Lecture One

DNA and Genomes

• The central dogma

  • DNA. RNA, Amino Acid, Protein,

• 1869- Friedrich Miescher,

  • CHaracterises pus.

• 1871- Pal Plosz

  • Redblood cells of birds and reptiles.

• Discovery of nuclein,

• 1909- Phoebus Levene

  • Determined the bases of Nucleic Acids and nucleotide structure.

• 4 requirements for genetic material

  • Genetic Code

  • Replication- Heritable

  • Mutation/variation

  • Gene function.

• Aat the time they doubted that it was heridatry/

Transformation

• Specific to bacteria,

• Dna is taken up by bacterial cells.

• Plasmid is circular to give stability.

• bacteria + HEat shock + Calcium chloride = competent bacteria, they then take up the plasmid.

Variants of streptococcus pneumoniae

• Griffiths Experiments

  • Virulent vs avirulentt

  • Virulent has a polysaccharide coat which makes it able to CAUSE disease.

  • He infected mice with both strains then looked at the tissues after death and was able to isolate the bacteria.

  • Boiled s straun and living r strain caused mouse to die, the living s strian was found in the mouse.m

  • He concluded thast a transforming principle, from the dead S bacteria converted some of the R bacteria into S bacteria.

Avery-Macleod-Mcarty

• Treated the dead s strain with enzymes

• Then introduces live r strain to look for transformed s strain.

• When dnase was added there was no chang.e

Erwin Chargaff- 1947

Hershey and Chase- 1952

• Used bacteriophages

Genomics

• Entire genome, all of the genes encoded.

Denaturation

• HEating the DNA in solution breaks apart theDA strands.

• Treating it with very high or very low pH.

• G-C and A-T separate with different amounts of force.

C-Value Paradox

• Amount of DNA in a haploid genome does not correspond strongly to the complexity of the organism.

• C-value is extremely variable.

• Much of SDNA is often repetitive sequences if varying length.

miRNA- regulates gnee expression at mRNA.

MAKE A GENETICS TIMELINE- 1959- Current DAY.

Key dates and key experiments.

Prokaryotic Genomes

Viruses

Structure and Function

• Viruses are smaller and simpler.

• They are essentially genes wrapped in a protein coat.

• They lack internal structures and machinery to replicate.

• They have 3 components

  • Nucleic Acid

  • Protein SHell

  • Envelopes

    • Only some viruses have an envelope

• They are not strictly alive as they are only able to reproduce and replicate when inside the host cell.

Replicative cycle of viruses

• Bacteriophages go through 2 different replicative cycles.

  • Lytic Cycle

  1. New Phage DNA and proteins are synthesised and self-assembled into phages.

  2. The cell lyses and releases phages.

  3. The phage will attach to a host cell and injects its DNA,

  4. Phage DNA circularises.

  5. Cycle continues.

• Lysogenic Cycle

1. Phage DNA integrates into the bacterial chromosome becoming a prophage with the use of integrase.

2. The bacterium reproduces normally, copying the phorphage abd transmitting it to daughter cells.

• Animal Cells

  • Retroviruses are important because HIV and Coronavirus are retroviruses.

  • Their genome consists of single-stranded RNA,

  • Reverse transcriptase and integrase create double-stranded DNA and insert it into the host genome.

Baltimore Classification of Viral Genomes

• Viral Genomes are divided into groups I through VII.

• Viruses are either; DNA or RNA

  • Linear or Circular

Single Stranded or DOuble Stranded

• Group I

  • DNA, Double Stranded, e.g Herpesvirus

• Group II

  • DNA +, Single Stranded e.g Bacteriophage M13

• Group III

  • RNA, Double Stranded e.g Reovirus

• Group IV

  • RNA (+), Single Stranded, e.g Poliovirus

• Group V

  • RNA (-), Single Stranded, e.g Influenza

• Group VI

  • RNA (+), Single Stranded, goes through reverse transcriptase to make a DNA intermediate, E.g HIV.

• Group VII

  • DNA (=/-), goes through intermediate. E.g hepatitis B.

Prokaryotes

• Prokaryotes are single celled organisms belonging to the domains bacteria and archae.

• Prokaryotic cells are much smaller than eukaryotic cells, have no nucleus and lack organelles.

• They come in different shapes such as spherical, rod shaped and spiral shaped.

• They are either Gram-Nrgative or Gram-positive

• Gram positive

  • They have a peptidoglycan cell wall, and stain a dark purple

• Gram Negative

  • They have a thinner peptidoglycan cell wall and a LPS membrane, they strain a light pink.

• Some prokaryotes also have capsules and/or appendages.

  • Fimbrae- helps cells stick to surfaces and objects in the environment

  • Pili- Locomotion and conjugation

  • Flagella - tail like structures aiding in mobility.

• Bacterial Genomes

  • Prokaryotic genomes lack complex internal organisation, they have considerably less DNA than eukaryotes.

  • The main chromosome is arranged in a dense clump known as the nucleoid.

  • Plasmids may carry beneficial genes for survival, such as antibiotic resistance, and so they can spread through populations via horizontal gene transfer.

  • The chromosome of E.cOli is several orders of magnitude larger than the cell itself, this is due to supercoiling allowing for DNA to be highly condensed.

  • Topoisomerases control the amount of DNA supercoiling,

  • DNA breaks cause the spontaneous untwisiting which relaxes the DNA as it has a dynamic structure.

  • The twisted loops of DNA are often anchored by a protein core.

• We can map genes (find the physical distance between any two loci) using genetic material that is transferred between bacteria.

• • This can happen in one of three ways:

  • • Conjugation

  • • Transformation

  • • Transduction

• Conjugation

  • In some bacterial cells, the plasmid can become integrated into the chromosome.

  • F plasmid integrates into the chromosome by recombination

  • Cells join via a pilus

  • A portion of F plasmid partially moves into recipient cell trailing a strand of donor DNA.

  • Conjugation ends with pieces of F plasmid and donor DNA in recipient cells, cells synthesise complementary DNA strands.

  • Donor DNA and recipient DNA recombine making a recombinant F cell.

• Conjugation allows for genetic mapping, Stopping the transfer of donor genes on limited nutrient agar mediums, allows us to determine the order of genes on the donor chromosomes.

• Bacterial Transformation

  • Unidirectional transfer of extracellular DNA by donors into recipient cells.

  • Can be used to determine if the genes are physically linked by distance to determine the distance to other genes and to determine the order of genes,

  • Recombination frequencies allows us to determine if genes are are close to each other as only genes within certain distances of each other will cross over.

Regulation of gene expression in bacteria

• Genetic regulation

  • Genes whose activity is dynamically controlled by the needs of the cell or organism

• Housekeeping genes

  • Genes that are always active in a cell or organism and are essential to its functioning,

• Inducable gene

  • A gene whose expression is turned on by adding a substance such as lactose.

• Genes that encode proteins which work together in the cell are organised into operons

  • This is a way of controlling multiple genes at once, they are adjacent to each other and are transcribed together.

Structure of Operons

• They have 3 regulatory regions

  • Promotor sequence

  • Operator sequence

  • Regulatory sequence

Mitosis & Meiosis

Cell Division

• All multicellular organisms develop from a single cell i,e the fertilised egg.

• Repeated division of the zygote by a process of cell division by mitosis. These give rise to all cells that make up a multicellular organism.

• The function of mitosis is to produce new cells, each of which receives a set of chromosomes identical to those of the original cell that gave rise to them; genetically identical cells are the result of mitosis.

• Mosacism/Chimeric - Where early on in embryonic development, a mutation occurs, a subset of chromosomes have the mutation but not all of the cell.

Cell Cycle

• Mitosis is Only a small part of the cell cycle.

• Occurs after G2 of Interphase is completed.

Mitosis

• Interphase

  • The cell carries out its normal functions but also prepares to fivifde

  • Chromosomes aer not visible under a light microscop

  • DNa unravels and is replicates

  • Organelles are also replicated.

  • Centrioles are tubular structures associated with nucleus, containing microtubules

• Prophase

  • CHromarin in the nucleus begins to condense and vecmoem visible in the light microscope

  • The chromatin starts to wind together and condense seeing thick chromosomes.

  • These consist of sister chromatids which are joined at the centromere.

  • The nucleus’ membrane breaks down.

  • Centrioles migrate to opposite sides of the nucleus

  • Microtubules attach to the chromatids at places called the kinetochores/

  • The nuclear membrane breaks down.

• Metaphase

  • Microtubules align the chromosomes along the equator of the cell/

  • The sister chromatids attach to these via kinetochores.

  • This helps the chromosomes are separated each nucleus will receive one copy of each chromosome.

• Anaphase

  • THe sister chromatids are pulled by the microtubules attached by the kinetochores.

  • The sister chromatids separate at the centromeres and move to opposite sides of the cell.

  • Ensure each daughter cell has one copy of each chromatid.

• Telophase

  • Chromatrids arrive at opposite poles of the cell and new nuclear membranes begin to reform arounf each centrosome.

  • 2 identical nuclei have been made

  • The chromosomes disperse and are no longer visible under the light microscope/

  • A new cell membrane begins to form.

• Cytokinesis

  • Cleavage furrow and actin pinches the new cell and new membrane is formed.

  • Each half of the cell breaks off from each other incurring that a copy has been made.

  • The DNA unwinds back into its chromatin form

  • Each cell then returns to interphase and the process starts again.

Mitosis and cytokineses produce two genetically identical daughter cells

Centromere-

Centrosome-

Asexual reproduction in eukaryotes

• Budding forms a new organism from a small projection growing on the surface of the parent.

• This is not a good thing as it does not allow for variation.

• Fragmentation is the splitting og the parent into a piece that each grow into a new organism. E.g starfish reproduce do this. Able to regenerate parts of the organism\

• Vegetative reproduction.

  • Forms a new plant from the modification of a stem or underground structure on the parent plant.

• Telomeres shorten each time they reproduce, this causes aging/

Meiosis

• 2 major differences

  • Homologus chromosomes, creates 2 non identical daughter nuclei via recombination

  • 2 daughter nuclei divide creatinf 4 unique gametes.

Stages

• Meiosis I & Meiosis II

• Interphase

  • Chromosomes duplicated

• Prophase I

  • Recombination

• MEtaphase I

• Anaphase I

• Telophase I

• Cytikineses

  • 2 non identical diploid cells/

• Prophase II

  • No Chromosome duplication

• Metaphase II

• Anaphase II

• Telophase II

• Cytokineses

  • 4 Haploid cells form

Recombination

• Increases genetic variation in DNA between individuals of the same species

• Prophase I & Recombination

• Homologous pairs find each other and form a tetrad

• Crossing over and recombination occurs

• REcombination multiplies the already huge number of different gamete types produced by independent assortment.

• This increases variation

• In the first division of meiosis the homologous chromosomes associate

• Chiasma- the point at which chromosomes cross over.

• Recombination between non-sister chromatids

  • Results in translocation

  • Balanced or Non-Balanced.

  • Can contribute to diseases

  • Monosmy & Trisomy syndromes.

  • Philadelphia Chromosome - 9:22,

    • Translocation between chromosomes 9 and 22, over 95% of people with myeloid leukaemia also have this chromosome recombination.

    • Is a balanced translocation gene.

  • Robertsonian Translocation

    • AN unbalanced translocation.

    • REsults from a breakage of 2 acrocentri chromosomes close to the centromeres. (13, 14.15., 21 and 22)

      • Very repetitive and similar to each other.

    • The two chromosomes fuse together to form a recombinant chromosome. This means they often lose a fragment of the chromosome as the centromere is also lost.

    • Example (14;21). If inherited in conjunction with Normal Chromosome complement the zygote will have trisomy 21, Down Syndrome leading to trisomy 13; Patau syndrome.

Random Alignment

• During metaphase I, the chromosomes can line up in any order.

• This means that each daughter cell can end up with a different combination of chromosomes and therefore genes,

• This explains why features come from both parents.

Independent Assesment

• Look Up

Nondisjunction

• When chromosomes are not separated equally during meiosis.

• One gamete will have an additional chromosome, the other will have none.

• At fertilisation, one will be trisomic and one will be monosomic.

• Only Trisomy 21, 13 and 18 are the only ones compatible with life.

  • Thry are the smallest one, only a few genes are present, so there is little extra genomic information.

Trisomy is not Triploidy

• Trisomy is 3 copies of the single chromosome

• Triploidy is 3 copies of the entire genome.

• A triploid fetus’ rarely survives to birth.

• Though to affect 1-3% of pregnancies.

• A result of 2 sperm fertilising the egg at the same time, or diploid sperm or ovum.

• Increases with maternal age.

Binary Fission vs Mitosis

Eukaryotic Genomes

Bw ux hw

The Nucleus

• The nucleus oversees the cells functions and regulatory mechanisms

• Controls cell growth via synthesis of structural proteins, energy and nutrient metabolism.

• Regulates the secretion of ribosomes.

• Is the site of gene transcription.

Nuclear Envelope

• A nuclear envelope is a phospholipid bilayer with nuclear pores.

• These pores control what enters and leaves the nucleus.

• They are selective of controlling the movement of RNA, ribosomes proteins between nucleus and cytoplas,.

• Membrane functions to keep the chromosomes contained.

Nucleolus

• HAs no membrane

• Site of protein synthesis.

Nucleoplasm

• Thick fluid containing chromatin and nucleolus.

• Maintains the shape of the nucleus.

• Complex mixture of proteins , enzymes ions

• COntains high free nucleotides for DNA and RNA synthesis.

Chromatin

• State that chromosomes are in when in the nucleus.

• Heterochromatin vs Euchromatin

  • Heterochromatin

    • Densley packed regions of chromatin which stains dark.

    • Transcriptionally Inactive

  • Euchromatin

    • Loosley packed regions of chromatin which stains light

    • Transcriptionally active chromatin.

Chromosome Staining

• During prophase chromatin condenses to form chromosomes.

• They are most easily seen during metaphase.

• To stain we;

  • Halt cell cycle

  • Stain with Giemsa

  • Drop cells onto slide, burst nucleus

  • Giesma stains dark in AT rich regions

  • Single G band is several million base pairs of DNA.

  • THis produces a Karyotype.

Chromosome Classification

• Metacentric

  • Centromeere is in the middle of the chromosome

• Submetacentric

  • Between Middle and End.

• Acrocentric

  • Close to the end

• Telocentric

  • At the End

  • Humans do not have any telocentric chromosomes.

Vectors and Molecular Cloning

DNA Cloning

• Massive amplification of DNA sequences.

• Stable propagation of DNA sequences.

• A single DNA molecule can be amplified allowing it to be

  • Studied- Sequenced.

  • Manipuated- Mutagenised or Engineered.

  • Expressed- Generations of Proteins.

• Cloning is the first stage in any genetic engineering project.

Vectors

• Vectors are needed to carry the DNA into a new organism.

• They must have the following properties

  • Ability to promote autonomous replication inside a host cell. (Ori)

  • Contains a genetic marker for selection (usually a dominant gene).

  • Unique restriction sites to facilitate cloning of insert DNA.

  • Minimum amount of non-essential DNA to optimise cloning.

• Vectors are of different types depending on the host

  • Bacterial Vectors

  • Yeast Vectors

  • Plant Vectors.

  • Animal vectors.

• There are various cloning vector systems.

  • Bacertiophages.

  • Bacterial plasmids.

  • Cosmids.

  • Bacterial/Phage/Yeast Artificial Chromosomes.

  • Ti plasmid.

  • Eukaryotic viruses

    • Baculovirus

    • SV40 virus

    • Retroviruses.

• Vectors allows the introduction of foreign genes into a host cell.

  • Host cell systems trend to be well-characterised model organisms.

    • Most commonly used host system is E-Coli

    • Yeast used for propagating genomic DNA libraries for expressing eukaryotic genes, mostly used for post-translational modifications.

    • Mammalian cells or insect cells are used for expressing eukaryotic genes,

    • Plant cells for expressing plant genes.

Bacterial Vectors

• E. coli is the most commonly used bacterium for gene cloning, though others are used.

Cloning Strategy

• There are 5 steps to cloning

1. Enzyme restriction digest of DNA

2. Enzyme restriction digest of Vector.

3. Ligation of DNA into the vector.

4. Transformation to the host cell/

5. Growth on media selecting for the resistance marker.

• DNA digestion

  • Using restriction endonucleases enzymes.

  • These enzymes were discovered in bacteria.

  • They help the bacteria destroy viral DNA.

  • They cut between specific bases of the double stranded DNA molecule

  • The DNA is then cut into pieces.

• Restriction enzyme sites

  • Specific restriction enzymes cut at specific DNA sequences to leave sticky ends.

  • Results in fragments of DNA called REstriction fragments.

• The “sticky ends”

  • When you cut two separate molecules of DNA with the same restriction enzyme the fragments will have matching sticky ends.

  • This is how recombinant DNA is created.

• Alkaline phosphates may be added prior to ligases. This digests 5’ phosphate and stops the plasmid from sticking together without an insert.

Plasmids

• Souble stranded circular DNA

• Relatively small/

• Self replicating, having a single origin of replication.

• Plasmids give an advantage to the host cell.

• For cloning purposes, usually, plasmids that contain an antibiotic resistance genes are used.

How to transform E Coli

• Physical-Electroporation

  • Creates an electrical pulse across the media containing the host cell and the plasmid.

  • Disrupts the phospholipid bilayer.

  • Uses electrodes in the solution.

  • REassembles spontaneously and will engulf any additional DNA in close proximity.

  • This tends to have a higher death rate.

• Chemical- CaCl2

  • The CaCl2 binds to cell walls neutralising negative charges disrupting the membrane.

  • Mixes with plasmid and heat shock at 42 degrees to create pores in cell membrane.

  • Return to 37 degrees and any plasmid in close proximity is engulfed into the host cell.

Phage (Insert Lambda symbol) is a temperate phage,

• The Lambda genome is 45-48.5 kilobase pairs.

• Of which some 15kbp or so is optional

  • Contains genes that are only needed for integration of the phage DNA into the E Coli Chromosoems.

• These segments can therefore be deleted without impairing the ability of the phage to infect bacteria and direct synthesis of new lambda particles by the lytic cycle.

By using Bam H1.

Directional Cloning for expression

• Often we want to insert foreign DNA in a particular orientation.

• This can be done by making two cleavages with two different restriction enzymes.

• Digest foreign DNA with the same two restriction enzymes.

• Foreign DNA can only be inserted in one direction.

• This ensure the sticky ends dont match so that they have to go into the plasmid the correct way.

COsmid Vector

• Clone Large inserts of DNA, around 45 kilobase pairs.

• COsmids are plasmids with one or two Lambda cos sites.

• Presence of the COS site permits in vitro packaging of cosmid DNA into Lambda particles.

• Strong selection for cloning of large inserts.

• Infection process rather than transformation for entry of chimeric DNA into E.COli host.

• Maintained in the host as a plasmid.

Artificial Chromosomes

• Both use E.Coli has the host.

• 100-300kb.

• Has a low copy number.

• Induce the lytic phase to increase DNA yield.

• Large insert size allows for some entire genes and control regions to be clones as one.

• Useful in the generation of genome libraries and sequencing projects.

Yeast Artificial Chromsoeomes.

• Autonomously replicating sequences necessary for replication.

• Telomeres

• A yeast centromere.

• Selectable markers that allow the easy isolation of yeast cell that have taken up the artificial chromosomes.

• Unique RE sites.

Expression Vectors

Phages are the vectors that cause plaques.

Cloning and Genetic Engineering 2

Expression Plasmids

• Expression plasmids are a great way to shuttle genes and their products between organisms.

Artificial Selection

• Selective Breeding/Hybridisation

• In-breeding.

• Breeders choose which organism to mate to produce offspring with desired traits.

• They cannot control what genes are passed.

• When they get offspring with the desired traits, they maintain the,

• Artificial Selection is NOT natural selection.

Mendel

Genotype-The types of alleles present

Phenotyoe-What the organism Looks like

Allele- Different forms of a characteristic

Homozygous - Two of the same alleles

Probability- Percentage chance that something occurs

Heterozygous- Two different alleles

Dominant- Traits that may be expressed

Recessive- Traits that may be hidden.

Punnet Square- Shows how crosses are made.

Mendels 1st Law of Dominance-

Hybrid offspring will only express the dominant trait in the phenotype.

The allele’s that are suppressed are called the recessive traits while the alleles that determine the trait are known as the dominant traits.

Mendels 3rd Law of segregation-

States that during the production of gametes two copies of each allele segregate so that offspring acquire one factor from each parent.

Law of Segregation- The two members of a gene pair segregate from each other in the formation of gametes. Half of the gametes carry one allele and the other half carry the other allele.

Punnet Squares (Alevel shit)

Homozygous F1 Mating from Homozygotes

Monogenic disorders

• Single gene disorders

Cystic Fibrosis

• A recessive mutation.

• 1 in 20 Caucasians carry the recessive gene.

• 6 Classes of CF dependent on the type of proteins the mutation affects.

• Compound heterozygots. Two different diseased alleles.

• Autosomal recessive

Huntintgtons

• Autosomanal Do,inant

• Single gene dominant disordar due to expansion of CAG repeat.

• Gene falls off chromosome and repeats are added into other parts of the chromosome.

• Affects offspring.

• Increases with generations causing ‘anticipation’

Mendels 2nd Law- Law of independent assortment.

• A pair of traits segregate independently of another pair during gamete.

• AS the individual heredity factors assort independently different traits get

• Only occurs on traits on different chromosomes.

Tall + Purple = 9

Tall + White = 3

Short + Purple= 3

Short + White = 1

Incomplete Dominance

• Genes show incomplete dominance when the heterozygous phenotype is intermediate.

• Dominant Allele’s expression is dampened.

Rr x Rr

= 1:2:1

Rr x rr

= 1:1

RR x Rr

= 1:1

Sex Linked Genes

Prokaryotic Transcription and Translation

Protein Synthesis requires 3 types of RNA;

• mRNA

  • Encodes proteins

• tRNA

  • Acts as adaptor between mRNA and amino acids.

• rRNA

  • Forms the ribosome.

Protein synthesis occurs in 2 major steps and requires 3 major types of RNAs;

• miRNA= regulated gene expression

• siRNA- silences gene expression.

• IncRNA - REgulates gene expression.

These processes are highly regulated. Not all genes are expressed at the same time.

• Consituitevely expressed genes are switched on all the time, a.k.a housekeeping genes. E.g Actin genes.

• Non-constiuitively expressed genes are switched on as and when needed.

Cells begin variable but then eventually become unipotent and cannot differentiate.

Transcription

• The goal; to synthesise a chain of RNAs (mRNA) via RNA synthesis.

• There are three stages

  • Initiation

  • Elongation

  • Termination.

• Transcriptional Initiation in prokaryotes.

  • RNA polymerase is the main transcription unit.

  • Transcription begins when RNApol binds to the promoter.

  • The association of sigma factor dramatically increases binding affinity and specificity.

  • Most important part, allows specific sites and genes to be transcribed.

  • The binding of RNA polymerase holoenzyme to form a closed complex/

  • The affinity of the RNA polymerase to the promoter represents how much the gene will be expressed.

• RNA Polymerase in Ecoli

  • Consists of two alpha subunits and two beta subunity

  • Sigma factor

    • Upstream and Downstream

  • The sigma factor gives the enzyme specificity, and is now called the holoenzyme.

  • Sigma factor allows the polymerase to get the correct start point.

• Prokaryotic promotors have two characteristic sequences.

  • TTGACA ~35 bases, before the transcription starting point

  • TATAAT ~10 bases before transcription starting point.

  • These are called consensus sequences because they must be similar in all promoters.

  • Sigma factor is essential for recognising the -35 and -10 regios/

  • Each sigma factor has its own factor that it recognises.

• Holoenzyme binding steps .

  • Holoenzyme binds loosely to -35 region in a closed complex.

  • The binds tightly to the -10 and the DNA unwinds forming the open complex.

  • RNA polymerase reoritnetatoes correctly, sigma factor is released and transcription can proceed at the next nucleotide.

• Sigma Factor Heterogenity

  • Not all sigma factors are the same.

  • They all recognise different consensus sequences.

  • E.g sigma factor 70, has two consensus sequences for -35 and -10, but sigma factor 54, regonises -24 and -12/

  • Sigma factor 54 requires ATP for stability.

  • 

  • This is specificity and regulation to be dynamic.

  • SIgma factors need to be regulated.

  • Regulation is achieved by anti-sigma proteins.

  • Typically consist of a sigma-binding domain and a sensory/signalling domain that responds to na outside signal either within or outside of the cell.

  • Stabilisint the sigma-facto by binding to the occluding RNApol recognition sequence.

  • Outer membrane proteins sense stress and activate the proteases DegS via C-terminal domain binding to PDZ resulting in site 1 cleavage.

  • RseP senses site-1 cleavage and cleaves site 2 erleasing a soluble sigmaE/RseA complex.

  • Proteases degrade RseA and sigma is released into cytoplasm.

• Elongation

  • RNA polymerase unwinds at the -10 box as it is rich in adenine and thymine.

  • This becomes the transcription bubble which moves along with the holoenzyme.

  • Rna polymerase moves from 3’ to 5’ direction and synthesis RNA.

  • mRNA is the product of transcription.

    • Its purpose is to encode information from chromosomes .

    • It tells the ribosomes which amino acids to string together and in which order/

    • Prokaryotic mRNA is polycistronic.

    • Information is encoded on mRNA in three letter words called codons. EAch codon codes for an amino aicd.

    • Coding regions start with a Start codon (AUG),, and end with a stop codon (UAA).

• Termination

  • Termination refers to the dissociation of the RNA polymerase holoenzyme complex from the mRNA.

  • Signalled by the terminator.

  • Terminators form hydrogen bonds between the base pairs of the ssRNA creating a hairpin loop structure.

    • Terminator region

      • GC rich area - complementary to the first GC region, so they come together.

      • This causes the RNA polymerase to stalls/

      • Trnascitption is terminated.

    • Poly U site.

      • A-U weakly bind to each other. Allows tracnript to be released from the DNA.

    • Rho-Dependant termination

      • Terminator contains as sequence encodes a Rho-binding site.

      • The RHO binds to mRNA and moves along the molecule until it reaches RNA polymerase.

      • Rho has helicase activity so it ids through it promotes RNA polymerase dissociation by unwinding the DNA double helix upstream

Translation

• Synthesis of polypeptide chians from single mino acids.

• The ribosome is the main translational unit thay synthesis the polypeprode. This process is directe d ny the information encoded on the mRNA.

• It requires mRNA, ribosomes, tRNA and amino aicds as well as other helper proteins.

• Initiation of Translation

  • The ribosome is protein synthesiseing machine.

  • Comprised of rRNA and ribosomal proteins and organised into large and small subunitys.

  • Several initiation facto proteins deliver the small subunit to the mRNA,

  • Prokaryotic mRNA contains a SHine-Delgarno sequence which encodes a ribosomal recognition and binding site.

• Elongation

  • After initiation, the ribosome begins to read along the mRNA strand building a protein one amino acid at a time.

  • Ribosomes have 3 active sites

    • A Site - Acceptor

    • P Site - peptide site, where the peptide is bound.

    • E site - exit site, where the proteins are released.

  • Other proteins also help this process along. Requires GTP

• Transfer RNA

  • Trnsfers the amino aicd to the growing peptide.

  • A d’ amino aic attachment site which binds to the amino acid.

  • An anticofon wjich recognises the codon on the mRNA.

  • Specific to specific amino acids.

• Aminoacyl-tRNA synthetases

  • These match the correct amino acid with the correct tRNA.

  • There is an aminoacyl-tRNA synthestease and a tRNA specific for each amino acid.

  • Needs ATP.

  • Amonoacyl-tRNA synthetase are enzymes which cataluse the attachment of the correct amino aicd to the correct tRNA.

  • Two Step Decoding process.

    • Linkage of AMino acid to its tRNA.

    • Requires ATP.

    • Then the matching of the anticodon to the codon.

• Protein folding and polysomes.

  • Once the ribosome has moved away from the initiation site., another will bind.

  • Typically several ribsosomes are translating a single mRNA at a given time.

  • AS the peptide chain grows it folds giving the protein its 3D structure.

  • 3D shape is stabilised by;

    • Disulfide bridges

    • Hydrogen bonds

    • Ionic bonds.

    • Hydrophobic interactions.

• Termination

  • When a stop codon is encountered, release factors recognise this codon and bind it to the A site.

  • This forces the ribsosome to release the finished polypeptide sequence from the P site.

  • This occurs in 3 steps

    • REcognition if the stop codon

    • Hydrolysis of the ester bond of the peptidyl-tRNA.

    • Dissociation of Release factors (RF1 & 2)with the help of release factor 3 (RF3)

• TRanscription and translation are coupled.

• REgulation of gene expression

  • Bacteria can regulate the expression of genes in response to external stimuli.

  • This is achieved through two component regulator systems which transduce signals from outside of the cell to the nucleoid.

  • Cell signals from outside the cell cause a signalling cascade.

  • Basic form of TCR

    • Membrnane bound sensor kinases.

    • DNA-bidning response regulator.

• The lac Operon

  • ABcteria can regulate gene expression in response to environmental signals,

  • One such signal they can respond to is the prescnece of substances that can be utilised for survival.

  • They need a source of carbon like glucose to survuve.

  • A useful adaptation is to be able to metabolise them only when present in your environment.

  • E.coli have a set of lactose metabolising genes that are only induced in the presence of lactose.

  • Z, y and a

  • LacY (permease, is required for the movement of lactose into the cell via transmembrane protein transport.

  • LacZ turns lactose into glucose and galactose.

  • LacA transfers an acetyl group from coenzyme A to the hydroxyl group of galactosidase.

• Induction of the lac operon.

  • In the absence of lactose, the regulator protein binds to the operator and inhibits transcription

  • In the presence of lactose, some of it is converted into allolactose which then binds to the regulator protein, making the protein inactive.

  • The regulator protein cannot bind to the operator and the structural genes are transcribed and translated.

• Positive regulation of the lac operon.

  • Ehn glucose is low cAMP is high, this represses the metabolite activator protein. This enhances transctopiron and repressor will not bind ot the DNA.

• Catabolite repression of lac operon

  • When glucose os present at the same levels as lactose glucose is used preferentially.

  • Glucose greatly reduces the amount of cAMP available in thec cell.

  • RNA polymerase cannot bind to promoter effectively/

Population Genetics

• Human evolution has been underlain by adaptive and non-adaptive changes in allele frequencies.

• Diseases are commonly due to effects of alleles and alleles interacting with the environment, there is a spectrum from single-locus disorders to polygenic disorders.

• Fucking hardy weinberg

• Population - Group of interbreeding individuals of the same species sharing a common geographical area.

• Species - Group of populations tha thave the potential to interbreed in nature and produce viable offspring.

• Gene Pool- The sum total of all the alleles within a population.

• Different populations of the same species can have different frequencies of alleles in their gene pool.

• Immigration + Births = Emigration + Deaths.

• Genetic Drift

  • Ina large population allele frequencies remain relatively constant.

  • In a small population, one allele may become extinct in a few generations.

• Factors affecting allele frequence in a gene pool

  • Evolution

  • Natural Selection

  • Migration

  • Mutation

  • Genetic Drift

    • Founder Effect

    • Bottleneck effect.

• Allele Frequency = number of alleles/total number of alleles.

• EMigration reduces genetic diversity, this may remove alleles from a population reducing a populations genetic diversity.

• Immigration increases genetic diversity - this may add new alleles to a population increasing a populations genetic diversity.

• Mutations

  • Small mutations creep into populations over time. Some are helpful, some harmful, many neutral may be beneficial or harmful later.

  • Mutations increase genetic variation in a population.

• Genetic Drift - Non random mating

  • Many living species have ways to compete for mayes, In some populations only a few individuals can mate.

  • Mate preference is non random, many animals will prefer mates that resemble themselves.

• FOunder Effect.

  • Creates a smaller population with different gene rations than the population it derives from - no effect on the original population.

  • Common in small populationes e.g Amish

• Bottle Neck

  • Catastrophic event reduces the population size, decreases genetic diversity in later generations-drastic effect on original population.

• Island of COlourblinf

  • Island which has a high frequency of achromatopsia.

  • Autosomal recessive trait.

  • A typhoon reduced the islands population drastically.

  • Geographical isolation meant inbreeding over subsequent generations which increase the frequency of the recessive allele.

• Mutation, migration, natural selection and genetic drift causes changes in allele frequencies.

• Selection

  • Selection occurs when a trait or set of traits gives a survival advantage or a disadvangtahe.

  • Traits increase in a population if they cause individuals to have more offspring. Traits decrease in a population if they kill off ondividuals before they can reproduce

  • Directional Selection-

  • Stabilising Selection-

  • Disruptive Selection-

• The essence o population genetics,

  • A gene by itself is a constant entity.

  • Alternative forms of a gene can exist at certain frequencies in a population

  • These frequencies can change via a genetic drift, selection mutation resulting in adaptation and evolution.

• Combination of Darwin, MEndel and AMthermatical population genetics (hardy Weinberg)

Hardy Weinberg.

• Used to estimate frequencies of genotypes and alleles for a single genetic locus which only has two alleles.

Must be;

• Able to mate at random.

• Stable environment.

• Large population.

• No migration.

• No mutation.

• No difference in allele frequencies between genders.

• 2 Alleles.

AA=pxp

Aa=2Pq

aa=qxq

p^2 + 2pq + q^2 = 1

Polygenic Traits & multifactorial Inheritance

• Non-mendelian

• A result of a cumulation of many genes.

• They display complex inheritance patterns, the offspring may be quite different to the parental phenotypes.

• They are measured on a continuous scale.

• Number of genotypes is = 3^n

• N= number of genes involved in the expression.

• More genes = more genotypes are expected.

Pollygneic inheritance of skin tone.

• Skin pigmentation is synthesised in ,elanocytes. Specialised melanin producing cells in the bottom layer of the skins epidermis.

• Differing skin tone is the result of melanin distribution rather than melanocyte numbers.

• Melanin production is dynamic and is triggered by exposure to UV.

• Melanin occuts in two for,s, eumelamin (dark-black insoluble polymer) and pheomelanin, (light red-yellow sulphur containing soluble polymer.

  • UV radiation stimulates POMC production from keratinocytes-converted to a-MSG.

  • a-MSH binds to MC1-R and activates cAMP pathway.

  • cAMP pathway activqtion positively regulates the MITF transcription factor.

  • MITF transvribes TYR, DPC and TYRP1 which are melanocyte specific proteins.

Multiple Alleles & Skin Pigmentation

• MC1-R has 15 different allelic forms, and each has different effects on skin pigmentation.

• Other genes involved with melanin synthesis has 9 transcript variants.

Ldlodm

Epigenetics

The Epigenetic landscape

• Waddington defines it as

  • A branch of biology that studies how genes and their products bring the phenotype into being.

  • The form which occurs in nature under the influence of natural selection is much less variable in appearance than the majority of the mutant races.

• CHemicla tags to DNA that regulate gene expression. - Modern application

Phenotypic Plasticity

• The ability of a single genotype to produce multiple phenotypes in response to environmental variation.

Modern Terms

• The srudy of heritable phenotypic changes that do not involve alterations in the DNA sequence.

• How the environment can cause changes that affect the way our genes work.

Control of Gene expression

• Every cell in a living organism contains identical genetic material ( a complete set of chromosomes).

• This contains all of the information to create everything the organism will need to function properly.

Human Genome Project

• Only 1% of DNA is made up of protein coding genes.

• The non-coding DNA accounts for Promoters, ENhancers, Silencers, Non-coding RNA.

Encode Project

• This project aims to explore the non-coding human genome and understand how the genome is regulated.

• This includes

  • Transcription factor binding sites,

  • 3d structure of the genome

  • Chromatin patterns.

  • Non-coding RNA species.

  • DNA methylation patterns,

Epigenetic Regulation

• REgulation of gene expression can be achieved broadly and specifically.

• At the broafest level. Whole genomic regions can be transcriptionallu activated of repressed through their location within the nucleus.

• REgions of chromosomes cnan be regulated through a variety fo reversible modifications tho histone proteins, this histone code hypothesis. (INSERT DEFINITION)

• Single genes of sets of genes can be transcriptionally regulated through tagging via methylation of cytosine nucleotides.

Nuclear Organisation’

• The genetic material inside of the nuclear envelope os highly organised.

  • Nuclear envelope is a double membrane that defines the area of the nucleus.

• The broadest organsisational structure of the nucleus is the state of chromatin.

  • Heterochromatin - genetic material is tightly packed and inaccessible.

  • Euchromatin- genetic material is loosely packaged and accessible.

• Transcription can only take place when chromatin is an open state.

• 3 states (Closed, persuastive, open)

• The landscape of accessibility changes dynamically in response to both external stimuli and developmental cues.

Chromosome Territoeries

• Within the nucleus chromosomes occupy specific territories.

• Inter-chromosomal territories are interspersed throughout the nucleus. (most transcriptionallyu active).

• Heterochromatin primarily occupies regions near the nuclear lamina.

• Genomic lamina associated domains are generally gene poor and have a low transcriptional activity.

• Domains are dynamic throughout development and differ between cell types.

The Histone Code Hypothesis

• Chromosomes are organised into tightly coiled nucleosomes, which form chromatin fibres.

• Nucleosomes made of four histone proteins.

• Histones are positively charges and have N terminal tails.

• Tails are where modifications are applied.

• The tails undergo a variety of PMT,.

  • Acetylation

  • MEthyl;ation

  • Phosphorylation

  • Ubiquitlianation

• Different combinations of modifications tpepresent a cpde of orts that defines transciptional states.

• Theis process is guided by histone modifying enzymes.

  • Two major groups

  • Histone acetylating enzymes.

  • Histone methylating enzymes.

• AKA chromatin remoddeling.

• Open chromatin will have acetylated.

• Closed chromatin, histones are methylated.

DNA methylation

• Hte majority of DNA methylation occurs on CpG sites, cytosines that precede a guanine nucleotide.

• They are dispersed across the genome and they are heavily methylated.

The exception is within CpG islands, These are regions of high CpG sire frequency and are often not methylated.

Core Promoters.

• Ahc gene within the genome is associated with regulatory elements which reside upstream of the gene coding sequence.

• The most important is the promoter sequences.

  • They contain,

• Mediator interacts with silencer proteins, and interacts

CpG islands and gene expression

• Most promoters are within CpG islands.

• Occurrence of these islands are highly conserved within species,

• CpG islands contain fewer nucleosomes than other stretches of FNA,

• Around 50% of these islands contains known transcription start sites.

DNA methylation enzymes.

• The DNA methylation reaction so cata;ysed by a family of enzymes by DNA meyhtltransferases.

• DNMT 1- copies DNA methylation pattern from parentla DAN onto newly syntheissed daughter strand. ALso has the ability to repair DNA methylation.

• DNMT3A and £b can establish new methylatuio patterns to unmodifies DNA.

• Occurs during gamete production, how epigenetic inheritance occurs.

Epigenetic Crosstalk.

• TRancro[tion is ultimately regulated by the interaction of multiple epigenetic mechanisms that co operate to activate or silence gene ecpression

• DNMT1 and DNMT3a both bind to a histone methyltransferase that restrict the gene expression by methylation on H3K9.

• Methyl binding proteins serve as rhe strongest link between DNA methylationa nd histone modification.

Non Coding RNA.

• Non coding RNAs play some role in epigenetic regulation.

• E.g dosage compensation via X inactivation.

Dynamic epigenetic reprogramming of the embryo

• In mammals active methylation of the parental genome occurs in the zygote shortly after sperm entry,

• This is follwe by more passice methylation and then de novo methylation.

• The de novo methylation re establishes the methylation patterns across the entire genome.

• The maternal genome is not subjected form the epigenetic reprogramming.

Genomic Imprinting

• Genomic imprinting is the process by which specific genes from each parent are epigenetically silenced.

• We inherit two copies of each gene but we only have one working copy, te other is silences,

• These tags are maintained throughout the life of the individual,

• These tags are reset during gamete production.

Transgenerational epigenetic inheritance.

• If they are not properly erased and/or incorrectly reestablished this would result in Non-genetic inheritance, or transgenerational epigenetic inheritance,

• This cna occur via the

  • Inheritance of epigenetic tags in the germline.

  • Inheritance of other non-genetic factors, proteins, hormones, ncRNA.

ENdoccrine Disruptors & Male fertility

• Ecperimant by exposing pregnent female rats to an endocrine disruptors during the period of gonafal sex determination.

• This led to increaed incidence of male fertility and decreased spermatogenic capacity in male offspring,

• These were transferred thofuh the male germ lin eto nearly all males of subsequent generations.

• Lookaed at methylation fo the ssperm.

Epigenetic Contril of MAternal behaviour