MCP 2 Flashcards
what is FISH and what is it used for?
fluorescent in situ hybridization
- DNA fragments with fluorescent labels are hybridized to chromosomes
- perform in metaphase or interphase cell
- either locus specific (single copy) or chromosome specific
- use control probe; 2 signals/chromosome in a normal person
what are the different types of FISH?
- repeat sequences: probes isolated from telomere or centromere region
- single copy: unique sequence; subtelomere is a specific type (high frequency of mutations there)
- chromosome painting: cocktail of probes, ID nonreciprocal translocation
restriction enzymes
based on ecoRI’s system of restriction and modification; recognition sequences can serve as landmarks in the DNA creating a DNA fingerprint; leaves sticky ends which can be ligated
gel electrophoresis
detect presence, size, and quantity of purified DNA; use polyacrylamide (smaller molecules and proteins) or agarose (larger molecules)
-anode attracts anions
DNA cloning steps
- restriction enzymes cut DNA fragments
- isolate with gel
- ligate into vector using T4 DNA ligase (bacteriophage enzyme)
- include amp so you can culture and see if DNA has ligated
types of cloning vector
- plasmids: 15 kb, circular DNA
- bacteriophage lambda: 20 kb, easy to purify because of viral size
- cosmid: 45kb, gutted lambda
- BAC: 100-300 kb, ds circular DNA, trick bacteria into thinking its their chromosome; low copy number; most applicable
- YAC: 100-2,000 kb, ds linear DNA; include telomere, centromere, and selectable marker; highly predisposed to recombination
cDNA library
contains only portions of the genome that becomes mRNA (introns removed)
-make RNA then degrade, reverse transcriptase to make DNA, DNA polymerase to make second DNA strand,
what is PCR
repeated rounds of mRNA primer directed DNA replication off a rare template used to amplify and detect specific sequences from a complex mixture of DNA
- heat to separate strands
- hybridize primers
- DNA synthesis from primers
- gel to see if it worked
multiplex PCR
amplify multiple specific sequences from a complex mixture in a single PCR reaction; used diagnostically (DMD)
reverse transcriptase PCR
make DNA copy of RNA genome then do PCR; so sensitive it can be used to detect presence of pathogen genes from patient samples (ex. HIV test)
southern blot
detects presence and size of a specific DNA sequence in a complex mixture; gel, transfer DNA to nitrocellulose using strong base, probe with hybridization for fragments with homolog to a sequence of interest
northern blot
purified mRNA is separated on a gel to see whether mRNA is expressed, what is the expression level, and are the mRNA of proper length
What is a microarray? What are some different types and what are they used for?
- compare two sets of DNA (normal and test, usually cDNA)
- miniaturized nucleic acid hybridization/detection
- isolate sequence, label, hybridize to DNA and read signal on microscope; each spot on gene chip is a probe that IDs a particular mutation
- first tier study in cases of unexplained developmental delay, intellectual disability, autism, and multiple congenital anomalies
- gene arrays: look for specific mutations or copy number variation
- chromosome arrays: used in clinical labs, look at locations and regions on chromosomes (peaks: duplications, valleys: deletion)
- expression arrays: look at mRNA to see heat maps of up regulation; characterize tissue types by the genes expressed; display by cluster analysis (genes that are coordinately regulated fall out into clusters)
denaturing vs. non denaturing gel electrophoresis
- denaturing: separates proteins by size, boil with SDS and ßmercaptoethanol (reducing agent) to create uniform (-), separate in polyacrylamide
- nondenaturing: use to compare change in charge or size between two proteins (ex. sickle cell)
isoelectric focusing
separate proteins by pI (proteins migrate until they carry no net charge)
western blot
detect presence, size, and abundance of a specific protein in a complex sample
-nitrocellulose incubated with primary antibody then washed and incubated with secondary antibody that has been coupled to an enzyme that fluoresces with substrate
primary antibody
antibodies raised in animals by injecting them with the antigen of interest
secondary antibody
antibodies raised against the constant regions of antibodies from different animal species; recognizes the primary antibody as foreign
monoclonal antibody
recognizes a specific epitope on a specific protein
immunofluorescence
detects presence and localization of a protein in a fixed tissue/cell sample
-use for analysis of dystrophin in muscle biopsies from normal vs. patients with DMD or beakers
GFP fluorescence
determine the localization and dynamics of a protein of interest after it has been fused to GFP; can look at living cells in real time
RNAi
introducing of dsRNA corresponding to a cellular mRNA induces degradation of mRNA and down regulation of the gene
- recognized by protein complex with RNA endonuclease “dicer” that cuts dsRNA into small pieces, RNA-protein complex destroys many RNA molecules
- morpholinos: ssDNA complementary to a transcript you want to knock down inhibits expression by blocking process of ribosome translation or by blocking splice site
fragile x (FRAXA)
disease from dynamic mutation, most common inherited mental retardation
- consider x-linked dominant with reduced penetrance
- CGG expansion in first exon and methylation of FMR1 gene
- anticipation (sherman paradox); means higher incidence in sons than uncles
- premutation females have mild symptoms, premature ovarian failure
- NTM: mild symptoms, 1/3 develop FX tremor/ataxia later
- NTMs may have FRAXA grandkids with full mutation but daughters will only have premutations
how do you test for fragile x?
southern blot
- repeats greater than 200 triplets become unstable; smear on southern blot
- premutation females: 4 bands because of x-inactivation
- full mutation male: only smear
sickle cell
structural varient hemoglobinopathy
- destruction of MnI site through mutation; do digest to distinguish S and C from A
- (HbA/HbS) asymptomatic carriers
- (HbS/HbS) sickle cell disease
thalassemia
imbalance of synthesis hemoglobanopathy, deficiency of one hemoglobin chain leads to inclusion bodies formed by excessive amount of the other chain
ßthalassemia
- reduced ß production; excess of aglobin chains form Heinz bodies in the RBCs, leads to hemolysis
- degres of allelic heterogeneity
- ß0 (severe) ß+ (reduced HbA detected)
athalassemia
- underproduction of aglobin chains
- homozygous deletion: hydrops fatalis
Hb Constant Spring
mutation results in tetramers of ßglobin chains which do not release O2 in peripheral tissues; aglobin is there but they don’t form tetramers
cystic fibrosis
autosomal recessive with allelic heterogeneity
- CFTR gene product is chloride channel across cell membranes in exocrine cells of lungs, pancreas, and sweat glands
- ∆F508 mutation is common
- therapy depends on class of mutation
fertilization
1 wk; in ampullary region of uterine tube, activates cleavage division; sperm and oocyte fuse and replicate (N–>2N; 23 mom, 23 dad)
cleavage division
mitotic divisions, increase number of cells, not total size
- cells are called blastomeres
- compaction occurs at 8 cell stage (segregates inner from outer cells, tight junctions stabilize
- 16 cell morula (totipotent)
blastocyst
occurs after morula undergoes cavitation; inner=ICM (pluripotent) and outer=trophoblast (placenta)
implantation
end of wk1; blastocyst expands in zone pellucida (zp prevents adhesion to oviduct), hatches in uterus and adheres to wall/begins implantation
- trophoblast differentiates into cyto (inner layer of mononucleated cells) and synctio (multinucleated layer that lacks distinct cell boundaries)
- ICM differentiates into epiblast and hypoblast to form bilaminar disc
gastrulation
- extensive cell movements and rearrangements of bilaminar disc form 3 germ layers
- node is organizer
- epiblast cells invaginate through primitive streak to form endo and meso
- body axes are establish before or during this time
neurulation
- neural crest–>delaminate and migrates to new locations, gives rise to many cell types; epithelial to mesenchymal transition
- neural plate–>neural tube–>brain and spinal cord; close cranial end (day 25) and posterior end (day 28)
- surface neural ectoderm–>skin (3 different ectoderm domains)
how do the neural crest cells give rise to characteristic cell types?
- 4 different regions of neural crest
- specific gene networks determine crest cell fate
- ex. cardiac neural crest cells enter the outflow tract of the heart to generate the septum between the great arteries
contiguous gene syndromes
regions in genome with clusters of closely associated genes whose normal functions are generally unrelated
william’s syndrome
- type of contiguous gene syndrome
- initially diagnosed as deletion of elastin gene on chromosome 7
- gene adjacent to elastin associated with learning and personality traits
velocardiofacial syndrome
- type of contiguous gene syndrome
- cleft palate and conotruncal heart defects
- chromosomes mispair because repeated sequences that flank the gene are similar (get micro duplication or deletion of 22q)
- variable phenotype
- wouldn’t be able to see this on karyotype
genome sequencing
ultimately want a genome-wide scan that will detect mutations with an individual’s medical concern; panels have been created that include all current data about particular genes or critical regions known to be associated with a specific disease
homologous genes
evolutionarily conserved; function interchangeably during development in different species
genome equivalence
all cells contain the same set of genes
differential gene expression
only a small percentage of the genome is expressed in each cell type; regulated at several levels and controls fundamental cellular processes
- proliferation
- specialization
- interactions
- movement
induction
one group of cells changes the behavior of an adjacent set of cells
- requires inducer and responders (must be competent)
- signals often transmitted between the two via paracrine (proteins secreted into ECM) or juxtacrine signaling
morphogens
paracrine signaling molecule that cause concentration-dependent effects; can specify more than one cell type by forming a concentration gradient
signaling cascades
many inductive molecules and morphogens transmit their signals through the cell membrane and to the cell nucleus via signal transduction pathways
- at every point in the cascade, receptors have to be competent
- transcription factor in nucleus binds DNA and alters gene expression
how is the left-right body axis formed?
-asymmetric signal cascade: cilia-driven fluid flow to left side triggers Nodal gene expression on the left side and establishes a morphogen gradient and signaling cascade in lateral plate mesoderm
–>activates downstream target genes (PITX2, Nodal, and nodal inhibitor Lefty)
odal gene codes for a member of TGFb superfamily, first expressed asymmetrically in the node region
-cilia generate fluid flow that
kartagener’s syndrome
immobility in what is meant to be motile cilia; accomplanied by a 50/50 chance of situs inversus or heterotaxy
mitochondrial diseases
- caused by mutations in oxidative phosphorylation, most series in CNS and muscle (neuropathies, encephalopathies, myopathies)
- may show autosome, x-linked or matrilineal inheritance
- must be a high proportion of mutants present to express dysfunction
- often progress with late onset
homoplasmy
populations o mitochondria that all have the same genetic composition; analygous to homozygosity in the nucleaus
heteroplasmy
when there are 2+ populations o fmitochondria present in a cell; analgoes to heterozygosity in the nucleus
replicative segregation
as cells divid, relative proportions of mutant mitochondria may change over time
acquired mutation
high mutation rate in mitochondrial DNA so it is possible for a mutation to occur as a new event and then proliferate in the population generating a subpopulation of mutants
what type of DNA should you use for specific identification?
use nuclear DNA
what type of DNA should you use for familial and evolutionary identification?
use maternal DNA; harder to degrade because it is circular
axenfeld-rieger
single gene mutation causes multi system genetic birth defect
-2 known genes (FOXC1 and PITX2) produce TFs in developing eye
holoprosencephaly
birth defect with multiple underlying causes (chromosome abnormalities, maternal diabetes, single gene mutations in HHS pathway)
- incomplete forebrain development
- broad spectrum of causation and severity
when is the most sensitive period in fetal development to teratogens?
weeks 3-8
rubella virus infection during pregnancy
measles linked to microcephaly, patent ductus arteriosus , and cataracts
hyperthermia during pregnancy
can interfere with neurulation and cause neural tube defects
thalidomide
example of how drugs can pass from mom to infant, causes limb defects (phocomelia)
fetal alcohol syndrome
- leading cause of congenital mental retardation
- dosage not clearly defined
- microcephaly, indistinct philtrum, narrow upper lip, flat mid face
- proposed mechanism: cell migration and adhesion, proliferation and survival, signaling and gene expression
SSRIs during pregnancy
associated with a low increase of septal heart defects, must weigh risks and benefits to mother by taking her off SSRIs
atrial septal defect
hole in septum that separates left and right atria, O2 rich blood is pumped back to the lungs
congenital heart defects
- majority are from complex interactions between genes and environment
- ASD, VSD, cardiac laterality defects, outflow tract defects
ventricular septal defect
hole in septum that separates ventricles, allow O2 rich blood to flow from left ventricle to right ventricle instead of flowing into aorta
cardiac laterality defects
- dextrocardia: heart positioned on right side of thorax
- double outlet right ventricle: aorta rises from right ventricle
- transposition of great arteries: pulm. and aorta are switched in position, O2 poor blood enters the right side of the heart and is pumped back to the body
22q11.2 Deletion Syndrome
heterogeneous multi system syndrome, leads to cardiac outflow tract defects (defects in cardiac neural crest development)
- truncus arterioles: single common blood vessel from heart instead of pump artery and aorta, blood mixes
- tetrology of fallout: VSD, pulm. stenosis, overriding aorta (increases flow), ventricular hypertrophy
types of noninvasive prenatal testing
ultrasound, MSAFP, MSQS, integrated testing, NIPT
types of invasive prenatal testing
amnio, chorionic villus sampling
prenatal ultrasound
- nuchal translucency (>6mm associated with Down’s)
- clefting
- neural tube defects
- anencephaly and encephalocele
maternal serum alpha-fetoprotein
15-20 weeks, take into account baby age and mom’s age/weight/race/diabetic status
- albumin-like protein produced by baby liver crosses placenta to mom
- low level: risk of Down’s
- high level: risk of ONTD
maternal quad screening
15-21 weeks, tests for 4 different substances, gives ~80% combined detection for Down’s
integrated testing
10-13 weeks, PAPP-A and nuchal translucency plus quad screening
NIPT
10-22 weeks, gives risk of chromosomal abnormality
- collect cell-free placental DNA from mom and use next gen sequencing to identify DNA fragments per chromosome compared to expected number for mom and fetus
- not diagnostic but very accurate for trisomy 21 and 13
amniocentesis
16-18 weeks
- amniotic fluid AFP: multiple pregnancies at once will mean high MSAFP but potentially normal AFAFP, small mom would also mean high MSAFP but potentially normal AFAFP
- acetylcholinesterase: confirmatory test for ONTD
- molecular diagnostics, metabolic assays, cytogenetics
chorionic villus sampling
10-14 weeks (can’t do earlier, at risk of disrupting vasculature)
- higher risk than amnio
- can do all same tests except AFP
- possible that placenta and fetus have different genetic make up based on when a mutation occurred
types of assistance reproductive technologies
- polar body analysis: allows for embryo selection when known that one/both parents carry gene mutation
- IVF: mix eggs and sperm, preimplantation genetic diagnosis at 8 cell stage before implantation
- ICSI: single sperm injected into egg
- ZIFT: IVF eggs transferred back to fallopians
- donor egg IVF with dad sperm
- enucleated donor egg with mom’s DNA swapped in for mitochondrial diseases, then IVF
heat shock proteins
folding machines, expression unregulated at high temperatures
- protect against aggregation by covering hydrophobic patches
- usually only folds large multi domain proteins
GroEL/GroES structure/mechanisms of action
- folding machine whose exact mechanism is unknown
- anfinsen box: protein folds by itself in isolation
- iterative annealing: unfolded/misfolded portion binds to GroEL cavity, lid binds, deforms cavity, changes interior non polar residues –> polar which mechanically disrupts proteins; GroES dissociates and protein is released to try to fold again
- active container where cavity size and charge affects folding rates (folds faster in box than in solution)
Ubiquitin-Proteosome Pathway
ubiquitination: proteins are targeted for destruction by 3 enzymes
–E1 activating: ATP drive reaction creates high E, covalent thioester E1-ubiquitin bond
–E2 conjugating: transfer activated ubiquitin to target protein bound to specific E3
–E3 ligating: repeated to generate polyU chains
proteosomal degradation
–polyU (at least 4) bind to proteosome, ATP hydrolysis to unfold protein
–20S particle cleaves protein into peptides
–peptides transported through ER for antigen presentation by MHC class 1 or recycled
diseases of ubiquitin-proteosome pathway
- cancer: increased degradation of tumor suppressors
- neurodegenerative diseases: observed accumulation as it attempts to clear plaques
- CF: cleaves misfolded ∆F508 CFTR
- autoimmune: improper processing of peptide antigens
what are the ways in which a residue mutation can affect a protein?
- direct knockout: mutation of a residue essential for protein function
- destabilization: shifts equilibrium to unfolded state
- toxic conformation: shifts equilibrium to an incorrectly folded state, aggregation is a common manifestation (e.g., amyloid diseases)
mutation in p53
tumor suppressor, transcription factor activated by DNA damage or conditions that are likely to cause DNA damage
- 50% of all tumors have a point mutation in p53
- destabilization: loss of thermodynamic stability causes proteins to be degraded more quickly as cell recognizes unfolded proteins and tags for destruction via U-P pathway
a1-anitrypsin (a1-AT) deficiency
- major protein in plasma, targets neutrophil elastase
- molecular mousetrap: a1-AT uses stored E to trap target
- target protease binds to serpin fold RCL and cleaves, RCL is frustrated ß-strand that inserts into the ß-sheet and drags protease with it, sheet splits in middle and opens up for loop to insert
- mutant: premature insertion into ß-sheet, can insert into identical sheet from neighbor causing polymerization (polymers build up in liver)
- small molecular-based therapy make a short peptide that mimics RCL and prevents RCL from inserting
Phe508 deletion in CFTR
-70% of CF caused by this deletion on surface of nucleotide binding domain
-mutation doesn’t effect function, just doesn’t get into the target membrane (folding pathway defect)
small molecular-based therapy: small organic molecules, over expressing chaperones, inhibiting degradation by U-P pathway, stimulating CFTR function
protein conformational disease: prion diseases
- 2 covalently identical forms of PrP that exist in 2 different conformations that interconvert (PrP: non pathogenic, soluble, protease sensitive, little ß-sheet; PrPsc: pathogenic, insoluble, protease insensitive, 45% ß-sheet
- nucleation: PrPsc come into contact and bind via ß-sheet interaction
- complex contains stabilized ß-sheet structure which does not readily dissociate and serves as a nucleus for subsequent addition
- potential therapies: siRNA silencing of PrPsc, stabilization of PrPsc making it less probably for nucleation
protein conformational disease: alzheimer’s
- Aß peptide produced by cleaved of APP by a, ß, y secretases into Aß40 and Aß42 (is this cause or effect?)
- amyloid fibers are virtually all Aß42
- amyloid hypothesis: mutations associated with early onset and protectivity are mapped onto sites of secretase complex that change ratio of 40:42 (based on rate of degradation and ratio)
- potential treatments: modulate y-secretase activity (made cognition worse), monoclonal antibodies that bind to Aß and work to reduce plaques (didn’t improve cognition) BUT could work if threshold of plaque accumulation has not been reached (otherwise it triggers other biochem pathways that end in cell death)
islet amyloid polypeptide (IAPP, amylin)
- cosecreted with insulin by ß-cells in islets of langerhans
- increased insulin production leads to increased IAPP; in late diabetes ß-cell crisis due to large scale apoptosis and IAPP amyloid in 90% of cases post-mortem
- structureless but has helical tendencies
- pore hypothesis/soluble oligomer hypothesis: soluble oligomers of IAPP are more toxic than mature fibrils (process of amyloid fibril formation is most harmful), makes membranes leaky
- potential treatments: fibrils are ß-sheets which grow by both side-chain interactions and peptide-peptide H-bonds so block extension by endogenous peptides or inhibitory peptides can cap ß-sheets
hallmarks of cancer
- mutation/loss of genes involved in cell control
- environmental influence
- acquired/inherited mutations
- chromosome instability
clonality
cell line acquires mutation and additional chromosomal abnormalities
- the more abnormalities, the more severe the disease
- karyotype evolution shows changes over time
oncogene
- dominantly acting gene involved in unregulated cell growth
- carried by viruses
- few oncognes in humans (PHV, EBV, HHV8, etc…)
protooncogenes
- normal genes involved in cell proliferation and development
- mutation is dominant, acquired, gain of function
- activation: change that converts it to an oncogene-like gene leading to tumorigenesis
- mutations often expressed as leukemias and lymphomas
chronic myelogenous leukemia
- translocation between chromosomes 9 and 12 creates chimeric protein, loss of regulatory control
- overproduction of tyrosine kinase
acute promyelocytic leukemia
- unbalanced translocation results in chimeric protein
- use FISH dual fusion probe for diagnosis and monitoring of bone marrow transplant (mixed sex transplants can be scored for 2Xs or an X+Y to determine relative proportions of different populations and track engraftment)
tumor suppressor
- genetic element whose loss/inactivation leads to neoplastic growth
- mutations are often solid tumors
- recessive, 1 mutation may be inherited
- diseases have primary role in specific target tissue but can be secondary to another cancer gene
- gate keepers and caretakers
difference between gate keeper and caretaker tumor suppressor genes
- gate keeper: suppress tumors by regulating cell cycle or growth inhibition (retinoblastoma, li-fraumeni syndrome, breast cancer)
- caretakers: repair DNA damage and maintain genomic integrity, do not present as gene mutation–>aberrant protein–>disease; malfunction in normal cellular process leads to accumulation of errors resulting in system dysfunction (chromosome breakage syndrome, hereditary nonpolyposis colon cancer)
retinoblastoma
- RB1: gatekeeper tumor suppressor regulates G1–>S
- tumor of retinoblasts (prenatal-5 yrs)
- knudson’s 2 hit hypothesis: single mutation is inherited but highly likely for second mutation, sporadic=unilateral, 1 tumor; inherited=bilateral, >1 tumor
- appears dominant
li-fraumeni syndrome
- inherited mutation of p53, a gatekeeper tumor suppressor, no one target tissue
- multiple neoplasia, increased risk of cancers
breast cancer
- BRCA1 and BRCA2 gatekeeper tumor suppressor, produce proteins that help repair damaged DNA
- in 80-90% of familial cancer but only 5-9% of all breast cancer
chromosome breakage syndrome
- group of 5 recessive disorders characterized by chromosome instability due to defective DNA repair mechanisms (caretaker tumor suppressor)
- chromosome instability: breakage or recombination leads to chromosome rearrangement
hereditary nonpolyposis colon cancer
- defect in mismatch repair process, gives rise to 2 cell populations that both proliferate, leads to additional mutations
- can be indirectly detected by micro satellites (small repeats that are highly polymorphic, mutation alters total number of repeats)
RNA polymerase vs. DNA polymerase
- similarities: synthesis of RNA vis phosphodiester bond formation 5’–>3’
- differences: DNA polymerase use NTPs as substrates, U vs T, transcript de novo (no primer required), no exonuclease activities
RNA pol II transcription
- RNA pol II: protein-coding genes
- TBP binds TATA box (not transcribed), TFs and RNA pol II recruited to form pre-initiation complex
- pol II phosphorylated to leave promoter and start transcribing
- pol II recruits RNA processing enzymes, mRNAs are processed cotranscriptionally (5’ guanine cap and 3’ polyA tail)
- termination site transcribed
control of transcription in bacteria
- genes are clustered in operons and are coordinately regulated
- turn on promoter and transcribe all proteins for biochemical pathway
- mRNA are polycistronic (multiple proteins from single mRNA)
- operators are binding sites for activators and expresser proteins (once bound RNA pol can’t bind)
control of eukaryotes
- gene activator proteins increase rate of transcription initiation
- act directly on the transcription machinery and/or change chromatin structure
- TFs are modular; DNA binding and transcription activation/repression are separable entities
- enhancers bind TFs far from promoter; DNA can loop and bring proteins together on promoter
- TFs can recruit chromatin modifying enzymes that loosen chromatin or chromatin remodeling complexes
- single TF can activate multiple genes
how does misregulation of hemoglobin synthesis lead to hemoglobinopathies?
-locus control region: unique regulatory region that control chromatin structure over entire domain; deletion of LCR silences entire ß-like glob in gene cluster by preventing chromatin decondensation
autoregulation
a given factor binds to its own promoter and either activates or represses transcription
epigenetic inheritance
- phenotype of a cell or individual is affected by which of its genes are transcribed thus heritable transcription states can give rise to epigenetic effects
- mechanisms of heritability of histone state are not well understood
degeneracy
the genetic code contains synonyms, there are more codon triplets than amino acids
how do silent mutations affect amino acid sequences?
they do not shift the reading frame (like insertions or deletions) and they code for the same amino acid as the unmated sequence BUT silent mutations may change the rate of translation and folding
wobble hypothesis
- some tRNAs can recognize more than one codon, last position is not very stable
- wobble codon base can code for a few possible anticodon bases
what is the role of amino acyl synthetases?
- activate amino acid using ATP to more aminoacyl adenylate
- transfer activated amino acid to 3’-OH of tRNA via C-terminus
- process is conserved from bacteria to man; differences are important antibiotic targets
what happens during step 1 of protein synthesis?
initiation: sequential binding of mRNA; cycles of IF association/dissociation ensure forward direction
1. 30S initiation complex: eIF-2-GTP, tRNA, small ribosomal subunit, mRNA
2. GTP hydrolyzed, releases eIF-2-GDP and drives assembly of large ribosomal subunit, initiator tRNA goes straight to Psite
what is the difference between start codon recognition in prokaryotes and eukaryotes?
- proks: shine-dalgarno sequence (G rich) recognizes AUG in the operon
- euks: recognition of AUG by 5’ cap at the 3’UTR (AUG is upstream of 5’ cap)
what happens during step 2 of protein synthesis?
elongation
- EF-Tu forms complex with GPT and aa-tRNA, complex binds to ribosome, kicks out tRNA in E-site
- Proofreading
- Transpeptidation: formation of peptide bond; does not require additional energy (aa-tRNA is high energy); reaction catalyzed by large ribosomal subunit
- Translocation: EF-G helps shit mRNA and tRNA by 3 bp to prepare for next aa-tRNA (requires GTP hydrolysis); unchanged tRNA left in Psite moves to Esite
what happens during step 3 of protein synthesis?
termination
- release factor carries bound GTP, bind A-site; recognizes STOP codon (no corresponding tRNA)
- peptide is hydrolyzed and released: binding of RF alters activity of peptidyl transferase, H2O takes role of incoming tRNA
- components dissociate: GTP hydrolyzes, changes conformation of ribosome
describe the fidelity of aa-tRNA synthetase fidelty
- have two active sites (synthesis and editing)
- incorrect aa costs 2 phosphoanhydride bonds (ATP or GTP)
how can efficiency of protein synthesis be regulated at the level of initiation?
phosphorylation of eIF-2 leads to inhibition of translation
-ex. synthesis of globin in response to heme availability: heme controlled inhibitor is a protein kinase that prevents glob in synthesis when there is no heme; inactive with heme bound; active when heme is not bound and phosphorylates eIF-2 to prevent initiation
how can efficiency of protein synthesis be regulated by sequence elements?
half life of mRNA is important in regulation
-ex. regulation of ferritin/transferrin receptor in response to Fe availability: aconitase binds IRE of ferritin mRNA; inhibits synthesis of ferritin; binding of aconitase to IRE on 3’ UTR of transferrin receptor mRNA stabilizes mRNA against degradation and translation occurs
how can efficiency of protein synthesis be regulated by micro and small interfering RNA molecules?
little pieces of mRNA match mRNA you want to destroy
-ex. interferon-regulated susceptibility of individuals to viral infections: interferons are intercellular messages that are activated by dsRNA produced by viruses; induces expression of enzymes that inhibit protein synthesis, prevents spread of viral infection
hardy weinberg assumptions
- large, randomly mating population
- individuals of all genotypes are equally capable of mating/passing on genes
- allele frequencies remain constant over time: no immigration, mutation, or selection
- only when there is a very sudden change do you find that HW doesn’t apply
rules for hardy weinberg problems
- autosomal: P=A/A; H=A/a; Q=a/a
- xlinked recessive: 2pq=female carrier, q=male affected
how does the ratio of carriers changes as the number of affected individuals decreases?
ratio of carriers to autosomal recessive affected individuals increases as frequency of disease decreases
prader-willi syndrome
- small at birth but prone to overeating; temper
- microdeletion on long arm of chromosome 15
- PWS deletion on dad’s chromosome
- or NO deletion but uniparental disomy of 15 (maternal)
angelman syndrome
- severely developmental delayed, bursts of laughter, seizures
- microdeletion on long arm of chromosome 15
- AS deletion on mom’s chromosome
- or NO deletion but uniparental disomy of 15 (paternal)
imprinting
differential modification of maternal and paternal genetic contributions; results in differential expression of parental alleles during development
-associated with methylation (epigenetic modification); results in inactivation of genes
rett syndrome
neurodevelopment disorder primarily affects females; disruption of motor functions
- MECP2 is x-linked TF that can activate or repress transcription
- normal function of MECP2 required for maturation of neurons and normal development
- epigentic control of x-inactivation leads to differing phenotype
pharmacogenetics
related heritable variation to interindividual variation in drug response; adverse drug reactions
pharmacogenomics
field of new drug development based on increasing knowledge of all genes in the human genome; adverse drug reactions result in FDA rejection (use genetics to match dry to specific genotype)
-ex. ADME core markers; CYP2D6 of Cyc p450 family
pharmacogenomic example: warfarin
- anticoagulant inhibits enzyme resulting in inhibition of vitamin K metabolism (essential for clotting factors)
- start with low dose and ramp up slowly
- polymorphisms of genes that directly affect warfarin metabolism are most common in caucasians)
- knowing genotype should make it possible to better estimate effective dose
pharmacogenomic example: 6-mercatopurine
- immunosuppressive cancer drug
- most metabolize quickly (need high doses); others metabolize slower (need lower dose to avoid toxicity)
microarrays for pharmacogenetics
- create gene chip that has genes/known mutations for critical genes, can do country-wide study to see what people are at risk for certain diseases and then intervene for that predisposition
- look for regions of homozygosity
- pros: early treatment might cure or lessen intensity
cons: what genes to include? who decides? GINA protects against discrimination based on genetic information
3 types of population screening
- prenatal: MSAFP, Quad, AFAFP
- newborn: for clearly defined and treatable diseases; usually mandatory
- carrier: detect carriers and prove counseling in hopes that couple will opt for prenatal testing (ex. tay-sachs testing in ashkenazi jews has lowered incidence of affected kids)
