Cell Biology Book 2023 PDF

Boards & Beyond: Cell Biology Slides Color slides for USMLE Step 1 preparation from the Boards and Beyond Website Jason Ryan, MD, MPH 2023 Edition B...

Boards & Beyond: Cell Biology Slides Color slides for USMLE Step 1 preparation from the Boards and Beyond Website Jason Ryan, MD, MPH 2023 Edition Boards & Beyond provides a virtual medical school curriculm used by students around the globe to supplement their education and prepare for board exams such as USMLE Step 1. This book of slides is intended as a companion to the videos for easy reference and note-taking. Videos are subject to change without notice. PDF versions of all color books are available via the website as part of membership. Visit www.boardsbeyond.com to learn more. Copyright © 2023 Boards and Beyond All rights reserved. i Www.Medicalstudyzone.com  This PDF was created and uploaded by www.medicalstudyzone.com which is one the biggest free resources platform for medical students and healthcare professionals. You can access all medical Video Lectures, Books in PDF Format or kindle Edition, Paid Medical Apps and Softwares, Qbanks, Audio Lectures And Much More Absolutely for Free By visiting our Website https://medicalstudyzone.com all stuff are free with no cost at all. Furthermore You can also request a specific Book In PDF Format OR Medical Video Lectures. Www.Medicalstudyzone.com  Table of Contents DNA Replication...................................................1 Flow Cytometry................................................. 35 DNA Mutations......................................................8 ELISA...................................................................... 37 DNA Repair.......................................................... 12 Microarrays and FISH..................................... 40 Transcription...................................................... 17 Cell Cycle............................................................... 42 Translation........................................................... 23 Cell Structure...................................................... 47 PCR.......................................................................... 29 Cytoskeleton........................................................ 53 Blotting.................................................................. 31 Connective Tissue............................................. 58 iii Www.Medicalstudyzone.com  DNA Replication DNA Contains genetic code Nucleus of eukaryotic cells Cytoplasm of prokaryotic cells Replicated for cell division/growth DNA Replication Jason Ryan, MD, MPH Wikipedia/Public Domain DNA Structure Base Pairing 1. Sugar (ribose) backbone DNA 2. Phosphate Adenine-Thymine Guanine-Cytosine 3. Nitrogenous base RNA Adenine-Uracil Guanine-Cytosine Antiparallel Wikipedia/Public Domain Wikipedia/Public Domain Nucleotides 5’ DNA Replication Synthesized as monophosphates Converted to triphosphate form 3’ 5’ 3’ Triphosphate form added to DNA 3’ 5’ Adenosine Cytosine Deoxy-adenosine Triphosphate Thymidine Guanosine 1 Www.Medicalstudyzone.com  DNA Replication DNA Replication 5’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ Adenosine Cytosine Adenosine Cytosine Thymidine Guanosine Thymidine Guanosine DNA Replication DNA Replication Helicase ATP Unwinds/opens double helix DNA Hydrolyzes ATP Helicase Single strand binding proteins 5’ 3’ Origin Assist helicase of Stabilize and straighten single strands of DNA Replication 3’ 5’ ssBP ssBP ssBP Origin of Replication DNA Polymerases Specific DNA sequences Bacteria (prokaryotes) Attract initiator proteins DNA polymerase I-IV Easy to unwind/open Polymerase III: Major DNA polymerase Fewer bonds A-T Polymerase I: Removes RNA primers “AT rich” sequences Eukaryotes Easy to open DNA polymerase α, β, γ, δ, and ε Polymerase γ: located in mitochondria Wikipedia/Public Domain 2 Www.Medicalstudyzone.com  Primers Primers DNA polymerase cannot initiate replication DNA Primase: Makes primers Primers: short nucleotide sequences Primers contain RNA Formed at point of initiation of new chain Ribonucleotides (not deoxy-ribonucleotides) Uracil instead of thymine Required by DNA polymerase to function Eventually removed and replaced with DNA Ribonucleotide Deoxyribonucleotide Replication Fork DNA Replication Directionality 5’ 3’ C 5’ A Adenosine-TP 3’ T 5’ G 3’ DNA Replication DNA Replication Directionality Directionality 5’ DNA Always occurs in 5’ to 3’ direction C Polymerase Nucleotides added to 3’ end of growing strand A T G 3’ 3 Www.Medicalstudyzone.com  Replication Fork Replication Fork DNA Polymerase 3’ 3’ 5’ 5’ 3’ 3’ DNA 5’ Polymerase 5’ Replication Fork 3’ Replication Fork 3’ DNA Polymerase 5’ 5’ 3’ 3’ DNA Polymerase 5’ 5’ Replication Fork Primer Removal Leading Strand Okazaki fragments synthesized until primer reached RNA primer removed and replaced with DNA Prokaryotes: DNA polymerase I Eukaryotes: DNA polymerase delta Okazaki fragments Masur/Wikipedia Lagging Strand 4 Www.Medicalstudyzone.com  DNA Ligase Topoisomerase Joins Okazaki fragments Prevent DNA tangling Creates phosphodiester bonds Break DNA then reseal to relieve tension/twists Topoisomerase I Break single strands of DNA then reseal Topoisomerase II Break double strands then reseal Wikipedia/Public Domain Topoisomerases Topoisomerase DNA Replication Clinical Correlations Key Points Quinolone antibiotics Leading strand replication is continuous Prokaryotic topoisomerases Lagging strand replication is discontinuous Chemotherapy agents Okazaki fragments Eukaryotic toposiomerases DNA ligase Etoposide/teniposide Irinotecan, topotecan Anthracyclines DNA Replication Proofreading Key Point Semi-conservative DNA polymerase can correct errors New DNA: one old and one new strand Synthesizes in new strand 5’ to 3’ direction Wrong nucleotide added: Can move backwards 3’ to 5’ direction Correct error Exonuclease activity: remove incorrect nucleotide DNA polymerase: “3’ to 5’ exonuclease activity” Significantly reduces error rate Adenosine/Wikipedia 5 Www.Medicalstudyzone.com  Replication Fork Replication Fork 3’ 3’ 5’ 5’ DNA DNA Polymerase Polymerase 3’ 3’ 5’ 5’ Adenosine Cytosine Adenosine Cytosine Thymidine Guanosine Thymidine Guanosine Replication Fork Telomerase Telomeres: nucleotides at end of chromosomes 3’ Contain T-T-A-G-G-G sequences No place for RNA primer on lagging strand 5’ Major problem eukaryotic cells (non-circular DNA) Telomerase enzyme DNA Recognizes telomere sequences Polymerase 3’ Adds these sequences to new DNA strands 5’ Adenosine Cytosine Thymidine Guanosine Telomerase Contains an RNA template Uses template to synthesize telomere DNA “RNA-dependent DNA polymerase” Similar to reverse transcriptase Uzbas, F/Wikipedia 6 Www.Medicalstudyzone.com  Telomerase Telomerase Extends 3’ end of DNA Found in hematopoietic stem cells Allows DNA polymerase to complete lagging strand Allows controlled indefinite replication Avoids loss of genes with duplication Other cells that divide indefinitely Epidermis, hair follicles, intestinal mucosa Implicated in many cancers Allows immortality Uzbas, F/Wikipedia 7 Www.Medicalstudyzone.com  DNA Mutations Protein Synthesis DNA Transcription DNA Mutations RNA Translation Jason Ryan, MD, MPH Proteins Codons Genetic Code 3 Nucleotide Sequences DNA T A C Transcription RNA A U G Translation Proteins Methionine DNA Mutations DNA Mutations Errors in DNA Germ line mutations Simple: One/few base(s) abnormal DNA of sperm/eggs Transmitted to offspring Complex: Gene deletions, translocations Found in every cell in body Somatic mutations Acquired during lifespan of cell Not transmitted to offspring 8 Www.Medicalstudyzone.com  Point Mutations Pyrimidines Wobble Transition (more common): Some transitions less likely to alter amino acids Purine to purine A to G Genetic code: often same AA with altered base Pyrimidine to pyrimidine (C to T) Cytosine Transversion: Purine to pyrimidine (A to T) UU-Pyrimidine Pyrimidine to purine (C to G) Same AA Thymine Purines Adenine Guanine Silent Mutation Nonsense Mutation Nucleotide substitution codes for same amino acid Nucleotide substitution Often base change in 3rd position of codon Result: Early stop codon Nucleotide triplet DNA A – A – A A–A–G Signals termination of translation of proteins RNA U – U – U U–U–C UGA, UAA, UAG PHE PHE DNA A – C – C A–C–T RNA U – G – G U–G–A TRY Phenylalanine Phenylalanine Missense Mutation Sickle Cell Anemia Nucleotide substitution Root cause: Missense mutation beta globin gene Result: Different amino acid Single base substitution 6th codon of β gene Adenine changed with thymine DNA G – T – A – G – T – A –G – T – A – G – T – A Substitution of valine for glutamate in beta chains RNA C – A – U – C – A – U –C – A – U – C – A – U HIS HIS HIS HIS DNA G – A – G G–T–G C–T–C C–A–C RNA G – A – G G–U–G GLU VAL DNA G – T – A – G – G – A –G – T – A – G – T – A RNA C – A – U – C – C – U –C – A – U – C – A – U HIS PRO HIS HIS Glutamate Valine 9 Www.Medicalstudyzone.com  Insertions and Deletions Insertions and Deletions Addition/subtraction of nucleotides Addition/subtraction of nucleotides Can alter the protein product of a gene Can alter the protein product of a gene Cystic fibrosis Cystic fibrosis Most common mutation: delta F508 Most common mutation: delta F508 Deletion of 3 DNA bases Deletion of 3 DNA bases Loss of phenylalanine Loss of phenylalanine Abnormal protein folding Abnormal protein folding Frameshift Mutation DNA G–T–A–G–T–A–G–T–A–G–T–A HIS HIS HIS HIS Insertion or deletion of nucleotides/bases Point DNA Size Unchanged Mutation Alters the reading frame G–T–A–G–C–A–G–T–A–G–T–A HIS ARG HIS HIS G–T–A–G–T–A–G–T–A–G–T–A HIS HIS HIS HIS Frameshift DNA Size Changed Mutation A–G–T–A–G–T–A–G–T–A–G–T–A SER SER SER SER Frameshift Mutation Frameshift Mutation Deletion/insertion not multiple of 3 Described in Tay Sachs disease Misreading of nucleotides downstream Frameshift mutations (insertions/deletions) Gene for hexosaminidase A Significant change to protein Many amino acids may change Duchenne muscular dystrophy Early stop codon → truncated protein Dystrophin gene Loss of stop codon → elongated protein Frameshift deletions → absence of functional dystrophin 10 Www.Medicalstudyzone.com  Slipped-Strand Mispairing Slipped-Strand Mispairing DNA Slippage DNA Slippage Occurs in areas of repeated nucleotide sequences Occurs with inadequate mismatch repair Insertions/deletions → frameshift mutations A–A A–A A–A A–A A–A T–T T–T T–T T–T T–T Slippage in template strand → deletion (DNA not replicated) Slippage in replicated strand → insertion (replicated strand longer) Trinucleotide Repeat Disorders Microsatellite Instability Occur in genes with repeat trinucleotide units Microsatellite Example: CAGCAGCAGCAG Short segments of DNA Extra repeats in gene → disease Repeated sequence (i.e. CACACACA) Key examples Mismatch repair enzyme failure → instability Fragile X syndrome Variation (instability) in size of segments among cells Friedreich’s ataxia Seen in colon cancer Huntington’s disease Myotonic dystrophy 11 Www.Medicalstudyzone.com  DNA Repair DNA Damage Occurs frequently in life of a cell Heat, UV radiation, chemicals, free radicals Rarely leads to permanent damage Numerous repair enzymes/mechanisms exist Without repair, genetic material quickly lost DNA Repair Jason Ryan, MD, MPH Types of DNA Damage Types of DNA Damage Depurination Depurination Occurs spontaneously thousands of times per day Results in loss of purine bases (guanine and adenine) Deamination Occurs spontaneously hundreds of times per day Guanosine Sugar Phosphate Base loses amine group (cytosine) Guanine Adenine Guanine Adenosine Adenine Types of DNA Damage Types of DNA Damage Deamination Free radicals or radiation damage base rings Cytosine Uracil Oxidative damage, methylation, hydrolysis Cytidine Oxidative attack Methylation Guanosine 12 Www.Medicalstudyzone.com  Repair Mechanisms Base Excision Repair Single strand Pathway for damaged DNA repair Base excision Recognize specific base errors Nucleotide excision Deaminated bases, oxidized bases, open rings Mismatch repair Numerous variations/enzymes used by cells Double strand Functions throughout the cell cycle (all phases) Homologous end joining Non-homologous end joining Base Excision Repair Base Excision Repair DNA glycosylases AP endonuclease Several different enzymes Recognizes nucleotides without a base Remove damaged bases Attacks 5’ phosphate end of DNA strand Glycosidic “Nicks” damaged DNA upstream of AP site Creates a baseless nucleotide Bond Create a 3'-OH end adjacent to the AP site “Apurinic” or “apyrimidic” nucleotide AP lyase Some DNA glycosylases also possess AP lyase activity Attack 3’ hydroxyl end of ribose sugar A T C G A C G T A G C T A G C A C G A C G T A G C T A G C Base Excision Repair Base Excision Repair DNA polymerase AP Adds new nucleotide (complementary to opposite base) Endonuclease Extends 3'-OH terminus DNA ligase seals strand AP Lyase A C G A T C G T A G C T A G C Wikipedia/Public Domain 13 Www.Medicalstudyzone.com  Nucleotide Excision Repair Nucleotide Excision Repair Cyclobutane Removes “bulky” DNA damage Pyrimidine Multiple bases Dimer Often pyrimidine dimers Thymidine Thymidine Commonly caused by UV radiation (sunlight) G1 phase (prior to DNA synthesis) Endonucleases removed multiple nucleotides DNA polymerase and ligase fill gap Public Domain Wikipedia/Public Domain Xeroderma Pigmentosum Mismatch Repair Defective nucleotide excision repair Identifies incorrectly placed bases/nucleotides Extreme sensitivity to UV rays from sunlight Insertions, deletions, incorrect matches Occurs when proofreading misses errors Signs appear in infancy or early childhood Very easy sunburning No damage to base – not recognized by repair systems Freckling of skin Occurs in S/G2 phase (after DNA synthesis) Dry skin (xeroderma) Newly synthesized strand compared to template Changes in skin pigmentation Nucleotide errors removed and resealed Very high risk of skin cancer May develop in childhood James Halpern, Bryan Hopping and Joshua M Brostoff Mismatch Repair Mismatch Repair Important for microsatellite stability DNA slippage can occur at repeats DNA has many repeating segments Results in a mismatch “Microsatellites” Repaired by MMR systems Result: number of repeats (microsatellites) stable A AAACCC GGTT C CCCAAA TTGG 14 Www.Medicalstudyzone.com  Mismatch Repair HNPCC Hereditary Non-Polyposis Colorectal Cancer/Lynch Syndrome Microsatellite instability Germline mutation of DNA mismatch repair enzymes Results if MMR systems deficient About 90% due to MLH1 and MSH2 mutations Seen in cancers cells (colon cancer) Leads to colon cancer via microsatellite instability About 80% lifetime risk Hallmark: cancer cells with microsatellite instability Double Strand Damage Homologous End Joining Commonly result from exogenous sources Homology = similar structure Ionizing radiation HEJ = uses sister chromosome template Caused by radiation therapy (cancer) Sister Chromosome Template Non-Homologous End Joining Fanconi Anemia Uses many proteins to re-join broken ends Inherited aplastic anemia DNA pol λ and μ re-extend the ends More than 13 genetic abnormalities identified Many other enzymes Many involve DNA repair enzymes No template used (non-homologous) Hypersensitivity to DNA damage Highly error-prone Cells vulnerable to DNA strand cross-links Also impaired homologous recombination NHEJ Double Strand Break (ionizing radiation) 15 Www.Medicalstudyzone.com  Ataxia Telangiectasia Ataxia Telangiectasia Clinical Features Defective Nonhomologous end-joining (NHEJ) Most children healthy for first year Mutations in ATM gene on chromosome 11 Begin walking at normal age but slow development Ataxia Telangiectasia Mutated gene Progressive motor coordination problems Repairs double stranded DNA breaks via NHEJ By 10 years old, most in wheelchairs DNA hypersensitive to ionizing radiation Other symptoms CNS, skin, immune system affected Recurrent sinus/respiratory infections (immune system) Telangiectasias (skin) High risk of cancer 16 Www.Medicalstudyzone.com  Transcription Transcription Synthesis of RNA Ribonucleotides (not deoxyribonucleotides) Uridine (not thymidine) DNA used as template Transcription Jason Ryan, MD, MPH Thymidine Uridine Transcription Transcription 3’ 5’ 5’ 3’ Adenosine Cytidine Thymidine Guanosine Transcription Transcription DNA GTCA Transcription RNA GUCA 17 Www.Medicalstudyzone.com  Types of RNA Types of RNA Messenger RNA Micro RNA (miRNA) Longest chains of RNA Regulate gene expression Nucleotides specify amino acids Target mRNA molecules → bind via base pairing Used the synthesize proteins Block translation into protein Ribosomal RNA Small interfering RNA (siRNA) Form ribosomes Also regulate gene expression Transfer RNA Cause degradation of mRNA Transfer amino acids to proteins Small nuclear RNA (snRNA) Splicing of pre-mRNA RNA Polymerase RNA Polymerase Synthesizes RNA from DNA template Prokaryotes: One RNA polymerase Does not require a primer (like DNA polymerase) Multi-subunit complex Makes all types of RNA Binds promoter regions of DNA Requires transcription factors (proteins) Eukaryotes: multiple RNA polymerase enzymes Binds DNA → opens double helix RNA polymerase I: most rRNA (5.8S, 18S, 28S) RNA polymerase II: mRNA RNA polymerase III: rRNA (5S), other RNAs RNA Polymerase Inhibitors RNA Polymerase Inhibitors Alpha amanitin Rifampin Protein found in Amanita phalloides (death cap mushrooms) Inhibits bacterial RNA polymerase Powerful inhibitor of RNA polymerase II Used to treat tuberculosis Liver failure (taken up by liver cells) Actinomycin D Used as chemotherapy Inhibits RNA polymerase Newnam/Wikipedia 18 Www.Medicalstudyzone.com  Transcription Factors Promoters Additional proteins required to initiate transcription DNA regions Prokaryotes Not transcribed Protein factor (σ factor) Bind RNA polymerase and transcription factors Eukaryotes Bound RNA polymerase opens double helix Multiple factors (“transcription factors”) Many bind RNA polymerase II TFIID, TFIIB, TFIIE, etc Transcription Start Point Promoter Gene 5’ 3’ Promoters Enhancers TATA Box DNA sequences that increase rate of transcription Very common eukaryotic promoter May be upstream or downstream of gene they regulate TATAAA Binds transcription factors called activators Binds transcription factors (TFIID) Because of DNA coiling, many are geometrically close CAAT Box but many nucleotides away from gene CCAAT sequence Stabilize transcription factors/RNA polymerase GC Box GGGCGG Silencers Untranslated Regions DNA sequence that decreases rate of transcription Portions of mRNA at 5’ and 3’ ends May be upstream or downstream of gene they regulate Not translated into protein Binds transcription factors called repressors 5’ UTR upstream from coding sequence Repressors prevent RNA polymerase binding Recognized by ribosomes to initiate translation 3' UTR found following a stop codon Important for post-transcriptional gene expression 5’ 3’ UTR mRNA UTR 19 Www.Medicalstudyzone.com  Protein Synthesis Protein Synthesis Prokaryotes Eukaryotes Introns = stay IN nucleus Exons = exit nucleus mRNA in Eukaryotes 5’ Capping Initial transcript: hnRNA Addition of 7-methylguanosine to 5’ end Heterogeneous nuclear RNA Added soon after transcription begins Also called pre-mRNA Distinguishes mRNA from other RNA hnRNA modified to become mRNA Three key modifications before leaving nucleus 5’ capping Splicing out of introns 3’ polyadenylation Zephyris/Wikipedia RNA Splicing RNA Splicing Occurs during transcription Primary transcript combines with snRNPs Introns removed from mRNA in nucleus Small nuclear ribonucleoproteins (snRNPs) Short RNA polymers complexed with proteins Introns always have two nucleotides at either end RNAs contain high content of uridine (U-RNAs) 5' splice site: GU Five different U-RNAs defined: U1, U2, U4, U5, and U6 3' splice site: AG 5’ 3’ mRNA Exon GU AG Exon 20 Www.Medicalstudyzone.com  RNA Splicing RNA Splicing snRNPs and mRNA forms “spliceosome” Loop of mRNA with intron is formed (“lariat”) Lariat released → removes intron Exons joined BCSteve/Wikipedia Antibodies Alternative Splicing Anti-Sm (anti-smith) Allows many proteins from same gene Antibodies against proteins in snRNPs DNA: Exon1 – Exon 2 – Exon 3 – Exon 4 … Exon 10 Seen in patients with SLE Protein 1: Exon1 – Exon 3 – Exon 7 Anti-RNP Protein 2: Exon 2 – Exon 5 - Exon 10 Antibodies against proteins associated with U1 RNA Strongly associated with Mixed Connective Tissue Disease Also seen in SLE, Scleroderma Alternative Splicing Splicing Errors Can lead to disease Loss of exons, retention of introns Incorrect joining of introns Beta thalassemia Many mutations described Some involve splice sites Oncogenesis Many splice site mutations/errors described Wikipedia/Public Domain 21 Www.Medicalstudyzone.com  3’ Polyadenylation 3’ Polyadenylation Occurs at termination of mRNA transcription Requires several RNA binding proteins Triggered by specific DNA/RNA sequences Cleavage and polyadenylation specificity factor (CSF) “Polyadenylation signal:” AAUAAA Binds AAUAAA AAUAAA followed by 10-30 nucleotides then CA Cleavage stimulation factor (CstF) Binds CA sequence Leads to termination of DNA transcription 5’ 3’ DNA AATAAA CA 3’ (coding strand) mRNA AAUAAA CA 5’ 3’ mRNA AAUAAA CA 3’ Polyadenylation Transcription Summary Enzyme: Poly-A polymerase (PAP) Transcription DNA Coding Strand Start Adds ~200 adenosine nucleotides to 3’ end mRNA 5’ 3’ No template CAAT TATA Exon Intr Exon Intr AATAA 3’ mRNA AAUAAA CA Promoter mRNA 5’ 3’ PAP Cap Exon Exon AAUAA AAAA 3’ mRNA AAUAAA AAAAAA Cytoplasm MicroRNA Processing Bodies miRNA P-bodies Important regulatory molecules for mRNA Some mRNA moved to P-bodies in cytoplasm Regulate mRNA expression to proteins Seen with less extensive miRNA binding Bind mRNA via base pairing mRNA sequestered from ribosomes Extensive binding can remove poly-A tail Often degraded Exposes mRNA to degradation by endonucleases Some evidence that mRNA may later be translated Modifies gene expression at mRNA level 22 Www.Medicalstudyzone.com  Translation Transcription Summary Transcription DNA Coding Strand Start 5’ 3’ CAAT TATA Exon Intr Exon Intr AATAA Translation Promoter 5’ Exon Exon AAUAA 3’ AAAA mRNA Cap Jason Ryan, MD, MPH Cytoplasm mRNA read 5’ to 3’ Translation Translation Synthesis of protein using mRNA as template Occurs in cytoplasm on ribosomes tRNA brings amino acids to ribosome for assembly Boumphreyfr /Wikipedia Ribosomes Ribosomes Some are “free” in cytoplasm Prokaryotes Also bound to the endoplasmic reticulum 70S ribosomes Forms rough ER Small (30S) and large (50S) subunit Small subunit: 16S RNA plus proteins Contain rRNA and proteins Large subunit: 5S RNA, 23S RNA, plus proteins Arranged as a large and small subunit Protein synthesis inhibitor antibiotics Size measured in Svedberg units Aminoglycosides, others Measure of rate of sedimentation by centrifugation Target components of bacterial ribosomes 23 Www.Medicalstudyzone.com  Ribosomes tRNA Eukaryotes Transfers amino acids to protein chains 80S ribosomes Synthesized by RNA polymerase III Small (40S) and large (60S) subunits Many bases are chemically modified Small subunit: 18S RNA plus proteins Large subunit: 5S RNA, 28S RNA, 5.8S RNA plus proteins N,N dimethyl Guanosine Guanosine tRNA Anticodon Cloverleaf shape (secondary structure) 3 nucleotides on tRNA Base pairing within molecule Pairs with complementary mRNA 70-90 nucleotides in length (tiny) Correct pairing → correct protein synthesis Key portions Anticodon D loop (part of D arm) T loop (part of T arm) 3’ end Boumphreyfr /Wikipedia Yikrazuul Genetic Code D loop Contains dihydrouridine tRNA recognition by aminoacyl-tRNA synthetase Dihydrouridine Uridine 24 Www.Medicalstudyzone.com  T loop 3’ End Contains a TΨC sequence Always ends in CCA T = Ribothymidine Hydroyxl (OH) of A attaches to amino acid Ψ = Pseudouridine C = Cytidine Needed for tRNA ribosome binding Ribothymidine Pseudouridine Uridine Yikrazuul Charging tRNA Aminoacyl Group Charged tRNA Process of linking amino acids to tRNA Each tRNA linked to one amino acid Catalyzed by Aminoacyl-tRNA synthetase Adds amino acid to tRNA Requires ATP ATP Amino Acid AMP AMP Aminoacyl-tRNA synthetase tRNA One enzyme per amino acid in most eukaryotic cells Many amino acids have similar structures i.e. one enzyme attaches glycine to correct tRNA Mischarged tRNA → wrong AA for mRNA codon Hydrolytic editing Aminoacyl-tRNA synthetase scrutinizes amino acid If incorrect → hydrolyzes from AMP or tRNA Increases accuracy of charging tRNA Dancojocari/Wikipedia 25 Www.Medicalstudyzone.com  Protein Synthesis Protein Synthesis Amino acids: N-terminal and C-terminal ends Three stages: Proteins synthesis: addition to C-terminal end Initiation Elongation Termination Protein Synthesis Protein Synthesis Ribosomes: Four binding sites A-site: Amino acid binding (charged tRNA) One for mRNA P-site: tRNA attached to growing protein chain Three for tRNA: A-site, P-site, E-site E-site: Exit of tRNA 5’ 3’ 5’ 3’ E P A E P A Initiation Initiation Begins with AUG on mRNA Uses GTP hydrolysis Codes for methionine or N-formylmethionine (fMet) In eukaryotes require initiation factors (proteins) Binds directly to P-site Assemble ribosomes and tRNA Usually removed later by protease enzymes fMET = chemotaxis of neutrophils (innate immunity) Methionine N-formylmethionine 26 Www.Medicalstudyzone.com  Elongation Protein Synthesis Usually divided into a sequence of four steps Step 1: Charged tRNA binds A-site Uses elongation factors (proteins) P-site and A-site next to one another Bacteria: EF-Tu and EF-G Eukaryotes: EF1 and EF2 Hydrolyze GTP to GDP NH2 EF2: Target of bacterial toxins Diphtheria toxin (Corynebacterium diphtheriae) Exotoxin A (Pseudomonas aeruginosa) Inhibits protein synthesis t t 5’ 3’ E P A Protein Synthesis Protein Synthesis Step 2: Amino acid joined to peptide chain Step 3: Ribosome moves down mRNA toward 3’ end Catalyzed by ribosome (“ribozyme”) “Translocation” Peptidyl transferase: Part of large ribosome (made of RNA) Protein moves to P-site Protein attached to A-site NH2 NH2 t t t t 5’ 3’ 5’ 3’ E P A E P A Protein Synthesis Termination Step 4: tRNA leaves E-site Translation ends at mRNA stop codons UAA, UAG, UGA Not recognized by tRNA Do not specific an amino acid NH2 Releasing factors bind to ribosome at stop codons Catalyze water added to protein chain NH2 t t OH 5’ 3’ E P A 27 Www.Medicalstudyzone.com  Posttranslational Modifications Posttranslational Modifications Creates functional protein Phosphorylation Folding Amino acid residue phosphorylated Protein kinase enzymes add phosphate group Addition of other molecules Glycosylation Formation of the sugar–amino acid linkage Many linkages: N-, O-, C-linked glycosylation Creates glycoproteins Posttranslational Modifications Posttranslational Modifications Hydroxylation Methylation Addition of hydroxyl (OH) groups Addition of methyl (CH3) groups Important for collagen synthesis Acetylation Hydroxylation of proline and lysine residues Addition of acetyl (CH3CO) group Acetyl Group Ubiquitination Addition of ubiquitin (small protein) Tags proteins for destruction in proteasome Proline Hydroxyproline Chaperones Proteins that facilitate folding Bind to other proteins → ensure proper folding Classic example: Heat shock proteins Family of proteins Also called stress proteins Constitutively expressed Increased expression with heat, pH shift, hypoxia Stabilize proteins; maintain protein structure Help cells survive environmental stress 28 Www.Medicalstudyzone.com  This PDF was created and uploaded by www.medicalstudyzone.com which is one the biggest free resources platform for medical students and healthcare professionals. You can access all medical Video Lectures, Books in PDF Format or kindle Edition, Paid Medical Apps and Softwares, Qbanks, Audio Lectures And Much More Absolutely for Free By visiting our Website https://medicalstudyzone.com all stuff are free with no cost at all. Furthermore You can also request a specific Book In PDF Format OR Medical Video Lectures. Www.Medicalstudyzone.com  PCR PCR Polymerase Chain Reaction Laboratory technique Amplifies (copies) DNA molecules in a sample Polymerase Chain Uses: Make more DNA from small amount Determine if DNA is present (i.e. does it amplify?) Reaction Determine amount of DNA (i.e. how quickly does it amplify?) Jason Ryan, MD, MPH PCR PCR PCR Ingredients Primer Technique A C TG Sample (DNA) Heat sample DNA polymerase DNA denatures into single strands Primer Cool sample Single-stranded DNA segment Primer anneals (binds) complementary DNA (if present) Complementary to DNA under evaluation Warm sample Nucleotides DNA polymerase elongates from primer Process repeated in cycles Each cycle generates more DNA PCR Real Time PCR Quantitative PCR PCR done in presence of fluorescent dye Amount of dye proportional to amount of DNA More DNA = more fluorescence Fluorescence detected as PCR ongoing Rapid increase florescence = more DNA in sample Enzoklop/Wikipedia 29 Www.Medicalstudyzone.com  PCR Uses Herpes simplex virus encephalitis DNA in CSF HIV Viral Load Uses reverse transcriptase to make cDNA Amplification of cDNA Amount of cDNA produced over time indicates viral load Standard tool for monitoring viral load 30 Www.Medicalstudyzone.com  Blotting Blotting Laboratory techniques Southern blot: Identifies DNA Northern blot: Identifies RNA Western blot: Identifies proteins Blotting Jason Ryan, MD, MPH Southern Blot Probe Named for inventor (Edward Southern) Single-stranded DNA molecule Uses a probe to identify presence of DNA in a sample Carries radioactive or chemical markers Binds complementary sequences Probe called “cDNA” “Hybridization” Once bound, markers reveal DNA in sample Probe 5’ 3’ DNA in Sample Southern Blot Southern Blot Step 1 Step 2 Gel Electrophoresis Restriction nucleases Size separation (enzymatically cleavage) DNA Sample 31 Www.Medicalstudyzone.com  Gel Electrophoresis Southern Blot Step 3 Blotting Transfer to filter paper Jeffrey M. Vinocur Southern Blot Southern Blot Step 4 Step 4 Add probe Often done with multiple samples Wash away unbound probe Sample 1 Sample 2 Only bound probe remains Filter paper exposed to film → bound DNA revealed Southern Blot Southern Blot DNA Clinical Uses Restriction fragment length polymorphisms Sickle cell anemia Electrophoresis DNA probe 32 Www.Medicalstudyzone.com  RFLP RFLP Restriction fragment length polymorphisms Restriction fragment length polymorphisms Restriction nucleases DNA cutting enzymes Cut DNA at specific base sequences (i.e. GTGCAC) Restriction fragment length polymorphisms Analysis of fragments of DNA from restriction nucleases Different genes = different length of fragments Southern blotting to detect lengths after fragmentation 1.5kb 1.3kb 1.0kb Gene A Gene B Wikipedia/Public Domain Sickle Cell Anemia Northern Blot RNA Useful for assessing mRNA levels Normal β-globin gene: Two fragments (gene expression) 1.15kb and 0.2kb Sickle cell: One fragment 1.35kb Electrophoresis This fragment seen only with HbS gene 1.35kb Probe 1.15kb Normal Carrier SS Western Blot Western Blot Protein Detection of antibodies IgG or IgM in Lyme disease IgG HIV-1 Electrophoresis Antibody 33 Www.Medicalstudyzone.com  Southwestern Blot Southwestern Blot Protein Used to study DNA-protein interaction Combines features of Southern and Western blots Proteins separated by electrophoresis (Western) DNA probe added (Southern) Electrophoresis Used for studying DNA-binding proteins Especially transcription factors DNA 34 Www.Medicalstudyzone.com  Flow Cytometry Flow Cytometry Flow = motion of fluid Cytometry = measurement of cells Flow cytometry = Analysis of cells as they flow in a liquid through a narrow steam Key point: Used to analyze cells Flow Cytometry By size By surface proteins Jason Ryan, MD, MPH Flow Cytometer Flow Cytometer Key components: Flow cell: moves cells through machine Laser: light scattered by cells Photodetector: detects light scatter Light Source Photodetector Biol/Wikipedia Flow Cytometer Flow Cytometry Granulocytes Light Forward Scatter Side Scatter Size Monocytes Lymphocytes Side Scatter Front Scatter Granularity 35 Www.Medicalstudyzone.com  Antibody Staining Antibody Staining Specific antibodies to surface/intracellular proteins Tagged with unique fluorochrome Flow cytometer detects fluorochrome Indicates presence of protein in cells CD4 CD8 Flow Cytometry Flow Cytometry Clinical Uses Clinical Uses Fetal maternal hemorrhage Paroxysmal nocturnal hemoglobinuria Fetal red cells cross placenta to maternal blood Fluorescently-labeled monoclonal antibodies Seen with placental failure/trauma Bind glycosylphosphatidylinositol (GPI) anchored proteins Presents as decreased fetal movement, abnormal fetal HR Decay Accelerating Factor (DAF/CD55) Can cause stillbirth MAC inhibitory protein (CD59) Flow cytometry: monoclonal antibody to hemoglobin F Reduced or absent on red blood cells in PNH Detects fetal hemoglobin in red cells Databese Center for Life Science (DBCLS) Øyvind Holmstad/Wikipedia 36 Www.Medicalstudyzone.com  ELISA ELISA Enzyme-linked immunosorbent assay Detects antigens and antibodies in serum Based on enzymatic color change reaction Several forms Direct Indirect ELISA Sandwich Competitive Jason Ryan, MD, MPH ELISA Direct ELISA Enzyme-linked immunosorbent assay Add serum to be tested Serum coats plate → antigen secured to surface Wash away fluid Jeffrey M. Vinocur Direct ELISA Indirect ELISA Add enzyme-labeled antibody specific to antigen Add serum for analysis (like direct) Wash away unbound antibodies Add antibody to antigen of interest Add substrate → color change Antibody not enzyme linked Enzyme-linked antibodies directly bind antigen Wash away unbound antibody S S S E E E 37 Www.Medicalstudyzone.com  Indirect ELISA ELISA Direct vs. Indirect Add enzyme-labeled secondary antibody Direct Substrate → color change → identification of antigen Fewer steps Specific antibody must be enzyme-linked Result: Identifies presence of antigen in serum Time-consuming to label antibodies to unique antigens Enzyme-linked antibodies indirectly bind antigen Indirect More steps Specific antibody NOT enzyme-linked S S S Specific antibody easier to acquire (i.e. mouse antibody) E E E Secondary antibody easier to acquire (i.e. anti-mouse IgG) Sandwich ELISA Sandwich ELISA Plate coated with capture antibody High specificity Sample added → any antigen present binds Two antibodies used Unlikely to bind wrong antigen Detecting antibody added → binds to antigen Direct: detecting antibody enzyme linked Works with complex samples Indirect: secondary enzyme-linked antibody added Antigen does not require purification Substrate added → color change Can use secondary antibody like indirect S S S E E E Competitive ELISA Competitive ELISA Primary antibody incubated with sample Mixture added to antigen coated plates Antigen-antibody complexes form Unbound antibody binds antigen More antigen = more binding = less free antibody Wash away antigen-antibody complexes Secondary antibody and substrate added More color change = LESS antigen in sample 38 Www.Medicalstudyzone.com  Competitive ELISA ELISA Uses Lots of antigen Less color HIV antibody detection Indirect method (many variants used) Wash away HIV antigen attached to well Mixtur