2nd half Flashcards
severe combined immunodeficiency disease (SCID)
Also known as bubble boy syndrome, caused by mutation in adenosine deaminase (ADA) gene. No normal response to infection. because no ADA means no other enzymes after ADA. Makes shit ton of Deoxy ATP which destroys t cells and prevents b cells from being activated
how is gene therapy done for SCID
remove ADA deficient lymphocytes from scid patient, culture cells in lab, infect cells with a retrovirus that has normal ADA gene, reinfuse ADA-gene back into SCID patient. the patients need some levels of t cells
what are the effects of gene therapy for SCID (negative and positive)
one patient developed immunity to gene transfer system, little to no ADA expression. no adverse effects otherwise.
how do you get dna into cells for gene therapy, pros and cons of each
viral vectors (easy, fast), electroporation (breaking membrane, harmful), direct injection into blood or other tissue (depends on target, inefficient), particle bombardment (harmful), liposomes (inefficient), crispr (in the future, may have off target effect). (even voldemort doesn’t like crack pipes)
adeno-associated virus (AAV) characteristics
specific chromosomal site, long term expression, nontoxic, infects dividing and nondividing cells, carries small genes. has an episome.
adeno-associated virus (AAV) applications
cystic fibrosis, sickle cell disease, thalassemias, canavan disease (cindy stole talia’s candy)
adenovirus (AV) characteristics
large virus, carries large genes, transient expression (not long), evokes immune response, infects dividing and nondividing cells
adenovirus (AV) applications
cystic fibrosis, hereditary emphysema, (also used for otc deficiency
herpes characteristics and applications
long term expression, infects neuroglia, used for brain tumors.
retrovirus characteristics
stable but imprecise integration, long term expression. most types only infect dividing cells, nontoxic. integrates into the genome itself, which may cause cancer. can deliver big genes
retrovirus applications
gaucher disease, hiv infection, cancers, scid (combined with adenovirus and herpes: cindy obsessively hates engineering but greatly hates computer science)
how to make sure enough transgene will be in the right cells at the right time??
Ex vivo is the best, strong promoters, tissue specific promoters. insulators (prevent transgene from silencing surrounding environment, prevents enhancers from activating the wrong stuff around transgenes)
immune responses to vector or transgene product cause what
reduction in transgene expression, adenovirus causes common cold, and since everyone has immunity against it, cells expressing transgene shit get deleted
what is the problem with transgene inserting into a functional gene, consequences?
common for DNA to go to actively transcribed parts of genome, and could promote proto-oncogenes or tumor suppressor genes
What is SCID X-linked mutations due to
deficiency in IL2 receptor gamma
How did the gene therapy trials in france aim to remedy SCID X-linked, what are the results
ex vivo gene therapy of CD34+ cells (which are precursors to lymphocytes, which are the missing B and T cells). the results are that 3 of 11 got t-cell lymphoblastic leukemia, but 10 developed functional immune system
What will happen if a transgene is put into LMO2 gene
it will have a ton of LMO2 protein because the transgene boosted expression rates
Why does moving LMO increase expression
you probably moved it next to an enhancer
leber congential amaurosis (LCA)
visual impairment in childhood, total blindness when 30-40. abnormal roving eye movements (nystagmus) and abnormal electroretinography (ERG). poor pupillary light reflexes
what genes does LCA affect
5% of LCA patients have muated RPE65. You can have homozygous or compound hetero mutations. RPE65 affects phototransduction and photoreception
how did briard dogs help us know about LCA
four nucleotide deletion (AAGA) makes a premature stop codon
What vector was used for RPE65 gene therapy, how was it
rAAV2, adult subjects remained healthy
What does prof nirenberg do to let blind people see
For people with no photoreceptor, they get optogenetic components put into retinal ganglion cells to let ganglion cells work as photoreceptors. They then wear glasses with an encoder that helps visualize for the ganglion cells, so the brain can construct an image
incomplete dominance
red and white get pink
codominance
black and white get spots
additive inheritance
independently segregating loci that adds up, a special type of incomplete dominance
two hypothesis of quantitative traits
segregation of alleles at many loci, loci have small, equal, additive effects. quantitative trait can be explained by few genes with large additive effects
quantitative traits are also called
polygenic/multifactorial
how is familial hypercholesteraemia an example of incomplete dominance
heterozygous phenotype distinct from either homozygous phenotype to get an intermediate
what is qtl (quantitative trait locus)
a trait that is affected continuously by the environment and multiple genes. can be masked by environment or the genes
why are complex traits shaped like a normal curve
because they are the sum of various phenotypes which form small hills
Vp =
Vg + Ve
Vg =
Va + Vd + VI
what does broad sense variation document
how much phenotypic variation is attributable to genotypic variation
narrow sens variation
family specific predictability of phenotype based on parental phenotype. additive effects are the only transmissible effects
what doesnt h^2 tell you
whats happening in other families, what the genes are
How to identify a QTL
inbreed two tomatos, one big one small. you get heterozygous f1. Then breed f1 to get a bunch of combined tomatoes, determine frequency distribution in F2,genotype individuals to find markers that cosegregate with trait (eg. a marker only appears in big tomato). then use a statistical method. calculatee lod
lod formula
probability of linkage given the recomb rates/probaility of no linkage
how to determine recomb rates for lod
see what the grandparents passed down to paretns, then use that to see if the same allele formed recomb or parental for children. if 1/11: (1/11) * (10/11)^10
whats the lod demominator
if 11 items. (0.8)^11
what are RILs
recombinant inbred lines, inbred for multiple generations so you get a ladder like mosaic of paternal and maternal alleles. this allows for fine mapping of traits
what do you do when you find quantitative trait loci
create recombinant inbred lines, identify specific candidate genes in the QTL interval with fine mapping. NILs used to map QTL to a gene (do all the big tomatoes have gene A? do all the small tomatoes?). examine candidate gene with pcr, to see if it is really expressed in big or small plants, then check to see if the function makes sense. use genetic engineering to test candidate gene
what are indel mutations
insertion deletion
subsitution mutation
base replaced by one of other 3 bases
reciprocal translocation
2 nonhomologous chromosomes change places
transition subsitution
a to g (purine to purine) or c to t (pyrimidine to pyrimidine)
transversion
purine to pyrimidine or vice versa
backgroundrate of mutation
2-12 * 10^-6 mutations per gene per gamete
depurination
guanine gone (cannot be adenine)
deamination
cytosine changed to uracil
x ray mutation
deletion
uv light mutation
thymine dimer
oxidation
guanine to go, which mispairs with A
what do indel mutations cause
loops in dna for the loop that has extra with respect to the other
how to know strength of selection for a mutation
compare nonsynonymous with synonymous. neutral genes have similar rates
what two types of mutation change length of protein
frameshift, nonsense
intragenic supressor mutations
a mutation that compensates for another (check)
how can mutations be outside the coding sequence
disrupts splice donor and acceptor site
null (amorphic mutation)
mutant has the same amount of activity as if its deleted
hypomorphic (leaky) mutation
mutant is less severe than straight up deleting the allele
what makes a loss of function mutation haploinsufficient or sufficient?
threshold for wt trait
hypermorphic mutation
too much prtein
neomorphic mutation
protein with new function, or normal protein made at wrong time and place
what does unequal crossing over in human vision do
natural variation in r/g vision
alkaptonuria
phenylalanine eventually makes homogentistic acid (HA) needs an oxidase to turn it into smth else. without it, HA accumulates and you have black pee
how do biosynth pathways work
if there is a plus, then the compound is after the point of mutation. because if its after the mutation, it makes what the mutation is supposed to but doesn’t
how do you get a partially yellow and purple corn
if there is wild type, it is purple. if the acentric fragment witht he dominant parts is lost, then its yellow. this is due to ac activated chromosomal breakage at ds
how do you get spotted corn
a ds element disrupts color gene, turning it recessive. however, ds is rebellious and randomly leaves sometimes. or you could have an ac instead of ds + ac in the color region of the DNA that jumps out
what is flanked by short repeats
transposed DNA
how much of the drosophila and human genome do TEs take up
12.5 and 34 respectfully %
after a p element is excised for transposons, how is the gap repaired
using a homologous chromosome. transposon will remain in original position if homologous chromosome also has it
What is a line
autonomous retrotranposon 1-5kb
What is a sine
nonautonomous retrotransposon 100-300kb
why dont TEs kill all of us
usually in introns. sometimes dont work because no repeats at the ends. heterochromatin prevent it.
what kinds of mutations could happen with TEs
unequal crossing over leading to deletions and duplications, or seeing 2 tes as one big te and yoinking that whole thing
how are transposable elements used in the lab
gene on a plasmid can be put into nuclei with a helper plasmid that can then be shown to express the gene. this can be done to germ cells of babies which then grow to be adults and childeren will exhibit the phenotype
how is x chromosome inactivated
coated by xist rna, hypoacetylation of lys in 2 histones. histone methylation.
euploidy
complete sets of chromosomes (total x 2 or /1)
aneuploidy
loss or gain of one more more chromosomes (i.e. one less in a set)
how does monoploidy work
parthenogenesis (development of unfertilized egg into embryo with no fertilization). single set of chromosomes, produce gametes by modified meiosis
lethality of monoploidy
ok in male bees, wasps, ants, lethal for other systems because it unmasks recessive lethals. if survives, no meiosis and sterility
uses for monoploid plants
visualize recessive traits, introduce mutations
how to get monoploid experimentally
haploid pollen treated and plated onto agar, grow embryoids and then treat with hormones
polyploidy
common in plants, associated with origin of new species, correlates positively with size and vigor
tetraploid plants
alfalfa, coffee, peanuts, large apples, pears, grapes
octaploid
big strawberries
how are autotriploids fertile
if there are even chromosomes from nondisjunct in meiosis 1
how are autotetraploids formed
2n to 4n from spontaneous doubling or drug like colchicine
how does tetraploid meiosis happen
1 can sortwith 2 or 3 or 4
homeologous
partially homologous chromosomes. like somewhat related plants
allopolypoids
hybrid of 2 or more closely related species
amphidiploid
douubled diploid (so 4n instead of 2n)
you have 2n = 18 parents from 2 different species, what happens when you breed them
gametes of parents are n = 9 and this forms a sterile f1 hybrid n + n = 9 + 9. 2n = 18. This spontenouslly doubles so everyone has a prtner, forming a fertile amphidiploid (2n + 2n = 18 + 18. 4n = 36)
nullisomy
2n - 2
monosomy
2n - 1
trisomy
2n + 1
tetrasomy
2n + 2
mitotic nondisjunction
results in a mosaic
monosomy 21
born with severe multiple abnormalities, dies shortly after birth
turner syndrome (XO)
99% of affected fetuses are not born, those that are born have developmental abnormalities
down syndrome
21 trisomy, females can be fertile, males infertile, average life expectancy 40-60
edward syndrome
trisomy 18
patau syndrome
trisomy 13
klinefelter syndrome
XXY, some genes expressed at twice the level of XY
other types of xy trisomy
XYY: usually fertile, extra y does not pair and not transmitted
XXX: usually fertile, extra x does not pair.
conditions not passed to progeny
genomic hybridization: microarrays
detects duplication and deletions of at least 50 kb
first trimester screening tests for prenatal testing
nuchal translucency (ultrasound), maternal serum blood tests (placenta hormone levels), noninvaseive prenatal testing (nipt), blood tests
diagnostic prenatal testing
chorionic villi sampling (10-13 weeks) using an ultrasound, 1% miscarriage. amniocentesis (16+ weeks) use a needle to get fluid from fetus 0.5%. look for abnormal karyotypes
preimplantation embryo diagnosis
screen for mutant alleles before implantation of test tube baby
polytene chromosomes
used to study changes in chromosome structure, duplicated a lot without meiosis to form these wiggly things. bandinding can be used to find genes
intragenic deletion
small deletion within gene
multigenic deletion
many genes deleted, homozygous are usually inviable. heterozygotes may be haploinsufficient
pseudodominance
not actully dominant but looks dominant because you deleted an allele which uncovered the mutant gene
deletion complementation tests
crossing deletion mutatns to get strains with deletions in differen tplaces. this reveals location of mutant gene
duplication
less likely to affect phenotype, most like dosage effect or genetic imbalance, genes may be in new location which modifies expression
x ray breakage causing duplication
nontandem duplication
unequal crossing over
different number of genes
breakpoints between genes
inverted alignment
breakpoint within genes
inverted alignment causing disrupted genes
breakpoint in 2 genes
fusion of the 2 genes
heterozogous inversion
inversion loopsp
paracentric inversion
dicentric and acentric fragments. reduced number of viable gametes, deletion products formed
pericentri inversion
no dicentric but you still get reduced number of viable gametes
translocations
most do not alter phenotype unless breakpoints occur within genes. unless genes are put in some new location that modifies expression’
robertsonnian translocation
exchange between acrocentric chromosomes to make a large metacentric and small chromosome that may be lost (basically a big one yoinks the big ar of another, and the small one gets small arm and disappears)
robertsonian translocation in madeiran mice
causes loss of a chromosome which causes different numbers of chromosomes
translocation homozygote segregation during meiosis
exchange of some genetic material causes the shared part to pair together. there are 3 ways for the 4 chromosomes to separate, and 2 will have deletions or duplications and will die
homozygote segregation results
semisterility. < 50% of the time there are viable gametes. pseudolinkage because no independent assortment
what is chronic myelogenous leukemia caused by
when c-able and bcr genes are spliced reciprocal translocationally, because they are oncogener and now you dont have normal c-able and bcr mRNA
epigenetics
heritable modifications in gene function not due to changes in base sequence of DNA
dna methylation at CpG islands, influence in mice
repression of gene expression. the more methylation at the cpg island in mice at iap (retrotransposase insertion), the more grey.shown by mothers diet during pregnancy
iap retrotransposase insertion is a what mutation
gain of function that needs to be silenced
why is epigenetics needed for normal effect
a cell needs to be able to differentiate
drosophila hox gene expression and prc
hox sets up body plan. pre binds to a repressor which binds to a prc. prc will bind to methylation and after several divisions, the methylation will remain where it is. without prc, the methylation will not be retained
imprinting epigenetics
methylated with special methylases, demethylated by special demethylases
in what phase are parental imprints erased
parents with maternal and paternal imprinted genes are passed onto a zygote which is passed onto BOTH the somatic and germ cells of the child. the child will have erased the old imprints in the germ cells, make new imprints based on maternal or paternal, and now eggs or sperm will have their imprints
maternal imprinting for igf2/h19
the igf2 is off, insulator has CTCF dimer on it which is like the donald trump wall, and igf2 is mexico. h19 is america because it has bald eagles and guns, and because ctcf doesn’tb lock it off. the activaotr binding to te enhancer can activate america (h19)
paternal imprinting for igf2/ h19
methylation at the insulator AND h19 which means no trump wall (CTCF) and also no activation of america (h19). but without the wall, mexico (igf2) can be activated by the enhancer
what does the igf2 do
growth and development of various types of cell tissue in utero
does ctcf silencing happen in somatic cells normally
no its a silly thing that only happens in placenta
do mutations on the same place for maternal and paternally imprinted chromosomes have the same effects
no, because they may not express the same gene. if its silenced, mutations dont do anything
delete igf2 from mother
normal levels of lgf2 because its not expressed anyways
delete igf2 from fahter`
no lgf2 and small size
prader-willi syndrome
paternal deletion (mutation) from chromosome deletion, obese, small hands and feet, eats uncontrollably, does not mature sexually. since maternal is imprinted, both inactive. if maternal not inactive, it may compensate.
angelman syndrome
developmental delay, mental retardation from maternal deletion in chromosome 15. mechanism vice versa for prader-willi
benefits for zebrafish
easier to rear for vertebrates, clear embryo, easy microscopy
how are living forms related, example with eyes
eyeless (drosophila), pax-6 (mice) and aniridia (humans) are homologous. adding pax-6 to drosophla rescues it from no eye gene.
aniridia in humans
partial or complete absense of the iris. sensitivity to light. homozygous is lethal, heterozygote gives you the condition
reverse genetics
knockout or mutate certain genes then analyze phenotype
forward genetics
need shit ton of mice. mutagenesis following exposure to mutated gene, examine phenotype
how is cloning done
donor gives unfertilized eggs, somatic cells’ nuclei removed from another animal and put into the egg, embryo grows and put back into surrogate mother to get cloned animal
multinucleate syncytium
when there are a shit ton of nuclei and they go out to the cortex. replication of nuclei but no cell division
syncytial blastoderm
nuclei go on pilgrimate to edges. pole cells (primordial germ cells) forming
how do you screen for mutagenesis to see if a gene is maternal effect or zygotic lethal
feed mutagen, cross with balancer chromosome fly with dominant marker on balancer. balancer inversions prevent recomb. f1 gen will have balancer and a mutation (recessive) and cross this again with the balancer except there is a dominant marker on both chromosomes so you know you don’t get the dominant marker instead of the mutation. cross the heterozygous (one wiggly balancer one mutation) and then if homozygous offspring die, it is zygotic lethal, else maternal effect if mothers sterile. check offspring for segmentation
gap genes
multisegment that affect many places early. single broad band. include kruppel and knirps.
pair rule genes
every other segment. visually, you see 7 segments. even skipped, odd skipped, paired, runt.
segment polarity
every segment, 14 of them. gooseberry and patched
what do segmentation genes code for
transcription factors for certain proteins
how are gap genes activated
maternally provided proteins.for example bicoid bind to cis acting regulatory elements which cause reporter expression
bicoid targets
activates hunchback, represses caudal.
where are the maternal effect proteins. list from anterior to posterior
bicoid (big range but shit ton on most anterior), giant, hunchback, eve stripe 2, kruppel
what activates eve-2
bicoid and giant, inhibited by hunchback ad kruppel
how do you find out what activates a specific pair rule or segment polarity gene
by looking at the activators and repressors. then you can put the cis acting regulatory elements onto a plasmid with a reporter gene like gfp that will glow and tell you where shit is. then you put it in am embryo and look at where the glow is :)
homeotic mutation
segment characteristic changes (e.g. antenna become legs)
how are hox genes normally regulated, where are they present
they are normally in spatially restricted domains, are found throughout the animal kingdom and the order is similar. they limit development of primordial germ cells
cancer uncontrolled growth
autocrime stimulation, contact inhibition, cell death, gap junctions.
genomic instability
not correcting mismatches
angiogenesis
new blood vessels for metastasis
multihit model of cancer development
to get a tumor you need several mutations which increases the chance of cells become malignant. (e.g. people get more cancer when they get older)
ras
oncongene
p53
tumor supressor (it sounds like a gun. guns have suppressors)
why do we know cancers are clonal descendants of one cell
get a and b types of tissue, culture them and electrophoresis to compare alleles, compare with tumor
evidence from cancer being due to environment
if one sibling or twin gets cancer, other does not mostly. if populations migrate, the chance of getting cancer becomes like the people in new location. cosplayers will get cancer and die, especially me
radiation mutagen
x rays and ultraviolet light
chemical mutagens
asbestos, cigarette tars
virus mutagens
epstein barr (EBV) and human papilloma virus (HPV)
protooncogenes
gain of function dominant mutation converts these into oncogenes. inhibit apoptosis and stimulate cell cycle. haploinsufficient
tumor-supressor genes
loss of function recessive mutations (DNA repair loss). haplosufficient
oncogenes
mutated receptor tyrosine kinase (RTK) genes, ras, bcr, c-abl
how do ras proteins promote cell division
when bound to gtp, it creates a map signal cascate and proliferation. it is chill when bound to gdp
receptor tyrosine kinases
you have 2, her2 and egf. when they are chilling, they are only activated when binding to signal. if the valine is mutated to glutamine, they will be always dimerized. in the case of EGF, it turns into ErbB oncoprotein which has the extracellular part gone. for the Her2, it turns into the neu oncoprotein
how does ras work
inactive binds to gdp, sos stimulates gdp to become gtp, which activates ras. gtp ras will activate downstream serine and threonine kinase. gap turns it off, without gap it is always on
how does rb stop tumors
it binds to E2F. phen phosphorylated by cdk4-cyclin d and cdk2-cyclin e, it no longer inhibits ef2. the not inhibited ef2 will start s phase more quickly
xeroderma pigmentosum
defect in nucleotide excision remair cause uv induced skin cancers (dimer excision)
hereditary nonpolyposis colorectal cancer
defect in mismatch repair. mutations in msh2 or mlh1 are more common, predisposition for colon and ovarian cancer
how you you get predisposed for retinoblastoma
by being heterozygous for it because there will be divisions that turn the other into mutant as well and that causes the tumor. it appears autosomal dominant but is actually recessive
