genes – activate genes required to specify organs and cell types (e
...
wing)
...

o Transcription factor code: body segment development within a drosophila embryo depends on which genes
are expressed
...

abdA+Ubx+Antp give no outgrowths

o Ultrabithorax (Ubx) – no Ubx in TS3, pair of legs and extra pair of wings, lose halteres
...

Similar homeobox (Hox) genes are also found in mammals – many more than in drosophila
...

o Mammalian embryos implant into the uterus/uterine horns: mice release a lot of eggs which are implanted
in the lining of the uterine horn, each has their own placenta for nutrients
...

Gastrulation: process which sets up 3 germ layers (ectoderm, mesoderm, endoderm) forming a trilaminar
embryo; starts with ingression, where cells become dissociated and move individually; signalling region,
node, at posterior position of epiblast layer signals for a primitive streak to form; cells come away from the
epiblast layer, move down the primitive groove, come into the primitive endoderm, and eventually
completely displace it, forming three germ layers; cells that move through the node become the notochord
– rod of cells forms at the end of gastrulation, acts as signalling centre for next step
...

Germ layers:
o Ectoderm: epidermis, brain and central nervous system
...

o Endoderm: epithelium lining of the gastrointestinal tract, epithelium lining of respiratory tract, liver,
lungs, pancreas
...

o Form a blastocyst prior to implantation
...

o Gastrulation occurs forming three germ layers: ectoderm, mesoderm, endoderm
...

o Germ layers eventually give rise to all the tissues and organs through the process of organogenesis
...

Differentiation – process of unspecified cells becoming specified
...


Genetic Manipulation (of the Mouse) – Reverse Genetics
o Development is similar in all mammals – features unique to mammals must be studied in mammals to be
more clinically relevant
...

o Can’t use experimental embryology: embryos smaller, development inside mother
...

o Method of overexpressing a gene product in mouse (transgenics):
o Clone gene you want expressed into vector with marker gene
...

o Held in place by holding pipette, DNA injected – easier into male pronuclei as they’re bigger
...

o Birth 19-20 days later
...

o Method of overexpressing a gene product in Xenopus: inject mRNA into embryo, lots of protein is then
made, the mRNA is gradually degraded
...

o Make a targeting vector/construct (plasmids made up of pieces of DNA stitched together:
homologous arms flanking gene, positive selectable marker, negative selective marker, reporter
gene) and purify from bacterial plasmid
...

o Implant recombinant embryos into a pseudopregnant surrogate mother, into uterine horns as at the
blastocyst stage
...

Pdx1 – example of a knockout: Pdx1 is a transcription factor, knockout has no pancreas

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