pos

basic structure of chromosomes-

• centromere at centre

• telomeres at ends- protective, repetitive sequence of DNA

• there are replication origins

• genes spread around

structure of a eukaryotic gene

• promotor region

• exons and introns

• TATA box in promotor- initiation of transcription

• transcription initiation site

• translation initiaton site in exon 1 (ATG)

• translation termination site

• transcription termination site

what is the central dogma

DNA to RNA to protein

(transcription, splicing, export, translation, folding)

what are RNA polymerases and what are the 3 types

• protein complexes made up of 8-14 subunits

• Pol 1 - ribosomal RNA

• Pol 2- mRNA for translation into protein

• Pol 3- small RNA (tRNA)

transcription initiation

• transcription factor binds to eukaryotic promotor

• formation of preinitation complex

• RNA pOL 2 begins elongation

what do transcription factors bind to

• gene specific transcription factors bind to regulatory sequences of the gene

gene definition

A locus (or region) of DNA, which is made up of nucleotides and is the molecular unit of heredity

chromosome definition

A thread-like structure of nucleic acids and protein found in the nucleus of most living cells, carrying genetic information in the form of genes.

genome definition

The complete set of genes or genetic material present in a cell or organism

initiation

• general transcription factors bind , eg TBP to TATA box

• pre initiation complex formed

• rna polymerase holoenzyme binds to the promotor region

where do transcription factors bind to?

regulatory sequences

what is a transcription factor homodimer

2 protein monomers that bind together to regulate gene expression

trans activating domain

regions of transcription factors which bind to coactivator complexes to activate transcription

helix turn helix motif

• two alpha helices joined by a short strand of amino acid

• recognition and binding of DNA is done by the 2 helices, one at n terminus and one at c terminus

• c terminal binds to major groove, n terminal helps to position the complex

other dna binding motifs

• zinc finger

• leucine zipper

• helix loop helix

elongation

• what does rna polymerase do

• what helps maintain the correct length

• what does the zipper do

• RNA polymerase travels along from the 5’ to 3’ end (upstream to downstream)

• rudder in the upstream end of the RNA-DNA hybrid helps maintain the correct length, etc

• the zipper is another protein loop within RNA polymerase and maintains boundaries of the transcription bubble especially at the downstream end

what are topoisomerases

regulate dna overwinding or underwinding

5’ cap addition

• 7 methyl guanosine triphosphate cap

• this is a modified guanine cap to the 5’ end of mRNA

• protection from degradation

• initiation of protein synthesis

3’ polyadenylation

• example of the sequence

• what recognises it

• what happens then to the mRNA

• what then happens at the 3 end

• what binds

• why does this happen

• sequences of the pre mRNA such as the AAUAAA sequence are recognised by the cleavage and polyadenylation specifity factor (CPSF)

• then an endonuclease cleaves mRNA at specific site

• enzyme poly-a-polymerase joins multiple adenine nucleotides to the 3’ end

• poly a binding proteins bind to the newly formed tail

• provides stability and facilitates exit of mRNA from the nucleus

xm2

nuclease that degrades remaining nacsent transcript and leads to termination of transcription

splicing

• introns always begin with the residues GU and end with AG and an A somewhere towards the middle

• phosphodiester bond is attacked between GU and A

• 3 prime end attacks phosphodiester bond between A and AG joining exons and removing the introns

how can proteins be altered for a specific function?

alternative splicing

what is the genetic code

• 20 amino acids

• 4 diff nucleotides

• if code is x nucleotides , number of possiblity is 4 to the power of x

• codons

charging of tRNAs with amino acids

• what does amino acyl tRNA synthetase do

• what does it then attach and what is released as a result

• what is created

• what does the enzyme bind this to

• what then happens and what kind of bond is formed

• what molecule is released

• amino acyl tRNA synthetase binds an amino acid and ATP

• the enzyme catalyses a reaction that attaches AMP to amino acid releasing pyrophosphate 

• this creates a high energy aminoacyl-adenylate intermediate

• The same enzyme, aminoacyl-tRNA synthetase, binds the activated aminoacyl-adenylate and its corresponding tRNA molecule.

• amino acid is transferred from AMP to the 3 end of tRNA which forms an ester bond

• AMP is released, as the amino acid is now covalently linked to the tRNA, creating a "charged" aminoacyl-tRNA. 

what are the site of the ribosome

• p site holds trna with growing polypeptide chain attached

• e site holdsthe trna which will exit

• a site holds aminoacyl trna

elongation cycle

• what is it

• what is delivered to the a site and what escorts it to where and where does it bind to

• what does this consume

• what catalyses formation of a peptide bond

• what happens and what 2 things does this need

• process of adding amino acids to a polypeptide chain

• aminoacyl trna is delivered to the A site. elongation factor escorts the trna and binds it to the ribosome consuming GTP (to GDP)

• peptidyl transferase catalyses formation of peptide bond

• ribosome shifts one codon along in the 3 direction. facilitated by elongation factor 2, this requires GTP.This action moves the tRNAs: the now uncharged tRNA from the P-site moves to the E-site, and the tRNA carrying the polypeptide chain moves from the A-site to the P-site, freeing up the A-site for the next cycle. 

translation initiation

• what do the eukaryotic initation factors bind to

• what does initiator tRNA carry and what does this do

• what does the small ribosomal subunit do

• what happens to the GTP on the tRNA and what does this cause and allow

• what sits on the p site

• eukaryotic initiation factors bound on the 5’ end join to the (poly a binding protein) bound on the poly a tail of the 3’ end

• initiator tRNA carries methionine, GTP and initiation factor 2, and the 40s ribosome both recognise the initiation factors bound to the 5 and 3 ends. They all interact

• small ribosomal subunit finds the start codon, using protein initiation factors. but some are easier to find than others (purine 3 residues upstream of A -Kozak sequence) 40s subunits slides down

• GTPase activity converts the GTP on the tRNA to GDP 

• causes conformational change, and GDP is release

• this allows the large ribosomal subunit to bind

• methionine sites on the p site of the ribosomal complex

• ready to begin elongation

termination

• stop codon do not have a corresponding trna to recognise

• instead when a ribosome reaches the stop codon the a site accepts a protein called release factor instead of tnra

• release factor hydrolyses the bond between trna in the p site and the amino acid in the polypeptide chain, freeing it

• two ribosomal subunits and other structures dissociate

antibiotics targeting translation

• chloramphenicol

• erythryomycin

• tetracycline

• streptomycin

• chloramphenicol binds to 50s rrna inhibiting formation of a peptide bond

• erythromycin binds to 50s of rrna and prevents movement along mrna

• tetracycline inhibits trna anticodon reading of mrna codon

• streptomycin changes shape of 30s rrna and causes mrna to be read incorrectly

functions of chromatin

• package and condense dna

• prevent dna damage

• control dna expression

what are nucelosomes

• how many base pairs

• what is the core comprised of

• what are the cores separated by

• what brings nucleosomes together and what does this do

• what do multiple stacked rings form

• what is formed when they are coiled further

• ‘beads on a string’

• around 146 base pairs of dna wrapped around 1.7 times around a core

• core is comprised of an octamer of histone proteins

• nucleosome core proteins are separated by linker proteins

• histone H1 brings nucelosomes together to condense chromatin further

• multiple stacked rings form the solenoid fibre/30nm fibres

• these are coiled further to form chromosomes

pcr

• heat to 94 to denature (separate) dna strands

• cool to 50-60 to allow primers to anneal

• 72 to allow elongation of new strands using taq polymerase

• so at the end of the first cycle 2 new strands

• begin cycle two by denaturing and annealing again

• elongation generates strands of different sizes depending on the template strands

sanger sequencing

• mix small amount dideoxy with deoxy nucleotides

• cycle through multiple rounds of pcr

• each ddNTP contains fluorescent tags

• separate by gel electrophoresis

• detect by laser

• (chain synthesis terminates when ddNTP is incorporated)

technical applications of PCR that could be used to an advantage in veterinary medicine

• VNTR

• RAPD

• AFLP

• they are all used to determine breed, pedigree and/or parentage

what components are essential for basic PCR?

• DNA polymerase

• all 4 deoxyribonucleotides

• dsDNA template

• a pair of primers which bind to opposite polynucleotide strands and have their 3 ends pointing towards each other

what is PGE characterised by

• diarrhoea/ weight loss

• poor weight gain

• hypoalbuminaemia

examples of parasitic nematode worms casusing bovine pge

• ostertagia (abomasum)

• cooperia and neatodrus (small intestine)

life cycle of ostertagia

• adult ostertaga in abomasum

• egg containing L1 larva passed in faeces

• eggs hatch on pasture

• develops to L3 larva, migrates from faeces to grass

• L3 larva ingested by cow

• this L1 to L3 development is temperature dependent. it takes up to 2 weeks or can be even slower

• the prepatent period is 3 weeks, or up to 6 months if the development is arrested

• typical strongyle eggs in faeces: oval, thin clear wall, bundle of cells. 80um

• 3rd stage larva is 750 um.

• adult around 1cm long, brown. they emerge from the gastric glands and live/mate on abomasal mucosal surface

pathogenesis

• where do larvae migrate to on ingestion

• when do adult worms emerge

• what happens if a large number of worms emerge at the same time

• on ingestion larvae migrate to gastric glands of abomasum to continue development

• adult worms emerge from glands around 18 days later

• if a large number of worms emerge at the same time

  • pH increases from 2-7 as less acid produced as gastric glands are affected

  • pepsinogen cannot be activated to pepsin

  • abomasal epithelium becomes leaky

  • plasma proteins lost into gut lumen causing hypoalbuminaemia, weight loss and diarrhoea

what can happen to gastric mucosa

• thickened, hyperpastic

• raised nodules

• some of the L3 larvae survive on pasture overwinter

• the new eggs deposited in early spring develop slowly to L3 as it is too cold

• as the weather warms some existing L3 die as drier conditions and trapped in cow pats. but rate of L1 to L3 dev increases.

• Then L3 increase furthe as more calves are infected and shed and eggs in faeces

what does rate of infection depend on?

• host appetite

• number of infective larvae (L3) on pasture

• so disease is most common in calves where they are grazing permanent pasture and kept at a high stocking density

what happens to L3 is ingested in late autumn or early winter?

• L4 arrest in gastric glands

• then L4 resume development and emerge from glands in waves (type 2 disease)

immunity to ostertagia

• slow to develop - takes the whole grazig season

• may dip over winter ad re established upon turnot (2nd grazing season)

• adult cattle solidly immune

epidemiology in beef herds- spring calving

• calves at foot with cows

• spring mortality of L3 occurs before calves eat much grass

• so immun cows eat most of grass and pass few eggs

• so very low disease risk

epidemiology in autumn calving

• calves turned out at start of year and will eat some outwintered L3 BUT cows eat most

• then lower autoinfection peak

• some risk but still low

type i disease

• typically calves in first grazing season

  • mid july onwards

• morbidity high, mortality low

• diarhoea common (larval damage to gastric glands)

• weight loss

type ii disease

• typically yearlings

• late winter/ spring following first grazing seasn

• prevalence low, only some affected

• mortality more likey

• ± diarrhoea with anorexia and thirst

• hypoalbuminea more marked, weight loss

control of type i disease

• use clean pasture

• delay turnout untul after spring mortality in L3

• strategic anthelmintic use

anthelmintic use

• macrocyclic lactone wormers last 5 weeks eg doramectin

• prepatent period for ostetagia s 3 weeks

• so dosing needed every 8 weeks

options

• dose and move to fresh pasture in july before autoinfection peak

  • but can still get disease if L3 high, increased risk of spreading resstant worms to fresh pastur

• dose every 8 weeks from july and keep on same pasture

• dose at spring turnout and 8 weeks later reducing autoinfection peak

• give an intraruminal device before turnout which reduces autoinfection peak

control of type ii

• cattle exposed to low challenge at pasture in late autumn

  • unlikely to require worming treatment at housing

• cattle exposed to medium/ high challenge at pasture in late autumn or cattle of unknown origin

  • likely to require worming treatment at housing

what is meant by a protein structure hierarchy

-primary, secondary, tertiary, quaternary(eg 2 beta and 2 alpha globin polypeptides with a heme group)

• what type of amino acids are mammalian proteins

• how many amino acids

• what is the peptide bond classified by

• how many amino acids makes a protein

• shape of peptide bond

• mammalian proteins are alpha amino acids

• 20 amino acids

• carboxyl group reacts with amino group to form a peptide bond and lose a water molecule

• catalysed by peptidyl transferase (28s ribozyme-ribonucleic acid enzymes)

• within a polypeptide chain, individual amino acids = residues

• when >50 amino acids are in a polypeptide is a protein

• peptide bond is planar (partial double bond between carbonyl O and N)

bonding determining protein structure

• covalent primary

• hydrogen secondary

• hydrophobic+VDW tertiary

• electrostatic within tertiary helps maintain

• VDW/ electrostatic quaternary

alpha helix

• what runs parallel to the helix axis

• what winds around the axis

• what does htis lead to

• how many amino acid residues per complete turn

beta sheets

• what orientation do H bonds run to the chain direction

• alpha helix- H bonds run parallel to helix axis. alpha carbon backbone winds around an axis so that each carbonyl O atom is H bonded to each amino N of the amino acid located 4 residues closer to C terminus

• The standard alpha helix has approximately 3.6 amino acid residues per complete turn

• beta sheets- H bonds run perpendicular to chain direction

• parallel and antiparallel

types of r groups

nonpolar, polar, electrically charged

cysteine

can form disulphide bonds as it has an SH group

examples of tertiary proteins

-catalase

-triose phosphate isomerase

-actin

tertiary domain

the smallest stable unit of a tertiary structure. a domain is defined as that region of a polypeptide chain that can fold into an autonomous stable tertiary structure

domain shuffling- when domains have been switched around between proteins through evolution

quaternary

1. haemoglobin- needs to deliver oxygen tissues. needs high enough affinity to pick up oxygen but low enough to release it.

2. t state- oxygen unbound

3. r state- oxygen bound. so that the 3 other units have higher affinity

4. small changes in oxygen concentration can dramatically affect binding to haemoglobin giving a sigmoidal curve of oxygen binding

collagen

• what type of protein

• what is the general structure

• extracellular fibrous protein but not an alpha helix

• 3 helical chains that wind around a central axis

• general structure is Gly-X-Y where X can be any amino acid especially proline lysine or hydroxyproline

• Glycine is small so glycines from each chain fit at the centre of each helix

• H bonds between the chains

skin and problems

• thick layer of cells and sebaceous gland secretions

• wounds

• vector borne pathogens

mucous membraness

• mucociliary escalator, peristalsis

• coughing and sneezing, vomiting and diarrhoea

• secretions- physical and anti microbial properties

• commensal microflora

what does the innate immune sstem use for detection

• uses pattern recognition receptors to detect microbial components that are itrinsically foregin

  • protein associated molecular patterns eg

  • lipopolysaccharide in gram negative

  • peptidoglycan in gram positive

  • mannose sugar in prokaryotic carbohydrate molecules

bacterial PAMPs

pathogen associated molecular patterns

pattern recognition receptor

• where can they be found

• what type in each

• can be found in the cytoplasm, cell membrane, inside vessicles or as soluble molecules int he tissue fluid/plasma to detect PAMPS

• cytoplasm: NOD receptors

• membrane bound: TLRs

• soluble: complement c3 protein, mannose binding lectin, c reactive protein

toll like receptors- what do these recognise

• 2

• 4

• 3 and 7

• what are they predominantly expressed by

• tlr2 recognises peptidoglycan- gram positive

• tlr 4 recognises LPS - gram negative

• tlr3 and 7 recognise viral nucleic acid

• predominantly expressed by neutrophils and macrophages

how does innate to viral work

• how are they detected

• how do cells respond and what effect does this have

• do not have strutural pamps

• are detected by presence of double stranded rna produced during replication (not present in mammalian cells) or DNA in the cytoplasm

• cells respond by producing type 1 interferon

  • this includes interferon alpha, beta, omega

  • interferons have a paracrine effect

function of type 1 interferon

• resistance to viral replication

• act on neighbouring

  • increased deradeation of viral mrna

  • inhibition of viral protein sythesis

  • increased antigen presentation of viral antigens

what is the paracrine effect

innate immunity to viruse

• viruses can infect any nucleated cells

• all nucleated cells can respond to viral infection by producing type 1 interferon

• interferon omega can be used to treat persistent viral ifnection of cat eg FeLV/FIV

natural killer cell- large granular lymphocyte

• what does it recognise

• what does it release

• recognise decreased levels of MHC molecules on host cells

  • there is decreased production during viral protein synthesis

  • some virus block transport to cell surface to prevent expression

• this decrease is a symptom of viral infection.

• the NK cell releases toxic granules killing the cell before the viral replication is complete

innate- cellular mechanisms

• what is the response

• how is the micro organism killed

• what helps and enhances

• recognition of pathogen (membrane, vesicular and cytoplasmic PRR)

• response is phagocytosis and inflammation

  • phagocytic organisms attach to the organism and use pseudopodia to capture it forming a phagosome

  • then it stimulates a respiratory burst and toxic metabolites (eg oxygen free radials, hydrogen peroxide) are pumped into the vesicles to kill the micro organism

  • subsequent fusion of the lysosomes with the phagosome (phagolysosome) releases proteolytic enzymes and anti microbial mediators (defensins and lactoferrin)

  • increasingly acidic pH results in digestion of the microorganism

  • enhanced phagocytosis (opsonisation) can be achieved with the help of the antibody IgG and or complement CC3b

  • destruction of endocytosed organisms can be enhanced by stimulation of cytokines released by T helper cells

innate- humoral mechanisms

• recognition of pathogen- soluble PRR

• response: killing of foreign organism, enhacned phagocytosis and inflammation

tlr

what could NOD2 receptor defect cause

• crohns in man

• ibd or anal furunculosis in german shepherd

what does recogn tiion of bacteria by macrophage tlr lead to

phagocytosis and inflammatory resposne

respiratory burst

• enhanced cellular aeorbic metabolism

• reactive oxygen intermediates are formed

  • superoxide anion

  • hydroxyl radicals

  • hydrogen peroxide

• this isthe oxygen dependednt mechanism of bacteiral killing

lysosomes

• 3 types

defensins

• cationic anti microial peptides that damage bacterial cell wall

lactoferrin

• binds and chelates free iron, which is required for bacteiral growth

acid proteases

• digestive enzymes active at low pH

inflammatory mediators-histamine

• what is it released by

• produced by mast cell degranulation in tissues

• anti histamine

pro inflammatory cytokines

• examples

• what are they synthesised by

• tumour necrosis factor a

• synthesised predominantly by wbc and macrophages

• corticosteroids

lipid mediators of inflammation

• prostaglandin and leukotrienes

• derived from arachidonic acid by action of cycloocygenase and lypoxygenase enzyme

• nsaid

localised effect

inflammation

systemic

hypothalamus

• fever

liver

• acute phase response

bone marrow

• neutrophil and monocyte mobilisation

• speed up production

acute phase response

• where are the proteins produced by in response to what

• examples

• what do they do

• are they specific or non

• acute phase proteins are produced by the liver in response to pro inflammatory cytokines

• serum amyloid protein, C reactive protein and mannose binding lectin stick to bacterial cell walls

  • act as opsonins to enhance phagocytosis and stimulating complement activation

• but are non specific

complement

• where are complement proteins found

• what steps do they form

• what is the end product

• complement prtoens are found in the blood

• series of enzyme activation steps forming an aomplification cascade

• small amount of activation is amplified to generate a large response

• similar in nature to clotting cascade but with a diff trigger and outcom

• the end product of the cascade is the polymerisation of C9 monomers to form a C9 polymer forming a membrane attack complex

  • a tube like structure that create holes in the cell walls of bacteria causing them to lyse

• inactive C3 in the blood dissociates into C3a and b in the presence of bactiera- it is a pro enzyme

  • C3b is deposited onto the surface of the microbe and acts as an enyzyme to catalyse the formation of the MAC and also act as an opsonin (phagocytic cells express c3b receptor)

  • C3a binds to receptors on local tissue mast cells triggering degranulation and stimuating a inflammatory response

what are 3 examples of physical barriers

• thick stratified squamous epithelium (skin and lower urinary tract)

• mucociliary escalator (resp tract)

• peristalsis, vomiting and diarrhoea when necessary (alimentary)

example of biochemical barriers

• lactic and fatty acis in sebum from sebaceous glands of skin

• enzyes

• acid in stomach

• antibacterial peptides eg defensin

how do commensal organisms provide protection

compete with organisms for space

provide natural antibiotic

what do gamma delta t cells do

react to stress proteins that are upregulated on the surface of infected mucosal epithelial cells

overall oucome of complement activation

• lysis of the bacteria by the MAC

• enhacend phagoytosis of bacteria coated in complement proteins C3b

• inflammation at the site of complement activation C3a

where are these prrs located

• TLR 2

• TLR 4

• TLR 5

• TLR 9

• NOD2

• peptidoglycan

• lipolysaccharide

• flagellin

• prokaryotic DNA

• muramyl dipeptide