MGD Sessions 7-11 Flashcards
What can be said about the specificity of bacterial endonucleases?
High
What do bacterial endonucleases commonly recognise and cleave?
Palindrome restriction sites
Which bond of the DNA sequence is cleaved by a bacterial endonuclease?
Phosphodiester
What are ‘sticky’ cuts?
Staggered cuts made by endonucleases which anneal w/complementary sequences to the overhang
What are ‘blunt’ cuts?
Double stranded ends created by endonucleases
How do bacteria protect their own DNA?
Methylation to block restriction enzymes
What is the general rule for the number of restriction sites present where n is the number of bases in the restriction site?
n^4
What is DNA ligase?
Enzyme that creates phosphodiester bonds b/w any sequences that have the same overhang or complementary blunt ends
Why will DNA move towards the anode in an electric field?
Phosphate group makes it negatively charged
What characteristic does DNA gel electrophoresis use to separate DNA fragments?
Size (also shape when plasmids-sorry!)
What are the 4 requirements for DNA gel electrophoresis?
Gel
Buffer
Power supply
Stain/detection
What type of plate is used in DNA gel electrophoresis to investigate long DNA w/larger size differences?
Agarose
What kind of plate is used to investigate DNA fragments with only one nucleotide differences?
Polyacrylamide gel
Why is restriction analysis used?
Clone DNA
Investigate mutations
Investigate size of DNA fragments
Investigate DNA variation
What can be used to identify the point mutation of one nucleotide as seen in sickle cell disease?
Restriction analysis
Why is a buffer needed in DNA gel electrophoresis?
Allows charge on the DNA samples across the gel
Describe how gene cloning is carried out.
Choose plasmid vector w/antibiotic resistant gene –> cut plasmid and gene of interest w/same restriction enzyme –> ligate –> recombinant DNA molecule –> transformation –> secreted by bacterial cells
What is the importance of the antibiotic resistance gene in the plasmid vector for gene cloning?
Can introduce antibiotic to see which bacteria have undergone transformation and taken up the recombinant plasmid
How is proinsulin synthesised by bacteria?
Mammalian proinsulin mRNA from pancreas –> reverse transcriptase –> proinsulin cDNA –> joint to plasmid –> recombinant plasmid –> infect E. Coli –> transformed bacterium synthesises proinsulin
Why do we clone human genes?
Gene therapy Make useful proteins Find out what genes do Genetic screening Smaller initial sample needed for analysis
What happens in the first stage of PCR?
Heat to 95 degrees
Add 2 oligonucleotides unique to region to act as primers
Add heat stable Taq polymerase
Why are oligonucleotides used in PCR to act as primers?
Small pieces anneal easier
Which direction do the primers in PCR work?
5’ –> 3’
What happens to the temperature of the PCR during the second stage?
Cooled to RT
How does DNA renature in PCR?
Complementary bases realign upon cooling
Why is PCR used?
Amplification
Investigate variation/genetic relationships
Identify small deletions/insertions
Investigate single base mutations
In which direction will proteins move if placed in an electric field?
Towards anode or cathode
What properties can be used to separate proteins?
Size
Shape
Charge
What four things are required for protein electrophoresis?
Gel
Buffer
Power supply
Stain/detection
Why must separation of proteins by electrophoresis be further tested?
Relies on intrinsic properties of proteins so can be attributed to folding or other factors which need to be narrowed down
Which characteristic of proteins does SDS page investigate?
Size
How does SDS-PAGE only consider one factor affecting protein separation?
Protein denatured to remove tertiary and secondary structure
SDS binds to give standard overall -ve charge
How are proteins separated by isoelectric focusing?
Uses basis of charge
How is isoelectric focusing carried out?
Stable pH gradient established in vertical tube w/electric field applied –> protein added and moves until it reaches pI –> stain
What is 2D-page?
IEF put on top of SDS-polyacrylamide slab which allows separation of complex protein mixtures
What is proteomics?
Study of proteins
What are the 5 stages in proteomics?
Digest protein w/trypsin Perform mass spectrometry Generate list of peptide sizes Use database of predicted peptide sizes for known proteins Identify protein
What method can be used for protein analysis but has fewer options than for DNA electrophoresis?
Specific cleavage of proteins
What does enzymatic cleavage recognise?
Proteins after certain amino acids
How many segments would you expect to see in proteolysis of normal haemoglobin when using Endo-Arg-C?
4
What method of protein analysis can cyanogen bromide and hydroxylamine be used for?
Chemical cleavage
What an epitope?
Name given to a.a. an antibody can bind with
How does the production of polyclonal and monoclonal antibodies differ?
Polyclonal from many B lymphocytes
Monoclonal from 1
How many different antibodies are there in monoclonal and polyclonal antibodies?
Monoclonal = 1 identical Polyclonal = multiple different
Are monoclonal or polyclonal antibodies specific to one antigen?
Both are
How many epitopes does a polyclonal antibody bind to?
Multiple
How many epitopes does a monoclonal antibody bind to?
1
How are monoclonal antibodies made using mice?
Inject mouse w/antigen 3-4 times at 2 week intervals
Mouse produces antigens which can be found in its blood
How are monoclonal antibodies formed using mice?
Fuse mouse spleen cells and myeloma cells
Select and grow either in vivo/vitro
Why are myeloma cells used to make monoclonal antibodies?
They are immortalised
What is the end product of Western blotting?
Immunoblot
What happens in Western Blotting?
Nitrocellulose replica of gel electrophoretogram
Bind primary antibody
Wash
Labelled enzyme-linked secondary antibody bound
Immunoblot
What is ELISA?
Enzyme-linked immunoabsorbent assay
What happens between each stage of binding in ELISA?
Wash
Describe the stages in ELISA.
Antigen coated well
Specific antibody binds to antigen
Enzyme linked antibody binds to specific antibody
Substrate added
What is proportional to antibody level in ELISA?
Rate of colour production
What indicates how many antigens are present when carrying out ELISA?
How much antibody is needed
What does a radioimmunoassay use in conjunction with the ELISA method to measure protein concentration in a solution?
Radiolabelled primary antibody
What do enzyme assays measure to assess if an enzyme is present and if so, how much?
Appearance of product/disappearance of substrate
What are two methods of continuous enzyme assay?
Spectrophotometry
Chemoluminescence
What are two methods of discontinuous enzyme assay?
Radioactivity
Chromatography
Why would an enzyme assay be used to measure serum enzymes?
Suspected metabolic disorders
Give examples of some serum enzymes that can be assayed for.
Alkaline phosphatase - bone
Gamma-glutamyl transferase - liver
Cardiac troponin I - heart (gold standard)
Amylase/lipase - pancreas
What can enzyme activity be used to measure?
Clinically important metabolites
What do bacteria produce that recognise and degrade foreign DNA which enters the cell?
Endonucleases
Is Western blotting a DNA hybridisation technique?
Nope
What is found in the 4 tubes used for Sanger Chain DNA hybridisation?
ssDNA template
dNTPs
DNA polymerase
One ddNTP per tube
What happens in the Sanger chain method of DNA hybridisation?
4 tubes with one ddNTP per tube
Incubate at 37 degrees Celsius
Separate lanes for separate tubes on DNA electrophoresis
How can a chromatogram be used for Sanger chain?
Fluorescently labelled ddNTPs detected by laser as they fall off thing capillary gel
What is the Sanger chain method useful of?
Easy and quick sequencing of large genes
What does Northern blotting use to detect RNA species after gel electrophoresis?
DNA
Why are dideoxynucleotide triphosphates used in DNA hybridisation?
Have a H at 3’ end so are a substrate of RNA polymerase but prevent elongation
Do DNA probes used for DNA hybridisation have to completely align to target sequence?
Nope, partial overlap at one end is enough
Why do probes not have 100% similarity to target sequence?
They would bind too tightly
What can ssDNA be labelled with to be used as a probe for DNA hybridisation?
Radioactive or fluorescent marker
What does Southern blotting use to identify DNA sequences after gel electrophoresis?
DNA probes
How is Southern blotting carried out?
Transfer DNA fragments to nylon/nitrocellulose
Hybridise filter w/labelled gene probe
Detect hybridisation by exposing filter to X-Ray film
How are the DNA fragments from gel electrophoresis transferred in Southern blotting?
Alkaline solution
DNA –> ssDNA
Capillary action onto filter
Voltage applied causing DNA to move
Why use Southern blotting?
Allows detection in v. small amounts of DNA Investigate gene structure Identify mutations in genetic tests Gene expansions Variation/genetic relationships
How would you use Southern blotting to test for SCD?
Use allele specific probes
How does the DNA examined in Southern blotting differ to that which is the end product of the preceding gel electrophoresis?
dsDNA after gel electrophoresis
ssDNA in Southern blotting
What do antibiotics that inhibit bacterial cell wall synthesis have in their molecular structure?
Contain beta-lactam ring
What is the function of glycopeptide transpeptidase?
Cleaves peptide bond b/w D-alanine residues for cross linking in cell walls
What can bacteria produce in order to be resistant to beta-lactams?
Beta-lactamase to break ring
How does mec-A gene expression allow for beta-lactam resistance?
Provides different, lower affinity penicillin binding protein so transpeptidase is unaffected
How can bacteria acquire resistance to beta-lactams via vertical transmission?
Mitosis
How can bacteria acquire resistance to beta-lactams via horizontal transmission?
Conjugation tube and resistant plasmid
Transformation
Transduction (bacteriophages)
How do beta-lactams have specificity?
Only peptidoglycan is not X-linked so only bacterial cell walls are affected
Give an example of a clinical use of narrow spectrum beta-lactams.
Antistaphylococcal penicillin
Give an example of a clinical use of broad spectrum beta-lactams.
Aminopenicillin
Natural penicillin
What is erythromycin an example of?
Antibiotic that inhibits bacterial protein synthesis
Why can erythromycin be used instead of penicillin in patients who are allergic to penicillin?
Spectrum is similar
What is the mechanism of action for antibiotics that inhibit bacterial protein synthesis?
Binds to large subunit so prevents translation by stopping tRNA moving along chain - no transfer from A to P
Does a usual dose of erythromycin kill bacteria?
Nope, prevents protein replication
How do bacteria adapt to become resistant to antibiotics that inhibit bacterial protein synthesis?
Methylated 50S subunit so drug cannot bind
How do bacteria acquire resistance to antibiotics that inhibit bacterial synthesis?
Random genetic mutation passed on by vertical transmission
How do antibiotics that inhibit protein synthesis have specificity?
Bind to 50S subunit which is specific to prokaryotic cells
What is the clinical use of antibiotics that inhibit protein synthesis?
Upper and lower RT infections
STDs
Off-label for gastric emptying
What mechanism of action do rifamycins use?
Inhibits bacterial transcription
How can antibiotics inhibit bacterial transcription?
Penetrate cell wall –> allosterically blocks beta-subunit of RNA polymerase –> elongation inhibited
Why are antibiotics that inhibit bacterial transcription broad spectrum?
Their RNA polymerase target is similar in lots of prokaryotes
How do bacteria become resistant to antibiotics that inhibit their transcription?
Mutations in beta-subunit so RNA polymerase cannot bind
How can bacteria have different levels of resistance to antibiotics that inhibit their transcription?
Different point mutations give different levels of resistance
How do bacteria acquire resistance to antibiotics that inhibit their transcription?
Spontaneous mutations
Naturally non-susceptible
Multi/cross resistance
What is multi/cross resistance?
If a bacterium has developed resistance to an antibiotic it is likely to be resistant to a similar antibiotic
How do antibiotics that inhibit bacterial transcription have specificity?
Mammalian cells have 5-subunit RNA polymerase complexes so it is not affected
What are clinical uses of antibiotics that inhibit bacterial transcription?
Readily absorbed in GI tract for TB, leprosy and other mycobacteria
What effect does rifamycin have on urine?
Colours it red
Give an example of an antifolate.
Methotrexate
Why mechanism of action does methotrexate use?
Competitively inhibits DHFR –> inhibits purine and pyrimidine biosynthesis –> no DNA, RNA or protein synthesis
What cellular effects do antifolates in chemotherapy cause?
DNA, RNA and protein synthesis inhibited Impaired cellular transport Reduced polyglutamate formation in CSM Increased drug efflux Increased levels of gamma-glutamyl hydrolase
How can a cell be resistant to antifolates?
Inherent/primary/acquired drug resistance
How can a cell have an acquired resistance to antifolates?
Transformation via vertical transmission
Transduction
Conjugation
How does somatic evolution allow for acquisition of antifolate resistance?
Causes higher genomic instability
Do antifolates have specificity?
Nope
Why is methotrexate not specific?
Mammalian and bacterial cells have highly similar DHFRs
What can low doses of antifolates be used for clinically?
Autoimmune disease e.g. RA and uncontrollable psoriasis
As well as being a competitive inhibitor for DHFR, how else do antifolates inhibit cell division?
Cofactor to methyltransferases which inhibit cell division
What is the active form of dihydrofolate which is not seen in cells susceptible to anitfolates?
Tetrahydrofolate
What are ethical consideration with genome sequencing?
Who would be interested in the genome - insurance?
Can the knowledge help prevent illness later in life?
Does it open up areas for discrimination?
Does the scientist or the participant own the sequence?
What types of primer must you have for DNA hybridisation in PCR?
Forward and reverse
Do both forward and reverse primers act 5’ to 3’ in PCR?
Yes
What happens if there is a mismatch between the 5’ end and primer in PCR?
Not critical reduction in how complementary it is
What happens if there is a mismatch between the 3’ end and primer?
Elongation is prevented
What does Northern hybridisation analyse?
RNA
Why is a copy of mRNA made regardless of sequence in reverse transcriptase PCR?
All mRNA have a poly-A tail
How does the cDNA formed in reverse transcriptase differ from genomic DNA?
Has no introns
What is real time PCR?
Fluorescent PCR products show how much is made in real time
How does reverse transcriptase PCR analyse RNA?
Lots of template indicates high levels of mRNA expression
What does microarray analyse?
1000s of genes at once
What tail is seen on the cDNA strand formed in reverse transcriptase PCR?
Poly-T
If a gene is only expressed in healthy tissue how will it appear in microarray?
Green
How do genes unique to cancer tissue appear in microarray?
Red
What is used to fluorescently label the genes in microarray?
Reverse transcriptase labelling
How does the resultant microarray indicate the genes present in a tissue?
Red and green dots appear in proportion to how much each tissue type expresses
Describe the process of microarray analysis.
Extract DNA Label w/2 fluorochromes Mix in equal quantities Read red and green fluorescence Work out red:green ratio and align to database
What does a green spot indicate in array comparative genome hybridisation?
Deletion
What does a red spot on array comparative genome hybridisation indicate?
Duplication
What is carried out after the first run through of array comparative genome hybridisation?
Dye swaps
Why use array technology?
Investigate 1000s of genes simultaneously
Investigate conditional gene expression
Investigate chromosome duplications/deletions
How many regions of DNA does DNA fingerprinting consider?
1
What shows copy number in DNA fingerprinting?
Mini satellites
What shows copy number variation in DNA profiling?
Small tandem repeats
Which gene technology does DNA profiling use?
PCR
Describe how karyotyping is carried out.
Arrest cells in mitotic metaphase
Isolate nuclei onto slide and stain chromosomes
Obtain pictures of chromosomes
Pair homologous chromosomes
How does karyotyping identify translocations, trisomies and monosomies?
Stain appears in places it isn’t expected
How is FISH carried out?
Prove DNA –> label w/fluorescent dye –> denature and hybridise
What is FISH?
Fluorescence in Situ Hybridisation
What indicates a difference in FISH analysis?
Absence so one of probe pair not seen
Why is FISH used?
Investigate chromosome number
Investigate chromosomes behaviour
Investigate chromosome structure
What aspects of chromosome structure can FISH analyse?
Translocations
Deletions
Duplications
What aspects of chromosome behaviour can be investigated by FISH?
Anaphase lag - fails to connect spindle/not included in reformed nucleus
What ethical considerations are there with using DNA analysis?
Abortion
Autosomal dominant with late onset: do older generations know/want to know they’re positive
Why are most mutations of the genome of little/no consequence?
Protein coding regions only make up 1-2% of all the DNA
Mutations at what location in the DNA are most likely to cause disease?
In or close to protein coding genes
What is the most common genetic mutation?
SNPs (snips) - single base substitutions
What base substitution makes up ~2/3 of SNPs?
C –> T
What is a transition base substitution?
Purine purine
Pyrimidine pyrimidine
What is a transversion single base substitution?
Pyrimidine purine
What base change forms the basis of the vast majority of heritable diseases?
CG –> TA
What is a silent genetic mutation?
Where there is no a.a. change
What happens if there is a single base substitution in the splice site?
Disrupts RNA splicing
What is a missense genetic mutation?
1 a.a. is substituted for another
What usually causes a missense mutation?
Single base change
Where do single base changes usually occur in the codon so that the substitution is silent?
3rd base
What is a nonsense genetic mutation?
a.a. to stop codon
What effects can a frameshift mutation have?
Reading frame altered
Premature translation stop
What genetic mutations can cause a frameshift mutation?
Insertions
Deletions
Splice site mutations
Stop codon mutation
What is conservative missense?
a.a. substituted with adjacent a.a. in the genetic code table which limit is the damage caused by mutation
How can small insertions or deletions affect the reading frame in genetic expression?
Multiples of 3 maintain
Non-multiples of 3 cause frameshift and premature termination codons
What are mRNAs with PTCs degraded by so little truncated or no protein is made?
Nonsense mediated decay
What happens if there is a mutation in an intron splice site?
Intron not present in mRNA
Skip exon immediately adjacent to mutation
What can occur during DNA replication to cause base changes?
Rare tautomer forms w/altered base-pairing
DNA strand slippage
How can chemicals cause base changes?
Direct DNA base alteration
Disruption of DNA base stacking
How can we be exposed to radiation which may cause base changes?
Exposure to UV light
Radioactive substances
What briefly changes position and allows a brief change in base-pair method?
Proton
What happens if the newly synthesised strand loops out due to imperfect base pairing during replication?
Addition of one nucleotide on the new strand
What happens if the template strand loops out during replication?
Omission of one nucleotide on new strand
What effect does nitrous acid have on nucleotide bases?
Replaces amino groups w/keto groups
How are the C, A and G bases altered with exposure to nitrous acid?
C –> U
A –> H
G –> X
What do the resultant U, A and H bases seen in nitrous acid exposure base pair with?
U w/A
H w/C
X w/C
What does ethyl methane sulphate cause removal of so that 25% of base pairing is right?
Purine rings so apurinic sites can be paired w/any base during replication
What type of molecule is identified in cooked meats and cigarette condensates which disrupts packaging of DNA bases and is a base stacking mutagen?
Heterocyclic aromatic amine
How can a heterocyclic aromatic amine cause a single base deletion?
Forces bases further apart on DNA strand so DNA polymerase misreads
Which cheap and effective DNA stain works in a similar way to the heterocyclic aromatic amine seen in cooked meats and cigarette condensates?
Ethidium bromide
What is the effect of ionising radiation on interaction w/cellular molecules?
Produces ions
What affect do UVA and B have on the skin?
Destroy vitamin A
What affect do UVA, B and C have on the skin?
Damage collagen fibres
What induces vitamin D in the skin?
UVB
What action do UV light photons have on DNA?
Cause adjacent thymine bases to base pair with one another rather than across helix - thymine dimer formation
How is thymine dimer formation usually resolved?
Spontaneously by photo-reactivation
How are most errors in DNA corrected?
Proofreading as polymerase detects mis-paired 3’ base in new strand
What are the two types of DNA repair?
Nucleotide mismatch
Excision
Give a brief description of nucleotide mismatch repair.
Reduces error rate further still after replication
Enzymes detect mismatched bases and replace them
Patch of DNA sequence replaced
What types of damaged bases can be accumulated by DNA in excision repair?
Oxidised bases
Alkylated
Uracil
De-animated
What is short patch repair?
Single nucleotide change in excision repair
What is long patch repair?
Multiple nucleotide change in excision repair
Which human genes encode mismatch repair enzymes?
MLH1
MSH2
MSH6
What are commonly mutated in cases of non-hereditary non-polyposis colorectal carcinoma?
Genes encoding mismatch repair enzymes
What do all cancer cells exhibit?
Chromosomal instability Microsatellite instability (unexpected expansion and contraction)
The probability of picking up 6 successive mutations necessary to cause tumour is very low so why do we see cancer?
Early mutations affect functions which raise the probability of successive mutations
What are BRCA1 and 2 involved in?
Detecting DNA damage and signalling cell-cycle checkpoints
What do sporadic breast tumours rarely have which is commonly seen in inherited breast tumours?
BRCA1 or BRCA2 mutations
How common are affected males in families affected by breast cancer?
Fairly
What are oncogenes?
Specific retro-viruses assoc. w/cancers
What do retrovirus studies show about oncogenes?
Contain genes that are able to transform cells to cancerous phenotype
What are human genes that exhibit sequence similarity to viral oncogenes called?
Porto-oncogenes
What functions do proto-oncogenes performs?
Range of cell cycle
What change can activate proto-oncogenes into dominantly acting cancer causing oncogenes?
Key a.a. substitutions
What mutations do inherited cancer genes tend to harbour?
Recessive
What type of inheritance does development of cancer display?
Dominant
What does initiation of a tumour require at the allele level?
Mutation to both copies or deletion of functional copy
Compare the progression of inherited and sporadic cases of cancer.
Inherited: all cells carry one mutation –> 2nd mutation –> tumour founder cells w/ 2 mutations
Sporadic: somatic mutation in one cell –> 2nd mutation –> tumour
Which type of cancer is more rare, hereditary or sporadic?
Sporadic
How can homozygous its arise?
Loss of wild-type chromosome
Deletion
Point mutation
Mitotic recombination
What is needed to b e able to advise relatives following a positive sweat test indicating CF?
Molecular test
Is it possible to be a compound heterozygote for two different genetic mutations?
Yes e.g. 2 CTFR mutations in CF
What is the most common genetic mutation that causes CF?
Delta F508 3 base pair deletion causing deletion of phenylalanine
What method of genetic analysis will give a mix of normal and mutated sequences following PCR if the individual is heterozygous for a mutation?
Single-strand conformation polymorphism
What change can be detected by silver staining in single-strand conformation polymorphism?
Single base mutation
What are the three methods of obtaining foetal DNA?
Amniocentesis
Mother’s blood
Chorion villus biopsy
When is amniocentesis carried out?
15-20 weeks
What type of cells are sampled by amniocentesis and can be cultured for 2 weeks?
Non-dividing
Which method of obtaining foetal DNA carried the highest miscarriage risk?
Chorion villus biopsy
How is chorion villus biopsy performed?
Trans-cervical
Trans-abdominal
When is chorion villus biopsy performed?
10-13 weeks
What must be separated from maternal tissue in chorion villus biopsy?
Foetal villi
What does multiplex ligation-dependent probe amplification (MLPA) principle perform?
Exon count
How does MLPA principle work?
It’s ability to ligate 2 single strand together only if target DNA is still present in sample
Why can PCR not be used for some inherited disorders such as osteogenesis imperfecta or Duchenne muscular dystrophy?
Arise through whole exon duplications or deletions so PCR gets product from remaining whole exons
What probes are used in MLPA principle?
Two adjacent for forward and reverse primers
One has a ‘stuffer’ region of any length and no relation to target sequence
What is the purpose of the ‘stuffer’ region in either the forward or reverse primer used in MLPA principle?
Varies fragment length for analysis by electrophoresis
How does the electrophoresis graph for MLPA principle show deletions in one gene?
Peaks half height of those in control
How are the highest order structures of DNA stabilised?
By hanging loops of DNA onto protein scaffold
What is epigenetic modification?
Switching of chromatin b/w active and inactive forms
What groups indicate chromatin is active?
Acetyl
What groups indicate chromatin is inactive?
Methyl groups
How can cells be accumulated at metaphase?
Spindle inhibitor
Hypotonic solution
Fixative
What types of growth can be used to culture cells in vitro for karyotyping?
In suspension
On substrate
What is used to describe abnormalities in karyotyping?
International System for Cytogenic Nomenclature (ISCN) 2013
What tissues can be used to give specimens for karyotyping?
Bone marrow Blood (T-lymph) Amniotic fluid CVS Solid tissue
Why is nothing expressed on the short arm of an acrocentric chromosome?
No euchromatin present
How are chromosomes assigned to groups A-G?
According to size and shape
What chromosomes are in the D and G karyotype groups?
Acrocentric w/satellites on their p-arms
How does the size of chromosome 22 relate to 21?
Bigger
What does solid staining show in karyotyping?
Shape and size of chromosomes
What is used in G banding of chromosomes to identify gene rich and gene poor areas for karyotyping?
Trypsin
What does a karyotype report of 47,XX,+21 indicate?
Female w/trisomy 21
What does a karyotype report of 46,XY,inv(7)(p11.2q11.23) indicate?
Make w/chromosome 7 inversion
What would the karyotype report of a girl with a chromosome count of 46, derivative 20 from translocation 2 and 20 which is maternally inherited look like?
46,XX,der(20)t(2;20)(q.27.3;11.2)mat
Why carry out cytogenetic analysis?
Prenatal diagnosis
Assess future reproductive risks
Better clinical management
Accurate diagnosis/prognosis of clinical problems
What constitutional abnormalities indicate referral for cytogenetic analysis?
Prenatal diagnosis Birth defects Abnormal sexual development Recurrent foetal loss Infertility
What acquired abnormalities indicate referral for cytogenetic analysis?
Leukaemias
Solid tumours
What is the main method of prenatal diagnosis in the first trimester of pregnancy?
Biochemistry and ultrasound looking at nuchal translucency
Give four broad types of birth defect.
Dysmorphism
Congenital malformations
Metal retardation
Developmental delay
How is trisomy 21 better known?
Down’s syndrome
What genetic defect causes Williams syndrome?
Deletion 7q11.23
What is the effect of the 22q11.2 microdeletion seen in Di George syndrome?
Significant heart defects
What is aneuploidy?
Loss or gain of whole chromosomes due to errors of cell division in meiosis
Which three trisomies are compatible with life?
Down syndrome (+21) Patau syndrome (+13) Edwards syndrome (+18)
What is the only viable fully monosomy syndrome?
Turner
What does Turner syndrome cause?
Inactivated X
What error occurs in the meiotic cell divisions during gamete formation?
Non-disjunction
What results if there is non-disjunction in mitotic cell division?
Mosaicism
What is mosaicism?
More than one cell population seen in an individual
What is polyploidy?
Gain of a whole haploid set of chromosomes
What is the most common cause of polyploidy?
Polyspermy
How common is triploidy?
Relatively, occurs in 2-3% of all pregnancies
Which polyploidy is often found as a cultural artefact on prenatal diagnosis?
Tetraploidy
Is diploid/triploid mosaicism seen in live births?
Yep
What is anaphase lag?
When chromosomes are left behind at cell division and are therefore not present in diagnosed cells
What happens in cell division to cause anaphase lag?
Defects in spindle
Defects in attachment to chromosomes
What can result from fertilisation by a normal gamete of a gamete which has undergone non-disjunction in meiosis I?
Trisomy
Monosomy
OK cell
What causes trisomy 18?
Maternal meiosis II error
What visible features indicate trisomy 18?
Rocker bottom foot Overlapping fingers Low-set ears Prominent occiput Small lower jaw
What is the modal lifespan of a neonate with trisomy 18?
5-15 days
What visible effects may be seen in trisomy 21?
Hypotonia
Characteristic facial features
Intellectual disability
Heart defects
What diseases are associated with trisomy 21?
Alzheimer’s
Leukaemia
What effects can be seen in trisomy 13?
Multiple congenital abnormalities
Polydactyly
Single lobed brain
What happens to the majority of neonates with trisomy 13?
Die in neonatal period
What characterises Turner Syndrome?
Mild learning difficulties Heart defects Neck webbing Infertility Hypotonia Short stature
What do phenotypic differences depend on in Turner syndrome?
Parental origin of X chromosome
What determines whether mosaicism occurs throughout the body or in specific tissues?
Time of mitotic disjunction
When would mitotic disjunction not cause mosaicism?
If it occurs after first zygotic division
Which cell line is usually lost when there is mitotic non-disjunction after the first zygotic division?
Monosomy
How is the trisomy conceptus created by mitotic non-disjunction after the first zygotic division rescued to give mosaicism?
Anaphase lag
When will the monosomy cell line not be lost in mitotic non-disjunction after the first zygotic disjunction?
If it involves X
What is uniparental disomy?
Presence of homologous chromosomes from one parent
What is heterodisomy?
Meiosis I error causing 2 homologous chromosomes from one parent
What is isodisomy?
Error in meiosis II causing 2 identical chromosomes from one parent
What is segmental uniparental disomy?
When only part of a chromosome is involved
Name two conditions which are uniparental disomy involving chromosome 15.
Prader-Willi
Angelman syndrome
Why do imprinted chromosomes show differential expression of specific genes in uniparental disomy?
Depends on parental origin
What can cause uniparental disomy?
Gamete complementation Mitotic error Trisomy rescue Monosomy rescue (2 separate must happen)
What are the 8 cytogenic structural abnormalities?
Translocations Inversions Deletions Duplications Insertions Rings Marker chromosomes Isochromsomes
What are reciprocal translocations caused by?
Two break rearrangements
How do balanced reciprocal translocations compare to unbalanced?
Balanced have all genetic material present just rearranged –> asymptomatic
Unbalanced will have abnormal phenotype dependent on the regions of trisomy and monosomy
What happens to create a normal/carrier phenotype in meiotic disjunction once the translocation has formed a quadrivalent?
Alternate chromosomes segregate together
What happens after the quadrivalent has been formed in meiotic disjunction to create an abnormal phenotype that will be full term?
Non-homologous centromeres segregate together
What happens in meiotic disjunction after the quadrivalent has been formed to create a massive degree of imbalance?
Homologous centromeres segregate together
What type of chromosomes fuse together in Robertsonian translocations?
Acrocentric
How does the chromosome count differ in balanced and unbalanced Robertsonian translocations?
45 in balanced
46 in unbalanced
What is there a risk of in Robertsonian translocation?
Aneuploidy
Why can homologous carriers of a Robertsonian translocation not have a normal pregnancy?
Only have Robertsonian gametes as empty gametes are not viable
How do deletions arise?
Uneven pairing and recombination during meiosis
What is the difference between a terminal and interstitial deletion?
Terminal - end of arm
Interstitial - middle of arms
How do deletions affect karyotype?
Cause unbalanced
What is needed to see micro deletions that can’t be seen by G-banding?
FISH
What is prenatal aneuploidy screening?
Interphase analysis used for prenatal diagnosis of common aneuploidies
What can be used with the FISH techniques to allow 2 analysts to blindly score 30 cells to look for abnormality?
Vysis aneuvysion probes
What method of interphase analysis looks for different types of abnormalities such as translocations, gene rearrangements or amplifications?
Leukaemia-FISH
What does microarray examine?
Whole genome at high resolution looking for copy number changes
What does excess red indicate in comparative genomic hybridisation slide scanning?
More reference DNA and deletion of test DNA
What does excess green indicate in comparative genomic hybridisation slide scanning?
More test DNA and duplication of test DNA
Why are copy arrangements visible but balance not in microarray methodology?
Uses ratios of test and reference DNA
What are the advantages of comparative genomic hybridisation slide scanning?
Examines genome at high resolution Targeted against known genetic conditions Can be automated Detailed info on genes Better phenotype/genotype correlation
What are the disadvantages of comparative genomic hybridisation slide scanning?
More expensive than karyotyping
Doesn’t detect balanced arrangements
Mosaicism may be missed
What is non-invasive prenatal testing?
Analysis of cell free foetal DNA in maternal plasma at 9 weeks gestation
Why is non-invasive prenatal testing tench only challenging?
DNA deteriorates rapidly
What is non-invasive prenatal testing already approved for?
Congenital adrenal hyperplasia
X linked disorders (not haemophilia)
Achondroplasia
Thenatophoric dysplasia
How is next generation sequencing carried out?
Sequence fragments of gDNA in parallel and realign to reveal entire reference genome
What is the problem with next generation sequencing?
Massive amounts of data are hard to store
What are the benefits of next generation sequencing?
Reduced cost and time - technology is quick but analysis is not
What is whole exome sequencing?
Targeted sequencing of all exons
What are the benefits of whole exome sequencing?
Reduced data in comparison to whole genome analysis
Easier to interpret
Has given rise to many reports of clinically significant de novo mutations
How many pairs of cranial nerves are there?
12
How many segmental/spinal nerves are there?
31 pairs
What make up nerves?
90% glia
10% neurones
What is the function of glia?
Create environment in which neurones can survive
When are micro glia seen?
Inflammatory insult to CNS
What is the function of microglia?
Act as phagocytic immune cells in the simple immune system of the CNS
Which cells are macroglia?
Astrocytes Oligodendrocytes Schwann cells Ependyma Satellite cells
What is the function of astrocytes?
Give brain and spinal cord shape Acts as phagocytes Form blood-brain barrier Glucose --> lactate for neuronal nourishment Support cellular matrix of NS
What is the function of oligodendrocytes?
Myelinated up to 250 CNS axons at a time
What is the function of Schwann cells?
Myelinated single PNS axons
What is the function of ependyma?
Line ventricles and central canal of spinal cord for circulation of CSF
What is the function of satellite cells in the PNS?
Physically support neurones
What is the structure of the cell body of a neurone?
4-120 micrometers Various shapes Contains centrally positioned nucleus and nucleolus Missouri substance Some have appendages called dendrites
What is Nissl substance?
ER and Golgi apparatus
How can a dendrite be identified if it is present on a cell body?
Widest part of cell
How does myelination of axons vary?
Thin are not
Thick are heavily
Level varies b/w neuronal types
How can conduction in an axon be expressed?
CV
6FD where F = level of atonal myelination and D = diameter
Where is the axon hillock found in a neurone if present?
B/w axon and cell body
What forms the grey matter of the nervous system?
Neuronal cell bodies
What are collections of cell bodies known as in the CNS and PNS?
CNS = grey matter PNS = ganglia
How does a Schwann cell myelinated an axon?
Wraps itself round
What diseases can demyelination axons?
Multiple sclerosis
Guillaine-Barre
Diabetes
Why are beta-lactams considered suicide inhibitors?
Undergo partial reaction to form irreversible inhibitor in glycopeptide transpeptidase active site
At what cross-sectional diameter do axons conduct faster if they are unmyelinated?
What forms white matter in the CNS?
Myelinated neuronal axons
What is the epineurium?
Ensheaths entire nerve
Interfacial binds attach adjacent fascicles
What is the perineurium?
Ensheaths a nerve fascicle
What is the endoneurium?
Ensheaths a single cell axon
What happens to the axon when an axon is severed?
Compression causes nerve to split
Proximal segment seals to prevent leakage of cell contents
Nerve stump formed
Distal segment cut off from nutritional support and dies
Wallerian degeneration
What happens in Wallerian degeneration?
Degradation of myelin sheath
Invasion by macrophages
Neurolemma remains and can reinnervate tissue if Schwann cells can reach
What happens to the cell body when an axon is severed?
Puffs up with increased contents
Nucleus displaced to periphery
Chromatolysis
What is an afferent neurone?
Neurone that carries signal from periphery to CNS
What is an efferent neurone?
Neurone that carries signals from brain/spinal cord to periphery
What forms the CNS?
Brain and spinal cord
What is the CNS characterised by?
Protection of the cranium and vertebral column, suspended in CSF
What is the CNS responsible for?
Sophisticated functions
Where do segmental nerves which are attached to the spine go?
To PNS
What are collections of neuronal cell bodies and axons called in the CNS?
Bodies = nuclei Axons = fibre tracts
What are the constituents of the PNS?
Axons of cranial nerves Axons of spinal nerves Ganglia Nerve plexuses Enteric nervous supply
What does the PNS connect?
CNS target organs
What are collections of neuronal cell bodies and axons called in the PNS?
Cell bodies = ganglia
Axons = nerves
Explain the appearance of the spinal cord in cross section.
Grey matter centrally w/butterfly shape
White matter peripherally fully surrounding grey matter
Are the CNS and PNS independent?
No, neurone can be in both at the same time
How is the efferent somatic nervous system laid out?
1 spinal/cranial nerpurone carries output and terminates directly on effector organ
How does the somatic nervous system progress during childhood?
Efferents underdeveloped at birth but are fully developed by puberty
What type of control is the somatic nervous system under?
Voluntary
What is the function of the autonomic nervous system?
Sub serves fundamental life functions incl. homeostasis
What is the ANS activated?
In utero w/organogenesis
How is the ANS controlled?
Not voluntarily
Change continuous output in two opposing systems
What are the effector organs of the ANS?
Viscera
Smooth muscle
Secretory glands
Where are the cell bodies of sympathetic nerve fibres located in the spine?
Thoraco-lumbar
T1-12 and L1-2
What do axon lengths in the ANS depend on?
Which division they belong to
Why is there lots of flexibility in the ANS?
Always 2 neurones, one w/cell body in CNS and one w/cell body in PNS
What are White Rami communicates?
Neurones w/cell body in CNS
What are Grey Rami communicates?
Neurones w/cell body in PNS
What are most organs innervated by?
ANS
Describe the ANS innervation of most organs?
Dual SNS and PNS which are mostly reciprocal so the balance determines the output
Whoa receives sympathetic drive only?
Sweat glands
What happens to the brain when there is over-activity of the PNS?
Dilates systemic blood vessels leading to shortage of substrates to brain
What happens to the body if there is over-activity of the SNS?
Constriction of systemic blood vessels leading to shortage of substrate to the tissues
What are all pre-ganglionic neurones?
Cholinergic
Where are the pre-ganglionic neurones in the SNS located?
Spinal cord
What do sympathetic post-ganglionic neurones express?
Nictoinic receptors
What do the effector organs of the SNS express?
Variety of alpha and beta receptors
What are the three modes of termination of signals in the SNS?
Synapse in chain at level of origin (paravertebral)
Synapse at different level up or down chain
Use specialised preganglionic nerves to not synapse in paravertebral chain
Why are preganglionic neurones in the SNS short?
Majority of efferents synapse immediately after exiting the spinal cord
Which receptors can exercise presynaptic inhibition in the SNS?
Alpha-1 + 2
Beta-1 + 2
What do the long postganglionic neurones of the SNS secrete?
Noradrenaline/adrenaline
Describe the neurones of the PNS.
Preganglionic: long and cholinergic
Postganglionic: short, in effector organ walls, cholinergic and express nitcotinic receptors
What do effector organs in the PNS express?
Muscarinic receptors
Which effector organ in the PNS behaves as a ganglion cell w/nitcotinic receptors?
Adrenal medulla
Which effector organs are exceptions in the SNS?
Sweat and ejaculatory mechanisms which are cholinergic
