Quiz 2 Review - Updated.pdf

** HUMAN GENETICS I & II Understand meaning of DNA sequence and amino acid polymorphisms and variants - variation often occurs outside the coding region of DNA: introns, intergenic region - polymorphism: anything that is a variation, in trait or DNA - genetic poly...

** HUMAN GENETICS I & II Understand meaning of DNA sequence and amino acid polymorphisms and variants - variation often occurs outside the coding region of DNA: introns, intergenic region - polymorphism: anything that is a variation, in trait or DNA - genetic polymorphisms: phenotype, genotype, frequency of minor alleles (less than 1%) considered "nucleotide variation" - DNA seq variation: common allele greater than 99% = polymorphism - "rare variant alleles" = less than 0.01 - mutation: any change in DNA sequence: can be silent, can be neutral mutations: single base pair substitution, an indel (del or insertion of 1 or + bp), rearrangement Recognize DNA seq variation: STR, SNP, SNV, CNV - STR: simple tandem repeat, or simple sequence repeat, 1-5 bp - SNP: single nt polymorphism - SNV: single nt variants -CNV: copy number variation; is a rearrangement - indel: small (few nt) insertion/del SNP/SNVs: most are "silent" and intragenic (between the same gene), promoters and regulatory seqs, introns, and exons (5' and 3' UTR, and sometimes in coding seq) CNV: big chunks of DNA that get deleted, duplicated, segmental duplicated, inverted. Avg: 250,000 bps, role in human disease *pic* Different DNA mutation classes: Point mutations, insertions/deletions, rearrangements Synonymous SNPs: silent sequence change (doesn't alter encoded AA) Nonsynonymous SNP: missense mutation (for another AA), nonsense mutation (stop codon), frameshift mutation, splice-site mutation (occurs usu. introns, alters RNA seq) Others: regulatory gene region, large dels/inserts, chromosomal translocation/inversion Understand how to distinguish disease-causing mutation from neutral DNA seq variation - DNA seq is gold standard: targeted region (known mutation), "whole" gene (unknown), whole exome/genome - large del/rearrang, frameshift, nonsense: likely disease-causing - functional analysis: express recomb protein, transgenic mice, phenotype tree w/ new mut - computer predictions - most are "variant of unknown significance" VUS **HUMAN GENETICS II The basic anatomy of the human genome - majority is non-coding and repetitive seqs (near 50% each), only a minor percent is protein-coding (1-2%) - What other functions exist? - Where are they encoded? - How are they encoded? Comparative genetics, inter-, intra- species comparison; intra-individual comparison - intra-individual seq comparison: DNA sequencing - across species 1 - within species - within an individual - complex diseases: hypertension, coronary artery, diabetes, obesity, cancer - harder to determine -SNPs: given three SNP positions, we have 8 possible haplotypes each could occur in. In practice, fewer (2) are observed, termed "linkage disequilibrium": non-random association of alleles at different loci in a population, aka, allele frequency is higher/lower than expected if the loci assorted randomly - SNPs are inherited in haplotype blocks across the genome, with 30 - 70 variants, and 10-20K bps - haplotype block: region of genome with little genetic recomb, with small number of distinct haplotypes. - longer blocks suggest more recent descendants from an ancestor, shorter time for genetic recombination - SNPs may be associated with "affected" phenotype or protective against "affected" phen - multiple observations: the probability of having at least 1 SNP is very high once there's large population Genome wide association studies (GWAS) to identify gene variants contributing to complex diseases/traits Ex (SNP not helpful): Type 2 diabetes: currently greater 200 significant loci, explains only 15% risk. Vs BMI and family history predictors (25%) - 90% GWAS are in non-coding regions - mutations in faraway regulatory seq; SNP can simply be nearby, or can be causative Ex (SNP helpful): Macular degeneration: common SNP - complement factor H polymorphism - leading cause of elderly blindness - Tyr 402 His: 1 variant predicted 20-50% risk Ex (SNP helpful): Venus thrombosis (VTE) "economy class syndrome" - Factor V Leiden (FVL) - associated w/ 5% frequency in Euros, prothrombin - MTHFR and PAI1 (cautionary tale): two common polymorphisms in 50% of pop. Bc we can't pick them up in GWAS, and they're super common, we can tell these are NOT risk factors for VTE Adding a polygenic risk score: makes things more predictive. Ppl with lots of SNPs in top percent have 5-fold risk in things like coronary artery disease The implications of GWAS findings for clinical care and "personalized medicine" - GWAS generally not helpful - Class I tests: actionable test result (PKU, sickle cell screening, cancers) - Class III test: no impact on med mgt: Huntington's, Alzheimer's - Class II: grey area - GWAS contributors to power: 1) how common the variant is 2) how big the effect is Ex) rare variant with big effect is harder to pick up; common variant w/ small effect is easier to pick up The implications of "Next-Gen" sequencing for future clinical medicine precision medicine: tailored Rx, ID disease & give Rx preventatively Ex) Chronic Myeloid Leukemia (CML): wildly successful! A chromosome 9:22 translocation in the Bcr-ABl fusion protein. Therapy converts CML cells back to normal. Gleevec Ex) Warfarin: no difference in dose-dept genotyping of outcomes Ex2) tumor profiling 2  **CYTOGENETICS, LECTURE I & II 1) Be able to describe basic anatomy of human chromosomes, basic nomenclature of karyotypes ➞ Chromosome morphology includes (1) metacentric, (2) submetacentric, and (3) acrocentric chromosomes ➞ Chromosomes can be identified by their morphology and G-banding (Giemsa staining) patterns ➞ Autosomes: 1-22 | Sex: X, Y | p short arm, q long arm | Translocation: t(14;21) | / mosaicism | ter terminal, der derivative, ins insertion, del deletion | + addition - loss of chromosome Submetacentric: centromere located near middle, p and q arms form L-shape bc unequal Acrocentric: centromere located such that one arm is much shorter than the other 2) Explain techniques for karyotype, FISH, CMA. Describe benefits/limitations of each technique Karyotypes: gross look at 23 chromosomes; can tell translocations, pattern-based FISH (Fluorescence in situ Hybridization) = identify specific chromosomes or segments without staining (dels, trans, duplications) ○ Probes 10-100 kb pairs long 1. Locus -specific probe ➞ detecting deletions, e.g. used to locate nucleus 2. Centromere repeat probe ➞ structural characterization, e.g. 48, XXY, +18 tells us that this is a boy with Klinefelter’s syndrome and trisomy 18 3. Chromosome -specific painting probe ➞ identify trisomy or monosomy ✓ FISH provides information about presence, absence and location of genomic material Clinical utility: gross look ✦ CMA (Chromosome Microarray) = refined method to assess gene dosage along chromosome, quantity of genetic material ➞ CMA is first-line diagnostic test for children with (a) Developmental Delay, (b) Intellectual Disability, c) Autism Spectrum, (d) Cognitive Anomalies Remember copy number variations (CNVs) can account for intellectual disabilities ➞ Must consider limitations of testing such as (i) benign variants bc sensitivity is so good, (ii) variable penetrance for CNVs and (iii) variants of uncertain significance (VUS) 3  ✗ Does not detect: (1) mosaicism, (2) inversions, (3) balanced translocations, (4) location of copy gains Clinical utility: fine detail at kb level, better visualization 3) Recognize consequences of both meiotic/mitotic disjunction Trisomy 5p ➞ Euploid = multiple of haploid number, N (e.g. 23, 46, 69) ➞ Aneuploid = trisomy or monosomy (e.g. trisomy 21 = Down syndrome) ✦ Aneuploidy errors occur during maternal meiosis, increasing maternal age raises risk Gene dosage is critical for correct development & physiological funx Mitotic disjunction could lead to mosaicism 4) Define/recognize meiotic consequences of reciprocal & Robertsonian translocations 5) Understand risks to offspring of translocation carriers for unbalanced karyotypes Ex) Down syndrome: microcephaly, dev delay, somatic abnormalities (flattened face, Brushfield spots), congenital heart disease | error of aneuploidy Uniparental disomy (MII nondisjunction): risk for recessive alleles, from 1 parent Nondisjunction: cause of trisomy DNA centromeric markers assign origin Other nondisjunctions: Heteroisodisomy = non-disjunction at meiosis 1 (different chromosomes from same parent) Isodisomy = Non-disjunction at meiosis 2 (same chromosome from parent), recessive risk ✦ TRANSLOCATION ✦ Reciprocal/Balanced = No observed loss or gain of genetic material ➞ Parental translocation increases risk of unbalanced offspring, not dependent on sex of carrier. ➞ Unbalanced offspring are partially monosomic and trisomic on diff chromosomes. 4  ✦ Robertsonian = Involves acrocentric chromosomes (13, 14, 15, 21, 22) fused centromeres ➞ Loss of short arms (tandem repeats of rRNA found elsewhere) not clinically significant ➞ Carriers can have balanced or unbalanced offspring (↑ risk for female carriers, sex-dept) 6) Recognize major clinical consequences of chromosomal disorders Many chromosomally abnormal fetuses are aborted, 70% of which are aneuploid 7) Explain CNV & genomic diseases concept ➞ Chromosome inversions influences the structure of meiotic products and risk of unbalanced offspring karyotypes ✦ Paracentric = no centromeres involved, breaks within same arm, low risk ✦ Pericentric = includes centromere, breaks on both chromosome arm, increased risk of abnormal offspring ➞ Chromosome must loop on itself to align genes, crossing over leads to inviable gene combinations ➞ Copy number variations are chromosomal segments with greater than 2 copies (or no copy at all) ➞ Intrachromosomal rearrangements facilitated by unequal crossing over by non-allelic homologous recombination (NAHR) ✦ Recurrent CNV = MEIOTIC | NAHR between low copy repeat (LCR) areas | rearrangement hot spots e.g. Charcot-Marie-Tooth (CMT) single or more copy gain of PMP22 gene for peripheral myelin protein, neuropathy affecting myelin sheath, progressive muscle weakness/atrophy HNPP = heterozygous loss of gene. ✦ Non-recurrent CNV = MITOTIC | Paternal basis from ↑ spermatogenesis divisions CMA can be used to identify 1 copy loss and 1 copy gain CNVs where LogR = dosage and BAF=B-allele frequency ➞ In addition to copy number, CMA allows us to assess loss of heterozygosity Labs can determine copy neutral absence of heterozygosity (AOH), tells us that relatives mated, pinpoints locus Mosaicism cells within one person’s body has different genetic makeup, can contribute/cause to disease. Meiotic or mitotic in origin. Epigenetic, Down Syndrome are exs 8) Explain chromosomal basis of sex determination The X-chromosome is large (5-6% of genome) and accounts for many genetic disorders, while the Y-chromosome is much smaller mostly for spermatogenesis Start w/ indifferent gonad → normal XX or XY causes regression of Wolffian & Muellerian, respectively Castrated XX or XY → Muellerian duct remains Castrated XX or XY + testosterone → promotes Wolffian duct 5 ➞ Sex-determining region Y (SRY) on Y-chromosome determines males sex traits and prone to recombination (pairing & recomb happens ONLY at pseudoautosomal region (PAR) Note: Recombination of SRY is what gives rise to XX males and XY females depending on retention of the SRY. XY-females have dels or pt muts in SRY (HMG domain) SRY necessary but not sufficient for sex determination SRY confers male dev in transgenic mice 9) Explain basis & consequences of X-chromo inactivation 10) Dosage compensation: equalizes expression of genes b/w the sexes, as females and males have different genes. Eg, females silence an X chromo ➞ Females are mosaic and each cell is functionally hemizygous because of x-inactivation Note: Female cat with different color fur on her body is evidence that different genes are expressed bc of x-inactivation X-inactivation in somatic cells: early, random, complete, permanent & clonally propagated Evidence: genetic (tortoise shell, x-linked coat color muts), cytologic (late replicating X, Barr bodies), biochemical (G6PD) G6PD: A and B alleles show diff in migration; in many cells, only 1 line is active. In addition, Barr body is a clump of chromatin as a result of the inactive X-chromosome (# Barr Bodies = Xn -1) n ➞ X Inactive-Specific Transcript (XIST) is an lncRNA molecule that causes x-inactivation, coded by the X-inactivation center (XIC) gene X-inactivation leads to greater variability of clinical manifestations in heterozygous females skewed x-inactivation can manifest X-linked genetic disease 11) Recognize major phenotypic features of X-chromo aneuploidy X-chromosome aneuploidy leads to genetic disorders: (1) Klinefelter syndrome and (2) Turner syndrome ➞ Klinefelter syndrome = XXY, result of nondisjunction in maternal or paternal meiosis I ➞ Turner syndrome = X, suggests that you may need X-linked genes that escaped inactivation Klinefelter syndrome (47, XXY): male phenotype. Small testes, infertility, social interaction difficulty, non-disjunction (NDJ) in paternal/maternal meiosis I, intellectual disability Likely that there’s skewed X-inactivation in Klinefelter’s Turner syndrome: amenorrhea, sexual immaturity, gonadal dysgenesis (eggs disappear) Karyotype abnormalities: 45, X (50%) - usu spontaneously aborted. 46,X (15%) - no p arm, q arms fuse together. Mosaicism (30%) 45X/46XX - mitotic NDJ, XX line mollifies syndrome, XY 6  line risk for virilization, important to look for Y chromo material in absence of cell line bc risk for sex cord tumors Why don’t males have Turner? ○ Need X-linked genes to 1) escape inactivation 2) have Y-chromo homologs 3) are essential to prevent Turner 12) Explain pathogenesis of Fragile X syndrome & how to assess family risk Fragile X syndrome is an X-linked dominant disorder, good example of a monogenic disease (single gene in all cells of the body) ➞ Due to mutation at chromosome "fragile site" FRAXA at Xq27.3 towards telomeric end of q arm of X-chromosome ➞ CGG repeats at 5'UTR region of FMR1 gene leads to methylation at promoter for gene that code for a protein that interferes with translation (leads to RNA toxicity, RNA is not made). Males always affected, ½ females cognitively impaired Repeats accumulate over generations (Sherman Paradox) where normal transmitting males propagate the repeats CGG repeats unstable, expand in female meiosis. Expansion risk decreases if: repeat length is less, presence of normal AGG seq interruption Note: Daughters of NTM are obligate heterozygotes but are not affected with the intellectual disability 13) Know indications for chromosome or chromosome microarray analysis known/suspected chromo abnormality (std karyotype) Multiple congenital anomalies/growth retardation/dev delay (CMA) Unexplained intellectual disability/autism (CMA) **MENDELIAN INHERITANCE I & II 1. Recognize, define key words Haplotype: “haploid genotype” combo of alleles transmitted together on one chromosome Compound heterozygote: having 2 or more heterozygous recessive alleles at the same loci, aka: hetero variation at one portion of gene, hetero variation at another portion of gene. A person can be compound heterozygote for 2 pathogenic variants (aka, both alleles are mutated) Double heterozygote 7  Probrand: index case Allelic heterogeneity Locus heterogeneity Variable expressivity Anticipation Triplet repeat expansion Gonadal mosaicism, heteroplasmy 2. Describe std pedigree symbols & how to use them 3. Recognize characteristics of different Mendelian patterns & define Autosomal dominant: vertical, can be de novo, see allelic heterogeneity (diff places in gene). Ex1) Neurofibromatosis 1 (NF1) - cafe au lait spots, vascular complications, point muts w/ dels and intragenic translocation interruption of gene), ex of pleiotropy (single gene effect) & var expressivity Lisch nodules (benign growths) plexiform neurofibroma (serious lesions that can grow into tumors) 8  Marfan syndrome - fibrillin1 mutation demonstrates variable expressivity & intrafamilial variability (valvular disease, scoliosis, myopia, dura ectasia, ectopia lentis (fibrillum protein can’t hold lens of eye in place), ascending aortic aneurysm, tendency to rupture w/o intervention) Erythermalgia Achondroplasia (80% are de novo) - almost always single AA alteration Gly -> Arg Ex2) non-Mendelian AD: Myotonic muscular dystrophy (DM1, DM2): triplet CTG repeats in 3’UTR of DMPK gene on 19q13.3. Polymorphic, unstable repeats, individuals are mosaic for diff repeat expansion lengths, expands in maternal meiosis, severity & age onset correlate w/ repeat size. an example of anticipation, where genetic disorder is passed along to generations and symptoms increase in severity & start earlier. Mechanism: DMPK gene produces toxic mRNA, which sequesters MBNL protein, an essential RNA splicing factor in the nucleus that then splices cell proteins incorrectly Autosomal recessive: horizontal, chance occurrence, identical by descent (IBD via consanguinity, view with CMA). confounded by pseudodominance, locus heterogeneity, & probability | Ex) congenital hearing impairment - has double heterozygotes, must consider loci heterogeneity X-linked recessive: males affected, carrier females, NO male-male transmissions, daughters of males are obligate carriers. Ex) Duchenne muscular dystrophy - males are hemizygous (person w/ one chromosome pair) | female X-linked R disease: hemizygosity for X (Turner syndrome, aka has partial X del), homozygosity for mut allele, or skewed X-inactivation (ornithine transcarbamylase deficiency, manifests as hyperanemia) X-linked dominant: more females affected, males severely affected, NO male-male transmission, all daughters of males affected Mitochondrial inheritance: maternal, 37 genes, mutation rate > in DNA. Mitochondrial defect in mitochondrial DNA or nuclear DNA: autosomal recessive, autosomal dominant disorders heteroplasmy: variation of degree of mitochondrial DNA, can influence disease penetrance, onset, severity homoplasmy Ex) MERRF (mitochondrial encephalopathy ragged red fibers, v debilitating) Genotype does NOT equal phenotype: incomplete penetrance, modifier genes, epigenetics, enviro, spec variant Triplet repeat diseases: affects ppl at the RNA or protein level Fragile X syndrome (CCG) - loss of funx Myotonic dystrophy 1 & 2 (CTG) - gain of funx Huntington’s disease (CAG) - loss & gain of funx 4. Interpret pedigree info to calculate risks of occurrence/recurrence in family members 5. Explain Bayes theorem / apply to analysis of pedigree & risk calculation **HUMAN POPULATION GENETICS Holotype: individual representative of species Cystic fibrosis: a Mendelian phenotype (CFTR normal gene is dominant to defective allele) 9 Most genetic disorders are “complex genetics”: don’t follow Mendel Phenotype variation = Enviro variation x Genome variation (PEG) Karl Landsteiner RBC experiment: agglutination if AB mismatch (type B led to serum A agglutination). A and B are co-dominant, O is recessive. ABO is a maintained variation among species - concept: balancing selection pressure can maintain 2 or more alleles. Hardy Weinberg Equilibrium tells us that dominant alleles don’t simply wipe out recessive alleles: 1) allele frequencies don’t change from generation to generation 2) genotype frequencies determined by allele frequencies at that locus 1 = p^2 + 2pq + q^2 7 assumptions: diploid orgs, sexual reprod, allele freq equal in sexes, (following are variable:) mating is random, pop size is infinitely large, no migration/mut/selection, generations non-overlapping Implications: allele freq constant b/w generations. Recessive allele means heterozygotes >> homozygotes. Selection for/against recessive alleles is inefficient Genetic drift: allele freq changes with no selection: skewing of genes leads to founder effect Types of selection, all are examples of evolution: stabilizing selection, directional selection (alters phenotype in one direction), disruptive selection (speciation) Admixed population: when previously isolated populations mix MULTIFACTORIAL INHERITANCE 1. Describe differences b/w Mendelian & multifactorial inheritance Mendelian traits: single genes where mut largely affects phenotypes Multifactorial traits: determined by interaction of genes w/ environmental factors. Variants in multiple genes has small effect on phenotype ex) height: highly heritable & affected by enviro (nutrition) 2. Explain the kinds of evidence that suggest genetic factors in disease causation, & studies used to ID genetic factors Genetic variation = susceptibility of disease rather than cause Thought to be heterogeneous; common variant, common disease OR rare variant, highly heterogeneous OR omnigenic (all genes expressed in relevant cell impact trait) Polygenic risk scores & hazard ratios aggregate data from genomic risk loci or SNPs to model complex disease Common birth defects: threshold model; increases risk if: more of family is affected, consanguinity, closer, distance to proband Sex bias: pyloric stenosis (M), idiopathic scoliosis (F), cleft palate, spina bifida 10  Familial aggregation: increased risk to families of affected individuals Twin studies: disease concordance shows it’s multifactorial w/ significant genetic component (100% > MZ >> DZ) Mendelian forms of common diseases: e.g. maturity onset diabetes of young (MODY), via vertical transmission and autosomal dominant inheritance ID genetic cause of disease: Linkage: co-segregation in families of a marker locus with disease Association: specific allele at a candidate locus with disease in pop (e.g. ankylosing spondylitis associated w/ increased HLA-B27) GWAS - common disease-common variant hypothesis Genome wide sequencing Implication: enviro triggers have greatest impact on genetically predisposed 3. Understand genetic view of disease, role of evolution, distinction b/w individual vs population care Genetic view of disease: disease results from mismatch b/w integrated, variable homeostatic systems PLUS variable environment and chance events Evolutionary tradeoffs: being bipedal + lower back pain! The thrifty gene + obesity **TRANSLATION Translation and degradation of mRNAs occur in the cytoplasm! Describe the generation of mature rRNAs and tRNAs via RNA pol I and III and accessory proteins and RNAs. Eukaryotic mRNA — nearly always encode a single protein species from one mRNA RNA Pol I, II, and II Similarities: o All have TBP (general transcription factor [TF]) o Conserved core topology Differences: different TF partners (i.e. DNA opening & start site selection differs) o Pol I = Rrn7 o Pol II = TFIIB o Pol III = Brf1 Explain the ribosome as a ribozyme comprised of RNAs and proteins. Ribosome = ribozyme (enzymatic activity) = 2/3 rRNA (in the core) + 1/3 protein (on surface) Ribosomes are assembled in the nucleolus Ribosome is 80S (60S & 40S subunits) o 60S = 5S rRNA + 28SrRNA + 5.85 rRNA 🡪 ~49 proteins o 40S = 18S rRNA 🡪 ~33 proteins RNA Pol I = ribosomal RNAs (rRNA) — 45S precursor is processed to 28S, 18S & 5.8S rRNAs Chromosomal organization of rRNA genes 🡪 rRNA genes (RNA Pol I) are in the nucleolar organizer regions (NORs) of the 5 acrocentric chromosomes o Heterochromatin isolates rRNA genes from other genes o NORs have tandemly arrayed copies of the rRNA genes 11  o Active and silent rRNA genes distinguished by DNA methylation and histone modifications (just like Pol II-regulated genes) Processing of 45S precursors rRNA 1) 2 chemical modifications occur in nucleolus: methylation of sugars isomerization of uridines ▪ Made by “guide”(sno)RNAs in snoRNPs ▪ Affect folding of rRNAs & perhaps their function in ribosomes 2) Cleavage of 45S precursor into 18S, 5.85S, and 28S rRNA ▪ 18S rRNA 🡪 incorporated into small ribosomal unit ▪ 5.8S & 28S rRNA 🡪 incorporated into large ribosomal subunit RNA Pol III = tRNAs, 5S rRNA (and some snRNAs) tRNA transcripts are extensively processed o Cleavage of 5’ end by RNase P o CCA added to 3’ end (aa-binding end) o Pseudouridine modifications o Intro removal by endonuclease and tRNA ligase o Dihydrouridine modifications Describe the generation of ribosome subunits in the nucleolus. Describe how tRNAs function to match AA’s to triple codons in mRNAs tRNAs match amino acids to triplet codons in mRNA — humans have ~500 tRNA genes The triplet mRNA code is degenerate for the 20 aa’s found in cells o 64 possible triple combinations ▪ 61 codons specify aa’s ▪ 3 codons are stop codons (UAA, UAG, UGA) o Wobble base-pairing: “wobble position” (5’ end of anticodon & 3’ end of codon) allows tRNA’s 48 anti-codons to recognize the 61 aa-specific codons Amino acyl-tRNA synthetases (AARS) — edits and synthesizes tRNAS (at different sites) o Synthesis site: uses ATP to charge tRNA with correct amino acid o Editing site: correct errors by hydrolysis of incorrectly attached amino acid o For most cells, 1 AARS per amino acid — i.e. 20 AARS per tRNA Describe how translation initiation occurs at different AUG sites in mRNAs Translation initiation (TI) occurs at an AUG start codon in the preferred context Steps for translation initiation, elongation, and termination 1) Initiator tRNA (bound to small subunit) moves along RNA (starting at 5’cap) searching for preferred AUG ▪ Kozack consensus sequence dictates which AUG is preferred 12  2) Once AUG found, eIF2 & other initiation factors dissociate (via hydrolysis of GTP) from initiator tRNA 3) Large ribosomal subunit binds to RNA Subunit has E, P and A sites ▪ A-site (amino-acyl tRNA) — where amino-acyl tRNAs bind to codon ▪ P-site (peptidyl-tRNA) — where growing peptide is transferred to the tRNA, making it a peptidyl-tRNA until the next peptide bonds Also where initiator tRNA is ▪ E-site (exit) — where uncharged tRNA exits the ribosome 4) Aminoacyl-tRNA binds to A-site 🡪 peptide bond forms between aa’s in A & P site ▪ Uncharged tRNA in P-site moves to E-site & peptidyl-tRNA moves to P site 5) Once ribosome reaches stop codon, release factor binds to A-site causing poly-A chain to be released and subunits to dissociate from RNA Internal ribosome entry site (IRES): RNA sequences with secondary structures (cis elements) that allow for protein translation to begin in the middle of an mRNA o = cap-independent recognition of AUG 🡪 IRES trans-acting factors (TAFs) allow ribosome to bind and start scanning at any site on the RNA ▪ Most initiation factors are cap-dependent o Mostly found in viral transcripts, but some are in cellular mRNAs Polyribosome: cluster of ribosomes bound to single mRNA — circularization of mRNA w/rapid recycling of ribosomes allowing for efficient protein production o = large amount of protein synthesis from single transcript in coordinated fashion o Poly-A-binding protein binding to 43S pre-initiation complex promotes circularization Explain how 5’ and 3’ UTRs modulate translation 5’ and 3’ UTRS regulate protein synthesis E.g., Regulation of intracellular iron (Fe) stores by coordinated regulation of expression of Ferritin & Transferrin receptor synthesis o Ferritin = intracellular iron-binding protein 🡪 stores iron Transferrin receptor = iron-binding plasma glycoprotein 🡪 transfers iron to systemic tissues o During iron starvation cytosolic aconitase is active: ▪ Binds to 5’UTR of ferritin mRNA, blocking translation 🡪 no ferritin made ▪ Binds to 3’UTR of transferrin receptor mRNA to stabilize mRNA, allowing translation 🡪 transferrin receptor made o When there is excess iron, iron binds to cytosolic aconitase (Fe acts allosterically), resulting in ferritin being made but no transferrin receptor made E.g., Hereditary hyperferritinemia cataract syndrome (HHCS): crystals of ferritin protein build up in tissues (including lens of the eye) — caused by mutations in 5’ UTR of ferritin gene, making it unrecognizable to cytosolic aconitase 🡪 results in ferritin production independent of iron levels in the body 13 miRNAs silence mRNA translation by binding to areas of RNA transcript Precise binding causes cleavage of poly-A tail, resulting in mRNA degradation Explain how nonsense-mediated decay (NMD) of mRNA works & NMD in disease Exon junction protein complexes (EJCs) are part of the mRNA when it’s exported into the cytoplasm, and EJCs are removed by the ribosome as it translates the mRNA NMD of mRNA: signaled by premature termination codon (PTC), resulting from abnormal splicing Translation of an mRNA with a premature termination codon (PTC) leads to interactions between EJCs & NMD proteins (UPF1), followed by endonuclease cleavage of mRNA then decay of the mRNA NMD in normal physiology o 2/3 of Ig and TCR gene rearrangements in B & T cells lead to PTCs o 45% of alternatively spliced mRNAs have one form w/PTC NMD in disease o 1/3 of all genetic disorders due to PTCs (result from nonsense or frameshift mutations) o NMD can aggravate disease phenotypes NMD Regulation of mRNA — Poly-A binding protein (PABP) & upstream frameshift proteins (UPFs) compete for mutually exclusive interaction with the release factor complex (eRF3/eRF1) at the terminating ribosome PABP binds 🡪 proper termination & mRNA persists UPF1 binds 🡪 NMD, mRNA degradation Duchene Muscle Dystrophy: X-linked recessive disorder that causes loss of walking in teens and death in 20s o 1 in 3,500 newborn boys — dx as toddlers o DMD gene is huge (>2.5 Mb) o Caused by variable spectrum of mutations (deletions, nonsense, frameshift), many of which lead to premature stop codons and NMD of DMD mRNA (no DMD protein production) **CYTOSKELETON Dynamic protein filaments in cytoplasm that gives cell shape + capacity for directed movement Explain the dynamics of actin filament and microtubule polymerization and depolymerization Actin Tubulin α-actin in skeletal muscle; ß and α ß dimer (ß has GTPase activity); gamma-actin in non-muscle cells stronger and thicker than actin 14  In vitro G-Actin + ATP or (α ß tubulin + GTP) + K+ + Mg2+ = Polymerization (in vitro) assembly (self-assembly! non-covalent interaction driven by ATP or GTP hydrolysis) Non-covalent G-actin monomers Non-covalent “head-tail” and “side-side” (asymmetric) associate into actin interactions of protofilaments -> 13 filaments -> polar filament filaments form helical cylinder Does not hydrolyze ATP -> in ATP Filament dimer is polar -> completed bound form in the cell -> filament is non-polar assembles into actin filament F-actin filament (symmetric) -> polymerized actin, steady state, treadmilling, hydrolyzes ATP (after ATP-G actin assembles into actin filaments) Microtubule associated proteins (MAP) non-covalent interaxn (bundling, capping, severing, deploy, sequester, GDP/GTP or ATP/ADP exchange) Major Barbed (+) end polymerization In vivo, - end is anchored to microtubule difference (ATP-G actin adds), pointed (-) end organizing center (MTOC) in the center of depolymerization (ADP-G actin falls the cell, so growth only happens at the + off) end In vitro treadmilling observed via hydrolysis on ß-tubulin (removal) and GTP-bound polymerization. In vivo dynamic instability -> + ends switch between phases of growth “rescue” and shortening “catastrophe” High GTP-tubulin: polymerization before GTP hydrolysis Low GTP-tubulin: polymerization slower than GTP hydrolysis -> depolymerization Both Actin and Tubulin have: 1. Lag phase: time taken for nucleation 2. Growth phase: actin monomers add to both ends of filament 3. Equilibrium phase: addition/subtraction of actin monomers (net length of filament stays the same) Describe the structure and function of intermediate filaments in normal and Progeria cells 15  o No common building block o Central region: α-helical segments -> coiled coil o Amino & Carboxy terminal ends vary among different intermediate filaments o Lateral, non-covalent interactions btw subunits -> strength o Resistant to harsh conditions o Non-polar o Do not bind/hydrolyze nucleotide o Dynamic o Nuclear lamina: filament disassembly/assembly is regulated by phosphorylation as cells enter/exit M phase of cell cycle o In axons: peripherin (type III) expression during rapid changes in cell shape accompanying nerve development -> neurofilament (type IV) assembled as axon diameter increases and cell reaches maturity, peripherin expression decreases Discuss the contribution of cytoskeletal filaments and molecular motors to membrane trafficking in normal and diseased states Normally: used to organize cell/tissue structure, protein/membrane trafficking, cell division/migration, cilia + flagella + microvilli Disease state Mechanism/Sx Cause Epidermolysis bullosa Blistering due to Hereditary keratin defect simplex (blistering skin dz) mechanical stress Nuclear structure Genetic disorders Laminopathies disruption (size, support, associated w/ lamin (Hutchinson Gilford mitotic spindle assembly,mutations (Lamin A -> progeria syndrome) DNA synthesis, progeria). transcription) -> premature Dominant-negative: aging progerin irreversibly anchors in the nuclear membrane and disrupts normal lamina function Motor neuron disease Neurodegeneration Mutations in dynein motor protein (muts in Cra1 and Loa dynein genes) Charcot-Marie-Tooth dz Most common peripheral Mutation in kinesin motor neuropathy (inherited) -> protein ATP binding pocket weakness/atrophy of distal -> kinesin cannot move to muscles. Depressed/absent + ends of microtubules as deep tendon reflexes, mild it’s stuck in the center sensory loss (loss of function mut in KIF1B gene) o ER -> golgi apparatus -> endosomes/secretory vesicles -> lysosomes (from endosome) or cell exterior (from endosome/secretory vesicles) 16  o Actin filaments and microtubules act as roads for molecular motor proteins (trains) o ATP hydrolysis drives movement of: dynein goes to (-), kinesin-1 goes to (+) Describe the roles of the cytoskeleton and motor proteins in cell division o Intermediate filaments disassemble -> “nuclear envelope breakdown” is transition mark b/w prophase and prometaphase ▪ Lamin phosphorylation -> cause of nuclear lamina disassembly + breakdown o Microtubules -> form mitotic spindle -> divides chromosomes ▪ Dynamic instability -> search and capture chromosome centromeres ▪ Kinesin motors capture mt + ends to slide mts into bipolar array o Chromosome congression: chromosomes must align at metaphase plate (metaphase checkpoint) o Actin makes contractile ring -> separates daughter cells during cytokinesis ▪ Myosin motors contract actin filaments and pull together the plasma membrane o Clinical: cancer therapeutics block mt dynamics Define the steps of cell migration and the role of actin polymerization o Cell migration driven by actin polymerization o Cell initiates actin polymerization at leading edge: ▪ Nucleation is the rate-limiting step; nucleation is driven by ARP 2/3 complex Binds to F-actin, nucleates at 70 deg angle Allows bypass of lag phase to go immediately to growth phase Severing protein cuts ARP 2/3 complex for recycling of actin units o Flagella and cilia (euk flagella) drive cell motility ▪ Beating driven by motor dynein ▪ Mts arranged in axoneme (9 doublets + 2 singles) stabilized by MAPs bundling protein 9+2 motile cilia 9+0 sensory cilia 17  ▪ Non-motile cilia sense extracellular environment (non-motile exception) o Microvilli: F-actin filament bundles increase cell surface area for digestion in the brush border of intestinal epithelial cells o Stereocilia: microvilli actin in sensory epithelium for sound transduction in Organ of Corti in inner ear Clinical Applications Cystic Kidney Diseases Single cilium defect does not Kidney responds by growing allow Ca2+ to pass through larger to sense Ca2+ → for sensing fluid flow (cilia results in cysts disease, aka ciliopathies) Situs inversus When motile cilium is unable Without directional to sweep cell fate movement, results in determination factors to incorrect internal symmetry correct side of embryo’s (L to R inversion of internal sensory cilia (cilia disease) organs, infertility, chronic bronchitis + sinusitis) Nonsyndromic senorineural Unconventional myosin Accounts for 80% of recessive deafness proteins result in short hereditary hearing loss stereocilia (microvilli disease) **HISTOLOGY OF EPITHELIA (Hortsch) 1. Be able to classify epithelial tissues 2. Know structure + function of junctions 3. Know structure of apical specializations + functions 4. Correlate different types of epithelia to functions 18 Junctions (from Apical to Basal) Barrier Occluding Junctions Function “belt” Zonula Occludens (Tight Cell-Cell Claudin, Actin Prevent passage of most Junction) Occludin materials between epithelial cells Structure Anchoring Junctions Function “belt” Zonula Adherens Cell-Cell Cadherin Actin Link epithelial cells together, (Intermediate/Adherent do NOT provide any barrier Junction) function “spot” Macula Adherens Cell-Cell Cadherin IFs Provide cell-to-cell adhesion (desmosomes) CAMs (Ca2+ like rivets connecting metal dep) plates Hemidesmosomes Cell-Matrix Integrin IFs Link basal domain of (anchors to epithelial cells to the BL) underlying basement membrane Focal Adhesions Cell-Matrix Integrin Actin Link basal domain of epithelial cells to the underlying basement membrane Signaling Communicating Function Junctions Gap Junction (Nexus) Cell-Cell 6 connexins None Allow for free diffusion of forming a small molecules (such as connexon Ca2+ ions) between two (2nm gap) neighboring cells **HISTOLOGY OF GLANDS (Hortsch) 1. Review secretary pathways of cells 2. Understand 3 modes of secretion & 3 cellular mechanisms of secretion 3. Learn general organization and cells of secretory acini 19  4. Differentiate bw different configurations of exocrine glands 5. Know difference bw serous and mucous exocrine cells 6. Learn about functions carried out by secretory ducts **EVIDENCE-BASED MEDICINE 6As: Ask, acquire, appraise, apply, agree (ACT) Background q’s: based on medical knowledge, context. Asked by less experienced. Who, what, when, where, why, how, + condition of interest Foreground q’s: used to inform clinical decisions. PICO: Patient/population/problem, Intervention, comparator, outcomes Therapy | Harm/Etiology | Diagnosis | Prognosis RCT: pts assorted into control and experimental groups (gold std) Cohort studies: prospective and retrospective following of a cohort through time to determine outcomes of control vs experimental group Case control study: compare cases and control groups that have already occurred Cross-sectional: snapshot in time; prevalence of disease in pop 6S Pyramid: Systems > Summaries > Synopsis of Synthesis > Syntheses > Synopsis of Single Studies > Single Studies Shared decision making: 1) 2-way exchange bw pt and physician 2) Discussion and deliberation 3) Consensus decision Pt-centered communication: 1) Patient perspectives (elicit & understand) 2) Unique psychosocial/cultural context (understand) 3) Shared solutions that match pt values 20