Microbiology 4 Flashcards
1
Q
List the important enteric viruses of veterinary species
A
- Reoviridae (Rotavirus)
- Parvoviridae (Parvovirus)
- Coronaviridae (Coronavirus and Torovirus)
- Paramyxoviridae (Paramyxovirus)
- Flaviviridae (Pestivirus, BVDV)
- Astroviridae (Astrovirus)
2
Q
Describe the structure of Reoviridae
A
- Icosahedral
- Non-enveloped
- Outer, middle, innre capsid
- Core contains segmented genome (dsRNA)
3
Q
Describe the epidemiology of Rotavirus
A
- Encodes own polymerase
- Cytoplasmic replication
- Segmented genome (reassortment)
- Multiple strains, no cross protection
- Very stable in environment
- Wide pH range, temp range
- May persist after outbreak if poor disinfection
- Major cause of diarrhoea in young farm animals
- Faecal oral transmission
- Minimal infectious dose
4
Q
Describe the pathology of rotavirus
A
- Infects enterocytes at end of villi
- Villi shorter
- Unable to absorb lactose due to loss of lactase
- Impaired Na transport, reduced water absorption
- Decreased digestion and absorption of milk in upper SI
- Undigested milk in lower SI and LI = bacterial overgrowth, osmotic diarrhoea, watery scour
- Pasty/watery diarrhoea
- Secondary infection with E. coli, other viruses, coccidia
- Dehydration
5
Q
How is rotavirus diagnosed?
A
- Sample faeces/gut contents
- Detection of viral antigen - ELISA or latex aggluniation
- Detection of viral RNA - polyacrylamide gel electrophoresis
- Post mortem examination
6
Q
Describe the structure of Coronaviruses
A
- +ve sense ssRNA
- Enveloped
- Spike proteins on envelope
- Contains nucleocapsid (ssRNA adnn protein)
7
Q
Describe the epidemiology of Coronaviruses
A
- Enteric and respiratory pathogens
- Cause of common cold in man
- Difficult to grow in lab
- Commonly mutate
- Survive well in environment
- Tolerate low pH
- Destroyed by common disinfectants
- Lots of strains
8
Q
Describe the pathogenesis of Coronaviruses
A
- Infect cells in middle of villi
- TGEV, CCoV, FCoV virus spike proteins bind to aminopeptidase N (highly expressed in mature enterocytes)
9
Q
Describe porcine coronavirus
A
- TGEV (transmissible gastroenteritis virus)
- EDV (porcine epidemic diarrhoea virus)
- Other strains exist but these are teh enteric ones
- Can be distinguished by serology
- TGEV highly contagious, young pigs, diarrhoea/vomiting, rapid dehydration, high mortality
- EDV - similar to TGEV, less severe
10
Q
Describe coronavirus in cattle
A
- BVC (bovine corona virus)
- Scour 4d-3wk of wage
- Dehydration, acidosis, depression, fever
- Recovery in 4-5d
- Winter dysentery in housed adult cattle
11
Q
Describe canine coronavirus
A
- Canine enteric CoV (CCoV)
- Mild self-limiting diarrhoea
- Novel genotypes may produce more severe disease (spontaneous mutation)
12
Q
Describe feline coronavirus
A
- Enteric virus but pathogenesis produces systemic sings
- Biologically distinct biotypes: feline enteric coronavirus and feline infectious peritonitis
- Following infection most will have transient infection, can shed for many months, some will acquire carrier state and shed for life
- Minority will get FIP
13
Q
Describe feline infectious peritonitis
A
- FIP
- Cahnges in virus lead to change of strain
- Mutation of virus, stress, viral load
- Dry or wet FIP
- Wet: chest full of sticky yellow fluid
- Dry: CNS fluid affected
14
Q
Describe Torovirus
A
- Rare
- Equine: uncommon cause of diarrhoea
- Bovine: diarrhoea in newborn calves
- Feline: diarrhoea and third eyelid syndrome
15
Q
Describe the structure of Flaviviridae (Pestiviruses)
A
- +ve ssRNA
- Enveloped
- Icosahedral capsid
- Cytoplasmic replication
16
Q
What diseases are caused by Pestiviruses?
A
- Bovine viral diarrhoea
- Border disease in sheep
- Classival swine fever
17
Q
Describe bovine viral diarrhoea
A
- Most important viral disease of cattle
- Diarrhoea
- Decreased fertility/milk yield
- Abortion, congenital defects, stunted calves (transplancental)
- Immunosuppression
- Mucosal disease
- 2 genotypes: BVDV-1 and BVDV2
- Isolates fo both associated with mild and severe disease
- 2 biotypes
18
Q
What are the 2 biotypes of BVDV?
A
- Non-cytopathic
- Cytopathic
- Exist for both genotypes 1 and 2
19
Q
Describe NCP BVDV
A
- Major cause of BVD
- For mucosal disease need NC then CP
- Can cause persistent infections
- Can cross placenta, immune system sees as self, immunotoleratn, if survives then shed NCP
- can survive without getting BVD but can get mucosal disease
20
Q
Describe mucosal disease of BVDV
A
- Infrequent consequence of BVDV infection
- Develops only in persistently infected animals
- Presence of NCP and antigenically related CP virus
- Mutation of NCP virus in PI animal, uperinfection of PI animal with another CP virus
- CP viruses show marked tropism for GALT
- Severe diarrhoea, invariably fatal
- Marked mucosal haemorrhage
21
Q
Describe the structure of Parvovirus
A
- Icosahedral capsid
- Non-enveloped
Small, linear ssDNA genomoe
22
Q
Describe the epidemiology of parvovirus
A
- Infect and kill actively replicating cells
- Persist for long periods in environment
23
Q
Describe the pathogenesis of parvovirus
A
- Infect and kill actively replicating cells
- Degrade villi
- INfect progenitor cells at base of crypts
- Villi tip cells turnover normally but not replaced
- Stunted villi, malabsorption, maldigestion
24
Q
What are the 3 important enteric parvoviruses?
A
- Feline panleukopaenia virus (FPV)
- Canine parvovirus (CPV)
- Porcine parvovirus (PPV)
25
Describe the epidemiology feline parvovirus
- AKA feline infectious enteritis (FIE) or feline panleukopaenia
- Faecal oral transmission
- Persists in environment up to a year
- Lots in faeces
- Lymph nodes of naso- and oro-pharynx
- Spread to other tissues
- Needs rapidly dividing cells to propagate
- Infects intestinal cells and bone marrow
- Young or older unvaccinated most affected
- Infected pregnant queens problematic
26
Describe the pathogenesis of feline panleukopaenia
- Decreased WBC count
| - Killing of lymphoid and myeloid stem cells
27
Describe the pathogenesis of feline infectious enteritis
- Killing of stem cells in crypts
- Dehydration
- Can be fatal
28
Describe feline cerebellar hyperplasia
- Infection in neonata kittens
- High risk secondary bacterial infection
- Perinatal infection
- Cerebellum controls coordination adn balance = wobbly kittens
29
Describe the diagnosis of feline parvovirus
- Faeces contain a lot of virus
- Detect viral antigen (ELISA) or viral DNA (PCR)
- Eidence of exposure to virus by detection of antibody in blood
- Supportive evidence through marked leukopaenia
30
What are the types of canine parvovirus?
- CPV-1 (minute virus, mild diarrhoea)
| - CPV-2 (serious pathogen of dogs)
31
Describe canine parvovirus 2
- Infection of actively dividing cells
- Generalised neonatal disease
- Myocarditis in neonatal puppies
- Bone marrow = leukopaenia
- Intestinal villi = enteritis = vomiting/diarrhoea
- Intestinal adn mesenteric lymphoid tissues = immunosuppression
- Mortality can be high even with appropriate treatment
- Myocarditis can follow infection if survive
- Due to myocardial necrosis, inclusion bodies in myocardial cells
- Inflammatory cells increase and fibrosis occurs
32
What are the important genera of Paramyxoviridae?
- Morbillivirus
- Rubulavirus
- Respirovirus
- Pneumovirus
- Metapneumovirus
33
Describe the virology of Paramyxoviridae
- Large enveloped virus
- -ssRNA
- Sensitive to heat, detergent, desiccation
- Cytoplasmic replication
- Release by budding
34
What diseases are caused by Morbillivirus?
- Measles
- Canine distemper
- Rinderpest
- Peste des petits ruminants
- Phocine morbillivirus
- Cetacean morbillivirus
35
Describe canine distemper
- Young dogs especially susceptible
- Direct contact transmission
- Replicates in URT
- Spread to tonsils/lymph nodes
- Viraemia and systemic spread to epithelia +/- CNS
- does not only infect GIT
- Pyrexia, depression
- Ocular and nasal discharge
- Cough
- Vomiting, diarrhoea
- Hyperkeratosis of nose/pads
- Solid immune response = recovery
- Poor immune response = development of neurological signs
36
Describe Rinderpest
- Cattle plague
- Highly infectious
- Respiratory and alimentary tract
- High morbidity and mortality
- High fever
- Nasal discharge
- Ocular diascharge
- Excess salivation
- Oral and nasal erosions and ulcerations
- Diarrhoea, with mucus, blood and debris
- Dehydration followed by death
- Mild signs if disease becomes endemic
37
Describe Peste de petit Ruminants
- Goat plague
- Similar to Rinderpest
- Mucosal erosions and profusediarrhoea
38
Describe Newcastle disease
- Avian Rubulavirus
- Notifiable
- Avian paramyxovirus-1
- Chickens, ducks, pheasants, geese, turkeys
- Shed in all excretions and secretions(aerosol)
- Stable for weeks on carcasses
- COmmon in wild birds (inapparent infection)
- Mild conjunctivitis in humans
- Strains vary in virulence and tropism
- Deterined by F glycoprotein
39
What are the strains of Newcastle disease?
- Lentogenic
- Mesogenic
- Neutropic velogenic
- Viscerotropic velogenic
40
Describe lentogenic Newcastle disease
- Mild, inapparent infection
| - Confined to it and respiratory tracts which trypsin like proteases are present
41
Describe mesogenic Newcastle disease
- Mild respiratory disease
| - Some death in young birds
42
Describe neutropic velogenic Newcastle disease
- Acute, severe, fatal with respiratory and nervous signs
- Can be cleaved in many tissues by furin-like (ubiquitous) proteases due to altered cleavage site
- Contain multiple basic AAs
43
Describe viscerotropic velogenic Newcastle disease
- Severe
- Fatal with haemorrhagic intestinal lesions
- Respiratory disease
- Can be cleaved in many tissues by furin-like (ubiquitous) proteases due to altered cleavage site
- Contain multiple basic AAs
44
Describe the virulence factors of Newcastle diseae virus
- F glycoprotein
- Haemagglutinin/neuramidase (HN) enables virus attachment to cell receptor
- Fusion protein (F) enables fusion and entry
- F protein cleaved to become active by host cell proteases (forms F1 and F2)
45
Describe the diagnosis of Newcastle disease
- Egg inoculation and testin shows presence
- Agglutination tests
- Virulence by intracerebral inoculation and demonstation of multiple basic AAs at F protein cleavage site
46
Describe Astroviruses
- Mild self limiting diarrhoea in many species
| - More severe in ducks where lethal hepatitis may occur
47
Describe the common features of enteric virus infection
- Small infectious dose
- Short incubation and life cycle
- Lytic
- Large amount shed in faeces
- Tough, able to survive pH of stomach and environment
- Secndary bacterial infection common
- Multiple viral infections
- Diarrhoea
- May have vomiting (dogs and cats usually)
- Death from dehydration can occur
- Maternally derived antibody protects newborn
- Disease as antibody levels fall or if there is failure of sufficient colostrum transfer/low antibody levels
- Occurence of disease influenced by amount of virus, viral virulence, host resistance
- High stocking density leads to outbreaks
48
Describe the common feature of enteric viral pathogeneis
- Denudation of microvilli
- Shortening, flattening and atrophy of villi
- Leads to malabsorption
- Food left in gut as not absorbed by SI leads to bacterial overgrowth
49
Describe the control of Rotavirus
- Colostrum important
- All-in, all-out management
- Good hygiene
- No vaccination for pigs in UK, available for cattle and horses
- Aim to vaccinate dam to increase colostral antibodies
- Can feed scour to sow during pregnancy but very risky
50
Describe the control of Coronavirus
- Removal of infected animals
- Maintain clean herd and housing
- Vaccine available for some (reduce, do not eliminate disease)
- Early weaning prevents
- Separate age groups
- Virus changes a lot, cannot vaccinate against all types
51
Describe the control of bovine viral diarrhoea
- Eliminate persistently infected animals from herd
- Maintian closed herd
- Maintain herd immunity so noo naiive cattle infected
- Vaccination
- Deliberate infection risky
52
Describe the control of FPV
- Maternally derived antibody wanes after 8 weeks
- Vaccinate
- Prevent exposure of kittens to FPV in environment
- Vaccination before colostrum protection wanes
53
Describe the control of CPV-2
- Live attenuated and inactivated vaccines
- Annual boosters
- Additional vaccines in outbreak
- Maternally derived antibody can interfere with vaccine so need multiple vaccinations or high titre vaccine
- Virus hardy - transmitted by faecal oral route and fomites
- Bleach effective at killing
- Isolate pups until vaccination complete
54
Describe the control of Newcastle disease
- Biosecurity
- Slaughter in event of sever outbreak with velogenic virus
- Vaccinaion in most commercial flocks
- Live attenuated and inactivated
55
What viral protein of Coronaviruses is important in mediating binding of TGE virus to a target cell?
Spike rpotein
56
Describe the binding and entering of rotaviruses to their target cells
- VP4 activated by trypsin (cleaved) to activate the virus, promotes cell entry
- VP8 adn VP5 remain non-covalently associated
- Cell entry multistep process
- Cleaved VP4 binds to sialic acid, integrins and other surface proteins in order to gain entry
57
Explain the role of the NSP4 viral protein in the pathogenesis of diarrhoea in rotaviruses
- Disrupts tight junctions
- Enterotoxigenic function
- Inhibits Na+-D-glucosesymport activity of the sodium-glucose linked transporter protein SGLT1
- Allows paracellular flow of waer and electrolytes
58
Give a basic outline of how Salmonella infects the host animal
- Initial interaction of Salmonella with enterocyte
- Bacterial internalisation
- Intracellular survival
- Dissemination in the host
59
Describe the initial interaction of Salmonella with enterocytes
- Salmonella interacts with enterocyte
| - Delivers Sop proteins into cell cytoplasm via TTSS-1 adn Sip dependent pathway
60
Describe Salmonella internalisation
- Promotes own invasion
- Sip, SopE and other Sops induve enterocyte membrane ruffling
- Promotes bacterial invasion
61
Describe Salmonella intracellular survival
- Bacteria reside within membrane bound vesicles
- Continue translocation of TTSS-1 secreted effeectors
- Replication of Salmonella within veiscles promoteed by TTSS-2
- Intracellular SopB protein affects inositol phosphate signalling events
- Transient increase of Ins(1,4,5,6)P4 can antagonise closure of chloride channels, influencing net electrolyte transport (and so fluid secretion)
- Salmonella infected cells secrete chemokines and prostagalndins, recruit inflam cells to foci of infection
- Release of some chemokines and prostaglandins affected by intracellular activity of Sops
- Salmonella interacts with inflammatory cells, stimulates release of proinflammatory cytokines, enhance inflammatory response
62
Describe Salmonella dissemination in the host
- Salmonella infected epithelial cells release pathogen elicited epithelial chemoattractant (PEEC) cross apical membrane
- Stimulates PMN transepithelial migration between enterocytes
- Infiltrating inflam cells phagocytose Salmonella, fate of tehse unclear
- Salmonella infected enterocytes become extruded from the villus, shedding of infected cells into intestinal lumen resulting in villues blunting and loss of absorptive surfaces
- Some infected cells migrate to drainig lymphatics, carrying Salmonella to systemic sites
63
What is the function of type 1 TTSS?
Used for invasion of cells by Salmonella
64
What is the function of type 2 TTSS?
Used for survival and replication of Salmonella within vesicles
65
What are SOPs?
- Salmonella outer proteins
- Effector proteins function to alter host cell physiology and promote bacterial survival in host tissues
- Contribute to formation of Salmonella induved filament formation
66
What are SIPs?
- Salmonella invasion proteins
- Translocated into cell by TTSS-1, bind to actin, stimulate actin polymerisation and counteracts F-actin destabilising proteins (SipA)
67
Explain the difference between S. gallinarium and S. typhimurium and poultry
- T not host adapted, can get subclinical infections
- G similar to Pullorum
- Lesions throughout lungs and focal necrosis of liver and spleen
- Sudden deaths
- Characteristic findings: enlarged, friable, bile-stained liver and enlarged spleen
68
Hwo does Salmonella survive inside cells?
- Formaiton of Salmonella infuced filaments
- Play role in intracellular replication
- Effector proteins protect intracellular organism from bactericidal compounds e.g. reactive oxygen intermediates
- Protect from phagolysosome fusion
69
Why has the incidence of transmissible gastroenteritis virus decreased?
- Reduced stocking density
- Improved hygiene
- Vaccine available (reduces incidence, will not eliminate)
70
How may diarrhoea be caused?
- Single strains of virulent pathogen
- Mixed infections
- Toxins
- Response of host to pathogen or irritants
- Disruption to nromal physiology
- Direct effects of the pathogen
- Stress e.g. shipping fever
71
What is the role of diarrhoea?
- Removal of infection
- Stops build up of pathogen/toxins
- May aid dissemination of pathogen
- Product of mucosal damage/effects
- Combination of all
72
In bacterial infections, what may cause dsisease?
- Adherence
- Secretion of exotoxin
- Invasion of target cells leading to cell death
- Invasion of host cells and lymphatics leading to systemic disease
- Triggering of inflammatory response
73
How to type III secretion systems work?
- type 1 and 2
- 1 used for invasion
- 2 used for intracellular survival
- Tubes that span inner and outer membrane of host cell
- Inject effector proteins through them
74
How does viral infection cause disease?
- Attachment
- Invasion
- Replication
- Release of virus
- Immune response to either viral infection or cell damage
- Macrophage migration to the area
75
What are the pathogenic mechanisms of diarhhoea?
- Hypersecretion
- Villous atrophy
- Infiltrative and proliferative distortion of mucosa
- Mucosal necrosis
- May be combination or partial effects
76
Describe how hypersecretion leads to diarrhoea
- Functional disturbance of intestinal epithelial cells
- Increased outflor or decrease in reabsorption of water
- ETEC
- Water moved into intestines thorugh specific action of crypt cells
77
Describe the specific action of crypt cells in hypersecretion diarrhoea
- Pump Cl- into crypt space
- Crypt cells actively draw water into intestine
- Cl- ions attract Na+ into crypt space, increases local osmotic pressure
- increased osmotic pressure, water pulled into intestine
- Some enterotoxins can trigger this
- Hypersecretion of water
- Lock system ON
78
How can villous atrophy cause diarrhoea?
- Destruction of epithelial cells on villi or in crypts
- Leads to malabsorption
- More water present in lumen
- Villi stunted or fused
- Attaching and effacing E coli, rotavirus, canine parvovirus
79
Describe how infiltrative and proliferative distortion of mucosa can lead to diarrhoea
- Chronic progressive immuno-inflammatory disease of adult ruminants
- Packed with cells, lose villous distinction
- Mucosa distorted
- Flat surface rather than villi, lose surface area
- Unable to resorb water
- Recruitment of large numbers of macrophages and T-lymphocytes in lamina propria and sub-mucosa
- Crypt compression and villous distortion
- Reduce absorptive capacity
80
Describe how mucosal necrosis can lead to diarrhoea
- Infection leads to cell death
- Secerity and extent depends on virulence of pathogen
- Majority of diarrhoea from reduced absorption
- Brachyspira hyodysenteriae, Clostridium perfringens typeC, some necrotoxigenic species fo E. coli and Salmonella
81
Describe Lawsonia
- Curved Gram -ve rod
- Obligate intracellular pathogen
- Microaerophilic
- Cannot be grown on inert media, requires growth in enterocytes
- Implicated in proliferative enteropathy f pigs and foals
- Diagnosis by clinical signs, demonstration in mucosa or faeces by immunofluorescence or PCR, staining of tissue sections, serological tests
82
What can molecular techniques be used for in veterinary medicine adn research?
- Pathogen detection
- Epidemiology
- Diagnosis of genetic diseases
- Quality trait loci (meat/wool quality)
- Forensic
- Public health (food testing for animal protein etc)
83
List some importnat molecular techniques used in veterinary diagnostics and surveillance
- Quantitative PCR
- Single nucleotide polymorphisms
- Variable numbers of Tandem Repeats
- Restriction Fragment Length Polymorphisms
- Random Amplification of Polymorphic DNA
- Multi Locus Sequence Typing
84
Outline the use of vectors in recombinant DNA technology
- DNA (gene) cloning
- Isolate and specifically amplify copies of a unique piece of DNA
- Amplify targeted pieces of DNA using PCR
- Specifically and reproducibly cut DNA into defined pieces (restriction enzymes)
- Joing individual pieces of DNA together (DNA ligase)
- Replicate specific pieces of DNA (cloning vectors derived from plasmids of phage)
85
What are important feature of a plasmid to be used for DNA cloning?
- Plasmid should have lots of different sites that restriction enzymes can be used at
- Want to cut and slot into a specific place
86
What steps are needed to amplify genes other than dsDNA by PCR?
- mRNA is expressed part of genoome at any one time
- Cannot be cloned directly
- Complementary DNA (cDNA) strand produced to allow integration into dsDNA plasmid
- From mRNA make complementary ssRNA stranf
- Destroy original mRNA strand
- Then make complementary to the first ssRNA strand
87
What are the important features of a cloning vector?
- Efficient means of introducing vector inot host cell (transformation, conjugation)
- Vector must be able to replicate within host cell so that many copies of the vector can be obtained
- Means to isolate vector DNA away from host DNA
- Restriction enzymes in vector that can be used for cloning an insert DNA
- Selectable marker on vector to indicate presence of vector in host cell
- Unique restriction enzyme sites within selectable or screenable marker useful
88
Describe bacteriophage derived vectors for DNA cloning
- Similar to plasmids
- Carrier molecules for DNA
- Can carry larger pieces of DNA
89
Describe M13 based vecors for DNA cloning
- Single stranded circular DNA
- Infection via F-pili
- Converted to replicative form after infection (dsDNA)
90
Describe cosmid vectors for DNA cloning
- Plasmids that contain cohesive ends of gamma phage and gamma DNA sequences required for packagin DNA into gamma particle
- Plasmid origin of DNA replication
- At least one selectable marker
- Unique restrictoin enzyme sites for insertine foreign DNA
- Can be used to clone larger pieces of foreign DNA
91
Describe YAC vectors for DNA cloning
- Yeast artificial chromosomes
- 2 copies of yeast telomere, yeast centromere, yeast ARS and appropriate selectable markers and unique restriction enzyme recognition sites
- Allows cloning in eukaryote
- Permits cloning of very large pieces of DNA (megabase range)
- Some problems with insert instability
92
Describe BAC vectors for DNA cloning
- Bacterial artificial chromosome
- Based on F-plasmid
- Permits cloning of up to 300kb inserts
- More stable than YACs
93
Describe single nucleotide polymorphisms
- Polymorphism: Having multiple alleles of a gene within a population, usually expressing different phenotypes
- SNP: site in DNA occupied by different nucleotide pair among significant fraction of individuals in a population
- e.g. susceptibility to scrapie
94
Describe QPCR
- Quantitative PCR (real time)
- Amplify and quantify targeted DNA molecule by measuring fluorescent signal at each PCR cycle
- Use of dsDNA dyes
- Use of fluorescently labelled probes (or TaqMan probes)
- Multiplex qPCR
95
Describe the use of dsDNA dyes in qPCR
- Bind to all dsDNA in PCR, causes fluorescence of dye
| - Intensity of fluorescence proportional to amount of dsDNA (not specific to one PCR product)
96
Describe the use of fluorescently labelled probes or TaqMan probes in qPCR
- Based on properties of Taq DNA polymerase (DNA polymerase, 5'-3' exonuclease - cleavage of dye labelled probes during PCR)
- Intenstiy of fluorescence proportional to number of PCR products
- More expensive but probe specific to target DNA
97
Describe multiplex qPCR
- Diagnostic test virulent/benign D nodosus
| - Muliple fluorescent probes in same assay
98
What are the applications of qPCR?
- Pathogen detection
- Quantification of gene expression
- Detection of gene mutations
99
Describe Variable Number of Tandem Repeats PCR technique
- Location in genome where short nucleotide sequence organised as tandem repeat
- Tandem repeat is patten of 2 more nucleotides repeated and repetitions are directly adjacent to each other
- Between areas that code for proteins there are regions with little importance - repeat nucleotides
- Often show variations in length between individuals
- Generally identified by PCR
- DNA finger printing
100
Describe Restriction Fragment Length Polymorphisms
- RFLP
- Identification of differences in seuence when they occur in or near a restrictoin site
- Presence or absence of restriction site
- E.g. genotyping scrapie genotypes
101
Describe Random Amplification of Polymorphic DNA
- RAPD
- Genotyping approach
- Sequence info not required
- DNA fragments from PCR amplification of random segmented of genomic DNA with single primer of arbitrary nucleotide sequence
- 10-mer primers will or will not amplify segment of DNA depending on positions thatare complementary to primer's sequence
- Mutation affedting primer binding will not produce PCR
- Different pattern in gel
102
Describe Multi Locus Sequence Typing
- MLST
- Classify bacterial/fungal isolates
- Epidemiolgoical studies/outbreak tracing etc
- 7 housekeeping genes
- Each strain has them
- Allelic profile
- If sequence different to all others, considered new allele and given new allele number
- Each unique allelic profile give one digit sequence type (ST)
103
What is meant by the term genome?
The complete set fo genes or genetic material present in a cell or organism
104
What is the coding region of a genoome?
The portion of a gene's DNA or RNA that is composed of exons (only a few % of total genome)
105
What is the non-coding region of a genome?
- Involved in regulating activity of coding regions
| - Much of this has no known function
106
What are the repetitive sequences of a genome?
- Repeated base sequences
- Most likely due to incorporation of invasive elements such as DNA transposons, retrotransposons and other repetitive elements
107
What are pseudogenes?
Defective copies of functional genes (truncated, fragments)
108
What are the transposable elements of a genome?
- Sequences of DNA that can move around to different positions within a genome
- Consists of DNA transposon, LTR-retrotransposons and non-LTR-retrotransposons
- Integrate into adn replicate as part of genome via transposition
- Interspersed reptitive non-coding DNA
- Can induce mutations by inserting near or within genes
109
Describe the mitochondrial genome
- Single circular chromosome
- Not all proteins necessary for mitochondrial function encoded by mitochondrial genome
- Most coded by genes in cell nucleus and corresponding proteins imported into mitochondrion
- Divide by binary fission
- Comes from egg only
110
Explain how genes are identified in genomic data
- Genetic mapping of genome
- Based on genetic techniques to construct maps showing positions of genes
- Cross breeding examination of historied
- RFLP, SSLP, SNP
- Restriction maps f genome and fluorescent in situ hybridisation
- Identify genome promoter, operator sequences, start codons, genes, stop codons, mutations, polymorphisms, repeats
111
What is a transcriptome?
- RNA copies of the active protein coding genes
- All the RNA present in a tissue (or cell) at any one time
- Very little of genoome actually encodes for proteins
- Most of it is transcribed
112
Describe RNAi
- RNA interference
- RNA mediated silencing of translation
- double stranded areas of RNA processed to small RNA molecules
- bind by base pairing to target nucleic acid
- Direct several effector proteins to this nucleic acid
- Protein containing complex named RNA induced silencing complex (RISC)
- Usually results in inhibition of transcription or translation
113
What is meant by epigenetics?
- Heritabe changes in genome function that occur without alteration to DNA sequence
- Methylated DNA best studied epigenetic mark
- Present in animal's offspring
- Not all inheritance if medelian
- Stressors across animals lifetime result in changes in methylation pattern
- Epigenetic marks may be lost in cancerous cells resulting in uncontrolled replications
- Methylation adn epigenetics very dynamic process
114
What is meant by maternal/genomic imprinting?
- Pattern of epigenetic marks you inherit from your parents
- Most epigenetic marks erased during embryogenesis
- Ones left over imprinted
- Erasure and resetting important in returnign cell to stem cell state
115
What is pre-mRNA
The transcribed gene including introns and exons
116
What is mRNA?
Messenger RNA, genes with introns spliced out
117
What is rRNA?
Ribosomal RNA, structural component of ribosome. 80% of RNA in the cell
118
What is tRNA?
Transfer RNA, adaptor molecule in protein translation
119
What is miRNA?
Micro RNA, regulates mRNA stability, translational role
120
What is piRNA?
Piwi interacting RNA, transposon regulation
121
What are CisNATs?
- Cis-natural antisense transcripts.
| - Modulate transcript levels
122
What is snRNA?
- Small nucelar RNA
- Aka uRNA
- Role in pre-mRNA splicing
123
What is sno-RNA?
- Small nucleolar RNA
| - Role in rRNA and tRNA maturation
124
Describe the mechanisms of RNA mediated control of transcription and translation
- RNAi: inhibits transcription or translation
