Molecular Bio - Comp Exam Flashcards

Question

Describe transposons.

Answer 1

Transposons

Answer 2

Conservative Site-Specific Recombination * Mediates rearrangements of other types of mobile DNA elements. * Break and join two DNA double helices on each molecule: * Depending on positions and relative orientations of recombination sites, can get: * DNA integration, DNA excision, or inversion. * Differ from transposition: * Need special sites on each DNA that serve as recognition sites for recombinase. * Only transposon sequence is required for transposition. * Form transient high energy covalent bonds and use this energy to complete DNA rearrangement. * No covalent protein/DNA intermediate in transposition. * Gaps must be filled by DNA polymerase and ligase. * Difference in outcome is in the relative orientation of DNA sites. * Many bacterial viruses move in and out of host genome by this mechanism: * ex. Bacteriophage lambda.

Answer 3

Meiosis * Gametes are haploid. * Arise from meiosis: * Involves two cell divisions but one round of DNA synthesis to produce half the number of chromosomes.

Answer 4

Fertilization * Released egg is surrounded by granulosa cells and an ECM rich in hyaluronic acid. * Capacitated sperm must penetrate granulosa cells: * Uses hyaluronidase. * Sperm binds to zona pellucida: * Acts as a species barrier. * Zona pellucida induces sperm to undergo acrosome reaction. * Contents help sperm to tunnel through zona pellucida. * Alters sperm so can bind and fuse with plasma membrane of egg. * Sperm binds egg plasma membrane first by tip and then side. * Microvilli on egg aide in the process. * Certain membrane proteins are crucial to binding: * ZP 1, 2 and 3. * ZP 2 and 3 form long filaments. * ZP 1 cross-links the filaments.

Answer 5

Gonadal Development * Sry in somatic cells direct differentiation into Sertoli cells instead of follicle cells. * Sertoli cells secrete anti-Mullerian hormone: * Suppresses female development. * Causes Mullerian duct to regress. * Induce Leydig cell differentiation in other somatic cells: * Secrete testosterone (responsible for 2ndary sexual characteristics. * In absence of Sry: * Genital ridge becomes an ovary. * PGC becomes an egg. * Somatic cells differentiate into: * follicle cells - support cells. * theca cells - estrogen-producing cells.

Answer 6

Central Dogma * DNA is transcribed to messenger RNAs which act as templates for protein synthesis in translation.

Answer 7

All Cells Replicate Their Hereditary Information by Templated Polymerization

Answer 8

Transcription

Answer 9

Translation * Translation proceeds in three phases: * **_Initiation:_** The ribosome assembles around the target mRNA. The first tRNA is attached at the start codon. * **_Elongation:_** The tRNA transfers an amino acid corresponding to the next codon. The ribosome then moves (translocates) to the next mRNA codon to continue the process, creating an amino acid chain (polypeptide). * **_Termination:_** When a stop codon is reached, the ribosome releases the polypeptide.

Answer 10

Proteins * Nitrogen and carbon containing compounds that consist of large molecules composed of one or more long chains of amino acids that are an essential part of all living organisms.

Answer 11

DNA Replication - Initiation * Prereplication complexes bind an origin of replication. Activated DNA helicases find origins of replication, polymerase and other supporting proteins are added, phosphorylation initiates DNA synthesis at the appropriate time in the cell cycle.

Answer 12

Short Telomere Syndromes * Short telomere syndromes (STSs) are accelerated aging syndromes often caused by inheritable gene mutations resulting in decreased telomere lengths. * Short telomere syndromes are multisystem disorders with widespread clinical manifestations. * **_Organs with high cell turnover, such as the bone marrow, liver, lungs, and immune system, are commonly affected._** * Key clinical cues to suspect short telomeres in a patient are a personal or family history of premature graying of hair (at age \<30 years), unexplained cytopenias, idiopathic pulmonary fibrosis, and cryptogenic cirrhosis. * Flow cytometry - fluorescence in situ hybridization is the initial screening test, followed by genetic sequencing. * Treatment requires a multidisciplinary approach.

Answer 13

Special Translesion DNA Polymerases Are Used in Emergencies * The cell recognizes that DNA is damaged and replication has stalled. * Specialized proteins cause the release of the DNA polymerase and bring in the correct translesional polymerase to allow the damaged DNA to be bypassed. * The polymerase clamp is then reactivated and replication proceeds. * **_Great potential for mutation._** * Despite being tightly regulated by a variety of transcriptional and posttranslational controls, the low-fidelity TLS polymerases also gain access to undamaged DNA where **_their inaccurate synthesis may actually be beneficial for genetic diversity and evolutionary fitness._**

Answer 14

Homologous Recombination in Crossing Over * Crossing over and gene conversion can occur in the same chromosome. * Multiple opportunities for genetic reassortment.

Answer 15

Effects of Radiation on Mutation Rates * Ionizing radiation (X-rays etc.) dislodges electrons in tissue causing free radicals which often damages DNA. * UV light induces the formation of pyrimidine dimer: two thymine bases covalently bonded that blocks replication. * SOS system in bacteria: SOS system allows bacteria cells to bypass the replication block with a mutation-prone pathway.

Answer 16

RNA Folding

Answer 17

Transcription * DNA is transcribed by RNA polymerase. The RNA moves stepwise along the DNA unwinding the DNA as the polymerase progresses. The incoming nucleosides (ribonucleoside triphosphate: ATP, UTP, CTP, GTP) supply energy needed for catalysis from their triphosphate bonds.

Answer 18

RNA Molecules - Categories

Answer 19

Transcription Start and Stop Signals

Answer 20

RNA Polymerase II * The TATA box is 25 nucleotides from the transcription initiation site. The protein TBP recognizes and binds to the TATA box providing the initial step to begin transcription. * A series of additional proteins are added until the entire complex of proteins are assembled and transcription starts. * The initiation of transcription usually requires multiple activator proteins along with proteins that unwind the chromatin. The activator proteins help coordinate the acquisition of the multiple different proteins needed for transcription initialization. They also play a role in modifying DNA shape which affects the rate of transcription

Answer 21

RNA Capping * As soon as the mRNA is ~25 nucleotides long it is capped. 3 enzymes work together to: 1. Dephosphorylate the 1st nucleotide, 2. Add a GMP in reverse linkage (5’ to 5’ instead of 5’ to 3’), and 3. Add a methyl group to the guanosine. * The cap allows mRNA to be distinguished from noncoding RNAs and is also important for establishing translation. * And sometimes another methyl group is added to the ribose of the 1st nucleotide.

Answer 22

RNA Splicing

Answer 23

Exon Size vs. Intron Size * Exons are much more similar in size among eukaryotes than are introns.

Answer 24

mRNA Splice Site Mutations * mRNA splice site mutations have serious consequences.

Answer 25

Generation of 3' End of mRNA - RNA-Processing Enzymes

Answer 26

Exosomes * The exosome is a protein complex that cleans up damaged RNAs before they leave the nucleus. * It is rich in RNAases and chops up RNAs for recycling. * Another definition: * Exosomes are cell-derived vesicles that are present in many and perhaps all eukaryotic fluids, including blood, urine, and cultured medium of cell cultures. * Exosomes contain various molecular constituents of their cell of origin, including proteins and RNA. Although the exosomal protein composition varies with the cell and tissue of origin, most exosomes contain an evolutionarily-conserved common set of protein molecules.

Answer 27

microRNA * microRNA are ubiquitous small noncoding RNAs. * A microRNA (miRNA) is a small non-coding RNA molecule (containing ~ 22 nucleotides) found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression. * The first miRNA was discovered in the early 1990s. However, miRNAs were not recognized as a distinct and important class of biological regulators until the early 2000s. * MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. * Biogenesis of miRNA is under tight temporal and spatial control. * Dysregulation of miRNA is associated with many human diseases, particularly cancer and neurodevelopmental disorders. * Regulation takes place at multiple levels including transcription, Drosha processing, Dicer processing, RNA editing, RNA methylation, uridylation, adenylation, Argonaute modification and RNA decay.

Answer 28

snRNPs * Biochemical modification of noncoding RNAs is implemented by snRNP (small nucleolar RNAs [snoRNA] + specific proteins). * The most common modifications are pseudouridylation and 2’-O methylation * Ribosomal RNAs have more than 100 of each type of modification. * Spliceosomal RNAs are also modified. * Evidence is accumulating that mRNAs are also modified in this fashion.

Answer 29

Nucleolus * The nucleolus is the site where rRNA is processed and assembled into ribosome subunits. The nucleolus is not membrane bound, but a dense assembly of pre and post processing – rRNA and the proteins needed to assemble the ribosome.

Answer 30

Nucleus * The nucleus has multiple aggregates including cajal bodies where most snoRNAs are assembled into snRNPs. * The protein Fibrillarin is present in both the nucleolus and cajal bodies. Identified with an antibody against with a red flourochrome. * Coilin is a protein found only in the cajal body. Shown here by immunohistochemistry with a flourochrome that appears pink in combination with the flourochrome for fibrillarin (arrows). * Interchromatin granule clusters (green) contain stockpiles of RNA processing components and are located near sites of transcription.

Answer 31

Genetic Code

Answer 32

tRNA - Matching to Amino Acids * tRNA have a complex 3 dimensional structure. Most tRNAs have extensive biochemical modification of nucleotides. * A. y = pseudouridine D = dihydrouridine. * B. and C. X ray diffraction projections of tRNA. * D. tRNA icon * E. The tRNA sequence, in this case the tRNA-Phe

Answer 33

Wobble Base Pairing * The nucleotide listed in the 1st column can base pair with any of the nucleotides listed in the 2nd column. * For example when U is at the 3rd codon position it can pair with 3 different nucleotides in the anticodon (I = deamination of A).

Answer 34

Incorporation of Amino Acids * 2 adapters help ensure the correct amino acid gets incorporated into a peptide. 1. aminoacyl-tRNA synthetase which helps ensure that the correct amino acid is coupled to the tRNA. 2. The tRNA is an adapter whose anticodon pairs with the codon in the mRNA to identify the correct amino acid to adds to the growing peptide.

Answer 35

Translation - Addition of Amino Acids * The fundamental reaction of protein synthesis is the formation of a peptide bond between the carboxyl group at the end of the growing peptide and the amino group on the incoming AA. * The formation of the peptide bond is energetically favored because of growing C terminus has been activated by the covalent attachment of the tRNA.

Answer 36

Translating an mRNA into a peptide. * There are 4 steps involved in protein synthesis: 1. New tRNA binds to A site pairing with codon. 2. Carboxyl end of growing peptide is released from tRNA in P site. Peptide bond formation between the previous AA added and the new one. tRNAs are in P site and A site. 3. Large subunit moves along mRNA held by small subunit, shifting tRNAs to P and E sites on small subunit 4. Small subunit moves 3 nucleotides along the mRNA and ejects the tRNA in the E site. * The entire complex moves 3 nucleotides along the mRNA as a result of the two translocation steps. The ribosome is thus reset and ready for another tRNA-AA.

Answer 37

Ribozymes * The Ribosome Is a Ribozyme – RNA capable of enzymatic function. * 2/3 of a ribosome is RNA. The 3D conformation of an entire ribosome was solved in 2000. * The structure helped demonstrate that the rRNA is responsible for the overall structure, the ability to position tRNAs and its catalytic activity. * **_The primary role of the proteins seems to be to stabilize the RNA core._**

Answer 38

Translation - Initiation * The initiation of protein synthesis requires the presence of several translation initiation factors. The poly A tail must be bound by poly A binding proteins that interact with eIF4G to ensure that both ends of the mRNA are intact. * 2 GTP hydrolysis steps provide the energy needed. The last 2 steps shown here are the ribosome has begun the standard elongation steps we discussed previously. * A consensus sequence flanking the start codon (ACCAUGG) tells the ribosome to begin translation on this AUG. If the flanking nucleotides differ very much, the ribosome will move to the next AUG. This results in alternative starting points for the protein.

Answer 39

Translation - Stop Codons * The binding of a release factor to an A site containing a stop codon causes termination of translation. The peptide is released and in a series of reactions the ribosomal subunits dissociate and are then ready to initiate translation again.

Answer 40

Polyribosomes * Synthesis of a protein takes ~20 seconds to several minutes. Upon completion of translation, the ribosomal subunits are near the 5’ end of the mRNA because of the association of the 5’ and 3’ ends of the mRNA. Thus they can quickly restart translation.

Answer 41

Antibacterial Compounds - Produced by Fungi * Inhibitors of Prokaryotic Protein Synthesis Are Useful as Antibiotics * Fungi produce many antibacterial compounds that exploit differences in ribosomal subunits. Bound tRNA is shown in purple and the antibiotic binding sites are shown with the colored spheres.

Answer 42

Translation - Quality Control Mechanisms * Quality Control Mechanisms Act to Prevent Translation of Damaged mRNAs – Nonsense Mediated Decay. * Incorrect splicing can result in premature stop codons. **_Nonsense mediated decay eliminates mRNAs with premature stop codons._** As the mRNA is being transported through the nuclear pore, a ribosome does a test run on it. Exon junction complexes (EJC) are bound at exon junctions and are removed by the moving ribosome. The stop codon should be in the last exon. If the ribosome hits a stop codon while EJCs are still present, the presence of EJCs will activate nonsense mediated decay and the mRNA will be rapidly removed to prevent any additional wasted energy. * This system may help prevent the translation of mutated genes with premature stop codons that could cause severe damage.

Answer 43

Molecular Chaperones * Molecular Chaperones Help Guide the Folding of Most Proteins. * Many energetically favorable pathways are available for protein folding. * Cells use additional proteins “chaperones” to ensure that peptides fold into the correct functional conformation or to refold them if they get damaged (for example after a heat shock). * Heat shock proteins (e.g. hsp70 family) recognize hydrophobic stretches of amino acids on the surface of a protein (generally these should be found on the interior of a protein) and bind to them along with hsp40 proteins using ATP hydrolysis to drive the reaction. Hsp40 dissociates and ATP rebinds inducing hsp70 dissociation. The process is repeated multiple times which helps the target protein to refold into the correct structure.

Answer 44

Protein Quality Control - Signals * Exposed Hydrophobic Regions Provide Critical Signals for Protein Quality Control. * Usually an exposed section of hydrophobic amino acids indicates a protein is misfolded. If not corrected the result is usually accumulation of protein aggregates that can be harmful. * Diseases of proteopathy include prion diseases, Alzheimer's and Parkinson's.

Answer 45

Proteasome * If the protein rescue process does not induce correct folding, the cell needs to eliminate the protein prior to aggregation. The proteasome searches for misfolded proteins in the nucleus and cytoplasm, ingests them and degrades them so that the AAs can be recycled. The cap (blue) binds damaged proteins that have been ubiquitylated, unfolds them (using ATP) and feeds them into the inner cylinder which is lined with proteases facing the inner chamber. * Only marked proteins can enter to prevent the random destruction of correctly folded proteins. Special proteins (E3) recognize misfolded proteins and add multiple ubiquitin peptides to the misfolded peptide. In this case the ubiquitin peptides are all linked at lysine 48 which is the key identifier of a damaged protein. * The cap has multiple functional domains: 1. Ubiquitin receptor 2. Unfoldase 3. Ubiquitin hydrolase

Answer 46

Proteins - Regulated Destruction * Protein lifespan is regulated by signals that direct the protein to the proteasome * Activating specific E3 ligases that target a protein for destruction. * Degradation signals within the protein can be induced leading to ubiquitylation and subsequent destruction.

Answer 47

DNA to Protein

Answer 48

Transcriptional Misregulation Leads to Disease * Abstract: The gene expression programs that establish and maintain specific cell states in humans are controlled by thousands of transcription factors, cofactors and chromatin regulators. Misregulation of these gene expression programs can cause a broad range of diseases. Here we review recent advances in our understanding of transcriptional regulation and discuss how these have provided new insights into transcriptional misregulation in disease. * Figure 1: * Transcriptional regulation: * A. Formation of a pre-initiation complex. Transcription factors bind to specific DNA elements (enhancers) and to coactivators, which bind to RNA polymerase II, which in turn binds to general transcription factors at the transcription start site (arrow). * B. Initiation and pausing by RNA polymerase II. RNA polymerase II begins transcription from the initiation site, but pause control factors cause it to stall some tens of base pairs downstream. * C. Pause release and elongation. Various transcription factors and cofactors recruit elongation factors, * D. Chromatin structure is regulated by ATP-dependent remodeling complexes that can mobilize the nucleosome, allowing regulators and the transcription apparatus increased access to DNA sequences. * E. Transcriptional activity is influenced by proteins that modify and bind the histone components of nucleosomes. Some proteins add modifications (writers), some remove modifications (erasers) and others bind via these modifications (readers). The modifications include acetylation (Ac), methylation (Me), phosphorylation (P), sumoylation (Su) and ubiquitination (Ub). * F. Histone modifications occur in characteristic patterns associated with different transcriptional activities. As an example, the characteristic patterns observed at actively transcribed genes are shown for histone H3 lysine 27 acetylation (H3K27Ac), histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 79 dimethylation (H3K79me2) and histone H3 lysine 36 trimethylation (H3K36me3). * Figure 2: * Master transcriptional regulators and reprogramming factors. * Transcription factors that have dominant roles in the control of specific cell states and that are capable of reprogramming cell states when ectopically expressed in various cell types.

Answer 49

Transport * Gated transport: * Transport between nucleus and cytosol through nuclear pore complexes (active transport and free diffusion). * Transmembrane transport: * Membrane protein translocators directly transport specific proteins from cytosol across an organelle membrane. * Vesicular transport: * Membrane-enclosed transport intermediates move proteins between various compartments via vesicles.

Answer 50

Protein Sorting Signals * Protein transfer/transport to various compartments guided by sorting signals.

Answer 51

Protein Sorting Signals * Stretch of amino acids, typically 15-60 residues long. * Localized on N or C terminus or within protein sequence. * Multiple scattered sequences in protein may form signal patch. * Signal sequences are both necessary and sufficient for protein targeting. * Physical properties of sequence (e.g., charge, hydrophobicity) more important than actual sequence. * If removed, the sorting signal can be removed by signal peptidase. * Signal sequences are recognized by complementary receptors.

Answer 52

Nuclear Pore Complexes * Perforate nuclear envelope in eukaryotic cells. * Molecular mass ~125 mill Da. * Composed of 30 different proteins or nucleoporins. * Arranged in octagonal symmetry with one or more aqueous pores. * Each nuclear envelope has 3000-4000 NPCs. * Transport molecules in both directions. * Passive diffusion of small molecules and facilitated transport. * Transport facilitated by binding of particles to fibrils extending from NPC.

Answer 53

Nuclear Transport * Gated, bidirectional, and selective. * Proteins needed in the nucleus are **imported** **from** **the** **cytoplasm** where they are synthesized. * Histones, DNA & RNA polymerases, topoisomerases, gene regulatory proteins. * tRNA and mRNA molecules synthesized in nucleus and **exported to cytosol.**

Answer 54

Nuclear Import * Nuclear import receptors (NIRs) recognize the nuclear localization sequence (NLS). * Each receptor recognizes a subset of cargo proteins. * NIRs are soluble cytosolic proteins that bind to NLS on protein and to NPC proteins present on fibrils that extend into cytoplasm. * NPC proteins have phenylalanine glycine (FG) repeats which serve as binding sites for import receptors. * Receptors plus cargo traverse NPC by binding, dissociating, and re-binding to adjacent FG repeats. * Cargo released inside nucleus and NIR return to cytoplasm.

Answer 55

Nuclear Export * Nuclear export works similar to import but in opposite direction. * Relies on nuclear export signals (NES) on molecules that need to go out of nucleus. * Need complementary nuclear export receptors (NER). * NER binds to cargo present in nucleus and NPC proteins. * Binding, dissociation and re-binding facilitates transport. * Cargo released into cytoplasm.

Answer 56

Nuclear Localization Signals * NLS are sorting signals that direct molecules to nucleus. * Short sequences rich in the positively charged AAs lysine and arginine. * Located on many different sites on protein. * Form loops or patches on surface. * Result in selective import of proteins into the nucleus. * Highly specific, changing one Lys to Thr prevents transport into nucleus.

Answer 57

Mitochondria - Signal Sequences * N terminal and internal signal sequences. * Signal sequence for matrix proteins is best understood. * Positively charge residues cluster on one end and uncharged hydrophobic on the other end to form amphiphilic alpha helix. * Specific receptor proteins recognize this **configuration** rather than precise sequence. * Multi-subunit protein complexes called protein translocators mediate translocation.

Answer 58

Mitochondria Translocators - TOM * Present in the outer membrane. * Required for import of all nuclear encoded proteins. * Inserts them in outer membrane. * 2 components: * Receptors for mitochondrial precursor proteins. * Translocation channels.

Answer 59

Mitochondria Translocators - TIM * Usually considered present in both inner and outer membrane, new evidence suggests only present in inner membrane. * 2 components: * Receptors for mitochondrial precursor proteins. * Translocation channels. * 2 TIM complexes: * TIM 22 * Mediates the insertion of a specific subclass of proteins (e.g., ATP, ADP and Pi transporter). * TIM 23 * Transports soluble proteins into matrix and helps insert membrane proteins in inner membrane.

Answer 60

Mitochondria Translocators - SAM * Translocates and inserts/folds beta barrel proteins in the outer membrane.

Answer 61

Mitochondria Translocators - OXA * Mediates insertion of proteins synthesized in mitochondria.

Answer 62

ER - Signal Sequence * ER signal sequences vary in AA sequence. * Have 8 or more non-polar AA at its center. * ER signal sequence guided to ER membrane by 2 components: * Signal recognition particle (SRP) * Rod shaped, made of 6 different polypeptides bound to a single small RNA molecule, with large hydrophobic pocket lined by methionines. * Cycles between ER membrane and cytosol and binds to ER signal sequence. * Pocket can accomodate hydrophobic signal sequences of different size, shape, and sequence. * SRP receptor.

Answer 63

ER - Co-Translational Translocation * SRP wraps around larger ribosomal subunit. * One end binds to ER signal sequence of emerging protein and other end to elongation factor binding site. * Transiently blocks protein synthesis giving protein time to enter the ER membrane. * SRP-ribosome complex binds to SRP receptor present in ER membrane. * Interaction brings the assembly to a translocator. * SRP and receptor released and protein translocated across the ER membrane into the lumen.

Answer 64

Mitochondria - Translocators * Translocase of the Outer Membrane (TOM) * Translocase of the Inner Membrane (TIM) * Sorting and Assembly Machinery (SAM) * OXA complex.

Answer 65

Vesicular Trasport * Proteins and other biomolecules are transported via transport vesicles. * Vesicles bud off from primary compartment and fuse with the next one. * Different shapes and sizes: small sperical, large irregular or tubular. * Contents of vesicle called cargo. * Transport is directional.

Answer 66

Vesicular Transport Systems * Biosynthetic-secretory. * Endocytic pathway. * Retrieval pathway.

Answer 67

Coated Vesicles - Types * COPI * COPII * Clathrin

Answer 68

Coated Vesicles - COPI * Mediates transport from Golgi cisternae.

Answer 69

Coated Vesicles - COPII * Mediates trasport from ER.

Answer 70

Coated Vesicles - Clathrin * Mediate transport from Golgi apparatus and from plasma membrane. * Each clathrin subunit: 3 large and 3 small polypeptide chains that form a 3-legged structure called triskelion. * Triskelions assemble into a basket-like structure of hexagons and pentagons that form coated pits on the cytosolic side of membrane. * Adaptor proteins form a second layer between the cage and membrane. * Trap various transmembrane proteins including receptors that capture soluble cargo inside vesicle.

Answer 71

Vesicle Targeting - Rab and SNARE * Specificity in targeting is acheived by surface markers on vesicles and complimentary receptors on target membrane. * Rab proteins direct vesicle to specific spots on target membrane. * SNARE proteins mediate fusion of vesicle with membrane.

Answer 72

Lysosomes * Membrane enclosed compartments filled with hydrolytic enzymes. * Heterogeneous - derived from late endosomes. * Important for intracellular digestion of macromolecules. * About 40 types of enzymes: proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases, and sulfatases. * Require acidic environment and proteolytic cleavage for optimal activation. * Lysosomal membrane protects cell against its enzymes. * Transporters in membrane pump out end products of digestion. * Vacuolar ATPase pumps H⁺ into lysosomes to maintain the acidic pH and to drive transport of small metabolites.

Answer 73

Lysosomes - Material Delivery * Endocytosis * Phagocytosis * Autophagy

Answer 74

Lysosome - Protein Sorting * Lysosomal hydrolases have the sorting signal mannose-6-phosphate (M6P) attached to them in the CGN. * M6P receptors in TGN recognize the sugar. * Receptors bind to hydrolases and to adaptor proteins in assembling clathrin coats. * Packaged into clathrin-coated vesicles that bud from TGN. * Contents delivered into endosomes and then to lysosomes.

Answer 75

Sub-Cellular Fractionation * Tissue: Mechanical blending * Homogenate: Suspension of different cell types. * Centrifugation to seperate different cell types, based on size & density. * Lysis of cells: osmotic shock, ultrasonic vibration, mechanical blending, forcing through small orifice. * Ultracentrifugation: separate organelles.

Answer 76

Column Chromatography * Separation of molecules by column chromatography. * A solution containing a mixture of different molecules, is applied to the top of a cylindrical glass or plastic column filled with a permeable solid matrix, such as cellulose. * Large amount of solvent is passed slowly through the column and collected in separate tubes as it emerges from the bottom. * Various components travel at different rates through the column and are fractionated into different tubes.

Answer 77

Matrices for column Chromatography * Ion-exchange chromatography * Insoluble matrix carries ionic charges that retard the movement of molecules of opposite charge. * Gel-filtration chromatography * Small beads that fill the matrix are inert but porous. * Molecules that are small enough to penetrate the beads are delayed and travel more slowly. * Affinity chromatography. * Insoluble matrix is covalently linked to specific ligand, such as an antibody or enzyme, that will bind to a specific protein. * Bound proteins are then eluted by dissociating the antibody-antigen complex with concentrated salt solutions or solutions of high or low pH.

Answer 78

Restriction Endonucleases * Enzymes isolated from bacteria that cut DNA at specific sites.

Answer 79

Ligase Reaction * "Glues' DNA ends together. * Much easier with compatible cohesive ends.

Answer 80

DNA Cloning in Bacteria * DNA fragment to be cloned is inserted in plasmid that has been engineered to serve varius functions i.e. carry and replicate manipulated gene products.

Answer 81

DNA Analysis * Agarose gel used instead of polyacrylamide gel. * DNA is already negatively charged, SDS not needed to add negative charge.

Answer 82

Cloning Vectors * Plasmids have been engineered to serve various functions i.e. carry and replicate manipulated gene products.

Answer 83

SNPs * You are 99.9% the same as the person next to you. * There are 3 million base pair differences between you and the person sitting next to you. * SNP variants can be neutral, pathogenic, or predisposing.

Answer 84

Embryo Development - Phases 1. Proliferation 2. Specialization 3. Interaction 4. Movement

Answer 85

Homologous Proteins * Basic machinery for development is similar for all organisms. * Homologous proteins are functionally interchangeable.

Answer 86

Gastrulation * Blastula consists of a sheet of epithelial cells facing the external medium. * This sheet gives rise to ***ectoderm***. * Ectoderm is precursor of nervous system and epidermis. * Part of epithelial sheet becomes tucked into the interior giving rise to ***endoderm.*** * Endoderm is the precursor of gut, lung and liver. * Group of cells move into the space between ectoderm and endoderm giving rise to ***mesoderm.*** * Mesoderm is precursor of muscles and connective tissue.

Answer 87

Development Program * Instructions for producing a multicellular animal is contained in non-coding regulatory DNA associated with each gene. * DNA contains regulatory elements that serve as binding sites for gene regulatory proteins. * Regulatory DNA defines the sequential program for development. * Coding sequences in DNA similar in most organisms but non-coding sequences make one organism different from another and provide uniqueness.

Answer 88

Development Decisions and Cell Fate * Cells make developmental decisions long before they show any outward signs of differentiation. * "Determined" - cells that are fated to develop into a specialized cell type despite changes in environment. * "Completely undetermined" - cells that can change rapidly due to alterations in environment. * "Committed" - cells that have some attributes of a particular cell type but can change with environment.

Answer 89

Inductive Signaling * Most important environmental cues are signals from neighboring cells. * Induction of a different developmental program in select cells in a homogenous group leading to altered character - inductive signaling. * Few cells closest to the source take on induced character - signal is limited in time and space. * Types of signals: * Short range: cell-cell contacts. * Long range: substances that can diffuse through the extracellular medium.

Answer 90

Morphogens * Morphogen - a long range inductive signal that imposes a pattern on a field of cells. * Exerts graded effects by forming gradients of different concentrations. * Each concentration can direct the target cells into a different developmental pathway. * Gradient formed by: * Localized production of an inducer that diffuses away from its source. * Localized production of an inhibitor that diffuses away from its source and blocks the action of a uniformly distributed inducer. * Morphogens need an 'on' and 'off' system. * Antagonists or extracellular inhibitors bind to the signal or its receptor and block interaction.

Answer 91

Gastrulation * Transformation of the blastula, a hollow sphere of cells, into a layered structure with a gut.

Answer 92

Phases of Neural Development * Phase 1 - different cell types (neurons, glial cells) develop independently at widely separate locations in embryo according to local program and are unconnected. * Phase 2 - axons and dendrites grow out along specific routes setting up a provisional but orderly network of connections between various parts of the nervous system. * Phase 3 - continues into adult life, connections are adjusted and refined through interactions with distant regions via electric signals.

Answer 93

Nervous System - Origin * Neurons are produced in association with glial cells (provide supporting framework and nutrition). * Both cell types develop from ectoderm from a common precursor. * CNS (brain, spinal cord, and retina) derived from neural tube. * PNS (nerves, sensory neurons) derived from neural crest.

Answer 94

Neuronal Migration - Molecular Mechanisms * A typical immature neuron has a cell body, a long axon and several short dendrites. * Axon and dendrites not distiguishable at first. * Tip of axon/dendrite has an irregular, spiky enlargement called growth cone. * Growth cone crawls through surrounding tissue, trailing the axon or dendrite behind. * Growth cone has the engine and steering apparatus that directs the process along the right path. * One of the growth cones starts migrating fast, becomes dominant, and develops axon-specific proteins - this will form axon.

Answer 95

Growth Cones * Growth cone behavior is dictated by its cytoskeletal machinery. * Growth cone throws out filopodia and lamelopodia. * Monomeric GTPases Rho and Rac control the assembly/disassembly of actin filaments, which control movement of growth cone. * Growth cones withdraw cells from unfavorable surfaces and steer them towards favorable ones where they persist for longer time.

Answer 96

Growth Cones - Migration * Growth cones travel towards target cells along predictable routes. * Exploit two major cues to find their way: * Extracellular matrix environment - sensed by receptors present on membrane. * Chemotactic factors - released by neighboring cells. Attractive or repulsive.

Answer 97

Mechanism of Commissural Neuron Guidance * First stage depends on secretion of netrin by cells of the floor plate. * Binding of netrin to its receptor causes opening of TRPC (Transient receptor potential C) channels. * Allow entry of extracellular Ca²⁺. * Leads to activation of machinery for extension of filopodia and movement of growth cone. * Non-commissural neurons in neural tube do not have netrin receptors, so do not migrate towards floor plate. * Midline cells secrete Slit. * Slit receptor Roundabout present on commissural neurons. * Slit repels growth cones and blocks entry to the midline. * Growth cones become sensitive to another repulsive signal called semaphorin. * Trapped between 2 sets of repellants, growth cones travel in a narrow track.

Answer 98

Activity-Dependent Synaptic Remodeling (Retinal/Tectal Neurons) * Each axon initially branches widely and makes multiple synapses with target cell. * Profusion of weak synapses. * Network subsequently trimmed by elimination of synapses and retraction of axon branches. * Accompanied by sprouting of axons to develop denser distribution of synapses that survive. * Synaptic remodeling dependent upon 2 rules that create spatial order: * Axons from cells in different regions of retina compete for tectal neurons. * Axons from neighboring sites which are excited at same time cooperate/collaborate to retain and strengthen synapses with tectal neurons. * Activity-dependent synaptic remodeling depends on electrical activity and synaptic signaling.

Answer 99

Growth Cone Migration - Chemotactic Factors * Secreted by cells, act as guidance factors at strategic points along path. * May be attractive or repulsive. * Examples: * Netrin * Slit * Semaphorin

Answer 100

Neurotrophic Factors * Axonal growth cones reach eventual target cells. * Halt, communicate and make synapses with target cells. * Signal from target tissue regulate which growth cones synapse and where.

Answer 101

Programmed Production of Cerebral Cortex Layers

Answer 102

Stem Cells - Characteristics * Not terminally differentiated. * Can divide without limit. * Ability to renew themselves. * Following division each new cell has ability to remain stem cell or become differentiated into a different cell type. * Undergo slow division.

Answer 103

Developmental Capacity - Totipotency * Ability of cell to give rise to all cells of an organism, including embryonic and extra-embryonic tissues. * Example: zygote

Answer 104

Developmental Capacity - Pluripotency * Ability of a cell to give rise to all cells of the embryo and subsequently adult tissues. * Example: embryonic stem cells.

Answer 105

Developmental Capacity - Multipotency * Ability of a cell to give rise to different cell types of a given lineage. * Example: adult stem cells.

Answer 106

Stem Cells - Maintenance * A steady pool of stem cell population maintained by: * Asymetric division - creates 2 cells, one with stem cell characteristics and another with the ability to differentiate. * Independent choice - Division makes 2 identical cells but the outcome is stochastic and/or influenced by environment.

Answer 107

Epidermis - Layers and Cells * Layers from deep to superficial: * Basal cell layer: * Attached to basal lamina and contains only dividing cells in epidermis. * Prickle cell layer: * Cells have numerous desmosomes that attach tufts of keratin filaments. * Granule cell layer: * Forms boundary between inner metabolically active strata and outer dead epidermis cells. * Squame layer: * Flattened dead cells, densely packed with keratin but no organelles.

Answer 108

Epidermis - Renewal * Continuous wear and tear needs constant repair. * Self-renewing process: * Basal cells divide with some maintaining the basal layer and others move to layers above. * Cells move through prickle cell layer, granule cell layer, etc. * Change in gene expression at each step of differentiation, acquiring phenotype appropriate for that layer. * Cells start undergoing partial degradation, losing nucleus and other organelles. * Depends on partial activation of apoptotic machinery. * Time from 'birth' of cell in basal layer to shedding from surface is 1 month.

Answer 109

Epidermal Stem Cells - Regulation * Regulation helps to control size of stem cell population. * ***Contact with basal lamina*** controls numbers of stem cells (***most important signal***). * Maintenance of contact preserves stem cell potential. * Loss of contact triggers terminal differentiation. * Stem cell markers not well known. * Proliferative potential of stem cells directly correlates with expression of beta-1 subunit of integrin (helps mediate adhesion to basal lamina). * Clusters of cells with high levels of integrin found near the basal lamina and in bulge of hair follicle.

Answer 110

Renewal of Epidermis - Governing Factors * Rate of stem cell division. * Probability that one daughter cell will remain a stem cell. * Rate of division of transit amplifying cells. * Timing of exit from basal layer and the time the cell takes to differentiate and be sloughed away from surface.

Answer 111

Olfactory Neurons * Humans ~40 million olfactory neurons. * Bipolar neurons with a dendrite facing the extracellular environment (interior space of the nasal cavity) and an axon that travels along the olfactory nerve to the olfactory bulb in the brain. * Many tiny hair-like cilia protrude from the dendrite. * Supporting cells present in between neurons - hold neurons in place and separate them from one another. * Basal cells in the epithelium - cells in contact with basal lamina. * Sensory surfaces of epithelium kept moist and protected by a layer of fluid (mucus).

Answer 112

Olfactory Receptors * The free surfaces of cilia have odorant receptor proteins (olfactory receptors). * A type of G protein coupled receptor. * Odorant receptor genes - 1000 in dog, 350 in humans. * Each neuron expresses only one of these genes enabling the cell to respond to only one class of odorant (organic smell molecules). * Structural features of odorant recognized by the receptor. * All olfactory neurons respond by a common mechanism. * A given olfactory receptor can bind to a single class of odor molecules (may include a variety of odor molecules). * Affinity variable depending on how well the ligand binds to the receptor. * Activated olfactory receptor in turn activates an intracellular G-protein (G_olf). * Activates adenylate cyclase in the plasma membrane which results in an influx of sodium and calcium into the cell. * This influx of positive ions causes the neuron to depolarize, generating an action potential.

Answer 113

Olfactory Receptors - Function * Action potentials relayed via the axon to the brain. * Relay stations in brain called glomeruli. * Located in olfactory bulbs one on each side of the brain. * 1800 glomeruli/bulb in mouse brain. * Although olfactory neurons expressing the same odorant receptor are located in different places on the olfactory epithelium, their axons converge on the same glomerulus.

Answer 114

Embryonic Stem Cells * Pluripotency well established. * Good growth properties in cell culture (in vitro). * Have ability to differentiate into a wide range of cell types if provided the right set of culture conditions. * Develop into different cell types with characteristics appropriate for that site (even germ cells). * Capable of proliferating indefinitely in culture with unrestricted developmental potential. * Unlike the zygote, ES cells are incapable of generating a full organism.

Answer 115

Embryonic Stem Cells - Derivation * Derived from the blastocyst stage of embryo. * When put back in blastocyst they can integrate well with the embryo.

Answer 116

Somatic Cell Nuclear Transfer (SCeNT) * Combines cloning methods with embryonic stem cell technology to produce cells which are custom-made for patient. * Steps: * Nucleus taken from somatic cell of patient and injected into oocyte of a donor replacing the oocyte nucleus. * Blastocyst generated from this hybrid oocyte and ES cells isolated.

Answer 117

Induced Pluripotent Stem Cells (iPSC) * Somatic cells can be reprogrammed to form iPSC by exposing them to defined, limited sets of transcription factors (genes for stem-ness). * Reprogramming adult cells: * Better if adult cells could be converted into ES-like cells by manipulating gene expression directly. * Biochemical comparisons of ES cells with other cell types suggest some candidate genes. * Key determinants of ES cell character are a set of 4 gene regulatory proteins: ***_Oct4, Sox2, Klf4, and Myc._*** * When injected into fibroblasts they form ES-like cells including the ability to differentiate into other cell types. * Yield low.

Answer 118

Olfactory Neurons - Regeneration * Individual olfactory neurons survive for only a month. * Neural stem cells residing among the basal cells in the olfactory epithelium generate replacements for the lost neurons. * Basal stem cells in contact with basal lamina divide and differentiate into olfactory neurons. * Odorant receptor proteins help in axonal guidance and allow the growth cone to migrate to and establish connection with correct glomerulus in olfactory bulb. * Regeneration of olfactory receptor cells is one of the only few instances of adult neurogenesis in the CNS. * Has raised considerable interest in dissecting the pathways for neural development and differentiation in adult organisms.

Answer 119

Human Embryonic Stem Cells - Alternative Approaches to Obtaining 1. Somatic Cell Nuclear Transfer (SCeNT) 2. Induced Pluripotent Stem Cells (iPSC)

Answer 120

Signal Transduction * Extracellular signaling molecules bind to receptors in target cells to initiate a chain of events. * Cell-to-cell communication. * Must traverse from outside of cell to inside of cell. * Important in development of organism and coordination of metabolism. * Required for multi-cellular organisms.

Answer 121

Signal Transduction * Extracellular signaling molecules bind to receptors in target cells to initiate a chain of events referred to as signal transduction. * Induce 2 types of response: * Change in activity or function of enzymes or proteins in cell (FAST RESPONSE). * Change in amounts of proteins by change in expression of genes (SLOW RESPONSE).

Answer 122

Cell Communication - Endocrine Signaling * Long distance signaling. * Freely diffusible signals. * Long lasting (long half-life) - takes time to find target through circulatory system.

Answer 123

Cell Communication - Paracrine Signaling * Acts locally on cells nearby. * Not as freely diffusible. * Short-lived. * Example: * NO - produces cGMP (a 2nd messenger), a small molecule can interact with other proteins and regulate activity. * Influences muscle relaxation in circulatory system.

Answer 124

Cell Communication - Synaptic Signaling * Acts locally. * Short-lived. * Uses neurotranmitters.

Answer 125

Cell Communication - Autocrine Signaling * Cells respond to their own signals or those released by same type of cell. * Signals cell growth, division, maturation. * e.g. growth factors in CA cells.

Answer 126

Cell Communication - Direct Cell Signaling * e.g. immune cells. * Ag-presenting cells to T cells.

Answer 127

Signal Transduction * Cytoskeletal changes. * Membrane changes. * Protein translation changes. * Cell cycle changes.

Answer 128

Cell Communication - G-Protein * Heterotrimeric G proteins are guanine nucleotide-binding proteins that assist in transmitting signals to intracellular targets. * Consist of 3 subunits - alpha, beta, gamma. * Receives input from receptor and impacts effector.

Answer 129

Cell Communication - GPCR Effector * Generates cAMP, an important second messenger that mediates cellular responses to a variety of ligands.

Answer 130

Cell Communication - GPCR Signal Sequence * Ligand binds to receptor. * Conformational change occurs in receptor. * Receptor binds to G protein. * Receptor acts as a GEF. * Conformation of G-alpha is changed, GDP released and GTP binds. * G-alpha becomes active and can bind to and activate effector. * Effector molecule then creates second messenger (which in this case adenylyl cyclase catalyzes cAMP). * Eventually, GTP on G-alpha is hydrolyzed to GDP (occurs after certain amount of time). * G-alpha returns to inactive step to be recycled through process again.

Answer 131

Cholera * Modifies G protein by keeping G-alpha in the active form indefinitely. * Makes pathway always active. * Pump Cl- and water out of cell in intestine and causes severe diarrhea.

Answer 132

Cell Communication - cAMP * Activates a cAMP-dependent protein kinase (PKA), which phosphorylates proteins, either activating or inactivating them. * PKA contains 4 subunits, 2 catalytic and 2 regulatory. * cAmp molecules bind to the regulatory subunits. * Releases active C-subunits. * Can also activate cAMP-gated cation channels.

Answer 133

Cell Communication - Receptor Tyrosine Kinases (RTKs) * Enzyme linked receptors. * Enzyme domain is in the cytoplasmic tail of the integral membrane protein. * Respond to growth factors and mediate signals. * 3 domains: * Extracellular * Transmembrane * Cytoplasmic (tyrosine kinase domain)

Answer 134

Cell Communication - RTK Function * Growth factor binds to extracellular domain. * Conformational change. * Dimerization of 2 receptors. * Autophosphorylation of tyrosines on cytoplasmic domain. * Cytoplasmic domain acts as scaffold and recruits other proteins. * Binds to proteins with SH2 (src homology) domain. * i.e. Grb2 (also has SH3 domain) * SH3 binds to prolines in son of sevenless (SOS) * SOS (a GEF) binds to Ras (small monomeric G protein - small GTPase) * Cascade: * Ras -\> MKKK (Raf) -\> MKK (Mek) -\> MK (Erk) -\> nucleus -\>increase gene transcription * Uncontrolled gene transcription = CA

Answer 135

Cell Communication - JAK-STAT Receptor * Ligand binds to Janus kinases (JAKs). * JAKs dimerize and phosphorylate each other. * Receptor binds and phosphorylates Signal Transducer and Activators of Transcription proteins (STATs). * STATs separate from receptor, dimerize, enter nucleus, and bind to DNA causing transcription. * Example: erythropoeitin

Answer 136

Cell Communication - G-Protein-Coupled Receptor * Composed of 3 parts: * Extracellular domain - binds ligand. * Transmembrane domain - anchors receptor. * Cytoplasmic domain - associated with G-protein. * 3 subunits: alpha, beta, gamma. * AKA 7 transmembrane receptor. * Affect olfaction, sight, taste.

Answer 137

Cell Communication - Adenylyl Cyclase * Generates cAMP as secondary signal that goes on to interact with its target proteins to cause a biological response.

Answer 138

Cell Communication - PKA * Regulates proteins by phosphorylating them. * Phosphorylation can: * Change conformation of protein by the addition of 2 negative charges. * Form part of a structure that other proteins recognize. * Cause activation or inactivation. * Cause alteration of intracellular localization of target proteins. * Cause alterations in abundance of target proteins.

Answer 139

Cell Communication - JAK-STAT * More direct route for impacting transcription.

Answer 140

Cell Communication - Serine-Threonine Receptor * Ligand binds to receptors (Type I and Type II). * Type II phosphorylates sites on Type I. * Type I then phosphorylates R-Smads (receptor specific Smad such as Smad1 and Smad2). * R-smad complexes with Co-Smad and migrates to nucleus. * Example: iron metabolism - hepcidin hormone.

Answer 141

cAMP Targets * cAMP interacts with it’s target proteins to cause a biological response. * cAMP activates cAMP-dependent protein kinase (PKA) - 4 subunits. * Inactive PKA: 2 catalytic subunits & 2 regulatory subunits. * Binding of 2 cAMP molecules to regulatory subunits of tetramer → release of active C subunits.

Answer 142

Errant Signaling * A point mutation in codon 12 results in a change from Gly to Val and constitutively activates Ras (Ras-G12V). * Mutations that result in constitutively activate Ras are frequently found in human cancer.

Answer 143

Cell - Structure and Function * All cells contain the same genetic material. * Differentiation depends on differences of gene expression.

Answer 144

Different Cell Types - Different Proteins 1. Common proteins (housekeeping proteins), i.e. glucose metabolism. 2. Specifically limited proteins, i.e. hemoglobin. 3. Typical human cell expresses 30-60% of its 21,000 structural genes (protein coding) but level of gene expression varies - fingerprint expression profiles e.g. microarrays or RNA Seq. 4. Other factors post transcription include: alternative splicing (dystrophin gene), post translational modification.

Answer 145

Gene Expression - Locations of Control 1. Transcriptional control 2. RNA processing control 3. RNA transport and localization control 4. Translation control 5. mRNA degradation control 6. Protein activity control

Answer 146

DNA Motif - Recognition * Regulatory proteins associate with, recognize specific DNA sequence, and bind to bases in the major groove. * Major groove presents a specific face for each of the specific base pairs. * Surface of protein is extensively complementary to the surface of the DNA region to which it binds. * Contacts made with the DNA involve 4 possible configurations: * Possible H-bond donors. * Possible H-bond acceptors. * Methyl groups. * H atom.

Answer 147

DNA Binding Motifs * Gene regulation requires: * Short stretches of DNA of defined sequence - recognition sites for DNA binding proteins. * Gene regulatory proteins - transcription factors that will bind and activate gene. * Recognition sequences for regulatory proteins e.g. GATA1: TGATAG * Logo: ex. TAATTGC * Recognition sequences can be proximal or distal to first exon (few base pairs - 50kb away).

Answer 148

DNA-Binding Motifs - Helix-Turn-Helix * Most simple and common DNA-binding motif. * 2 alpa-helices connected by short chain of AA that make the "turn" (turned at fixed angle). * Longer helix = recognition module - DNA-binding module - fits in major groove. * Side chains of AA recognize DNA motif. * Symmetric dimers: bind DNA as dimers.

Answer 149

DNA-Binding Motifs - Zinc Finger Module * DNA-binding motif includes Zn atom. * Binds to major groove. * Zn finger domains found in tandem clusters. * Multiple contact points.

Answer 150

DNA-Binding Motif - Leucine Zipper * 2 motifs: * Type I: * 2 alpha helical DNA binding domain. * Grabs DNA like clothes pin. * Activation domain overlaps dimer domain. * Interactions between hydrophobic AA side chains (leucines) * Type II: * Dimerizes through leucine zipper region (homo- / hetero-). * Interactions between hydrophobic AA side chains (leucines). * Leucine residue every 7 AA down one side of alpha-helix in dimerization domain: forms zipper structure.

Answer 151

DNA-Binding Motifs - Helix-Loop-Helix * Consists of a short and a long alpha-chain connected by a loop. * Can occur as homodimers or heterodimers. * Three domains or modules to this protein: * DNA binding * Dimerization * Activation

Answer 152

Transcriptional Activation * DNA looping and a mediator complex allow the gene regulatory proteins to interact with the proteins that assemble at the promoter. * Mediator serves as an intermediary between gene regulatory proteins and RNA polymerase II.

Answer 153

Post-Transcriptional Regulation of Gene Expression - RNA Stability * Most mRNAs have 1/2-life 30 mins (Globin 1/2-life is 10 hours). * Poly-A tail confers stability and gradual shortening by an exonuclease acts as a timer. * Once reduced to 25 nt, 2 pathways converge leading to degradation. 1. Decapping - 5' cap removed and exposed mRNA degraded from 5' end. 2. mRNA degraded from 3' end through poly-A tail and into coding region.

Answer 154

Post-Transcriptional Regulation of Gene Expression - Protein Activity Control * Post translational modifications required by proteins to be functional. * Phosphorylated * Glycosylated * Bind to other subunits or partners. * Modified by enzymes (i.e. thrombin cuts fibrinogen to form fibrin). * Proteins must fold into their 3-D conformations. * Molecular chaperones help proteins fold. * Heat shock proteins (Hsp60 & Hsp70) * Bind co-factors.

Answer 155

Post-Transcriptional Regulation of Gene Expression - Protein Degradation * Ubiquitin identifies unfolded or abnormal proteins for destruction. * Ubiquitin binding process: * E1 ubiquitin activating enzyme links ubiquitin to cysteine side-chain. * Transfered to E2 ubiquitin conjugating enzyme (with accessory protein E3 ubiquitin ligase). * E3 activated by phosphorylation, ligand binding, or protein subunit addition. * Transferred to lysine side chain of proteins with degradation signal. * Degradation signal activated by phosphorylation, unmasking of signal by protein dissociation, or creation of destabilizing N-terminus. * Ubiquitin chain is recognized by proteasome. * Aberrant protein destroyed.

Answer 156

Cell Cycle - 4 Phases * S phase - DNA synthesis * M phase - separate chromosomes and divide cells. * GAP phases - allow more time for growth: * G₁ - between M & S * G₂ - between S & M

Answer 157

Cell Cycle - Transition Check Points * Checkpoint I: START - cell commits to cell cycle entry and chromosome duplication (also called restriction point). * If conditions are not conducive to mitosis, cell cycle will not proceed. * Once started, cells will continue cell cycle even if conditions change. * Checkpoint II: G₂/M - chromosome alignment on spindle in metaphase. * Checkpoint III: metaphase-to-anaphase transition - trigger sister chromatid separation and cytokinesis.

Answer 158

Cell Cycle - Cdks * Cyclin dependent kinases (Cdks) govern cell cycle, ***_phosphorylate proteins downstream to activate them and regulate cell cycle events._*** * Causes cyclical changes in phosphorylation of substrates that regulate cell cycle events. * Activities of Cdks rise and fall during cell cycle. * Levels of Cdks remains constant.

Answer 159

Cell Cycle - Cyclins * Proteins that regulate Cdks. * Levels vary and cycle during the cell cycle (hence name 'cyclins'). * Expression controls what step of the cell cycle the cell is in (cyclin present -\> cyclin-Cdk complexes formed triggering cell cycle events). * Cdks are dependent on cyclins - must be bound to cyclin to have protein kinase activity. * Direct Cdks to their specific target.

Answer 160

Cyclins - 4 Classes 1. G₁/S cyclins: * Start cell cycle * Activates Cdks in late G₁, helping trigger progression through START. * Levels drop in S phase. 2. S cyclins: * Stimulates DNA duplication by binding Cdks after progression through START. * Levels remain high until mitosis. 3. M cyclins: * Initiate mitosis by binding to Cdks that stimulate entry into mitosis at G₂/M checkpoint. * Removed at mid-mitosis. 4. G₁ cyclins: * Govern activity of G₁/S cyclins, controlling progression through START checkpoint.

Answer 161

Cdk - 4 Types * 4 different Cdks that form cyclin-Cdk complexes: * G₁/S-Cdk * S-Cdk * M-Cdk * G₁-Cdk

Answer 162

Cyclin-Cdk Complex - Formation & Activation * Cdk is inactive without cyclin being bound. The active site is blocked by a region of the protein called the T-loop. * Binding of cyclin causes T-loop to move out of active site, partially activating Cdk. * Phosphorylation of Cdk at T-loop, by Cdk activating kinase (CAK), fully activates enzyme ("cave site").

Answer 163

Cdk - Regulation by Inhibitor * Wee1 - a kinase inhibits M-Cdk by phosphorylating the "roof" site, inhibiting the M-Cdk complex (ensures lots of primed M-Cdk by end of G₂). * Cdc25 - a phosphatase that dephosphorylates the "roof" site of M-Cdk, reactivating the M-Cdk complex to initiate mitosis. * Double-circuit positive feedback ensures fast M-Cdk activation. * Activated M-Cdk activates more Cdc25. * Cdc25 inhibits Wee1. * p27 - a Cdk Inhibitory Protein (CKI) that binds to both Cdk and cyclin to inactivate complex. * Primarily used for control of G₁/S-Cdks & S-Cdks early in cell cycle.

Answer 164

APC/C - Transition from Metaphase to Anaphase * APC/C is a ubiquitin ligase that initiates progression from metaphase to anaphase in mitosis. * Levels rise in mid-mitosis. * Adds ubiquitin to: * Securin - protects cohesin protein linkages that hold sister chromatid pairs together in early mitosis by inhibiting separase. * Cyclins * Activated by Cdc20. * Targets S-cyclins and M-cyclins. * Leads to addition of polyubiquitin to M-cyclin in M-Cdk complex. * Cyclins destroyed, Cdks dephosphorylated. * In anaphase S-cyclins destroyed.

Answer 165

DNA-Binding Domain Structural Motifs * Helix-turn-helix * Zinc finger motif * Leucine zipper * Helix-loop-helix * Homeodomain * Beta-sheet

Answer 166

Apoptosis * Programmed cell death, or cell death under physiological conditions. * Used to eliminate unwanted cells. * Important for removal of abnormal, non-functional, potentially dangerous cells. * Lymphocytes after destroying and ingesting microbes. * Cells with DNA damage that cannot be repaired. * Used to maintain correct organ size (cell division = cell death). * Clean form of cell death: * Tightly controlled and regulated. * No spread of damage or initiation of inflammatory process. * Most common cell death.

Answer 167

Apoptosis - Critical for Development * Removes unwanted cells. * Sculpts hands, feet, etc, during embryonic development. * Examples: * Tail of tadpole. * Development of mouse paw.

Answer 168

Apoptosis - Caspases * Caspase = cysteine aspartyl specific protease. * Cysteine in active site. * Cleaves proteins at aspartic AA residues. * Proteases that mediate intracellular proteolytic cascade of apoptosis. * Activation of caspases is key event. * 2 Classes: * Initiater caspases - activates multiple procaspases into executioner caspases. * Executioner caspsases - destroys actual targets: * Cleaves downstream proteins. * Cleaves inactive endonuclease. * Targets cytoskeleton. * Attacks cell adhesion proteins * Cells roll up into ball.

Answer 169

Apoptosis - Procaspases * Initial, inactive, precursor of caspases. * Activated by protease cleavage. * Procaspases cleaved at specific sites to form a large and small subunit which form a heterodimer.

Answer 170

Apoptosis - Intrinsic Pathway * Cells activate apoptosis from inside cell in response to injury, DNA damage and lack of oxygen, nutrients, or extracellular survival signals. 1. Translocation of cytochrome c from the intermediate space of mitochondria is key event. 2. Released into cytosol and will bind to Apaf1 (procaspase-activating adaptor protein). 3. Apaf1 forms apoptosome which activates caspase-9. 4. Caspase-9 activates downstream exectutioner caspases - caspase-3 (common to both pathways).

Answer 171

Bcl2 Family of Proteins * Regulate intrinsic pathway. * Bcl2 controls release of cytochrome c into cytosol (Bcl = B cell lymphoma) * 2 types: * Anti-apoptic (pro-survival) - blocks release of cytochrome c (example: Bcl2) * Pro-apoptotic - promotes release of cytochrome c. * 4 distinct Bcl homology domains (BH1-4) * Proapoptotic proteins include BH-123 or BH-3 only.

Answer 172

Apoptosis - BH123 * Pro-apoptotic - triggers the intrinsic pathway. * Become activated. * Form aggregation in mitochondrial outer membrane. * Induce release of cytochrome c. * Apoptosome formed by binding to Apaf1

Answer 173

Apoptosis - Bcl2 Proteins * Anti-apoptotic proteins. * Includes Bcl2 and Bcl-XL (Both BH1234) * Mainly located on cytosolic surface of outer mitochondrial membrane. * Prevent apoptosis by binding to pro-apoptotic proteins (e.g. BH123) and prevent aggregation into active form. * Can be inhibited by BH3-only protein.

Answer 174

Apoptosis - BH3-Only Protein * Pro-apoptotic. * Activated BH3-only protein is cytosolic. * Translocates to mitochondria after apoptotic signal activates it. * Inhibits anti-apoptotic Bcl2 protein from inhibiting aggregation of BH123, to release cytochrome c.

Answer 175

Inhibitors of Apoptosis (IAPs) * Bind and inhibit caspases. * Some IAPs add ubiquitin to caspases to mark them for destruction by proteasome. * IAPs block apoptosis by binding to caspases.

Answer 176

Anti-IAPs * Pro-apoptotic * If there is apoptotic stimuli or apoptosis signals, this triggers the release of anti-IAPs from mitochondria to block the activity of IAPs. * Allowing executioner caspases to be activated.

Answer 177

Other Controls of Gene Expression * Coordinated expression of genes. * Decision for specialization. * Methylation and genomic imprinting (which genes get expressed or repressed from mom and dad). * X-chromosome inactivation.

Answer 178

Condensin * Five subunit protein complex. * Related to cohesin. * Contains 2 SMC subunits (structural maintenance of chromosomes). * Contains 3 non-SMC subunits. * Forms a ring-like structure and uses ATP to promote compaction and resolution of sister chromatids.

Answer 179

Apoptosis - Phenotype * Overall shrinkage of cell volume and nucleus. * Loss of adhesion to neighboring cells. * Formation of blebs on surface. * DNA fragmentation. * Cytoskeleton collapses. * Nuclear envelope disassembles. * Rapid engulfment of dying cell by phagocytosis.

Answer 180

Apoptosis - Biochemical Cahracteristics * DNA fragmentation visible on agarose gel. * Endonucleases cleave DNA in linker regions of nucleosomes. * Cytochrome C released from mitochondria is a marker of apoptosis.

Answer 181

Apoptosis - Caspase Cascade * Machinery always in place. * Irreversible. * 2 pathways: * Internal: * Stimuli - DNA abnormalities. * Mitochondrial dependent. * External: * Stimuli - removal of survival factors and proteins of tumor necrosis factor family. * Mitochondrial independent.

Answer 182

Cancer * It takes time to accumulate the mutations necessary for cancer to develop. * Graph: Incidence colon cancer vs. age.

Answer 183

Cancer - Genetic Causes * Two broad types of mutations in cancer. * Mutations of genes that regulate cell proliferation. * Overactivity mutations: gain of function – oncogenes – involves single mutation event and activation of gene causing proliferation (dominant) “The Gas Pedal.” * Underactivity mutations: loss of function – tumor suppressor genes - involve genes that inhibit growth. Mutation event: one gene – no effect; second mutation causes problems (recessive) “The Brake Pedal.”

Answer 184

Activation of Oncogenes * These mutations are dominant, only one allele needs to be present for activity.

Answer 185

Tumor Suppressor Genes * Tumor suppressor genes generally encode proteins that inhibit cell proliferation. * 2 major categories of tumor suppressor genes: 1. Proteins that normally restrict cell growth and proliferation. 2. Proteins that maintain integrity of the genome

Answer 186

Retinoblastoma * Two forms of retinoblastoma: hereditary form and sporadic form. * 40% of retinoblastoma is familial in which both eyes are affected (tumors). * 60% of retinoblastoma is sporadic (no family history) – single tumor one eye.

Answer 187

Rb - Hereditary Form * Loss of function or deletion of one copy of Rb in every cell - because defect is inherited. * These cells are predisposed to be cancerous. * But have one good copy of Rb gene. * Somatic event occurs – eliminates one good copy and tumor forms. * Loss of heterozygosity.

Answer 188

Rb - Sporadic Form * Non-hereditary * Non-cancerous cells fine – no mutation of Rb. * Cancerous cells have both copies of Rb mutated. * Two hit hypothesis – first Rb gene obtains mutation then need second mutation Rb. * Familial: already have one mutation – predisposed to cancer. * Sporadic – 2 normal Rb genes, one hit then 2nd hit so more rare than hereditary form. * Rb protein regulator of cell cycle.

Answer 189

Tumor Suppressor Genes - Inactivation or Loss * These mutations are recessive, both copies of the gene (alleles) need to be inactive/lost.

Answer 190

Tumor Progression - Polyp * Polyp is precursor of colorectal cancer. * Slow disease progression: 10 years. * Cut off polyp – cure. * If left alone, malignant tumor develops from adenoma (polyp).

Answer 191

Colorectal Cancer - Common Mutations * Important loss is Apc mutation: loss of function – Apc is a tumor suppressor gene. * 40% of colorectal cancers have point mutation in K-Ras. * 60% inactivating mutation of p53.

Answer 192

Cytoskeleton - Functions * Represents bones of the cell. * Important in organization of cell. * Maintains correctly shaped cells. * Insures cells are properly structured internally. * Moves cell. * Re-arranges the cellular compartment. * Supports plasma membrane. * Provides the mechanical strength - resistance to the stress without being ripped apart. * Pulls chromosomes apart during cell division. * Defect can lead to chromosome # abnormality. * Splits dividing cells during cell division. * Guides intracellular traffic of organelles. * Vesicles move around by using cytoskeleton as a sidewalk. * Cells like sperm need to swim - cytoskeleton acts as motor. * WBCs and fibroblasts need to crawl. * Muscle cell contraction.

Answer 193

Disease occurs.

Answer 194

RBC - Cytoskeleton Function * Responsible for shape of cell (biconcavity). * Gives strength to the cells. * RBCs once released from bone marrow, go through 1/2 mil passages in the circulation. * Must be flexible and strong enough to squeeze through capillary passages. * Must resist shear forces of heart contraction. * If defective, leads to Hereditary Spherocytosis. * RBCs spherical not bi-concave. * Fragile * Causes hemolytic anemia which can be severe and lethal.

Answer 195

Cytoskeleton - Types * Actin filaments.(like Mardi-Gras beads) * Microtubules (slinky of life) * Intermediate filaments (like girders in building)

Answer 196

Cytoskeletal Filaments - Actin Filaments * Two stranded helical polymers of the protein actin. * Subunits are compact, globular and self-associate using a combination of end-to-end and side-to-side interactions. * Flexible structures 5-9 nm in diameter. * Determine shape of cell's surface and are necessary for whole-cell locomotion, secretion, endocytosis. * Can form cell surface projections that help move cells over solid substrate: * Lamellipodia - flat protrusive veils. * Filopodia or microvilli, spiky bundles. * Catalyzed by two types of regulated factors: * ARP complex * Formins

Answer 197

Cytoskeletal Filaments - Microtubule * Forms long, hollow, tube-like structure (25nm). * Made of tubulin subunits that are compact and globular. * One end attached to a single microtubule-organizing center called a centrosome. * Determine the positions of membrane-enclosed organelles. * Directs intracellular transport. * Make up centrioles and mitotic spindle. * Found in cilia and flagella. * Lack of function in cilia = cystic fibrosis.

Answer 198

Cytoskeletal Filaments - Intermediate Filaments * Rope-like fibers (10nm) * Formed by smaller elongated, fibrous subunits from large heterogeneous family. * Extend across cytoplasm to provide mechanical strength. * Span from one cell-cell junction to another to strengthen the epithelial sheet. * Staggered side to side binding of filaments, forming rope-like structures, allowing filaments to tolerate bending and stretching * Allows formation of hair and fingernails.

Answer 199

Actin - Dynamic and Adaptable

Answer 200

Microtubule - Dynamic and Adaptable

Answer 201

Actin - Polymerization * Actin monomer contains a binding site for ATP or ADP. * ATP-actin polymerizes * ADP-actin depolymerizes * Actin filaments consist of two protofilaments that twist around each other in a right hand helix. * Held together by lateral contacts. * Arranged head-to-tail to generate structural polarity (plus and minus ends). * Plus end - fast-growing or fast-shrinking * Minus end - slow-growing or slow-shrinking. * Flexible and easily bent.

Answer 202

Microtubules - Polymerization * Tubulin is a hetero-dimer of alpha and beta tubulin with non-covalent bonds. * Both have binding site for GTP. * Tubulin subunits form helical assemblies. * Self-associate, using a combination of end-to-end and side-to-side interactions. * Form stiff, hollow, tube made of 13 proto-filaments aligned parallel with alternating alpha-beta subunits. * Tubulin is polar: * Plus end - fast growing and shrinking. * Minus end - slow growing and shrinking.

Answer 203

Nucleation - usually requires random collision of 3 subunits.

Answer 204

Accessory Proteins - Spectrin * Attaches to membrane. * Provides durability and stability in RBCs. * RBCs circulate 1/2 million times. * Tight capillary spaces. * If defective, fragile RBCs result causing hemolytic anemia (Hereditary Spherocytosis).

Answer 205

Hereditary Spherocytosis * Hemolytic anemia characterized by spherical and fragile RBCs that lyse and release Hgb. * Most common hemolytic anemia in people of North European Descent (incidence 1/2000). * Clinical Presentation: hemolysis, anemia, splenomegaly. * Ranges from mild to severe anemia and can be fatal. * Caused by mutations in genes for the erythrocyte membrane skeleton of RBCs.

Answer 206

Hereditary Spherocytosis - Cause * Erythrocyte membrane skeleton confers property of durability and stability to RBCs. * Failure of the EMS leads to mis-shapen and fragile RBCs. * EMS is dependent upon: * Ankyrin - attaches spectrin to Band 3 in PM. * Spectrin - filaments of the EMS. * Band 3 - membrane bound protein that is bound by ankyrin which also binds spectrin. * Band 4.2 * Deficiency in any of these proteins can cause fragility.

Answer 207

Spectrin - Architecture

Answer 208

Hereditary Spherocytosis - Testing * RBC EMS Protein Gel * Osmotic fragility test

Answer 209

Osmotic Fragility Test * A few drops of blood placed into slightly hypotonic saline. * Water rushes in by osmosis. * Normal cells will swell but not break. * HS cells will swell and, because they are fragile, will break releasing Hgb. * Can also be done using increasingly hypotonic solution to develop osmotic fragility curve.

Answer 210

Actin Filament - Life-Span * Intestinal cells - must last a few days, actin filaments are replaced every 48 hours. * Actin bundles in hair cells of inner ear must last a lifetime.

Answer 211

Listeria * Pathogenic bacteria that invade your intestinal cells. * Ubiquitous in soil. * Found on: * Unwashed lettuce, animal products, dairy and meats. * Worst case 1988 outbreak from hotdogs.

Answer 212

Listeria - Infection * Causes serious infection. * Symptoms: * HA * Stiff neck * Confusion * Loss of balance * Convulsions * Fever and muscle aches * Treatment requires antibiotics. * Attaches to receptors on enterocytes. * Enters and replicates in intestinal cells. * Moves using actin and accessory proteins such as Arp2/3. * moves at approx. 0.2 micrometers per second. * Leaves actin track (comet trails) * Bacterial surface causes local nucleation of actin filaments by presenting ActA (activating factor). * Cofilin makes branched actin disassemble. * Causes problems by smashing through organelles.

Answer 213

Listeria - Infection * More likely to develop food poisoning if immunologically impaired or immunocompromised. * Pregnant women 10x more likely to get Listeria infection. * About 1600 Listeria infections in US annualy. * Most require hospitalization. * 1 in 5 die. * Infection in pregnancy can cause miscarriage, stillbirth, or newborn death. * Fetal loss - 20% * Newborn death - 3%

Answer 214

EMS Mutations in HS * Spectrin/Ankyrin defect - 63% * Band 3 defect - 22% * Protein 4.2 defect - 3% * Other defects - 2% * No known defect - 10%

Answer 215

Muscular Dystrophy * Duchenne - complete absence of cytoskeletal protein dystrophin. * Becker - dystrophin protein made but abnormal.

Answer 216

Duchenne Muscular Dystrophy * Most common fatal neuromuscular disorder. * Severe, progressive muscle degeneration. * Loss of ability to walk - in wheelchair by 12 years of age. * Loss of lung and cardiac function. * Scoliosis * Death 20-30 years of age from respiratory failure or cardiomyopathy. * No cure.

Answer 217

Duchenne Muscular Dystrophy - Treatment * No medical treatment that can significantly alter course of disease. * Maintain patient's general health. * Improve quality of life. * Gluccocorticoids (prednisone) slow the decline in muscle strength but the effect is relatively short (18-36 months) and do not alter the overall clinical course of disease.

Answer 218

Duchenne Muscular Dystrophy - Outcome * Relentless * Only supportive measures available: * Physiotherapy * Physical aids: wheelchair dependent at 12. * Respiratory assistance. * Many patients now live into 20's. * Abnormal hearts and/or respiratory failure causes death.

Answer 219

Duchenne Muscular Dystrophy - Genetics * One of the most common neuromuscular genetic diseases in man (1/3500 male births) * X-linked recessive. * Caused by dystrophin gene mutations. * Largest gene known: produces 427 kDa protein; 14 kb RNA; 79 exons. * Genetic defect present at birth but does not show symptoms until about 3 years of age (most DMD boys not recognized at birth).

Answer 220

Dystrophin Protein * Main function is to provide structural stability to the muscle cell membrane during cycles of contraction and relaxation. * Localized to inner surface of muscle membrane. * Loss results in destabilization of entire dystrophin-glycoprotein complex. * 4 functional domains: * N-terminus * Long spectrin-like repeat domain (the cytoskeletal portion) * Cysteine rich and C-terminus domains: * Bind to syntrophin proteins (linking proteins) * Bind to dystroglycans/sarcoglycans (dystrophin-associated glycoprotein complex) - transmembrane proteins

Answer 221

DMD - Clinical Presentation * Dystrophic myopathy = progressive muscle degeneration with loss of functional muscle tissue over time with resulting weakness. * Onset early in childhood. * Mean age of Dx: 4y 10m. * Wheelchair by 12-13y. * Slow walking & generalized weakness. * Elevated CK in blood (50-100x normal, greater than 1,000 U/L) * Necrosis of muscle fibers occurs with replacement of fat or connective tissue. * Leads to pseudohypertrophy: replacement of muscle with adipose and fibrous CT (example: enlarged calves). * Changes in stature/movement: * Waddling run/walk. * Difficulty in climbing steps. * Use of handrail to pull. * Walk on tiptoes. * Lordosis (excessive inward curvature of spine). * Kyphosis (curvature of upper back(humpback)) * Scoliosis * Frequent falls. * Gower maneuver (walking hands up legs to stand).

Answer 222

DMD - Testing * Electromyography (EMG) - records electrical activity of muscles. * Complex repetitive discharges (CRDs) - abnormal spontaneously firing action potentials associated with membrane instability. * Hallmark abnormality in dystrophinopathies. * Blood work - elevated CK (50-100x normal) * Muscle biopsy - histology: proteins * DNA gene deletion studies - PCR * Quantitative dystrophin analysis - western blot * Prenatal DX using fetal DNA (amniocentesis, Chorionic villus sampling (CVS)) * Preimplantation genetics - in IVF, blastomeres can be tested prior to embryo transfer.

Answer 223

Dystrophin - Deletion Hotspots * Exons 3-19 and 42-60

Answer 224

Genetic Mutations * Large deletions - 50-70% * Small deletions, insertions, nt changes (stop codons) - 25-30% * Large duplications - 5-10% * BMD associated with in-frame deletions: * Dystrophin protein of abnormal size. * DMD associated with frameshift mutations:

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Becker Muscular Dystrophy * Milder form than Duchenne. * Incidence 1/18,000. * Similar symptoms. * Loss of walking after 16y. * Muscle pain, dilated cardiomyopathy. * Increased workload on left ventricle leads to enlargement and potentially failure and death.

Answer 226

Human Retinal Dystrophin * Retinal dystrophin (DP260): * Similar to muscle dystrophin but smaller in size (260kDa vs 427kDa) * Has 3 of the functional domains of muscular dystrophin (spectrin, Cys, C-term) * Mini-dystrophin (instead of micro-dystrophin) * Endogenous in 30% of DMD patients. * No immunogenicity issue. * Dystrophin gene has many products driven by different promotors: * Every cell contains complete gene, thus potentially the ability to express other forms. * Expression of retinal dystrophin is driven by muscle creatine kinase (MCK) promoter/enhancer. * If it can be triggered in muscle cells, retinal dystrophin can function in place of regular dystrophin. * Has been shown effective in mice.

Answer 227

DMD vs. BMD * Both X-linked recessive. * DMD: * Worse than BMD. * Incidence: * 1/3 of case are de novo mutations. * 2/3 of cases are familial (female carrier). * Complete lack of dystrophin. * Do get frameshifts leading to stops * Wheelchair-bound by 12yo. * Average life span: 25y. * BMD: * Variability in symptoms. * No frameshifts: protein made. * Some dystrophin, but abnormal in quantity and size. * Male still walking at 16yo. * Average life span: 45y.

Answer 228

Muscular Dystrophy * Muscle membrane susceptible to mechanical injury. * Injury to sarcolemma. * Ca²⁺ influx/oxidative stress. * Degeneration of fibers. * Cycles of degeneration/regeneration. * Irreversible necrosis/loss of fiber. * Replacement of fiber by fat & CT.

Answer 229

DMD - Treatment Strategies * Growth factors: * Myostatin inhibitors = muscle cell growth (more muscle). * Hypothetically could work, but would make more defective muscle. * Gene therapy: * Replace full-length dystrophin gene. * Problem - too large for adeno-associated virus. * Utrophin (similar to dystrophin gene not on X chromosome). * Try to over express, does not work. * Microdystrophins. * BMD shows that large portion of spectrin-like repeats can be deleted from dystrophin and still be functional. * Works, but issue with immunogenicity. * Exon skipping: * Deletions cause frame shift mutations, skipping exon would put reader back in frame and generate BMD-like dystrophin. * Nonsense (stop) codon read-through: * Works...but problem with immunogenicity. * All approaches have problems.

Answer 230

Mitochondria * Maternally inherited - mitochondria in eggs. * Self replicate, segregate during cell division by chance. * Rate of mtDNA mutation is higher than nuclear genes. * Provide cellular energy in the form of ATP for the cell by: * ETC * Oxidative phosphorylation. * Have their own DNA / genome. * Contains 37 genes for 13 proteins and 24 parts of the machinery to make proteins (rRNA and tRNA). * Replicates and gets passed on to new mitochondria. * If mtDNA mutation occurs, a mixture of normal mitochondria and mutant mitochondria occurs in one cell - called heteroplasmy. * Need certain level of aberrant mitochondria vs. normal mitochondria for disease to occur. * Threshold effect of mutant mitochondria are required for disease manifestation.

Answer 231

Mitochondrial Myopathies * A muscle disease caused by mitochondrial dysfunction. * Characteristics of mitochondrial disorders: clinical variability and age related progression of disease. * 4 major types of Mitochondrial Genetics Diseases: * MERRF: myoclonus epilepsy with ragged red fibers (tRNA mutation). * MELAS: mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (tRNA mutation). * KSS: Kearns-Sayre Syndrome (mtDNA rearrangements). * CPEO: chronic progressive external opthalmoplegia (mtDNA rearrangements). * LHON - Leber Hereditary Optic Neuropathy - blindness in late adolescence, muscle not affected (SBP substitution in mtDNA coding for Complex I).

Answer 232

Mitochondrial Myopathies - Ragged Red Fibers * Mitochondrial myopathy where aggregates of abnormal mitochondria form red sarcolemmal blotches called Ragged Red Fibers.

Answer 233

Mitochondria - Mutant Segregation * Miotochondria segregate, during cell division, by chance. * Ratio of mutant mitochondria in daughter cells is random and may or may not meet the threshold needed for disease.

Answer 234

Mitochondrial Myopathies - Clinical Characteristics * Muscle weakness * Excercise intolerance * Lactic acidosis * Neurological signs: mitochondrial encephalopathies. * Other associated abnormalities: * Vomiting, seizures, dementia * Stroke-like episodes * Ptosis * Opthalmoplegia (paralysis of extraocular muscles) * Blindness * Cardiomyopathy

Answer 235

Mitochondrial Inheritance * Passed from mother to offspring.

Answer 236

MERRF - Myoclonus Epilepsy with Ragged Red Fibers * Clinical features: * Myoclonus - involuntary jerking of muscle, often first symptom. * Myoclonic epilepsy * Ataxia (lack of coordinated muscle movements). * Ragged Red Fibers (muscle tissue) * Seizures, dementia. * 90% caused by 2 mutations of tRNA^Lys. * Mutations: * 85% due to A to G mutation in the mtDNA tRNA^Lys gene at nucleotide position 8344. * 5% due to G to C at position 8356 in tRNA^Lys mtDNA gene.

Answer 237

MELAS * Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes. * Clinical presentation: * Seizures * Stroke-like episodes of hemiparesis (weakness on one side of body). * Blindness * Headaches * Anorexia * Recurrent vomiting * Lactic acidosis * Ragged Red Fibers * AOO: 2-10 years old (60%) * Mutation: * Typically caused by A3243G mutation in tRNA^Leu.

Answer 238

Mitochondrial Genetics in Disease * Heteroplasmy: mixture of normal and abnormal mitochondria. * Threshold - if cells carry too many mutant mitochondria - disease results. * Need a mutation to occur AND a certain percentage of the mitochondria to be aberrant. * Some tissues require more energy than others - e.g. neural and muscle tissue. * Neurological and muscle tissues affected by mitochondrial genetic diseases. * Brain/CNS, heart, skeletal muscle.

Answer 239

CPEO * Chronic Progressive External Opthalmoplegia * Mild to moderate mitochondrial myopathy. * Clinical presentation: * Ptosis * Red Ragged Fibers observed in skeletal muscle. * Mutation: * mtDNA rearrangements.

Answer 240

LHON * Leber Hereditary Optic Neuropathy * Mitochondrial mutation that only affects optic nerve. * No muscle involvement. * Clinical presentation: * Acute or subacute, bilateral, central vision loss. * Onset and progression typically rapid. * Initially affects one eye, but eventually both eyes affected at same time. * Degeneration of the retinal ganglion cell layer and optic nerve. * AOO: 20-30 years old. * Mutation: * Single base pair substitutions of mtDNA coding genes of Complex I proteins. * No tRNA or mtDNA rearrangements.

Answer 241

Mitochondria - Mutations * 2 major divisions: * Point mutations in mtDNA tRNA genes lead to MELAS and MERRF. * mtDNA genome deletions and rearrangements lead to KSS and CPEO.

Answer 242

Heteroplasmy - MERRF * Characteristics have clinical variability and age related progression of disease due to heteroplasmy. * Example: MERRF - there is a genotype/phenotype correlation. * 25yo - 95% mutant tRNA^Lys has severe clinical presentation. * 25yo - 85% mutant tRNA^Lys is normal and healthy, but develops disease later in life.

Answer 243

Kearns-Sayre Syndrome * Clinical presentation: * Retinitis pigmentosa (degenerative eye disease leading to blindness) * At least one of the following: * Cardiac conduction abnormality. * Cerebelar ataxia. * Cerebral spinal protein level above 100 mg/dL. * May present with optic atrophy, hearing loss, dementia, seizures, cardiomyopathy, lactic acidosis. * Red Ragged Fibers seen in skeletal muscle. * AOO: before 20 years old * Mutation: * 85% of KSS due to mtDNA rearrangements including duplicated mtDNA, deleted mtDNA and insertions. * 200 different deletions.

Answer 244

Erythrocytes * Typical person has 25 trillion RBCs. * Last 120 days. * Old, damaged RBCs are removed from the circulation and they need to be constantly replaced. * Produce 2.4 million RBCs per second. * Can carry 500-1,000+ L of O₂ per day, depending on activity. * Hb occupies 33% of the volume of an RBC and 90% of the cell's dry weight. * 2 lbs of Hb in normal person.

Answer 245

Erythropoiesis * 65% of Hgb is synthesized before the extrusion of the nucleus. * Reticulocyte makes remaining 35% of Hgb. * In reticulocytes, 95% of all protein synthesis is globin synthesis.

Answer 246

Erythropoeisis - Tissue Switching

Answer 247

Hemoglobin - Structure * Multi-subunit protein (tetramer): * 2 α-globin chains * 2 β-globin chains * 8 helical segments to a globin subunit and they are labeled A-H. * AAs are named according to the helical segment and AA# in that segment (e.g. F8 His is 8th residue in F helix which is 6th segment). * Heme: * Prosthetic group - a non-amino acid group of a protein. * Nestled in a hydrophobic crevice of the protein chain. * One per subunit. * Gives blood its color. * 75% of globin chain forms α-helix. * Has Fe atom that carries O₂. * 4 protoporphyrin IX rings.

Answer 248

Hemoglobin - Function * Best molecule, due to structure, for transporting O₂ from the lungs to tissues. * "Hb is a lung miniature - breathing as it allows the body to respire."

Answer 249

Hemoglobin Switching * Not fully clear how hemoglobin switching occurs. * Switch from HbF to HbA is closely related to gestational age - time controlled. * Premature infants switch later after birth to HbA than full-term babies.

Answer 250

Hemoglobins - Types * 3 types: * Embryonic: * Expressed in yolk sac but not after 8 weeks gestation. * Embryonic Hb (ζ2ε2) * Fetal: * Hbf made predominantly in liver (and bone marrow) * In fetus, 90-95% HbF until 34-36 weeks gestation. * HbF (α2γ2) * Adult: * HbA (α₂β₂) production starts after birth. * Reaches adult levels at 1y. * 4 types of globin chains: * α * β * δ * γ * Forms: * HbA (α₂β₂) - 97% (predominant form). * HbA2 (α₂δ₂) - 3% * HbF (α₂γ₂) - 0.5%

Answer 251

Hemoglobin - HbF * Fetus requires Hb that has a higher affinity for O₂ than mothers Hb so that O₂ can pass from mother to fetus. * HbF (α₂γ₂) * Does not bind well to 2,3-BPG: * Allowing increased affinity for O2. * Keeping HbF mostly locked in R form.

Answer 252

Hemoglobin - Chains * α-like chains: * ζ (zeta) embryonic * α (alpha) * β-like chains: * ε (epsilon) embryonic * γ (gamma) fetal * δ (delta) * β (beta)

Answer 253

Hemoglobin - Globin Genes * Chromosome 11 - β globin genes: * ε, γ^g, γ^a, δ, β * γg = glycine 136 * γa = alanine 136 * Chromosome 16 - α globin genes: * ζ, α₂, α₁

Answer 254

Hemoglobin - Sickle Cell and HbF * Hbs - E-\>V substitution at position 6 in β globin. * Causes polymerization. * Sickle shaped RBCs impede circulation - hemolytic anemia. * Pain, organ damage, strokes, increased infections, etc. * Research involving HbF expression is ongoing. * Currently using hydroxyurea to induce HbF and address inflammation, but this is a chemotherapeutic agent.

Answer 255

DNA-only Transposons * Exist only as DNA in their movement. * Predominate in bacteria (largely responsible for the spread of bacterial resistance). * Use cut-and-paste transposition or replicative transposition. * DNA only transposons contain: * Gene encoding transposase. * Sequences recognized by the enzyme necessary for movement.

Answer 256

Retrovirus-like transposon transposition steps: * Entire transposon is transcribed by host * RNA transcript contains reverse transcriptase enzyme * Translated by host cell * Reverse transcriptase makes a DS DNA copy of RNA molecule via hybrid DNA/RNA intermediate. * DS DNA integrates into site on chromosome using integrase, encoded by element. * Integrase cuts one strand at each end of the viral sequence * Each exposed 3' OH ends attacks a phosphodiester bond of target DNA. * This inserts the viral DNA into target, leaving gaps to be filled/ligated. * Leaves short repeats on each side of integrated DNA segment.

Answer 257

Transposition by Nonretroviral Transposons * Endonuclease and reverse transcriptase bind to L1 RNA * Endonuclease nicks the target DNA at insertion point * Releases 3' OH to serve as primer in reverse transcription step. * SS DNA copy of L1 directly liked to target DNA. * Insertion of DS DNA copy of L1 at target site.

Answer 258

Conservative site specific recombination can be used to turn genes on and off by removing or reinserting segments containing the genes of interest.

Answer 259

Nonretroviral Transposons * Comprise a large portion of our genome * Repeated sequences are mutated and truncated nonretroviral transposons * Most are immobile but few retain ability to move. * L1 element (LINE or long interspersed nuclear element) * Require endonuclease and reverse transcriptase to move. * Do not encode the enzymes * Use enzymes from other transposons * Make up **40%** of the human genome.

Answer 260

5 phases of prophase: * Leptotene - homologs begin to condense/pair. * Zygotene - homologs pair and synaptonemal complexes form. * Pachytene - Synapsis is complete; crossing over occurs. * Diplotene - Synaptonemal complex begins to break down; homologs begin to separate but remain attached at chiasmata. * Diakinesis - Reach maximum condensation; separation of homologs and transition stage into metaphase.

Answer 261

Retrovirus-like transposons * Resemble retroviruses, but lack a coat. * Move in an out of chromosomes the same way but unable to leave the resident cell.

Answer 262

Transposons are: * Mobile genetic elements also called transposable elements, "selfish DNA" and "jumping genes" * Specialized segments of DNA that move from one position in the genome to another. * Range in size from 100s to tens of thousands of nt pairs. * Each with unique set of genes and encodes enzyme that catalyzed movement of transposon. * Can provide benefits to the cell (i.e. antibiotic resistance in bacteria) * Can produce genetic variation or spontaneous mutations. * No sequence homology required - can insert anywhere in the genome. * Move infrequently: 1 in every 10⁵ cell divisions in bacteria. * Transposase - enzyme encoded by the transposon itself * Acts on specific DNA sequence on each end of the transposon * Allows insertion into a target DNA site.