HMB265 Notes PDF

Lecture 12 Additive ○ depends on how many doses of alleles present ○ can have a phenotypic effect of 0, 1, or 2 Frequency is higher in the middle = bell curve shaped graph ○ additive inheritance - contributions of each loci add up with no inter...

Lecture 12 Additive ○ depends on how many doses of alleles present ○ can have a phenotypic effect of 0, 1, or 2 Frequency is higher in the middle = bell curve shaped graph ○ additive inheritance - contributions of each loci add up with no interactions between them (e.g. no dominance/epistasis) Genetic origins of a quantitative trait: ○ Hypothesis 1: segregation of alleles at many loci, small equal and additive effects ○ Hypothesis 2: a few genes, large additive effects ○ Polygenic - only genetics, multiple genes contribute to their variation ○ Multifactorial - genetic and environmental factors (multi factors) affect influencing observed trait ○ C an also have environmental factors Environmental modifications ○ phenotype = genotype + environment ○ E.g. Siamese cat warm temp = enzyme nonfunctional = no melanin = light fur Cool temp = enzyme functional = melanin = dark fur Incomplete dominance ○ heterozygous phenotype is distinct from either homozygous intermediate phenotype Quantitative Traits ○ segregation of alleles at multiple loci with additive effects - affected by genotype E.g. complementation, law of segregation, linkage ○ Individual phenotypic classes are masked by the environment - quantitative trait locus (QTL) - affected by environment Lecture 13 How much of the phenotypic variation is attributable to genetic variation versus environmental variation? - determining what genetic effects that are buried within a quantitative trait ○ P = G + E (phenotype) ○ Vp = Vg + Ve (variation) Variants of phenotype ○ Vg/Vp (how much of variation is bc of genotype) Broad Sense heritability ○ The amount of phenotypic variation attributable to genetic variation ○ H2 = Vg/Vp (population or family specific) if variation is all due to genotypic then H2 = 1 if its all due to environment then H2 = 0 ○ H2 - NOT predictive , tells us why it is that phenotype  ○ H2 tells us if its high the phenotype of an individual is likely to be attributable to its genotype in that family or population ○ H2 does NOT tell us the phenotype of progeny due to their parent’s phenotype individual’s precise phenotype cannot be predicted on the basis of its parents’ phenotypes Why H2 is not predictive ○ Vp = Vg + Ve ○ Vg = Va + Vd + Vi Va - variation due to additive effects - PREDICTIVE Vd - variation due to dominance effects - NOT PREDICTIVE Vi - variation due to epistatic effects - NOT PREDICTIVE Vg - variation due to genetic effects ○ Dominance effects - phenotypic: can show dominant trait, but can be heterozygous therefore their progeny can homozygous recessive (progeny will differ in phenotype from parents) ○ epistatic effects: crossing two parents and can rescue a masked effect leading offspring to have a different phenotype ○ if its due to additive effects —> it is predictive Because each genotype has its own phenotype Narrow Sense Heritability ○ Phenotypic variation due to additive genetic variation ○ h2 = Va/Vp All phenotypic variation due to additive variation, h2 = 1 All phenotypic variation due to other genetic and environmental effect, h2 approaches 0 2 ○ h tells us that if its high, phenotype of individual is predictable based on phenotype of its parent IN THAT FAMILY ○ h2 does not tell us tell us what is happening in other families OR what the genes are E.g of narrow sense is height Lecture 14 Quantitative trait loci can be identified using genetic mapping and association of genetic markers with the trait Dissection of quantitative traits ○ step 1 cross between individuals that are inbred relative to each other and differ at the trait of interest purebred (Vg = 0) parents give offspring of F1, F1 are all identical and have no genetic variation, F1 offspring are crossed and now you have genetic variation ○ step 2 Determine the frequent distribution in F2 graph them in order  tomatoes: in order of size, smallest to largest ○ step 3 use molecular markers to genotype the individuals, attempting to find markers that cosegregate with the trait tomatoes: homozygous is small or large, heterozygous is medium (2 markers on electrophoresis) see which genetic markers match with the phenotype, see if there a segregation pattern ○ Step 4 use a statistical method to determine if markers are cosegregation (associating) with the trait or not Association = trait pairs with a genotype No association = variety of trait (fruit size) among each genotype graph as to have a slope ○ step 5 plot the degree of association (LOD score) on a linkage map odds ratio (OR) means he probability of cosegregation of trait and alleles at a locus =probability of linkage / probability of no linkage numerator: (probability of parental)^(how many parentals) * (probability of recombinant)^(how many recombinant) denominator: ½ ^ (how many progeny) LOD = log OR If LOD is 3 or greater, it is considered linked LOD = a marker may be associated with a trait - association does not equal causation There may be a marker of interest to look into With LOD alone you CANNOT say that a particular marker plays a role on a particular trait this proves association dissection of the cause of size ○ step 1 QTL controlling tomato fruit size is mapped chromosome is labelled etc ○ step 2 create recombinant inbred lines (RILs) Single seed descent for plants Inbred selected individuals down a lineage 1-2 siblings are inbred - creates single descent from each original F2 individual Recombination at each generation creates a “ladder-like mosaic” of maternal & paternal alleles down length of the chromosome Inbreeding for many generations results in each lineage having a unique mosaic, homozygous for either the maternal or paternal allele at each locus  More inbreeding = less heterozygosity Allows for fine mapping ○ step 3 candidate genes in QTL are identified by fine mapping recombinant chromosomes (NILs —> near isogenic lines) are used to fine map QTL to a single gene (picks the gene that associates with weight) ○ step 4 the expression of the candidate gene was examined using PCR results show a greater expression in small plants ○ step 5 the sequence of the protein was analyzed to see if the proteins function matches the role in tomatoes ○ step 6 genetic engineering used to prove its role inserting small allele into large tomato to see if it ends up small Marker plays role in trait this proves causation Lecture 15 Mutations ○ Mutations affecting phenotype is rare ○ Fwd mutation (WT —> mutant allele) ○ Reverse mutation (mutant allele —> WT) Rate of fwd is always higher than rev rate increases after exposure to mutagen Classifications ○ Base Substitution - one replaced transition: purine to purine (Pure silverA, Gold) or pyrimidine to pyrimidine (C, U, T the Pie) transversion: purine to pyrimidine, vice versa transitions are more tolerable, more frequent ○ Deletion - one or more deleted ○ Insertion - one or more added Indel mutations (insert deletion) ○ Inversion - dna segment flipped 180 degrees ○ Reciprocal translocation - parts of 2 nonhomologous chromosomes change place Studying mutation ○ Mutations act as markers for genes mutations disrupt gene fxn, so study how the WT gene works and what happens without WT gene causes of muts ○ Spontaneous Arise in absence of known mutagen, random  depurination loss of purine base from backbone hydrolysis of purine base loss identity of entire nucleotide deamination Removing an amino group from a base cytosine to uracil identity loss of amino group ○ Induced Mutagens by geneticists that alter nucleotide sequence Caused by x rays causes breakage - deletions UV light thymine dimer form and kink DNA —> trouble reading dimer and can’t translate properly ○ Oxidation (induced AND spontaneous) causes mispairing oxidative damage E.g., guanine oxidized -> 8-oxodG (GO) = oxygen added ○ Indel mutations Addition Strand slips and extra base pairs poking out addition of base pairs to shorter strand to even strand out Deletion Strand slips and extra base pairs poking out Deletion of base pairs to longer strand to even strand out Molecular consequences of mutations in coding sequence ○ silent point mutation Base pair altered but no change to protein function Synonymous = silent mutation Neutral - no affect on protein - no significant effect on fitness ○ missense point mutation Nonsynonymous = non silent Can be beneficial (positive effect on selection), harmful or neutral alters to protein function Base pair altered but there is change to protein function In western and northern blots the speed of band stays the same because only changing the base pair - no extra insertion or deletion (that would affect size) conservative subs in a similar amino acid, less likely messes up the fxn nonconservative subs in a different amino acid, more likely to mess up the fxn ○ Northern blot = mRNA  ○ Western blot = protein ○ Point mutations (silent and missense) The ratio of synonymous and nonsynonymous mutations provide a measure of the strength and type of selection acting on a gene (some are more favoured than others - drive evolution) ○ Nonsense Mutation: Switch base pair that turns it into a stop codon Protein is stopped not mRNA, amino acid sequence stays the same length Northern blot stays the same, western blot changes bc protein now its stopping prematurely and it will be smaller ○ frameshift mutation Inserts or deletes base pair to shift reading frame (groups of 3 amino acids) for rna polymerase Coding for dif codons can code for diff proteins or stop codons - making protein longer or shorter Western blot can change depending on size of protein ○ intragenic suppressor mutations downstream of the original frameshift to recover the reading frame mostly restored still have original mutation but the intragenic mutation is the second mutation that recovers most WT phenotype mutations outside the coding sequence ○ Splice donor/ acceptor site mutations Base pair switched, excision site not recognized and cannot be excised anymore so it stays and leads to larger rna (or vice versa) Larger mRNA leads to slower down northern blot and western blot Leads to large additions or deletions that may cause frameshifts ○ loss of fxn alleles - typically recessive Null (amorphic) - deletion causes mutant phenotype Mutation just as severe as deletion - no activity for both Hypomorphic (leaky) mutation - less severe (deletions or point mutations) Loss of gene activity by mutation is less severe that of the deletion mutation is recessive —> haplosufficient haploinsufficient ○ Gain of function mutations usually dominant Hypermorphic mutations Excess protein produced compared to normal by mutant allele - cause abnormal phenotype Neomorphic mutations Proteins produced with new functions or normal protein produced at inappropriate time or place - supposed to be expressed only in  hearts but mutant expresses in other places it is not supposed to be Disease mutation - vision ○ 1 blue, 1 red, dif amounts of green ○ Crossing over = red green colorblindness Pathway mutations ○ Mutations in pathways can halt the pathway from proceeding to final products Prevent gene from producing enzyme to continue path ○ mutations provide information about gene function biosynthetic pathway can be discovered ○ If supply compound mutant will grow biosynthetic pathway ○ compounds at the end of the pathway will support the growth of the most mutants (will have the most + in their columns) ○ compound at the start of the pathway will support the growth of the least mutants (will have the most - in their column) ○ Each mutant has mutation at one loci (gene), all other loci are WT the mutant (enzyme) at the start of the pathway will have the most + in their row the mutant (enzyme) at the end of the pathway will have the most - in their row ○ Genes encode for enzymes which are responsible for converting one compound to another compound (E to A) ○ Identify which mutated enzyme that the compound does not grow with Lecture 16 Transposition ○ Movement of small segments of DNA called transposable elements from one position to another in the genome Discovered by Barbara mcclintock McClintock's experiment ○ one strain of corn usually broke chromosome 9 Ds element caused break Ac required to activate Ds —> breakage ○ Chromosome 9 controls several phenotypes of corn breakage would cause altered phenotypes Unusual phenotypes caused by the Ds element ○ Genes control dif phenotypes ○ chromosome breakage Ac activated = breakage at DS whole segment is gone with dominant allele reveals recessive alleles doesn't happen in the entire cell —> patchy (colourless in certain part)  kernel can show the recessive phenotype ○ new unstable alleles (spotted) Ac activated = Ds loss from c gene Left with dominant C allele Ds can be inserted into c gene (attached together), Ac activates Ds loss and dominant allele restored transposable elements in corn ○ Ds: nonautonomous not automatic, needs other element for mobility ○ Ac: autonomous mobile on their own, can excise itself as well Autonomous encode info required for own movement and movement of nonautonomous elements ○ Ac or ds inserted makes the phenotype spotted (ac can be inserted to excise ds, or ac can be inserted and excise itself restoring WT phenotype) Mechanism of transposition of corn ○ transposable elements are surrounded by repeat sequences repeats are recognized by transposase ○ cleaves off in a “loop” can be inserted to target sequence ○ inserted elements surrounded by a short repeat Because of the staggered cut, host repairs gap Transposable elements are common ○ 12.5% of drosophila genome ○ 34% of human genome ○ two main classes in eukaryotes retrotransposons dna transposons retrotransposons ○ never leaves the cell ○ RNA intermediate ○ long term repeats, gag, pol, LTR gag: maturation of rna pol: codes for reverse transcriptase ○ instead of making protein the mRNA will get reverse transcribed into DNA Transcribed in nucleus, sent out, reverse transcribed into Dna (by reverse transcriptase), sequence put back into DNA DNA transposons ○ dna sequence will have a p element that will be excised out with transposase, can either 1) repair gap using a sister chromatid or homologous chromosome (template) containing a P element 2) repair gap using a homologous chromosome lacking a P element transposable elements in humans  ○ humans have large genomes, therefore we have a lot of transposable elements resulting in a c value paradox (the observation that genome size, or C-value, does not correlate well with organism complexity) Have such a big genome relative to how many genes we have DNA content can highly vary in vertebrates several types of transposable elements which explains why ○ Vast majority of repetitive sequences and transposable elements form 2 classes: LINEs: long interspersed elements Similar to retrotransposons, has their own reverse transcriptase used to move around in genome SINEs: short interspersed elements Doesn’t have their own reverse transcriptase Alu - highly repetitive DNA seq - elements in the human genome are a type of SINE (retrosposans) ○ Named Alu because their target sites contain the same recognition seq that the Alu restriction enzyme uses ○ More than 10% of Human genome is made up Alu DNA transposons autonomous nonautonomous ○ How do animals and plants survive with so many mobile elements Transposable elements are inserted into introns so they dont code for anything anyways often defective - cannot move around unable to transpose due to mutations mutations in inverted repeats or lack of active transposase lots of epigenetic changes is why there’s many mechanisms to regulate how active transposons are, helps prevents transposable elements from jumping around Examples of transposable elements in humans: lines affecting factor 8 gene - result of hemophilia A, insertion of Alu into brca2 causes breast cancer Transposable elements can ○ generate chromosomal rearrangements unequal crossing over between TEs misalignment causes duplication or deletion ○ Relocate genes two transposons an form a large, composite transposon can be incorrectly excised if it loops to the next transposon - large portion excise if looped incorrectly laboratory use of transposable elements ○ allows for the transfer of genes ○ rosy- x rosy - can give off rosy+ offspring if a rosy+ P element is inserted through a plasmid into the parents embryo  some of the germ line cells will contain this gene through transposition p element helps the rosy+ plasmid insert into germ line cells turns into transgenic fly (altered genome) only affects the germ line of parent Lecture 17 Chromosomal packaging ○ nucleosomes in heterochromatin are tightly packed transcription requires remodelling of them as promoters are hidden Chromatin remodelling ○ histone tail mods ○ alter chromatin structure X chromosome inactivation ○ heterochromatin formation inactivates entire X chromosomes ○ Random x-chromosome inactivation in females early development - hereditary through cell division Reactivated in germ cells to have both copies for meiosis Due to dosage compensation - only want one copy of X Mechanism of x inactivation ○ coating of chromosome with XIST RNA ○ hypoacetylation (condenses chromatin, represses genes) of Lys in two histones ○ histone methylation (blocks transcription) in inactivated X changes in chromosome number ○ euploidy: complete sets of chromosomes OIDY Diploid: 2N Monoploidy: one set of chromosomes N polyploidy: more than the normal number sets of chromosomes triploid: 3N Tetraploid: 4N ○ aneuploidy: loss or gain of one or more chromosomes OMY Nullisomy = 2n - 2 Monosomy = 2N -1 Trisomy = 2N+1 Tetrasomy = 2N+2 Monoploidy ○ pathogenesis: development of unfertilized egg into an embryo (w/o fertilization) single set of chromosomes produce gametes by modified meiosis ○ usually lethal unmasks recessive lethals sterility - no meiosis ○ example: male bees wasp and ants ○ can be produced experimentally  Monoploid plants used for visualizing recessive traits directly and into of mutations haploid protein grains are planted into agar, growth, embryoids treated with plant hormones and it grows sterile polyploidy ○ Common in plants ○ Associated with origin of new species ○ May positively correlate with size and health ○ coffee , peanuts, large apple, pears, grapes - tetraploid ○ Large strawberries octoploid ○ Autopolyploids (type of polypoid) originate within a species Additional copies come from the same species diploid (2n) (failure in meiosis) x monopolid (n) = autotriploid (3n) likely results in a new species, prob infertile bc unbalanced gametes (won't evenly split) - form aneuploid gametes Occasionally can also produce balanced gametes Autotetraploids: doubling of 2n chromosome complement to 4n Spontaneous doubling Induced by a drug called choline ○ instead of cel separating into two diploid cell, membrane grows around duplicated chromosomes and forms one tetraploid (4n = 8) ○ Allopolyploids Hybrid of two or more closely related species Partially homologous chromosomes (homeologous) ○ **homologous - same ancestor, homeologous - originated from speciation and brought back together in same genome amphidiploid: doubled diploid, doubling in germ cells ○ Individual that is a hybrid of two different species and that possesses four sets of chromosomes (2 from each parents) ○ Sum of parents chromosomes Tetraploid meiosis ○ 4n, in gametes = 2n (halved in gametes) ○ since there’s 4 chromosomes, lots of different arrangement (bivalent —> pairs of synapses homologous chromosomes) new ratio is 1 (AA):4(Aa):1(aa) so to get an aa phenotype after mating —> ⅙*⅙ = 1/36 Meiotic Nondisjunction ○ Normal division in meiosis 1 or 2 Nondisjunction in 1st division - both sets go into one cell and those split so that there are two in each cell The resulting 4 cells are: n+1, n+1, n-1, n-1  Nondisjunction in 2nd division - both sets go into separate cells (NORMAL) and those split so that one cell splits so that there are one chromatid in each cell The other cell splits two chromatids into one cell and none in the other ○ n+1, n+1, n, n monosomy ○ 2n-1 ○ usually lethal in utero in humans or will have birth defects monosomy 21: born with severe multiple abnormalities and dies shortly after birth turner syndrome (XO): 99% of affected fetuses are not born. But isn’t one x inactivated anyways?: this has abnormalities because X inactivation does not occur until the 100 cell stage in development (still need xx in beginning of development) some of the genes on the inactivated X chromosome are expressed trisomy ○ 2n+1 ○ often lethal in animals trisomy 21: Down syndrome (three chromosomes at chromosome 21 instead of 2) females can be fertile males are infertile lives to about 40-60 years risk of having a baby with DS increases as you age because your gametes have been sitting paired up for so long (prophase 1) that they can get stuck together XXY: Klinefelter syndrome infertile male, too much X chromosome expressed One X is inactivated some X genes are expressed at twice the level of XY male XXX fertile x pairs with one x, third does not pair and isn’t transmitted not passed to progeny XYY fertile x pairs with one y, other y does not pair and isn’t transmitted not passed to progeny genomic hybridization: microarray ○ Used to detect duplication/deletions ○ staining patterns show you  prenatal testing ○ screening (not diagnostic) in first trimester nuchal translucency —> ultrasound maternal serum blood test (placental hormone levels) noninvasive prenatal testing (NIPT), blood test ○ diagnostic tests chorionic villi sampling (10-13) Catheter taking tissue samples risk of miscarriage as you have to shove a cathedra through your stomach into the babies personal bubble amniocentesis (16+ weeks) Test results available 1-3 weeks later Risk of miscarriage due to the procedure = 0.5% ○ Fetal testing: Look for abnormal karyotypes Screening for biochemical and molecular disorders Tests are done in combination with blood tests for certain fetal proteins and maternal hormones, and with ultrasound Tests ○ Preimplantation embryo diagnosis Screen for mutatnt alleles prior to implantation First used for CF allele Looking at eggs, if mutant or normal, and choosing which you want Lecture 18 Polytene chromosome used to study changes in chromosome structure ○ Model structure because they have chromosomes that duplicate many times without sister chromatids separating ○ Can easily visualize banding patterns Deletions ○ X Rays: breaks both strands of DNA ○ Intragenic: small deletion within gene ○ Multigenic: many genes deleted ○ Del (Df) homozygous usually inviable ○ Gene imbalance in del heterozygotes can result in haploinsufficiency ○ Can be lethal, larger the deletion the more lethal Deletion loop ○ When a segment is deleted on one strand it will pair up with the homologous chromosome and since the normal homolog doesn't have a deletion, the “extra” will form a loop Pseudodominance ○ Not actually dominant but since the dominant allele was deleted, it will unmask the recessive allele Deletion mapping  ○ Complementation test: Del mutant crossed with a known mutant gene Del heterozygote reveals location of mutant gene Duplication ○ Tandem: adjacent to original gene ○ Nontandem: different part of the chromosome ○ Less likely to affect phenotype as there is no loss of genetic material ○ You can have a dosage issue or genetic imbalance Ex: Too much of the gene expresses too much of a certain molecule ○ Gene can be placed in new location that alters their expression How duplications arise ○ X ray breaks: a double strand break of a chromosome occurs and chunk removed, chunk inserted to another chromosome that has a double stranded break at a different site ○ Unequal crossing over: misalignment and mispairing Bar eyes: unequal crossing over during meiosis results in different copy numbers, too much 16A in the gene that expresses eye phenotype can have overexpression of it Inversions ○ Pericentric: included the chromosome ○ Paracentric: does not include chromosome ○ Most inversions don't alter phenotype unless breakpoint occurs within gene Breakpoints between genes ○ Break in DNA → inversion → joining of breaks ○ Does effect too much because each gene still has a promoter and 2 proper strands Break points within one gene ○ Has one break between genes and one break within genes ○ Unable to translate and transcribe the whole gene as promoter is lost with one half of the gene ○ You have a mutated gene Break points within two genes ○ Break points in both genes → each half from the different genes pair to each other ○ This gives rise to different phenotypes Heterozygosity for inversion ○ Reduces the number of recombinant progeny ○ It has to form an inversion loop to pair up properly with homologous chromosome Paracentric inversion loop (no centromere) ○ Dicentric (two centromeres) bridge causes a break and acentric fragment is lost ○ Crossing over in inversion results in formation of deletion products causing reduced number of viable gametes Pericentric inversion loop (contains centromere) ○ No dicentric bridge  ○ Reduced viable gametes Translocations ○ Nonreciprocal intrachromosomal translocation (same chromosome) ○ Nonreciprocal interchromosomal translocation (different chromosome) ○ Reciprocal interchromosomal translocation (exchanges entire segments) ○ Most translocations don't alter phenotype unless there a break within the gene Robertsonian translocation ○ Acrocentric chromosomes (most genes on one arm) swap material and one gene will contain most of the genetic material (large metacentric chromosome) while the small chromosome is lost ○ If extra pair of chromosome 21, can result in down syndrome If the large metacentric pairs with a normal chromosome it can have too much info/an extra chromosome ○ Mouse populations possess different chromosomal translocation and fusion resulting in different numbers of chromosomes, if crossed they are infertile therefore they are “separated” Normal segregation of translocation homozygote during meiosis ○ Red and blue chromosomes are not homologous, but have randomly paired up and exchanged genetic material ○ Since they share genetic material, now they will pair up and segregate different ways Alternate: two WT chromosomes with segregate to one side and the other two will go to the other side (all gametes are viable as there is no genetic material lost) Adjacent-1: one red WT and one blue with a bit of red will segregate together and the other two will segregate together as well (since theres duplications and deletions, there will be genetic imbalance and they will all be non surviving) adjacent -2: both chromosomes go one way, this is rare as the chromosomes from the same pair will go to the same spindle pole (as there is unbalanced pairing, there will be non surviving) Result of three segregation patterns: Semisterility: since embryogenesis —> larva —> adult ○ Takes ~4 days to grow to adult ○ Skeleton on the outside making it easy to screen for mutants in skeleton ○ Pathway Zygote: diploid zygotes nucleus Multinucleate syncytium: multiple rounds of nuclear division without cell division (one big cell) Syncytial blastoderm: most neuclei migrate to cortex and membrane grows inward Cellular blastoderm: primordial germ cells (pole cells) and segmentation starts Gastrulation: embryo cells move and germ layers form into basic body plan of animal Segmentation: the process of dividing the body of an animal or plant into repeated sections or parts Terms ○ Dorsal: back ○ Ventral: stomach ○ Anterior: head ○ Posterior: butt Screenings for drosophila mutants ○ balancer chromosome prevents recombination on chromosome ○ when there’s hétérozygote mother crossed with a hétérozygote father, both can pass on mutation and offspring is homozygous for mutation. If the mutation is maternal, the female will live but be sterile. If the mutation is zygotic, it will be lethal classes of drosophila segmentation  ○ gap: effects entire segment of embryo, large chunk ○ pair rule: expressed in every other segment ○ segment polarity: expressed in band of every segment in different levels Gap genes are activated by specific maternally provided proteins ○ In oocyte: bicoid at anterior nanos at posterior hunchback and caudle uniform ○ in embryo: bicoid at anterior (repress caudle) and hunchback at anterior nanos at posterior (represses hunchback) and caudle at posterior analysis of regulatory elements with reporter genes ○ replace coding region with reporter gene so that you can visualize expression ○ clone fragments a b or c and inject them, see at which part of the cell expression is shown hox gene ○ expressed in spatially restricted domains ○ they do not determine segmentation but determine what segment will become ○ how genes regulate the identity of body parts ○ order of genes on chromosomes match the order of expression on drosophila Lecture 21 Changes found in cancer cells ○ Uncontrolled growth autocrine stimulation: normal cells absent, cancer cells present Contact inhibition: normal cells present, cancer cells absent cell death: normal cells present, cancer cells absent gap junctions: normal cells present, cancer cells absent ○ genomic instability defects in genes cause mutations and become cancerous if not repaired ○ potential for immortality normal cells plateau at some point and stop growing cancer cells never stop growing ○ ability to distrust local tissue and invade distant tissues Metastasis, cancer cells migrate through blood stream multi hit model ○ cancer required several mutations, more mutations increases the cells chances of becoming malignant evidence of multihit model ○ incidence of cancer in humans increases with age ○ human colorectal cancers: later stages have more mutations ○ cancers are clonal descenders of one cell (if you do electrophoresis a normal cell and tumor cell can have the same gene bc it came from once a normal cell) most cancers result from exposure to environmental mutagens  ○ one sibling or twin gets cancer, the other one doesnt ○ populations that migrate develop cancers that are likely of that environment ○ exposure to mutagens increases the rick of developing cancer (cigs, sun, viruses) An individual can inherit a mutated gene and increase the probability that cancer will occur cancer genes ○ proto-oncogenes: gain of function dominant mutation converts these genes to oncogenes ○ tumor suppressor genes: loss of function recessive mutations, inhibit tumor supressors genetics of oncogenes and tumor suppressor gene ○ dominant oncogene With one abnormal gene activated, mutant protein is expressed ○ mutant tumor suppressor gene with one mutated gene, normal protein is expressed with both mutated genes, no normal protein is expressed (cell proliferation) examples of oncogenes ○ mutated receptor kinase genes will be phosphorylated will have continuous active tyrosine kinase and have cell division ○ RAS oncogene inative ras is bound to GDP SOS interaction stimulated GDP to GTP exchange and activates it, cell division is present GAP (cuts GTP to GDP to inactivate it) will be ignored and will have continuous cell division ○ Bcr/c-abl examples of tumor suppressor genes ○ RB (retinoblastoma) unphosphorylated Rb inhibits E2F CDK4 and Cyclin phosphorylated Rb and will let go of E2F E2F is now going to help progression of cell cycle (G1 to S phase) DNA repair defects ○ Xeroderma pigmentation Defect in nucleotide excision repair Prone to UV induced skin cancers ○ Hereditary nonpolyposis colorectal cancer Lecture 22 Somatic Gene Therapy  Only affect your own somatic cells Not hereditary - cant pass down Ex vivo - Remove cells from body, add therapeutic gene, return cells back into body In vivo - deliver wild type genes directly to somatic cells in body ○ Corrects diseased phenotype in affected somatic cells First gene therapy Trial (1990-1992) Severe combined immunodeffieciency disease (SCID) ○ caused by mutation in adenosine deaminase (ADA) gene - deficiency in ADA gene To be good target must be: Single gene causing disease Involves white blood cells - easy to obtain from blood, insert gene and return back to body Small recovery of ADA function can restore immune function - get much better ○ unable to mount a normal immune response to infection Effect of a Defect in ADA gene: Without ADA enzyme to break down deoxyadenosine, toxic Deoxy ATP builds up in T cells (T cells important for immune response) and B cells not activated ○ severe combined immune deficiency which can be fatal Gene therapy aimed at treating diseases where there is no other cure Gene Therapy protocol: 2 young girls (4 and 9), still had lymphocytes that could be harvested ○ Lymphocytes harvested ○ Cultured in lab ○ Infected with retrovirus (containing WT ADA gene) ○ Reinfuse ADA-gene-corrected lymphocytes back into SCID patient ○ Infused over 2 years over and over Long Term Outcome After 10 years of infusion: ○ Patient 1: 20% had ADA gene and gene expression ○ Patient 2: 0.1% of lymphocytes have ADA gene (despite all of treatment) with no detectable ADA expression. Also has immunity to components of the gene transfer system. Gene Therapy Challenges 1) How to get DNA into cells? viral vectors - method of choice for now (fast efficient)  ○ Herpes - can only affect certain cells ○ Viruses are extensively modified before they are used as vectors Electroporation - hurt cells direct injection into blood or other tissue - ineffficient depends on tissue particle bombardment - can be detrimental liposomes Future - CRISPR? - editing where you did not want editing to occur 2) Will the transgene be expressed in the correct cells at a high enough level for an appropriate period of time? ex vivo gene therapy helps to ensure that correct cells are targeted - remove and then edit is an advantage, only correct cells are treated strong promoters are generally used to drive transgene expression - ensuring its being expressed at high enough level to have effect tissue-specific promoters can be used if want to localize expression - tissue specificity in expression insulators can be added to transgene - help with inappropriate gene expression - dont want integration to be influenced enhancers that have expression at inappropriate place 3) What are the consequences of immune responses to the vector or transgene products? 1) Reduction in transgene expression e.g. adenovirus—causes common cold, known target for immune system- everyone has immunity for it, immunity for transgene too everyone has developed immunity against adenovirus cells expressing the transgene and viral components are killed by the immune system 2) Exaggerated immune responses can be lethal Example of lethality of gene therapy: Deficiency in ornithine transcarbamylase (OTC - liver enzyme that removes excess nitrogen) inherited as an X-linked recessive mutation OTC normally breaks down amino acids present in protein  Lack of OTC—build-up of ammonia, which damages brain function Low-protein diets and ammonia-binding drugs are used to treat OTC deficiency Clinical trials to treat OTC deficiency were established using adenovirus as a vector for the normal OTC gene Adenovirus has been used in over 330 gene therapy trials in 4,000 patients ○ E.g. Jesse Gelsinger had a mild OTC deficiency. He volunteered for the OTC gene therapy trial. ○ 4 days after gene therapy Jesse died of massive immune reaction and associated complications. He had related virus that probably sensitized his immune response to the adenovirus used in the treatment The adenovirus didnt target the liver cells, but became widely distributed - entered circulation Components of vector triggered some sort of inflammatory response Previous infection sensitized immune system to vector 4) What is the risk of the transgene inserting into a functional gene, and what are the consequences? More common for DNA to insert into actively transcribed regions of the genome Problems: ○ transgene promoter drives expression of a proto- oncogene ○ transgene disrupts a tumour-suppressor gene Gene therapy trial (France): SCID X-linked due to a deficiency in IL2 receptor gamma Protocol: 11 boys ex vivo gene therapy of CD34+ cells (precursor to lymphocytes) 1-6 X 106 cells/kg; 5-30 X 106 viral insertions Results: 10 boys developed a functional immune system 2-3 years later: 3 of them developed T-cell lymphoblastic Leukemia Explanation: Insertion of transgene into LMO2 gene leading to an increase in expression of the LMO2 protein Boosted proliferation too much Leber congenital amaurosis (LCA) LCA inherited form of blindness - no treatment - heterogeneous mutation, involves single gene mutations important in visual system Identified by Leber in 1869 Severe visual impairment in childhood—usually total blindness by 30-40 years of age Abnormal roving eye movements (nystagmus)  Abnormal electroretinography (ERG) Poor pupillary light reflexes ○ 5% of cases are mutated RPE65 - mutations in both copies of gene in order to have disease The same mutations in the gene are P65 Or two dif mutations but in the same gene (compund heterozygotes) Or novel B mutation Candidate gene approach. Families with Leber congenital amaurosis and retinitis pigmentosa. Examined all 14 exons in 207 individuals ○ RPE65: Only expressed in retinal pigment epithilium Associated with hereditary retinal degeneragtion - associated with blindness Tissue expression patterns shown of genes associated with LCA - genes affection photoreception ○ Function of RPE65 RPE65 is isomerase thats essential for regenerating from the all trans version to 11 cis version (needed for vision to occur) Delivered to rhodopsin When light perceived - isomerizes from the CIS to the trans in rhodopsin Animal model of LCA ○ Congenital stationary night blindness in Briard dogs. - spontaneous mutation, 5/9 had had mutation - inbred Narfstrom K et al. (1989). British J. Ophthalmology 73:750-6 ○ Loss of function mutation in RPE65. Four nucleotide (AAGA) deletion introduces a premature stop codon. Aguirre GD et al. (1998). Molecular Vision 4:23 ○ Adenoaddociate virus was used to deliver RPE65 via eye injection in 3 of these dogs with visual defects It was found that some aspects of their vision, e.g. electroretinograms could be restored using gene therapy trials of the wild-type RPE65G Human clinical trials ○ Human RPE65 Gene Therapy for Leber Congenital Amaurosis: Persistence of Early Visual Improvements and Safetv at 1 Year ○ "Human gene therapy with rAAV2-vector was performed. At 12 months after treatment, our voung adult subiects remained healthy and without vector-related serious adverse events. The remarkable improvements in visual sensitivity we reported by 3 months were unchanged at 12 months.' Cidecivan AV et al. (2009). Human Gene Therapy 20: 999-1004 ○ Injecting with WT in eye gene that wont go anywhere else - safer with less vector adverse effects If photreceptors too comprised its not going to work - no lasting solution  Future of gene therapy – prosthetics & optogenetics ○ Lost photoreceptor function - can no longer be treated ○ Developed gene therapy targeting retinal ganglian cells (output cells - final line of defense) ○ Developing therapy that combines viral insertion of optogenetic components in the retinal ganglion cells of the eye Introduce ganglion cell components that allow for photosensitivity Activate retinal ganglion cells using light The light then activates ganglion cells and therefore tells the central nervous system that light has been perceived ○ Glasses that have on board computer that processes visual information the way that layers of retina would Camera records scene, computer processes info (as retina would - mimics better), plays movie/ligh to ganglion cells so signal can be transmitted to the brain Better representation of normal processing in retina ○ 1) development of retinal prosthetics ○ 2) viral delivery of optogenetic components tht allow retinal ganglion cells to actually respond to light that os projected in little movies