Human Molecular Genetics Flashcards
what is eugenics
political and social movement in 20th century aimed at improving human species by controlling breeding
positive eugenics
encourage breeding by those with ‘good’ traits
negative eugenics
discourage breeding by those with ‘bad’ traits
theory behind eugenics movement
prevent feebleminded (n) from breeding caused artificial selection against n which reduced frequency of n so less feeblemindedness etc
argument against eugenics
medical rights, privacy, reproductive autonomy
huntington’s disease and eugenics
Acceptable to refuse to test women who do not give complete assurance that they will terminate a pregnancy where there is an increased risk of Huntington Disease. If test positive, must abort
theory of evolution
- all life is descended from common ancestor
- evolution is due to natural selection
- Overproduction of offspring + limited resources = competition for survival
factors required for natural selection
- Variation: individuals within a population vary from one another
- Inheritance: parents pass on their traits to their offspring genetically
- Selection: some variants reproduce more than others
- Time: successful variations accumulate over many generations
sexual selection
Individuals with certain inheritable traits are more likely to mate and pass on the alleles for those traits
process of DNA sequencing
- Mix known template strand with primer and DNA polymerase which will attach deoxyribonucleotides dATP, dCTP, dTTP and dGTP
- Then modified nucleotides “dideoxynucleotides” are attached which prevent further extension. These are fluorescently tagged - each dideoxynucleotide has a different fluorescent tag
- The DNA fragments are passed through a capillary to separate them by size
- Sequence is read by a detector which sees the fluorescent tags
challenges when sequencing large genomes
- Used to be very expensive - technology required
- Used to take very long - technological advances makes it much faster (20 years vs almost instantaneous)
- Mistakes can occur so the entire genome is sequenced several times over
- Confusing parts such as repetitive regions can be resolved by comparing to a “reference genome” or maps, if they exist
how to find protein-coding genes in genome
- Open reading frames (ORFs)
- Transcriptome analysis
- Comparative genomics
process of aligning DNA sequences in modern genome sequencing
- Break up many copies of the chromosomal DNA into overlapping chunks with ultrasound.
- Add ligate pieces of known sequence to each end (linkers) - so every unknown fragment has a known fragment attached to it so can be identified
- Sequence using primers that bind to the linkers
- Use the same known linker as a primer to “shotgun” sequence all the unknown bits of DNA from the genome, using massive parallel sequencing platforms.
- Computer records all sequences
- Assemble the reads into “contigs” - continuous sequence of DNA
- Computer ignores the linker sequences and tests each sequence for overlap with every other sequence. The aim is to produce a single contig for each chromosome.
open reading frames
- Find start (Met) codon (ATG)
- Find TATA box upstream of Met
- Most genes have STOP codon as final exon
Transcriptome analysis
- Instead of sequencing genomic DNA, we can make a DNA copy of all the mRNA within a cell or tissue, and sequence that.
- If we collect all the different sequences we should end up with a representation of the transcriptome (genes that are being actively transcribed) of that particular cell or tissue.
- These sequences often called expressed sequence tags or ESTs
comparative genomics
Line up sequences of related genomic DNA e.g. human vs. ape, and look for conserved (highly similar) regions
ways in which mutations can be acquired
- Inherited mutations are called germline mutations and are passed on via gametes
- Can be acquired by somatic cells if DNA gets damaged or is copied incorrectly. Somatic mutations - not passed on to next generation
types of mutations
- silent mutation
- missense mutation
- frameshift mutation
- triple repeat expansion
- chromosomal rearrangement
silent mutation
- base-pair substitution producing a codon that still codes for the same amino acid
- no effect on phenotype
- Often occur in non-coding regions such as intergenic regions and introns, but can also occur in exons
missense mutation
Missense mutations change an amino acid which can alter the proteins ability to function, as in sickle cell anaemia (in haemoglobin)
frameshift mutation
- When you add or subtract a base pair from a DNA sequence
- a lot of damage
- Changes DNA sequence along the rest of the molecule, creating extensive missense and nonsense mutations
triple repeat expansion
- Some genes contain stretches of triplet repeats
- These repeats sometimes undergo catastrophic expansion leading to dysfunction
- Can alter protein function (Huntington disease) or destabilise chromosome (fragile X syndrome)
chromosome rearrangements
Mutation effects can be larger scale and affect whole chunks of chromosomes rather than just a single base or repeat sequence
- e.g. translocation, inversion, aneuploidy
finding potential disease genes
- sequence genome
- map to Hg19 reference
- compare to common variants
- if not common then it is a novel variant - use knowledge of types of mutations to predict whether it will be harmful or benign
haemophilia A and B
- disorder of blood clotting
- Haemophilia A (classic one) is most common affecting 1/5000 males worldwide
- Results from impaired or absent clotting factor VIII protein
- Haemophilia B clinically indistinguishable, affects factor XI
- If untreated, high risk of death from uncontrolled bleeding
- Pain and tissue damage from internal bleeding
- Treated by intravenous infusion of missing protein
haemophilia inheritance pattern
- Haemophilia A caused by mutations (most commonly an inversion) in factor VIII gene found on X-chromosome
- Mutations are “loss of function”
- One intact copy prevents against disease so women rarely affected as have 2 X chromosomes
- Sons of carrier women have 0.5 probability of inheriting disease
- Around 30% of cases have no family history (sporadic)
- If only males affected in family tree, then assume X-linked recessive
huntington’s disease
- progressive tremor, involuntary movements, neurodegeneration
- onset in midlife so usually have kids without knowing
- no effective treatment
- caused by expansion of CAG triplet in huntingtin (HTT) gene
- protein has long polyglutamine tract so becomes unstable and fragments, clumping together in nerve cells
- genetic testing
- use PCR to determine length of CAG repeat
- 10-35 copies of CAG = normal
- 27-35 = risk of descendants developing HD
- 36-40 = risk of developing HD
- 40+ = disease develops
inheritance pattern of huntington disease
- autosomal dominant inheritance
- probability that individual 1 will get HD = 0.5
- probability that individual 2 will get HD = 0.5x0.5 = 0.25
cystic fibrosis
Symptoms:
- Lung infections, pancreatic insufficiency, congenital absence of vas deferens in males, salty tasting skin
Range from mild to severe
- Severe form has frequent infections and reduced life expectancy
Autosomal recessive
Caused by mutations of chloride ion transporter (CFTR = cystic fibrosis transmembrane regulator)
- Reduced function of CFTR protein causes thickening of cell secretions
Many different mutations in CFTR gene can cause CF
- Most common is 3bp deletion of deltaF508
polygenic disorders
- appear to run in families but do not follow straightforward inheritance patterns
- involve several genes acting together or combined with environmental factors
- e.g. asthma, diabetes, multiple sclerosis
showing conservation of genes in related species
- align sequences up and mark points where sequences are the same - aligning
- Things in common are ‘conserved’ - encode biology in common within a species
- Different things - may encode organism specific biology - our ability to do different things to other organisms
- By comparing order of genes on chromosomes in one species with another you can identify syntenic ‘blocks’ (= when gene order is conserved between species)
how to find function of genes
Inhibit them in an organism using induced mutation, and study the resulting phenotype of the offspring, compared to a normal genotype with a normal phenotype
advantages and disadvantages of mouse as a model organism
advantages:
- Mammalian - share more biological functions with humans
- Human and mouse genomes are the same size and many genes are in the same order (synteny)
- 99% of mouse genes have human homologues
- Similar immune system
- Small, easy to maintain, fast breeding
- Inbred lab strains reduce variation
disadvantages:
- Mouse physiology can differ from human
- Still more expensive than other models
- Ethic concerns
purpose of genetic screening
- accelerate rate of mutations and increase phontype discovery
- drosophila (fruit flies) are ideal as they are small and have short life cycle
genetic screening in drosophila
- Feed male flies chemical mutagen (ethyl methane sulfonate)
- Establish inbred lines from single sons
- Inbreed siblings to try and make homozygous offspring
- Do homozygous embryos die?
process of genetic screening
- Decide what process or structure you are interested in.
- Perform a genetic screen
- Pick mutants that have defects in the thing you want to study
- Use these mutants to discover what the mutant genes normally do.
- Using this technique you know the function of a gene (phenotype) before you know its sequence (genotype
transgenic process
- Put jellyfish gene coding for protein for green fluorescence into male pronucleus of mouse fertilised egg
- Replace fertilised egg into foster mother
- Offspring will have protein for green fluorescence
purpose of “knockout mice”
- We can remove (or replace) the gene we are interested in by genetically modifying an organism.
- By examining the GM organism we should be able to work out what the gene normally does
- can be used as a model for human cystic fibrosis to research effectiveness of drug therapy and gene therapy on damaged CFTR gene
- however not a great model as mice die of gastrointestinal problems, males not infertile as they are in humans, pancreatic disease not replicated
how do cells develop during development
- Embryo begins as a small number of naïve, totipotent cells
- Embryonic stem cells can give rise to all cell types except trophectoderm(pluripotent)
- Progressive restriction of cell fate until terminally differentiated, and can only give rise to same type of cell.
what makes a stem cell special
- Can divide without limit
- Undifferentiated
- Can divide to give rise to both stem cells and cells which will go on to differentiate into functional tissue cells
embryonic stem cells vs adult stem cells
embryonic stem cells
- pluripotent so can differentiate into almost all cell types in the body - essential for growth and development
adult stem cells
- thought to only differentiate into one or a few cells e.g. bone marrow contains haematopoietic stem cells which differentiate into blood cells, as well as mesenchymal stem cells which give rise to bone and cartilage cells
gene therapy
- way of correcting single gene disorders (such as cystic fibrosis) by inserting correct allele into cells of affected tissue
- best applied to stem cells because they will keep dividing and passing on corrected gene to cell progeny
- possible method is to use retroviruses containing corrected allele to infect cultured bone marrow cells from a patient then replace cells
how are mammals cloned
why is cloning hard
- Many mammalian cloned embryos and foetuses die before birth
- Long term health effects to cloned mammals and their offspring are not yet understood
selection pressures against HIV
Immune system, drugs, tropism (different environments) between different tissues etc select against HIV
what is a contig
computer assembled section of genome sequence made by aligning overlapping smaller sequences reads
germline mutation
when mutated DNA sequence is present in cells that produce gametes and can therefore be inherited by next generation
what is synteny
refers to the similarity in content and organisation between chromosomes of different species; similar blocks of DNA are found in genomes of different species and genes are retained in same order on chromosome
whats is the transcriptome
group of mRNA’s transcribed by a particular tissue or cell, representing genes that are coding for proteins required in that particular cell or tissue
nonsense mutation
base-pair substitution or frameshift mutation that creates a stop codon and prematurely terminates translation
therapeutic cloning
- Therapeutic cloning involves the creation of ‘personalised’ embryonic stem cells
- A donor nucleus is transferred into an enucleated zygote as for reproductive cloning
- The embryo is cultured until the blastocyst stage
- Embryonic stem cells are made from the blastocyst and cultured under various conditions to make cell types to order.
