MBB11004 -Genetics 1 Flashcards
What is genetics?
the study of genes and the way they are passed on (inherited)
What is heredity?
the inheritance of traits from parents to offspring, making offspring SIMILAR to parents
What are the three approaches to looking at genetics?
-molecular/developmental genetics (genes transmitted from DNA to affect cell function and phenotypes)
-transmission genetics (genes transmitted from parents to offspring)
-population/evolutionary genetics (genes transmitted over many generations within a large population)
How is genetic material organised differently in eukaryotes and prokaryotes?
eukaryotes:
-linear chromosomes in nucleus
-chromatin (DNA + histones)
prokaryotes:
-single, circular chromosomes condensed in nucleoid region
-no nucleus
What is chromatin?
DNA wrapped around histones (highly conserved proteins)
What is the structure of a linear chromosome?
-centromere (binding site for kinetochores)
-p arm (shorter arm of chromosome)
-q arm (longer arm of chromosome)
-telomeres (highly conserved, repeated DNA sequences at end of arms which protect ends from being degraded)
What is the p arm of a chromosome?
the shorter arm
What is the q arm of a chromosome?
the longer arm
What is a locus?
(pl loci)
set position on a chromosome which a gene can be found at
What is cytogenetics?
the study of chromosomes
What is a karyotype?
the chromosome complement of an individual
-cytogenetic techniques (eg. G-banding) are used to come up with a karyotype
What is a typical human karyotype like?
-22 pairs of autosomal chromosomes
-1 pair of sex chromosomes (XX = female, XY = male)
46 chromosomes total
What does diploid mean?
2 homologous chromosomes (same gene, diff alleles)
-2 copies of each chromosome
-2n
What does haploid mean?
1 copy of each chromosome
-n
What is a gene?
a unit of hereditary information that occupies a specific position (locus) on a chromosome
-determines phenotype
-passed from parent to offspring
What is an allele?
one form of a specific gene that exists at a single locus
-alleles can differ by one nucleotide or by hundreds of nucleotides
Why is most of the mitochondrial and chloroplast DNA from the egg?
-mitochondria and chloroplasts are located in the cytoplasm
-egg has larger volume of cytoplasm than sperm
What is euchromatin?
loosely packed chromatin which can readily be transcribed
What is heterochromatin?
condensed (tightly packed) chromatin which is not readily transcribed so not expressed
How is euchromatin/heterochromatin altered to the alternative?
via the addition of methyl and acetyl groups to histones
What is epigenetics?
the study of phenotypic changes caused by modifications to the chromatin structure to alter gene expression/activity without changing the DNA sequence itself
-addition of methyl and acetyl groups to histones
-euchromatin and heterochromatin
What is the asexual cell cycle?
cell divisions to give genetically identical daughter cells
-mitotic nuclear divisions
What is produced from unicellular organisms undergoing the asexual cell cycle?
clones
-causing a genetically identical population
What happens in binary fission?
-DNA duplicates
-cells increase in size
-replicated chromosomes move apart
-protein Fts2 marks middle of the cells, where a new cell wall forms
-2 daughter cells form
What are the stages of the cell cycle?
interphase
-growth 1 phase (increase in cell cycle, prod ribosomes and RNA)
-synthesis phase (chromosomes duplicate)
-growth 2 phase (DNA checed, prep for nuclear division)
mitotic phase
-mitosis (prophase, metaphase, anaphase, telophase)
-cytokinesis
What happens in terms of chromosomes during the cell cycle?
-chromosomes replicate in the S phase to produce sister chromatids
-sister chromatids separate in mitosis
What happens during prophase in mitosis?
-chromosomes condense, becoming visible thread-like structures
-nuclear membrane breaks down (end of prophase -known as prometaphase)
What is mitotic spindle?
structure of cytoskeleton made of lots of microtubules (polymers of small tubulin subunits) which separate sister chromatids into different daughter cells during mitosis
What is a centromere?
specialised chromosome regions at a fixed point along the chromosome (determined by specific epigenetic molecules) which direct the segregation of chromosomes in mitosis
-connected to microtubules by kinetochore (protein complex)
-sister chromatids are joined by centromere
What happens during metaphase in mitosis?
-centromeres align at equator of cell
-microtubules attach to centromeres and to ends of each pole, creating tension which keeps chromosomes at centre
What happens during anaphase in mitosis?
-cohesin (between sister chromatids) breaks down, causing the chromatids to become separate chromosomes
-microtubules contract, starting to move centromeres to opposite poles (chromosomes form V-shapes as chromosomes are dragged behind centromere)
What happens during telophase in mitosis?
-chromosomes arrive at poles
-chromosomes recondense
-daughter nuclei form
What happens during cytokinesis?
-cleavage furrow forms between 2 poles
-constriction
-2 daughter cells produced
-can be symmetric or asymmetric
How can mitosis go wrong?
-bridge chromosome (chromosome with 2 centromeres, pulled to both poles and breaks)
-acentric chromosome (chromosome missing a centromere, can’t separate properly)
What is a bridge chromosome?
error in mitosis where a chromosome has 2 centromeres, which both attach to centromeres, meaning the chromosome is pulled to 2 poles at once so breaks
What is an acentric chromosome?
error in mitosis where a chromosome lacks a centromere so can not separate properly
What is the sexual cell cycle?
meiosis
the halving of chromosome number through 2 successive nuclear divisions
What is a tetrad?
the four haploid products of meiosis
What is the nuclear cycle like in Saccharomyces cerevisiae?
-haploid most of life
-has 2 mating types: a and α
-one of each mating types combine to give transient diploid state
-meiosis to give 4 products
What happens in meiosis 1?
-homologous chromosomes are replicated to produce dyads (prophase 1)
-2 dyads join together to form a bivalent (metaphase 1)
-dyads move to opposite poles (with centromere kept intact) (anaphase 1)
-by end, have 2 nuclei, each with both sister chromatids of one of the parent’s homologous chromosomes
What is a dyad?
2 sister chromatids with a single centromere
(half a tetrad)
What happens in prophase 1 (in meiosis 1)?
-replicated chromosomes start to contract (leptotene)
-chromosomes line up in homologous pairs (synapsis) held together by synaptonemal complex (zygotene)
-crossing over occurs between non-sister chromatids (pachytene)
-chromosomes separate slightly (diplotene)
-chromosomes contract further (diakinesis)
What happens in meiosis 2?
-like mitosis
-sister chromatids line up
-chiasmata breaks down so that centromere splits and chromosomes move to opposite poles
-by end, have 4 nuclei, two of which contain 1 copy of one homologous chromosome and two of which contain 1 copy of the other homologous chromosome
How does meiosis generate variation?
-independent assortment of chromosomes (random how bivalents are orientated on meiotic spindle -all equally likely)
-crossing over (sections of chromosomes swapped via deliberate double-strand breaks by specialised enzymes to allow homologous recombination to give new allele combinations)
What are cross overs?
site of genetic exchange between homologous sequences on non-sister chromatids
What are chiasma?
site of crossover
When are cross overs visible in meiosis?
diplotene stage of meiosis 1
-can see chiasma
What is a mutation?
a gene/chromosome that differs to the wild type
(or the process that results in the different gene/chromosome)
What is the result of a spontaneous mutation in single-cellular organisms?
population is no longer clonal
What is the result of a spontaneous mutation in multi-cellular organisms?
mixture of genotypes
organism is “genetic mosaic”
mutation is only passed on if germline (in egg/sperm)
What is polyploidy?
an unusual number of chromosome sets
eg. monoploidy, triploidy, tetraploidy
What is aneuploidy?
when one/a few chromosome sets are missing/extra
eg. resulting in Downs syndrome, Edwards syndrome and Patau syndrome
sex chromosome aneuploidies like Turner syndrome and Klinefeller syndrome
What large scale chromosomal rearrangements are there?
-deletions (part of chromosome missing)
-inversions (part of chromosome flipped)
-translocations (part of chromosome moved)
What are the sources of mutation causing individual genes to differ from the wild type?
-mistakes in replication (point mutations, small insertions, small deletions)
-transposons interrupting genes
-incorrect repairs of DNA breaks
What are the types of point mutations?
-silent (no change in amino acid seq)
-non-sense (stop codon introduced in chain)
-mis-sense (changes in amino acid seq)
What are auxotrophic mutants?
mutants that can not synthesise essential compounds (eg. adenine, serine)
-study them by growing in minimal growth media and complete media
What is minimal growth media?
growth media which only contains nutrients organisms can not make for itself
-wild type can grow in it, auxotrophic mutants can’t
What is complete growth media?
growth media containing extra nutrients, making up for defects in metabolic pathways so that mutants can grow
-both wild type and auxotrophic mutants can grow in it
What are allelic mutations?
mutations where both mutations are on the same gene/step of pathway
What are non-allelic mutations?
mutations in different genes
How can complementation be used to determine whether mutations are allelic or non-allelic?
non-allelic mutations complement eachother (so that diploid contains one wild type allele so can grow on minimum media)
whereas allelic mutation don’t
Why were peas good for Mendel’s experiments?
-phenotypes are all simple traits controlled by a single gene
-no co-dominance
-genes controlling phenotypes are on different chromosomes
-peas are either cross-pollinated or self-pollinated
What are pure breeding lines?
when all the offspring from mating within the breeding lines have the same character
What are Mendel’s three laws?
-law of equal segregation
-law of independent assortment
-law of dominance
What is Mendel’s law of equal segregation?
during gamete formation, members of each gene pair separate equally
-each gamete carries 1 allele for each gene, meaning you end up with same allele ratio as at the start
What is Mendel’s law of independent assortment?
during gamete formation, chromosome pairs separate independently of eachother randomly
-large number of combinations possible -including recombinants
What is Mendel’s law of dominance?
alleles can be dominant or recessive
How can genetic hypotheses be designed and tested?
using Punnett squares or probability trees
How can you determine which allele is the dominant allele?
-cross pure-breeding parental lines with each genotype (100% of the offspring will have dominant phenotype)
-self-fertilise F1 generation (75% F2 gen will have dom phenotype)
-cross-fertilise F1 generation with homozygous recessive aka test cross (50% F2 gen will have dom phenotype)
What are dihybrid crosses used for?
considering 2 genes on different chromosomes to eachother
What are the two ways of coming up with a genetic hypothesis?
-predicting numbers of different types you expect before experiment is done
-look at experimental data and come up with ratios which closely fit
How can you go about testing a genetic hypothesis?
-start with biological hypothesis (predict numbers)
-expect numbers of observed and expected to not differ much if hypothesis is correct
-test using chi-squared
Chi-squared test
x^2 =
Σ (O-E)^2
_________
E
What are the degrees of freedom for the chi-squared test?
n-1
What is linkage?
genes positioned close to eachother on a chromosome to be more likely to segregate together at meiosis, and therefore be inherited together
-strength of linkage depends on the distance between the two genes
Where are linked genes in relation to eachother?
on loci next to eachother
-are carried and inherited together
recombination frequency (%) =
recombinants
_________________________ x100
total meiotic frequency
What is the recombination frequency?
the percent of meioses where two loci are exchanged by homologous recombination
-RF is roughly proportional to the distance between the two loci (closer the genes, the less likely recombination is)
-RF can be used to create maps showing the order of genes on a chromosome
What value is recombination frequency always between?
0 and 50%
0 = loci next to eachother, no recombinants
50 = loci on separate chromosomes, half recombinants
What does a recombination frequency of 0% mean?
loci are next to eachother
no recombinants
What does a recombination of 50% mean?
loci are on separate chromosomes
50% recombinant, 50% parental
What are the different types of tetrad?
-parental ditype (all segregants have parental genotypes)
-non-parental ditype (all segregants are recombinants)
-tetratype (2 parental and 2 recombinant genotypes in segregants)
What is a parental ditype?
a tetrad where all the segregants (products of meiosis) have parental genotypes
-all of them the same as one of their parents (2 of each parental genotype)
What is a non-parental ditype?
a tetrad where all the segregants (products of meiosis) have genotypes different to their parents
-all recombinants
What is a tetratype?
a tetrad where half of the segregants (products of meiosis) have parental genotypes and half have non-parental (recombinant) genotypes
-all 4 meiotic products are different
If there are more parental tetrads than non-parental tetrads, what does this suggest about the genes?
the genes are linked
-genes are inherited together
If there are roughly equal numbers of parents and non-parental tetrads, what does this suggest about the genes?
the genes are unlinked
-independent assortment can occur
recombination frequency for 2 loosely linked genes =
non-parental ditype + 1/2 tetratype
___________________________________________________ x100
parental ditype + non-parental ditype + tetratype
Why is it important to study viruses?
-discover new enzymes encoded by viruses (research, pharmaceuticals)
-phage therapy (treating disease)
-viruses can kill algal cells (agal blooms)
What is a virus?
genetic element that can only replicate inside of a living host cell
-can exist as viral particles outside host
-are small!
What is a virion?
an inert viral particle outside of a host made up of protein surrounding DNA or RNA
-has a limited number of hosts
-injects nucleic acid into host, protein coat generally stays outside
-intracellular form is replicative form
What are viral genomes like?
-very small (reliant on host machinery)
-DNA or RNA
-ss or ds
-linear or circular (can switch)
What does the tobacco mosaic virus (TMV) have in place to get round having a small size?
-only 1 kind of protein (capsomers) so only one gene needed for all its protein structure
-capsids can self-assemble so no machinery needed
What are early proteins for (in viral replication)?
replicating viral nucleic acids (transcription and translation)
What are late proteins for (in viral replication)?
packaging (coat proteins)
How does the T4 phage ensure viral transcription occurs?
-modifies host’s RNAP so that it binds to phage promotors (rather than host promotors)
-has sigma factors for early proteins
-T4 phage genome codes for anti-sigma factor, which binds to host sigma factor to prevent host transcription
How is the switch to middle proteins achieved in the T4 phage?
-some early phage proteins modify host RNAP α subunits
-other early proteins bind to RNAP, changing the complex
-these alter the RNAP specificity to recognise middle promotors
-early protein MotA recognises seq in middle promotors and guides RNAP to these sites
What are lysogenic viruses?
viruses which can integrate into the host genome, rather than escaping from the host
What happens in the lytic pathway of viruses?
(after attachment and injection)
-viral DNA replicates
-coat proteins are synthesised and viral particles assemble
-host cell lysis and viruses are released
What happens in the lysogenic pathway of viruses?
(after attachment and injection)
-viral DNA is integrated into host DNA (producing a prophage)
-lysogenized cell undergoes cell division
What is induction?
the switch between the lysogenic and lytic pathways
What is lysogeny?
the stable relationship between virus and host
-viral gene expression repressed
-prophage acts as part of host (viral genome is replicated in synchrony with host genome)
How does a viral genome exist during lysogeny?
integrated into host genome or as a plasmid
Why are repressors needed during lysogeny?
-repressors prevent lytic pathway and induction
-prevent viral gene expression
How does the lambda (λ) phage genome integrate into host DNA?
-viral genome integrated at attachment site attλ which requires λ integrase
-viral genome alters from being linear to circular inside host (due to cohesive ends)
-circularised DNA is nicked, creating staggered ends (same as host DNA) by site-specific nucleases
-λ DNA is integrated and gaps are closed by DNA ligase
How is a viral genome replicated in the lytic pathway?
by rolling circle replication
What happens in rolling circle replication?
(lytic)
-circular plasmid/genome (being replicated) rolls
-replication starts at origin, where RepA dimer makes nick
-polymerase moves along from nick, progressing in only one direction
-as it is replicated, a long single-stranded (continuous) concatemer is made
-this concatemer is used as a template to produce a double-stranded concatemer
-this is cut into genome-sized lengths at the cos sites (gives adhesive ends)
What happens in the lifecycle of eukaryotic viruses?
-binding
-fusion
-reverse transcription
(-integration)
-transcription
-translation
-assembly
-budding
-release
What are the issues for viruses living in eukaryotes?
-polycistronic mRNA can’t be translated in eukaryotes
-eukaryotic mRNA processing
How does Poliovirus undergo protein synthesis inside eukaryotes?
-genomic RNA acts as mRNA
-host translates it, synthesising one long polypeptide strand
-proteases cleave this polypeptide into diff proteins (structural coat proteins, proteases, RNA replicase, etc)
How does Poliovirus undergo replication inside eukaryotes?
-RNA replicase (made from translation) makes a -ve strand of viral RNA, which is used to make a +ve strand
-can occur in cytoplasm (no DNA invovled)
-host cap-binding protein is destroyed to inhibit host RNA and protein synthesis
What is the stucture of poliovirus genome?
-linear ssRNA (+) strand genome
-mimicks mRNA -has polyA tail at 3’ and Vpg (protein mimicking cap) at 5’
-RNA folds into stem-loops
What is the Rabies genome like?
one linear (-) strand RNA genome
-virus has its own polymerase (animal host can’t transcribe - strand to + strand)
What is the Influenza genome like?
(-) strand ssRNA genome segmented into 8 linear fragments
What is the structure of Influenza like?
-genome is (-) strand RNA segmented into 8 fragments
-has accessory proteins (eg. RNA endonuclease, RNA viral polymerase)
-envelope
-membrane-bound proteins neuraminidase and hemagglutinin
What does neuraminidase do?
cleaves neuraminic acids so virus can break through mucus
What does hemagglutinin do?
bind to receptors on host envelope to allow the virus to be internalised
How do Rabies and Influenza replicate their genomes inside host cells?
-use viruses own polymerase to transcribe (-) strand RNA to (+) strand RNA
-(+) strand RNA is used to produce copies of genomic (-) strand RNA and is used to translate viral proteins
-(-) strand RNA and structural proteins form progeny virus
What are the consequences of Influenza genome changes?
-antigenic drift (mutations in genes encoding surface proteins)
-antigenic shift (re-association of portions of RNA genome from genetically distinct strains leads to different combinations of surface proteins)
What is the HIV genome like?
2 identical (+) strand of ssRNA
(is a retrovirus)
-has R terminal repeats (essential for replication)
-has genes gap (structural proteins), pol (reverse transcriptase, integrase) and env (envelope proteins)
How is HIV replicated?
-viral entry
-uncoating
-reverse transcription to produce dsDNA
-dsDNA moves into host nucleus and is integrate into host DNA, becoming a provirus
-is transcribed, producing viral genomic RNA and viral mRNA
-viral mRNA is used to translate viral proteins
-encapsidation
-budding and release
How does HIV gene expression vary?
-when in provirus form (combined with host DNA), genome can be expressed or can remain latent
-activation of promotors in long terminal repeat regions (at ends of viral genome) causes capping and polyadenylation of mRNA transcript
-viral mRNAs can be encapsulated or translated
-polyproteins are synthesised and processed
What is the simian virus 40 genome like?
one dsDNA circle
-has overlapping genes
-very small (doesn’t encode viral DNAP, uses host’s)
How does the simian virus 40 replicate its genome?
using host cell machinery bidirectionally
-synthesis is initiated by T antigen protein binding to origin of replication
What are coronavirus genomes like?
single (+) strand RNA viruses
What is the structure of coronaviruses like?
-envelope
-glycoprotein spikes (giving “crown” appearance, hence “corona”)
-large (~30kb)
What happens in the infection cycle of coronaviruses?
-genome has 5’ cap and poly(A) tail so can act directly as mRNA
-only translated gene encoding replicase
-RNA replicase makes copies of (-) strand of RNA
-(-) strand generated synthesises (+) strand copes of whole genome (progeny genomes) or monocistronic mRNAs (go onto translate proteins)
Why is bacterial genomics important?
-mol bio dev -discovers in DNA rep, etc
-w/o genetic systems can’t clone genes, express proteins, etc
-synthetic bio and bact systematics
What are the similarities/differences between eukaryotic and prokaryotic chromosomes?
-proks mainly have coding genes (v. lil space between) while euks have a lot of non-coding sections (introns) and genome-wide repeats
-both have pseudogenes
What is a pseudogene?
sequence of DNA which resembles a functional gene but has been mutated into an inactive form over time so has no functional counterpart in wild-type populations
How are genes organised in prokaryotes?
-in operons (can code for a monocistronic or polycistronic message)
What is a monocistronic operon?
an operon consisting of only one gene so only codes for one protein
-rare
What is a polycistronic operon?
an operon containing multiple genes so codes for multiple proteins
(eg. has 3 genes, codes for 3 proteins)
How is a nucleoid structured?
-has 30-100 loops (domains) -generated by DNA-binding proteins
-helical within each loop
-supercoiled (supercoils can be same or opposite direction to helix) -generated by topoisomerases
How are loops within a nucleoid generated?
by DNA-binding proteins
(relatively small anchoring proteins similar in function to histones)
How are supercoils within a nucleoid generated?
-topoisomerases make double breaks
-another bit of DNA is passed through the break
-break is resealled
What happens in DNA replication in bacteria?
-replication starts at origin
-replication bubble forms and DNA is replicated in opposite directions of fork (bidirectional)
-at each fork, 1 strand is synthesised continuously (from 5’ to 3’) and the other is synthesised discontinuously (from 3’ to 5’) in Okazaki fragments
-replication ends at terminus
How is DNA replication terminated in bacteria?
-at termination regions (have specific seq)
-clockwise replication forks stopped by TerB, C, F, G and J
-anticlockwise replication forks stopped by TerA, D, E, H and I
(not all Ter sites necessary -more than needed so still functions is they are lost)
How is the issue of a catenane of linked rings being produced in bacterial DNA replication solved?
either
-enzymes XerC and XerD recognise different sites on both molecules and catalyse them to be cut and re-joined
or
-by topoisomerase 5
What is the chromosome of E.coli K-12 like?
-circular
-contains 4288 genes
-replicated bidirectionally from origin
-has restriction sites for Not1
-has some Hfr origins
How can plasmids be visualised?
using metal shadowing
-EM technique where very thin elements can be visualised (also used for proteins and DNA)
How are plasmids replicated?
by rolling circle replication
What plasmid-conferred phenotypes are there?
-antibiotic production
-antibiotic resistance
-metabolic functions (v. specific)
-virulence
How are resistance mutations in plasmids seen and selected?
-antibiotic resistant colonies grow around filter disc impregnated with compoound
-visible differences (eg. diff colour from diff pigment being produced)
How is a bacterial genotype written (Nomenclature)?
-gene name 3-4 letters; all italics, all lowercase except 4th is capital (eg. cydA)
-subscript + for wild type, no subscript for mutant
-add number afterwards if multiple alleles (eg. cydA1)
How is a bacterial phenotype written (Nomenclature)?
-only 1st letter capital
-presence or absence of property shown by + or -
-not in italics
How is a bacterial encoded protein written (Nomenclature)?
-only 1st letter capital
-not in italics
-no subscript
eg. CydA protein
How can mutants be screened?
-master plate (containing colonies) pressed onto velveteen
-velveteen (now with colonies imprinted onto it) is used to transfer colonies to fresh media (minimal and complete media -on diff plates duh)
-plates incubated
-all colonies will grow in complete media, mutants won’t grow in minimal media
What is a mutagen?
an agent which increases mutation rates
-have diff modes of action eg. UV radiation, intercalating dyes
How is genetic material transferred from donor to recipient in bacteria?
-transformation (uptake of naked DNA)
-transduction (phage-mediated)
-conjugation (plasmid mediated)
What happens in the mechanisms of transformation?
-DNA binds via protein outside cell
-one strand enters using DNA translocase, whilst the other strand is degraded
-internalised single strand is bound by RecA protein (prevents it being degraded)
-RecA holds ssDNA and dsDNA together
-RecA-ssDNA complex stretched dsDNA to increase complementarity recognition (known as conformational proofreading)
-branch migration (bps on homologous DNA strands are exchanged)
-homologous recombination
What is the role of RecA protein?
-prevents ssDNA being degraded (as cell will try to degrade ssDNA as it detects it as a sign of phage infection)
-holds ssDNA and dsDNA together to enable conformational proofreading to occur during transformation
What is competency?
a cell’s ability to take up extracellular DNA from environment via transformation
-not all cells are naturally competent -can be induced by electroporation or chemical treatment
What was the original experiment done to discover conjugation?
Lederberg and Tatum experiment
-2 auxotrophic E.coli strains each with diff mutations deficient for some factors
-alone, neither strain grew any colonies
-mixture of both strains, colonies grew (deficiencies overcome) -must be some transfer of DNA between cells
Davis U-tube experiment proved physical contact was needed between cells
What experiment proved physical contact was needed between cells for conjugation to occur?
Bernard David
-used a U-tube with fine pore filter between 2 strains
-no genetic transfer
-therefore contact must be needed between cells
What is an F plasmid?
Fertility plasmid
What regions are found on a fertility (F) plasmid?
-tra region for conjugate transfer
-origin of transfer in conjugation (where it starts being taken up into recipient cell)
-insertion seqs (allow to recombine with chromosome)
In what forms can fertility (F) plasmids exist?
either free as plasmid (F+ cell) or recombined as part of chromosome DNA (Hfr cell)
What happens in the mechanism of conjugation?
-pili male contact between with cell
-pilus contracts by dis-assembling subunits, inducing contraction so that cells move closer to eachother and merge(?)
-strand moves from one cell to other (+ to -)
-DNA is synthesised by rolling circle mechanism to replace transferred strand
What are Hfr cells?
cells with high frequency of recombination for chromosomal markers
-Hfr cells differ depending on the order genes are transferred and which regions they are inserted into
How were Hfr strains used to map chromosomes?
-conjugation was disrupted at diff times to see how much had been transferred, which identifies their insertion sites
-map can be made (minute charts)
(not really done anymore)
How is transformation used in labs today?
to move diff plasmids/DNA frags into diff bacterial cells (some are naturally competent, others need chemical treatment or electroporation)
How is transduction used in labs today?
to move small DNA frags or genes from one cell to another
How is conjugation used in labs today?
not used today (used to be used for chromosome mapping using minute charts)
How does genetics affect health throughout lifespan?
embryonic: half of embryos have chromosomal defects
newborns: 5% have birth defects
later on in life: 2/3 diseases have genetic component
What are mitochondrial disorders?
-when there is a mutation in one copy of mtDNA (out of the hundreds of copies present in cell)
-resulting in mixture of genotypes (heteroplasmic)
-causes mitochondrial disorder
-are maternally inherited (don’t follow Mendelian inheritance patterns)
What are single gene disorders?
disease caused by mutation at a single gene
-follow Mendelian inheritance pattern
-high penetrance
-predictive tests
What are complex disorders?
disease caused by mutations at multiple alleles (polygenic)
-susceptibility but not deterministic
-no reliable tests
-influenced by environment
What is the human genome like?
-3.2Gb bps
-appox 1.1% genome is coding -20,500 protein-encoding genes (proportionally not very many)
-non-coding areas still functional, like regulatory elements (eg. enhancer), promotors, etc
What is ENCODE?
Encyclopaedia of DNA Elements
-international research collaboration aiming to build list of functional elements (non-coding) in human genome including regulatory elements and elements acting at protein and RNA level
-achieved through genomic, functional and bioinformatic approaches
Why was it technically challenging at the time to sequence the human genome(/large genomes)?
-length of genome
-enough clones needed to be generated to ensure complete coverage of genome
-sequencing needed to be repeated (to be accurate)
-genome had to be reassembled from individual seqs (especially difficult with repetitive regions)
What is the difference between whole genome sequencing and whole exome sequencing?
-whole genome sequencing sequences entire genome, which generated masses of data and is more expensive
-whole exome sequencing is targeted to only the coding region of genome (1%)
Where are mutations of single gene disorders identified in next generation sequencing?
all mutations in coding seq
Where does next generation sequencing identify mutations of complex gene disorders?
some inside, some outside of coding seq
What has next generation sequencing allowed?
-reduced cost
-reduced time
What are the types of single gene disorders?
-autosomal dominant disorders (eg. Huntingtons disease)
-autosomal recessive disorders (eg. Cystic fibrosis)
-X-linked disorders (eg. Duchene Muscular Dystrophy)
What are autosomal dominant disorders?
disorders where one copy of a mutation is sufficient for an individual to be affected
-wild-type allele is recessive, defective allele is dominant
-inherited in dominant pattern or new mutation arises in a copy of the gene
-phenotype appears in every gen
-affected parent can pass it onto any progeny, following Mendelian inheritance (typical ratios not always seen due to small progeny sample size)
Eg. Huntington’s disease
What is Huntington’s disease?
a late-onset autosomal dominant disorder which is progressively degenerated brain neurones (causing neuronal dealth, CNS disorder, decreased cognitive function, etc)
-affects every generation
-follows Mendelian inheritance patterns
-caused by more than 36 CAG repeats in HTT prot
Mutations in which gene cause Huntington’s disease?
HTT gene
-gene encoding huntingtin protein with poly glutamine tract (endoced by CAG codon)
-normally <36 CAG repeats, when >36 repeats Huntington’s disease is caused
-the more CAG repeats, the earlier the onset of disease
What is the correlation between the number of CAG repeats and the onset of Huntington’s disease?
The more CAG repeats, the earlier the onset of disease
-CAG repeats are unstable and prone to expansion, including during parental transmission -meaning signs and symptoms appear at earlier age as disease is passed on from one generation to the next
What has positional cloning been used to identify?
diseases including Huntington’s disease and cystic fibrosis
What are autosomal recessive disorders?
disorders where two copies of a mutation are required for an individual to be affected
-wild-type allele is dominant, defective allele is recessive (inherited in recessive pattern)
-phenotype doesn’t appear in every generation
-both parents must be carriers for disease phenotype
-follows Mendelian inheritance patterns
Eg. cystic fibrosis
What is cystic fibrosis?
an autosomal recessive disorder characterised by build-up of mucous, which can damage organs
-caused by CFTR gene and mutations
What symptomatic therapies are there for cystic fibrosis?
-physiotherapy
-DNase (to reduce mucous viscosity)
-antibiotics and anti-inflammatories
-mannitol spray (to increase mucus osmolality)
Mutations in which gene cause cystic fibrosis?
CFTR gene
-encodes transmembrane protein which transports Cl- ions across membranes of cells lining lungs, ensuring hydration of surface layers of airways
-can be mis-sense, nonsense or frame-shift mutations (most common mutation is deltaF508 mutation, other mutations referred to as private mutations)
-diff mutations result in diff molecular phenotypes (protein phenotypes)
-sufferers of cf can be homo or heterozygous to CFTR mutations
What treatments are there for cystic fibrosis?
treatments depend on mutation/molecular phenotype
Diff drugs such as…
-production correctors
-correctors
-potentiators
-mutations may display multiple classes of phenotype ∴ require multiple drugs
-mutation specific targeted therapies -specific to individual genetic profile
What are X-linked disorders?
disorders where genes on the X chromosome are affected
-mostly recessive
-males can inherit from carrier female parent, females can only inherit when both parents are carriers
-females generally unaffected but act as carriers
-progeny of affected males never show diseased phenotype (female progeny will be carriers though)
-male progeny of female carriers have 50% chance of getting diseased phenotype, female progeny of female carriers have 50% chance of being a carrier
Eg. Duchene Muscular Dystrophy
What is Duchene Muscular Dystrophy?
a X-linked recessive disorder (gene responsible on X chromosome)
-onset is passed on by mother
-causes muscle weakness and degeneration
Mutations in which gene cause Duchene Muscular Dystrophy?
gene on X-chromosome which is the largest human gene (2.4Mb) containing 79 exons and encodes dystrophin protein
-dystrophin is part of a protein complex in skeletal and cardiac muscle which strengthens the muscle by linking actin to connective tissue
-most mutations are deletions
How can Duchene Muscular Dystrophy be treated?
using anti-sense oligonucleotides to block splicing machinery around the exons containing premature stop codons, so that a partially functional protein can be produced (rather than producing no protein)
How does Beckers Muscular Dystrophy vary from Duchene Muscular Dystrophy?
-both caused by mutation in gene encoding dystrophin
-Beckers has in-frame mutation (no stop codon midway along) so produces a partially functional protein ∴ is less severe
-Duchene has stop codons part way along seq so doesn’t produce protein ∴ is more severe
What has identification of gene and variants of single gene disorders allowed?
-diagnostics and carrier status
-prognostic info (likely outcome, recovery)
-scientific knowledge (eg. of mechanisms)
-tailored treatments to be developed
in future…
-identification and analysis of genetic determinants in novel conditions, characterised disorders, atypical presentation of disease and complex disorders
What are complex diseases?
common diseases affected by polgenic and environmental factors
-familial
-not deterministic
eg. obesity, diabetes, psychiatric conditions (Alzheimers, depression, etc)
Why do genetic components need to be identified for complex diseases?
-early diagnosis and treatment
-to identify lifestyle changes that can be made to lower risk
-develop molecular understanding for developing therapeutics
What are the different possible forms of complex diseases?
-small number of dominant alleles causing a large increase in risk (eg. Parkinsons)
-many alleles causing a small increase in risk (common disease, common variant model) (eg. type 2 diabetes)
-one main allele causing a large affect and several other alleles causing a small increase in risk (eg. breast cancer)
What is the common disease common variant (CDCV) model?
if a disease is common in a population, the genetic contributors will also be common in the population
What are single nucleotide polymorphisms (SNPs)?
germline substitution of a nucleotide at a specific locus
-found in coding and non-coding regions
-common variants
-unevenly distributed in genome
What is minor allele frequency (MAF)?
the frequency which the second most common allele exists in a population
-used as a measure as to how common/rare a single nucleotide polymorphism (nucleotide substitution at locus) is
What is the minor allele frequency (MAF) of a common single nucleotide polymorphism (SNP)?
> 5%
What is the minor allele frequency (MAF) of a rare single nucleotide polymorphism (SNP)?
<5%
How can complex disease alleles be identified?
-using linkage analysis (linkage between mapping markers and occurrence of disease in families)
-using genome-wise association studies (GWAS) (searching for alleles in population that occur more in disease cases than in matched controls)
What are genome-wide association studies (GWAS)?
studies looking for alleles in a population which occur more in disease cases than in matched controls
Vp =
Vg + Ve
What is phenotypic variation?
the sum of genetic and envrionmental variation
What is heritability?
the degree of variance in a particular phenotype in a population due to genetic variation
-not fully explained by risk alleles
heritability =
Vg/Vp
Why are twin studies useful?
can study the effect of genetics (heritability) and environment on phenotypic variance
What are monozygotic twins?
identical twins
-share ~100% of their genes
-shared environment
What are dizygotic twins?
non-identical twins
-share ~50% of their alleles
-shared environment
What is the ACE model of phenotypic variation?
model used in twin studies to show what proportion of variance is genetic and environmental
A = genetic variance
C = common environmental variance
E = specific environmental variance
What is genetic variance (A in ACE model) like in monozygotic and dizygotic twins?
constant in MZ twins
variable in DZ twins
What is common environmental variance (C in ACE model) like in monozygotic and dizygotic twins?
constant in both MZ and DZ twins
What is specific environmental variance (E in ACE model) like in monozygotic and dizygotic twins?
variable in both MZ and DZ twins
How can the ACE model show the extent a phenotype in one twin correlates in the other twin?
A + C
in MZ twins, A = 100%
in DZ twins, A = 50%
What are the two models complex disease may exist, looking at phenotypic variation?
-disease with continuous phenotypic variation
-disease with threshold for development of disease
How is a continuous phenotype seen in polygenic diseases?
large number of loci -the more loci, the more distribution, the more phenotypes
What is the liability threshold model of disease susceptibility?
that when an individual reaches a specific liability (the threshold), they will have the disease
-liability increases with genetic and environmental factors
-discontinuous disease phenotypes (either have disease or don’t)
How does the threshold model of disease susceptibility change with relatives?
increased risk
-threshold shifted
What affect do SNPs have when they occur at coding regions?
synchronous (no change to aa encoded) or asynchronous (mis-sense or non-sense mutations)
What affect do SNPs have when they occur at non-coding regions?
affect regulation/expression of associated genes
What do having combinations of multiple SNPs lead to?
a complex disease
What is Exome Aggregation Consortium (ExAC)?
a database containing exomes of unrelated individuals sequences as part of disease-specific and population genetic studies
-variants mapped, allele frequencies recorded, rare mutants documented
How are genome-wide association studies (GWAS) used to identifiy risk alleles?
-fixed number of SNPs in genome examined for association with disease phenotype
-search for alleles that occur more frequently in diseased cases than matched controls
-need lots of participants (with and without disease, with and without allele)
-only subset of panels looked at rather than whole genome (to save money and time) -hence association
-identifies association with disease, not necessarily risk alleles -need to integrate with functional data to find causality
What is linkage disequilibrium?
non-random association of alleles at different genomic sites
-deviation in haplotype frequencies from their expected frequencies in a population if loci defining the haplotypes are randomly associated
-SNPs correlate with risk alleles due to LD
What are haplotypes?
a set of DNA segments on a chromosome defined by alleles on locus
-usually inherited together
-haplotype blocks summarise linkage disequilibrium -are regions with high LD separated from other regions from historical recombination events
What is done in haplotype mapping?
groups of alleles clustered so that a SNP (tag SNP) can identify them
What is an odds ratio?
statistic which quanitfies the strength of association between two events
-used to measure the likelihood that a single nucleotide polymorphism is associated with a disease
1 = events independent
<1 = events are negatively correlated
>1 = events are correlated
What does an odds ratio of 1 mean?
events are independent of eachother
What does an odds ratio greater than 1 mean?
events are correlated
What does an odds ratio less than 1 mean?
events are negatively correlated
What is a Manhattan plot used for?
-to show the frequencies of points on a chromsome
-can determine which alleles are risk alleles -threshold line drawn on plot -alleles above line are risk alleles (risk of lots of false -ves!)
How have GWAS helped identify low risk alleles for type 2 diabetes?
-increased statistical power has enabled low risk loci to be identified -have odds ratios between 1.06-1.27
eg. FTO (intronic variant) and CDKN2A/B (non-coding region variant) discovered -both low risk alleles
TCF7L2 also visible (v large peak) -highest risk allele for type 2 diabetes
How have GWAS helped identify low risk alleles for breast cancer?
-identified 66 common low risk alleles in non-coding region
-can also see BRCA1/2 which are autosomal dominant genes responsible for 5% cases of breast cancer (mapped by linkage analysis before GWAS)
What is the principle of missing heritability?
the identification of risk alleles can not alone explain the heritability and phenotypes of diseases
What can the missing risk (missing heritability not explained by risk alleles) arise from?
-false -ves in GWAS
(the following are not detected in GWAS…)
-rare variant alleles with a MAF of 1-5%
-genome structural changes
-epigenetics
-3D genome organisation
What gives rise to different cell fates in development?
cell differentiation
-stem cells leading to diff lineages
-diff gene expression and proteome (but same DNA)
What gives rise to different cell positions in development?
pattern formation
-cells acquire positional values, which develop to give spatial patterns
-established by polarity
What gives rise to cells having different positional values in development?
polarity
-defined by molecular events
What are stem cells?
undifferentiated cells (of a multicellular organism) capable of giving rise to indefinitely more cells of the same time and which can other cells can differentiate from
How is the population of stem cells maintained?
via self-renewal
How do progeny with different fates arise?
via asymmetric divisions of progenitor cells
What are the different forms of stem cells?
-totipotent (can form whole new organism)
-pluripotent (can form most cell types but not all)
-multipotent (can form diff cell types but fewer that pluri)
How are embryonic stem cells generated?
-inner cell mass (ICM) cells are extracted from a blastocyst
-extracted ICM cells are grown on specific tissue culture
-embryonic (but not totipotent) stem cells are produced
What are the uses of embryonic stem cells?
-introduce new genes
-for gene knock-out
-chromosomal rearrangements/deletions can be done to see impact in model organism
-specific gene editing (CRISP)
How are transgenic mice produced from embryonic stem cells?
-ESCs are introduced into a blastocyst
-implanted into pseudopregnant mother
-chimeric mouse produced (from 2 diff zygotes -ICM and ESC)
How can somatic nuclei be reprogrammed to be totipotent?
extracting somatic cell nucleus and inserting it into an enucleated egg using UV irradiation
How can pluripotent stem cells be induced?
incubating adult cells in tissue culture with transcription factors
What are the genetics of Drosophila melanogaster like?
-4 pairs of chromosomes (X and Y, 2, 3, 4)
-16,000 genes (relatively small genome)
-~140Mb
How is the Drosophila body plan determined?
by hierarchy of developmental genes
What type of genes are involved in Drosophila development?
-maternal effect genes
-gap genes
-pair-rule genes
-segment polarity genes
-homeotic genes
What is the role of maternal effect genes?
establish pattern
-maternal genotype determines progeny phenotype
How is anterior-posterior polarity determined before fertilisation in Drosophila?
-by oocyte
-maternal effect gene transcripts present (just transcription, not translation)
-bicoid mRNA present at anterior and nanos mRNA present at posterior
-polarity informs system
-bicoid and nanos regulate gap genes
What does a bicoid mutant affect?
anterior region
What does a nanos mutant affect?
posterior region
What can be seen in the Drosophila oocyte before fertilisation using in situ hybridisation?
mRNA localisation
-bicoid at anterior, nanos at posterior
What can be seen in the Drosophila oocyte just after fertilisation using in situ hybridisation?
protein localisation
-conc gradient of bicoid protein with highest concs at anterior
-conc gradient of nanos protein with highest concs at posterior
sets up polarity
What is the role of gap genes?
further compartmentalisation
What is ultrabithorax?
homeotic mutant of Drosophila where there are 2 sets of wings (instead of 1)
-second segment is duplicated, replacing the third
How are homeotic genes organised?
often into complexes/clusters
Drosophila: Hom-C complex containing antennapedia and bithorax complexes
Humans and mice: 4 hox complexes (instead of 1 in Dros)
What is the relationship between the expression of genes and sequence of genes along the chromosome in hox complexes?
the same order
Why are homeotic genes known as homeodomain transcription factors?
homeodomain = have 60aa homeodomain, which is involved in DNA binding
transcription factor = binds to DNA and regulates gene expression (homeotic genes give rise to particular organs)
What is syndactyly?
mutant in human hox gene causing 2(+) digits to be fused
-caused by dominant mutation in Hox-D13
How are homeotic transformations seen in floral development?
2 organs lost (out of petals, sepals, stamen and carpel)
Why is it important to score random nuclei when counting cells in different mitotic stages?
so proportions reflect the asynchronous population of cells and the relative lengths of each of the stages
How many cross-overs result in chromosomes appearing circular during meiosis?
2
How many cross-overs result in chromosomes appearing cross-shaped during meiosis?
1
What is a pericentric inversion?
a chromosomal mutation where a segment containing the centromere is inverted
What is a paracentric inversion?
a chromosomal mutation where a segment not containing the centromere is inverted
What chromosomal rearrangement leads to Edwards syndrome?
trisomy 18.
47, XX or XY, +18
What chromosomal rearrangement leads to Patau syndrome?
trisomy 13
47, XX or XY + 13
What chromosomal rearrangement leads to Downs syndrome?
47, XX or XY +21
What chromosomal rearrangement leads to Klinefeller syndrome?
sex chromosome aneuploidy
47, XXY
What chromosomal rearrangement leads to Turner syndrome?
sex chromosome aneuploidy
45, XO
What is a null allele?
a mutant allele which has complete loss of function
What happens in leptonene (during prophase 1)?
replicated chromosomes start to contract
What happens in zygotene (during prophase 1)?
chromosomes line up in homologous pairs, held together by synaptonemal complex
What happens in pachytene (during prophase 1)?
crossing over occurs between non-sister chromatids
What happens in diplotene (during prophase 1)?
chromosomes separate slightly
-chiasma is visible
What happens in diakinesis (during prophase 1)?
chromosomes contract further
