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BS1007 Notes

by: Chong Yik Yan

BS1007 Notes BS1007

Chong Yik Yan
GPA 4.3
Molecular and Cell Biology I
Associate Professor Thirumaran THANABALU

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Summary for the NTU BS1007 Molecular and Cell Biology I course. Good for last minute revision and crash course studying.
Molecular and Cell Biology I
Associate Professor Thirumaran THANABALU
Study Guide
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This 89 page Study Guide was uploaded by Chong Yik Yan on Tuesday September 15, 2015. The Study Guide belongs to BS1007 at Nanyang Technological University taught by Associate Professor Thirumaran THANABALU in Summer 2015. Since its upload, it has received 325 views. For similar materials see Molecular and Cell Biology I in Biological Sciences at Nanyang Technological University.

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Date Created: 09/15/15
B81007 Molecular and Cell Biology I Notes DNA and RNA Contains genetic material proven by Frederick Griffith 1928 Structure determined in 1953 by Watson and Crick DNA transforming principle experimentally verified by Avery McLeod and McCarty rough and smooth strain bacteria and Hershey and Chase radioactive phosphorus and sulfur labeled bacteriophage Replication experiment by Meselson and Stahl replication determined to be semi conservative o Grow bacterial DNA in 15N medium 9 DNA appears as a single band 0 DNA transferred to 14N medium and replicated 9 DNA appears as single band intermediate between that expected for 15N and 14N o Continual replication 9 second round of replication yields DNA in two bands one in position of hybrid DNA and one in position of that containing only 14N Trinucleotide codon verified by Brenner 1961 Almost all DNA of eukaryotes contained in nucleus 0 Nucleus I Surrounded by 2 lipid bilayers I Large nuclear pores allow transport in and out I Nuclear envelope allows concentration of proteins that act on the DNA in the nucleus Carries genes that specify proteins and RNA molecules Arranged into chromosomes which contain long strings of genes Human DNA 0 Length 32 x 109 nucleotide pairs 0 104 exons per gene 0 145 nucleotide pairs per gene 0 Chromosome 22 48 x 106 nucleotides 15cm 2 pm in mitosis 10000 fold Synthesis of DNA only occurs 5 to 3 9 template strand runs from 3 to 5 Presence of ribose makes RNA alkali labile 9 OH group on RNA makes it more unstable than DNA especially in the presence of alkali disintegrates under alkaline conditions DNA STRUCTURE Can be double or single stranded Bases o Purines Adenine and Guanine O Pyrimidines Cytosine and Thymine Building block of DNA nucleotide sugar phosphate base DNA duplex double helix consists covalently linked sugarphosphate backbone and hydrogenbonded base pairs Each strand serves as template for synthesis of a new DNA strand Has polarity O O 5 end has a phosphate group 3 end has a hydroxyl group Polarity of DNA is important for gene expression Each linear DNA consists o Telomere protects ends of linear DNA 0 Replication origin 0 Centromere I Essential role during cell division I Consists short repeated DNA sequences alpha satellite I Some chromosomes have two regions with alpha satellite DNA but only one will serve as centromere I H3 histone is replaced by CENPA H3 variant which is centromerespecific I Sequence in CENPA allows it to bring in unique proteins with are important for the assembly of the kinetochore DNA PACKAGING Highly condensed DNA chromosomes 0 Chromosomes are partitioned into daughter cells at each cell division 0 DNA exist in different state during the various cell cycle stages 9 most compact form exists during mitosis easy to partition 0 Structure is dynamic 9 decondenses during gene expression Histones 0 Present in large quantities o Responsible for nucleosome formation 0 Small proteins 102135 amino acids positively charged 0 Core histones I H2A I HZB I H3 I H4 0 H2AHZB and H3H4 dimers formed H3H4 dimers combine to form tetramer which combines with 2 H2AHZB dimers 0 Each core histone contain an Nterminal tail which is subject to several forms of covalent modification I Largely unstructured I Extends out of the nucleosome I May help pack nucleosomes H1 histones I Linker histones I Larger than core histones I Less conserved during evolution I Changes the path of the DNA as DNA exits the nucleosome o Covalent modification I Modification of certain amino acids eg phosphate methyl acetyl group in proteins affects the protein structure I Histone regulation is carried out by modifications Acetylation enhance Phosphorylation enhance Methylation repression Ubiquitylation activation I Large scaffold protein which has protein modules that bind to specific histone modifications read the histone code 9 histone modifications are recognized by these codereader complexes which bind to specific modifications Codereader complex attracts other proteins with catalytic activities and additional binding sites Attachments to other components in the nucleus will lead to gene expression gene silencing or other biological function Nucleosomes 0 DNA binding proteins histones and nonhistones complexes with DNA to form chromatin ie chromatin refers to the DNAprotein complex 0 Nucleosomes refers to the repeating unit of a chromatin ie length of DNA wrapped around a histone histone o Packaged on top of one another to account for condensed form in cells 0 Unfolded chromatin can be digested with nuclease 0 High salt can be used to dissociate DNA from protein 0 DNA length around a histone is 147 base pairs 0 Linker DNA is up to 80 nucleotides long 0 Contains octameric histone core I 2 copies each of H2A HZB H3 and H4 histones 0 Dynamic in order for successful transcription I Presence of ATP dependent chromatic remodeling complexes and chaperones invivo Nucleosome can slide along Allows for exchange of histones Lampbrush chromosome 0 O Identified in interphase chromosome in oocytes female germ cell Consists extended loops of chromatin with chromomeres at each end of the extended chromatin loop adjacent chromomeres are joined by chromatin I Chromomeres are serially aligned beads or granules of a eukaryotic chromosome resulting from local coiling of a continuous DNA thread o A given loop always contain the same DNA sequence 0 Produces large amounts of RNA DNA TRANSLATION Stop codons o TGA o TAA o TAG Start codon 0 AUG Translation of DNA takes place on the ribosome o Ribosome moves along mRNA and captures tRNA matching codons in mRNA It then joins amino acid to generate the protein 0 Ribosome I Assembled in nucleolus I Made of 2 subunits large and small I Prokaryotes SOS and 308 Eukaryotes 608 and 408 8 refers to svedberg units 9 time unit 103913 seconds 9 offers a measure of particle size based on its rate of travel in a tube subjected to high gforce GENE ORGANIZATION A fragment of genetic information corresponding to one protein is one gene RNA from the same DNA segment can give rise to alternative versions via alternative splicing Expressions of genes are regulated when cell adjusts transcription and translation to suit its needs Coding and noncoding region 0 Noncoding regions consists regulatory and junk DNA pseudo genes etc Gene function is revealed by mutations GENETIC INNOVATION Intragenic mutation 0 Changes in DNA sequence due to error during replication Gene duplication 0 Two genes may then diverge during evolution DNA segment shuf ing 0 Two or more genes can be broken and rejoined to make hybrids Horizontal transfer 0 Common among bacteria 0 Addition of a particular gene of another species to genome GENE EXPRESSION Regulated by external and internal environment also includes temporal regulation timebased Inheritance of chromatin structure 0 So that all genes are passed from mother to daughter cell 9 daughter cell must express the appropriate set of genes 9 chromatin components are distributed to each daughter chromosome Structure used to regulate genes can be inherited 0 Genetic VS Epigenetic inheritance I Genetic inheritance suggests DNA sequence change 9 sequence change will be present in all multiplications of the somatic cells germ cells will also re ect this change in sequence I Epigenetic inheritance suggests chromatin change sequence of nucleotides are still the same 9 multiplication of somatic cells will re ect chromatin change germ cells however may not necessarily re ect epigenetic change 0 Heterochromatin highly condensed form Euchromatin less condensed form I Heterochromatin region is prevented from spreading by barrier DNA I Genes packaged into heterochromatin are turned off 9 position effect Eg Drosophila 9 red eyes mutation in gene gives rise to white eyes 0 White gene in normal location gives rise to red eyes 0 If white gene is moved near a heterochromatin region via chromosome inversion mottled uneven spots colour is observed Chromatin location within the nuclear envelope determines gene expression 0 Interior of nucleus is heterogenous I Different nuclear neighbourhoods affect gene expression 0 Movements of chromatin is driven by diffusion 9 position of gene in nucleus is not fixed I Movement of genes to different nuclear neighbourhoods results in active or silenced genes GENE STRUCTURE Promoter o Upstream from start site 0 Right promoter has to be present yeast promoter will not work for humans Transcription start site 0 Start of mRNA transcription RNA coding region 0 Downstream from start site 0 Contains translational start methionine 0 First ATG will determine how the protein is made reading frames depends on how the ATG is read 9 ATG has to be read as one codon Terminator Transcription termination site 0 mRNA drops off at termination site Template VS nontemplate strand Reading frame 0 Six different types of proteins can be produced from different reading frames I 1 2 3 refers to frames of reading direction for 5 to 3 I 1 2 3 refers to frames of reading direction for 3 to 5 0 Reading frames of insert has to be maintained note ATG of insert CLONING Cloning of gene occurs so that the gene can be sequenced 0 Nucleotide sequence of a gene can be used to predict the amino acid sequence of a protein 0 Amino acid sequence can then be compared with other proteins of known function 9 Function of gene can be derived from its DNA sequence Restriction enzymes 0 Named according to the source of the enzyme I EcoR I 9 Escherichia coli strain RY103 1St enzyme isolated I PstI 9 Providencia stuartii 1St enzyme isolated I Hind III 9 Haemophilus in uenza strain RD 3rd enzyme isolated I Taq I 9 Thermus aquaticus 1St enzyme isolated 0 Type I 9 recognizes site but cuts DNA elsewhere not as useful 0 Type II I Recognition sequence 4 8 nucleotides I Used widely in recombinant DNA technology I Have palindromic sequences I Different types of ends created Blunt ends Sticky ends 0 5 overhang o 3 overhang I For an ncutter n number of bases recognized frequency of cutting is 11 0 Restriction sites can be blocked using DNA methylases I Protects host genome from restriction endonucleases I Methylation of base residues will prevent recognition by restriction enzymes and therefore protects own DNA from being cut up DNAdependent DNA polymerase o All adds deoxyribonucleotides to the 3 end 0 E coli DNA polymerase I I 5 9 3 polymerase activity adds nucleotides from the 5 to 3 I 3 9 5 exonuclease removes nucleotides from 3 Serves as proofreading 9 reads the DNA strand backwards to check and removes nucleotides if necessary I 5 9 3 exonuclease removes from 5 Activity can be removed from DNA polymerase 1 results in E coli DNA polymerase I Large fragment or Klenow fragment 0 DNA polymerase Klenow fragment I Generates blunt end fragments I When DNA digested with restriction enzymes which generate 5 or 3 overhangs are incubated with dNTPs all 4 nucleotides and enzyme polymerase treats 5 overhangs by adding nucleotides 3 overhangs by cutting off the overhang 9 because polymerase can only add from 5 to 3 Klenow RNAdependent DNA polymerase 0 Reverse transcriptase from retrovirus such as AMV Avian Myeloblastosis Virus and MMLV Moloney Murine Leukemia Virus 0 Consists I RNAdependent DNA polymerase activity 9 uses RNA template to make DNA I DNAdirected DNA polymerase activity 9 slow I Degradation of RNA in RNADNA hybrid 9 3 to 5 or 5 to 3 RNase H activity 0 Useful for generating complementary DNA cDNA DNAdependent RNA polymerase O O 0 Makes mRNA Useful for generating transcripts invitro I Uniformly labeled probe I Invitro translation of proteins I Invitro generation of labeled proteins Two classes I E coli RNA polymerase 9 terminates prematurely transcripts gt 500 difficult to obtain not very specific I T7 T3 and SP6 RNA polymerases 9 very processive transcripts gt1000 bases rapid initiation at promoter sites extremely specific T7 RNA polymerase 0 Makes a lot of mRNA transcripts from the T7 site on the cloned DNA 0 If radioactive nucleotides or amino acids are added to the reaction mixture during transcription or translation radioactive mRNA or proteins will result 9 can detect protein or mRNA via radioactivity probe Deoxyribonuclease I 0 Used when differentiation of DNA and RNA is required 0 DNaseI from bovine pancreas usual source of DNase is an endonuclease that degrades D S doublestranded DNA to produce 3 OH oligonucleotides in the presence of Mg2 9 chops up DNA 0 Application I Nick translation I Cloning random DNA fragments for insertion into plasmid if restriction enzyme use is not desired I Removing DNA from cell lysates Ribonucleases o Recognizes RNA and degrades it o Ribonuclease A RNaseA from bovine pancreas is an endoribonuclease 0 Application I Hydrolyzing RNA that contaminates DNA preparation DNA ligases 0 DNA ligases catalyse the formation of phosphodiester bonds between 5 PO4 and 3 OH terminus in DS DNA 0 T4 DNA ligase usually used as it can ligate the different types of cuts works for most DNA fragments Ligation reaction 0 O O 0 Used to join DNA fragments together DNA ligase and restriction enzymes allows us to cut and join DNA fragments to generate hybrid DNA molecules Vector insert molar ratio 13 usually Inserts will anneal based on complementary base pairs 0 Usually two restriction enzymes are used so that we always get the direction of DNA insert that we want 9 if only one restriction enzyme used two types of hybrid DNA will be produced palindromic cutting site only one required 0 Products I Vector selfligation generates the original vector 9 forms colonies not wanted problem solved by treating with phosphatase I Vector insert ligation wanted forms colonies I Fragments self ligation concatemers formed 9 linear piece of repeating DNA no colonies formed I No ligation no colonies formed 0 Use of alkaline phosphatase will remove the vector selfligation product Alkaline phosphatase 0 Prevents vector selfligation from happening 0 Bacterial alkaline phosphatase BAP or calf intestine phosphatase CIP o Removes 5 phosphate groups I Normal vector insert ligation has 4 phosphates I In phosphatasetreated vector insert ligation two phosphates are missing I After ligation there are two nicks which will be repaired in E coli Restriction mapping 0 O 0 Used to check if the desired ligation product has been obtained Generate a map of restriction sites present in a vector or DNA fragment I Digest DNA with restriction enzymes either single enzyme or multiple enzymes I Use electrophoresis to size the fragments I Put fragments together like a jigsaw puzzle Compare new plasmid map with the original map of the unaltered plasmid Vectors O O A bacteriophage plasmid or other agent that transfers genetic material from one cell to another Usually plasmids or viruses that have been engineered to accept DNA insertions DNA fragment is ligated into an appropriate vector can be introduced into cells for propagation Cloning is the process of inserting DNA of interest into a suitable vector then establishing it as a stable part of a cell Vector Form Insert size Use Plasmid DS DNA Up to 15 kb CDNA llbfary subclon1ng Virus linear GenomiccDNA A phage DNA Up to 25 kb library Cosmid DS DNA 30 45 kb Bacterial BAC artificial 100 500 kb Genomic library chromosome Yeast artificial 250 1000 YAC chromosome kb o Bacteriophage A phage 485 kb I Cleave with restriction enzymes I Replace 20 kb phage fragment with 20 kb foreign DNA fragment ligate I Incorporate into virus capsules I Infect E coli host cells Cosmids I Combination of plasmid and phage behaves like plasmid but can take in larger fragments BAC I Circular in nature I Has all the characteristics of plasmid I Genes required to maintain a low copy number YAC I Behaves like chromosome becomes linear in yeast Usually bacterial artificial chromosome BAC and yeast artificial chromosome YAC used for human genomic library typical human gene has about 9 exons but biggest know gene has 178 exons distributed over 80 780 bp 9 plasmid vector is not adequate for genomic library Plasmids O O O 0 Closed circular DNA molecules Replicates independently of the bacterial chromosome Codes for functions involved in their own life cycles Copy number number of plasmids per cell varies from 1 2 to gt200 Size range from 2 kb to 100 kb 9 larger plasmids are more difficult to use easier to manipulate larger plasmids break more easily during manipulation Can be introduced into competent cells 9 competent cells are usually chemically treated so that efficiency of taking up the plasmids are high competent cell transfer will allow high efficiency Common characteristics I Origin of replication Stretch of DNA where DNA replication begins I Selectable marker eg antibiotics Plasmids produce enzymes to help cells break down antibiotics 9 encode resistance to antibiotic 9 only cells with the plasmid will be able to survive in antibiotic medium Usually dominant Necessary of maintaining the plasmid in the cell I Cloning sites Endonuclease cleavage site where foreign DNA fragments can be inserted without interfering with plasmids ability to replicate Antibiotics O 0 Chemicals that kill microorganisms but are relatively nontoxic to eukaryotic organisms Important for recombinant DNA methodology Genes encoding resistance to antibiotics on vectors allows us the ability to select for cells which have taken up the vectors Temperaturesensitive and are added when the temperature of media is below 50 C Alpha complementation O E coli Lac Z encodes for Bgalactosidase I Digests lactose to glucose galactose I Bgal can be divided into n and a fragment 9 need both fragments for activity I LacZ encodes Bgalactosidase I LacY encodes Bgalactoside permease I LacA encodes Bgalactoside transacetylase I LacI encodes a repressor that represses expression of lacZYA Part of lacZ encoding for the smaller enzyme fragment was put on pUC19 plasmid 9 part of Bgalactosidase is produced by plasmid which combines with the other fragment which is produced by the bacterial cell Cloning sites infused into pUC19 containing the part of lacZ and into the lacZ gene If no inserts present functional fragment will be produced by the part of the lacZ gene in the plasmid 9 inserts present will break up the lacZ gene and therefore produce no fragment pUC19 I Small plasmid 27 kb 9 small size can insert large fragments I Circular and nongenomic DNA I Has origin of replication I High copy number gt100cell I Ampicillin resistance gene degrades ampicillin I The lacZ a fragment is engineered into the plasmid Has MCS multiple cloning site with many unique restriction sites Introduction of this plasmid into strains with 1 fragment will result in a blue colony functional lacZ Insertion into any of restriction sites within the lacZ gene in the plasmid will results in a nonfunctional a fragment 9 recombinants will result in white colony Transformation o E coli cells are made competent by chemical treatment 0 Competent E coli cells are incubated with DNA on ice 0 Heat shocked 42 C 0 Selection for recombinants I Ampicillin kills all bacteria that lack the plasmid I XGal 5bromo4 cloro3indoylBDgalactoside a lactose analog which turns blue when split by Bgalactosidase I IPTG isopropylBD thiogalactopyranoside induces expression of lacZ 0 Selection of pUC19 recombinants I The transformation mixture is plated on LBagar plates with ampicillin and AGal Ampicillin prevents cells without plasmid from forming a colony Both plasmid vector and recombinant vector will support colony formation I Cells with functional lacZ will convert XGal colourless to a blue colour product Blue vector with no inserts White vector with inserts Shuttle vectors 0 Plasmids which can replicate in two different organisms two replication origins two of everything 0 Genetically engineered with two selectable markers and two replication origins I To check if insert is present Subcloning 0 Moving DNA of interest from one vector to another vector 0 Uses I Tag the protein of interest with epitope tags Eg add 6 histidine tags use antihistidine antibodies to do western blot I Express the protein in appropriate host Functional studies I Fuse the protein of interest with GFP etc Live cell imaging I Site directed mutagenesis Structure function analysis I Sequencing Cloning strategies 0 Two different cohesive ends two blunt ends Don t have to treat with phosphatase 9 two different enzymes will not selfligate 0 One cohesive and one blunt I 5 cohesive and 3 blunt I 5 blunt and 3 cohesive 0 Two same cohesive ends Treat with phosphatase to prevent selfligation Two possible orientations produced 0 Two blunt ends GENE AMPLIFICATION Polymerase Chain Reaction PCR is the InVitro amplification of DNA fragments o It requires two synthetic oligonucleotides one at each end of the fragments to act as primers Applications 0 Fast possible to perform with small samples 9 powerful technique with many uses 0 Forensic science DNA finger printing pathogen detection genetic diagnosis gene searching 9 PCR used to clone and increase the number of DNA fragments which can then be studied 0 Screen for inserts 0 Site directed mutagenesis random mutagenesis inframe deletions 9 PCR used to add remove nucleotides 0 Quantitative PCR 9 number of templates determine the number of cycles required to reach the exponential phase of emplification Reagents required 0 Buffers 0 Primers 0 Template DNA 0 dNTPs 0 DNA polymerase Procedure 0 Process of PCR repeated for 25 to 40 cycles to achieve desired amount of product 1 Denaturation of the doublestranded DNA 95 C 9 Disrupts the hydrogen bonding between complementary bases allowing for strand separation 2 Annealing ofthe DNA primers type of primer used determines the temperature required 9 length of primer and the sequence of the primers are taken into account 3 Extension of the nucleotide strands 72 C 9 synthesis of the complementary DNA strand by DNA polymerase Primer design 0 Specific primers yield specific products 0 Usually 20 30 nucleotides long 0 Avoid repetitive sequences within primer o 3 end nucleotide must base pair with template 9 ensure no ipping of primer off the template strand 0 Melting temperature Tm is midpoint in transition from D S to S S DNA 9 correct temperature to ensure that strands containing the primers can be split and replicated again I Tm number ofA and T x 2 C number of G and C x 4 C 0 Can be used to addfacilitate restriction sites for insertion of genes 9 add remove nucleotides in primers to facilitate restriction Polymerase o Heatstable polymerase required 9 ensure that no refilling of polymerase is required 0 Taq polymerase I From Thermus aquaticus I No proofreading activity no 3 to 5 exonuclease I 3 A o Tli polymerase Vent I From Thermococcus litoralis I Has proofreading activity exo I Blunt end 0 Pfu polymerase I From Pyrococcus furiosus I Has proofreading activity exo I Blunt end 0 Error probability Pfu lt Vent Taq Reverse transcriptasePCR RTPCR o Isolate mRNA 9 add primer 9 synthesise 1St strand of DNA 9 2nd primer added 9 PCR BIOMEMBRANES Provides an enclosure to cells Selective barrier water small polar molecules organic molecules can cross Allow for exchange of material Sits of biochemical reactions that include photosynthesis electron transfer oxidative phosphorylation Facilitates cell motion Provide cell recognition and cell fusion Cell membranes are dynamic uid structures and their molecules move Continuous lipid bilayer of 5 nm primarily phospholipids impermeable barrier to most water soluble molecules Transmembrane proteins mediate nearly all of the other functions of the membrane 30 of eukaryotic genes encode membrane proteins Phosphoglyceride molecule 0 Animal cell membrane 50 lipids by mass rest mostly proteins 0 Major lipids in cell membrane phospholipids gt sterols gt sphingolipids 0 Two fatty acids attached through ester bonds to adjacent carbon of glycerol 3rd carbon of glycerol attached to phosphate group I Typically one unsaturated and the other saturated 9 cis formed saturation allows for kink of tail allows loose arrangement in membrane uidity of membrane Major phospholipids in mammalian plasma membranes 0 O O O O Phosphatidylethanolamine Phosphatidylserine Phosphatidylcholine Sphingomyelin I Sphingosine fatty acyl tail phosphocholine Sphingosine I Long acyl chain with one amino and two hydroxyl groups Cholesterol I Amounts in plasma membrane gt golgi apparatus gt ER I Reduces membrane uidity at high temperatures interacting with hydrocarbon tails of phospholipid molecules increases Tm I Maintains membrane uidity at low temperatures helps prevent membranes from freezing Fluidity of a lipid bilayer depends on its composition 0 O O 0 Phase transition Tm liquid state to 2D rigid crystalline state or gel at a characteristic freezing point Sphingomyelin DECREASES membrane uidity Cholesterol acts as a bidirectional regulator Regulated by interactions of the lipids with each other varying lengths of I Fatty acid chain I Double bonds I Head group size Lipid microdomains and lipid rafts O O Thicker layer on membrane 9 proteins localized on lipid rafts Eg occurs when phosphatidylcholinesphingomyelincholesterol are at 111 ratio Asymmetry of the lipid bilayer is functionally important 9 phosphatidylserine is the only one with a negative charge negatively charged inner membrane cytosol side Signaling functions 0 O Extracellular signal triggers phosphorylation of inositol phospholipids in the cytosolic lea et of the plasma membrane 9 allows for docking of intracellular signaling protein to relay signal Extracellular signal docks in transmembrane receptor protein 9 activates phospholipase C which hydrolyses phosphorylated inositol phospholipid head group 9 head group of phospholipid becomes signal which relays and stimulates the release of Ca2 from the ER Signals include I Cell growth I Proliferation Glycolipids Differentiation Motility Survival Intracellular trafficking Stimulation of release of Ca2 from the ER 0 Present on the surface of all plasma membranes 0 May help protect membrane against harsh conditions 0 Allows for cell recognition and cellcell adhesion Membrane proteins 0 Includes Single a helix Multiple a helix Most transmembrane domains are ahelices as predicted using hydropathy plots Multipass proteins are synthesized in membrane then assembled 3 barrel Transmembrane channels a helix anchored protein Lipidanchored protein Lipid anchors control the membrane localization of some signaling proteins Myristoylation myristoyl anchor amide linkage between terminal amino group and myristic acid Palmitoylation palmitoyl anchor thioester linkage between cysteine and palmitic group Prenylation farnesyl anchor thioester linkage between cysteine and prenyl group Oligosaccharide linker eg GPI anchor Noncovalent interactions with other membrane proteins inner and outer leaf of plasma membrane 0 Many are glycosylated 0 Many diffuse in the plane of the membrane Shown by fusing a mouse cell and a human cell creating a heterocaryon and studying the protein diffusion using labeled antibodies against mouse and human membrane proteins Rate of lateral diffusion can be measured by photobleaching techniques Fluorescence Recovery After Photobleaching FRAP 9 instantaneous photobleaching and Fluorescence Loss In Photobleaching FLIP 9 continuous photobleaching 0 Proteins and lipids can be confined to specific domains within a membrane Methods Self assembling into large aggregates Interaction with macromolecules outside or inside the cell Interaction with proteins on the surface of another cell 0 Protein diffusion can be restricted by cortical cytoskeleton which also gives membranes mechanical strength MEMBRANE TRANSPORT Ion concentrations Components Intracellular concentration Extracellular concentration mM mM Na 5 15 145 K 140 5 Mg2 05 1 2 Ca2 104 1 2 H 7 X 10395 10 M or pH 72 4 x 105 1074 M or pH 74 3139 5 15 110 Membrane permeability o Lipid membrane contains hydrophobic interior 9 nonpermeable to charged polar molecules and neutral molecules that are gt150 Da 0 Permeability decreases hydrophobic molecules eg 02 CO2 N2 steroid hormones gt small uncharged polar molecules eg H20 urea glycerol gt large uncharged polar molecules eg glucose sucrose gt ions Transporters and channels 0 Each transports a particular class of molecules 0 Multipass membrane proteins eg aquaporin 9 hydrophilic environment within the protein 0 Defects in transport causes a variety of diseases eg cystinuria 9 transport defect of cystine cyscys from urine 0 Active transport is mediated by transporters coupled to an energy source eg ATP hydrolysis ion gradient 0 Driving force of passive transport facilitated diffusion transporter or channelmediated specific to molecules moving through can be concentration gradient or membrane potential 0 Conformational changes of the protein changes the affinity of various particles to the protein which in turn changes conformation of protein again allowing for release of substance 0 Active transport Ways to drive active transport Coupled transporter o Harvests energy from diffusing solute to allow for active transport via the same transporter ATPdriven pump 0 Phosphorylation of pump changes conformation 0 Types I Ptype 9 phosphorylation of phosphoprotein ATP to ADP I Ftype and Vtype 9 ATPase harvest energy from proton produces ATP I ABC transporter 9 ATP binding cassette Lightdriven pump Ion gradients o Uniport I Transportation of one type of solute selectively from one side of the membrane to another 0 Symport I Both transported and cotransported ion moving to the same side of the membrane I High to low concentration I Movement of one solute drives the movement of the other 0 Antiport I Similar to symport except that the substances move in opposite directions Na gradientdriven glucose transporter o Symport o Transporter allows 2 Na and 2 glucose molecules to bind 9 four bonded substances allow conformational change to transporter 9 transporter ips to allow for substances to be released 0 Against glucose gradient for Na gradient Ca2 pump 0 Ptype ATPase NaK pump 0 Establishes the Na gradient across the plasma membrane 0 3 Na out 2 K in o Ki gtgtgt Ko 0 Animal cell uses 13 of energy on this pump 0 10 ms cycle 0 Model I Binding of intracellular Na and phosphorylation I Transfer Na to extracellular I Binding of extracellular K and dephosphorylation I Transfer of K across membrane Vtype ATPase 0 0 Used to acidify a wide array of intracellular organelles maintains the low pH oflysosomes and other acidic vesicles pH 50 Uses ATP hydrolysis energy to pump H o Acidify compartments to promote ligand dissociation from receptor in endosomes o Activates lysosomal enzymes to facilitate degradation Ftype ATPase 0 Drives ATP synthesis by allowing the passive ux of H o Vtype ATPase is similar to Ftype ATPase 9 opposite hydrolyses ATP into ADP to allow for transportation of H against concentration gradient ABC transporters o Largest family of membrane transport proteins 0 2 ATP used to facilitate transport of molecules 0 Cystic Fibrosis CF 9 disorder for the protein cystic fibrosis transmembrane conductance regulator CFTR single residue deletion of phenylalanine at position 508 9 abnormal transport of chloride and sodium across epithelium leading to thick viscous secretions Ion channels 0 O Hydrophilic pores More efficient than carriers 105 times faster 9 no conformational change required Cannot be coupled to energy source passive Resting membrane potential due to K equilibrium potential caused by K leaking out of the cellquot through K channel 9 slight negative charge in the cell Function to allow specific inorganic ions to diffuse 9 Na K Ca2 Cl Essential in nerve cells Gating I Voltagegated I Ligandgated intracellular and extracellular ligand I Mechanically gated Eg bacterial K channel 9 exactly the size of the ion passing through conducts K 10 000 times more efficiently than Na Na entry is not energetically favoured because it is too small to interact with the edges of the channel ions are hydrated but get dehydrated before entry Families I Voltagegated cation channes Na o K o CaZ I Transmittergated ion channels Excitatory o Acetylcholinegated cation channels 0 Glutamategated Ca2 channels Inhibitory o GABAgated Cl39 channels 0 Glycinegated Cl39 channels Aquaporins O Permeable to water but impermeable to ions Passage of water molecules one at a time Water molecules rotate while going through channel to interact with the channel specific selection Neuron 0 000000 0 Receive conduct and transmit signals Up to 1 m long Cell body contains the nucleus Typically one long axon conducts signals away from the cell bdy Dendrites increase surface area for receiving signals Terminal branches of axon can pass message to many cells Voltagegated sodium channels generate action potentials 1 Action potential triggered by depolarization of plasma membrane 2 Voltagegated Na channels open 9 small amount of Na enters the cell gets depolarized more so more Na channels open selfamplification process or positive feedback From resting potential of 70 mV to 50 mV 4 Na channels have an automatically inactivating mechanism 9 channels close rapidly even though membrane depolarized 9 so that depolarization will not go in all directions ensures that signal can be passed inactivated channels so that the signal will not travel backwards 9 stays closed until membrane is repolarized Myelination I Formation of myelin sheath formed by specialized supporting cells called glial cells 9 ensures that current goes through without losing potential I Myelin sheath interrupted at regular intervals nodes of Ranvier 9 almost all Na channels concentrate in the region 9 reduces the amount of energy required to go from one neuron to the next I Action potential jumps from node to node saltatory conduction I Multiple sclerosis loss of myelin sheath 9 Voltagegated cation channels 0 O Evolutionarily and structurally related 9 highly conserved Mutations within genes of ion channels can cause a variety of diseases I Eg epilepsy genetic mutation in Na or K channel in brain resulting in excessive synchronized firing of neurons Transmittedgated ion channels 0 O 0 Convert chemical signals into electrical ones at chemical synapses Target ligand gated Neuromuscular transmission I Nerve impulse 9 open voltagegated Ca2 channel 9 Ca2 ows into nerve terminal 9 vesical fusion I Acetylcholine released 9 binds acetylcholine receptor 9 transiently opening cation channel 9 Na in ux I Local depolarization 9 opens voltagegated Na channels 9 more Na entering 9 more depolarization I General depolarization of muscle plasma membrane 9 activates voltagegated Ca2 channel I Transiently opens Ca2gated Ca2 release channel 9 Ca2 in cytosol increases 9 myofibrils contract INTRACELLULAR COMPARTMENTS AND PROTEIN SORTING Organelles serve as intracellular compartmentalization of eukaryotic cells 0 Plasma membrane is too small to accommodate all functions 0 Increases membrane area 0 Specialized functionally distinct o Membrane enclosed 9 allows for different pH and classes of enzymes within the cells eg lysosome 0 Major compartments I Cytosol I Nucleus DNARNA synthesis transcription I Endosome I Peroxisome free radical metabolism I Smooth ER lipid synthesis I Rough ER protein synthesis I Lysosome I Golgi apparatus protein modifications I Mitochondrion energy metabolism I Plasma membrane I Chloroplast photosynthesis 0 Amounts of each organelle present in the cell differs with the type ofceH Signal sequences direct proteins to the correct cell addressquot 0 Sequence can vary between proteins 9 note pattern of signal 0 Sequence amino acids can be separated 0 Typically found at the Nterminus 9 removed by signal peptidases after translocation into appropriate organelle 0 Internal stretch signals can be recognized by signal patch Import into nucleus ProProLysLysLysArgLysVal 5 positively charged residues Export from nucleus LeuAlaLeuLysLeuAlaGlyLeuAspIle Hydrophobic residues Import into H3NMetLeuSerLeuArgGlnSerIleArgPhePheLysPro mitochondria AlaThrArgThrLeuCysSerSerArgTyrLeuLeu Between 10 and 70 aa Nterminal signal Hydrophobic net charge positive Import into plastid H3NMetValAlaMetAlaMetAlaSerLeuGlnSerSerMet SerSerLeuSerLeuSerSerAsnSerPheLeuGlyGlnPro LeuSerProIleThrLeuSerProPheLeuGlnGly Import into SerLysLeuC0039 peroxisomes Specific Cterminal signal does not change Import into ER H3NMetMetSerPheValSerLeuLeuLeuValGlyIleLeu PheTrpAlaThrGluAlaGluGlnLeuThrLysCysGluVal PheGln Mostly Nterminal can be internal Return to ER LysAspGluLeuC0039 Cterminal retention signal 9 ensures that proteins returned to the ER stays in the ER Transport of molecules between nucleus and the cytosol o Nucleus I Nuclear envelope Encloses DNA 2 concentric membranes Inner and outer membrane continuous with distinct protein compositions Outer membrane continuous with ER I Nuclear lamina binds to DNA and membrane 9 keeps DNA together and close to the nuclear envelope I Nuclear pore complexes NPC 3000 to 4000 per nucleus 1000 proteins together to form Gated diffusion barrier o Aqueous channel of 9 x 15 nm but can dilate up to 26 nm 0 The larger the size of protein the increase in difficulty of movement through channel 9 proteins gt 60 000 require a carrier 0 Nuclear localization signal NLS I Type I short stretch of basic amino acids I Type II bipartite 9 two clusters of basic amino acids separated by 10 amino acids 9 comes together when folded to allow for recognition 0 Nuclear import receptors I Bind to both nuclear localization signals and NPC proteins I Bind both cargo proteins and nucleoporins I Bind to FGrepeats present in fibrils and many nucleoporins I Recognizes cargo by signal I All proteins found in nucleus are first synthesized in cytosol then transported to nucleus I Nuclear export identical but reverse 9 nuclear export signal contains 4 hydrophobic residues 0 RAN dependent nuclear transport I RanGAP Ran GTPase activating protein 9 localized in cytosol hydrolyses GTP to GDP I RanGEF Ran guanidine exchange factor 9 localized in nucleus bound to chromatin exchanges RanGDP for Ran GTP I Nuclear import receptor proteins are exible binds either RanGTP or cargo and moves to fit the cargo I Nuclear import nuclear proteins RanGTP has a higher binding affinity to receptor as compared to the cargo 9 receptor releases cargo and does not allow cargo to bind back Nuclear import receptor binds to protein with nuclear localization signal Complex enters nucleus through NPC FG repeats RanGEF binds RanGTP receptor cargo is released Receptor and RanGTP leaves nucleus RanGAP hydrolyses RanGTP to RanGDP RanGDP dissociates from receptor and receptor is recycled I Nuclear export RNA molecules and ribosomal subunits RanGDP and cargo both binds to nuclear export receptor Receptor leaves nucleus via NPC moves to cytosol through FG repeats RanGTP is hydrolysed into RanGDP cargo is released into the cytosol Receptor moves back into the nucleus 0 Control of nuclear import during T cell activation I Regulates transcription of gene 1 High Ca2 in activated Tcells 2 Nuclear factor of activated Tcells NFAT is dephosphorylated by calcineurin protein phosphatase in the cytosol 9 nuclear import signal is exposed NFAT travels into nucleus activates gene transcription 4 When no more gene transcription required low Ca2 in resting Tcells releases calcineurin from factor and phosphorylates NFAT 9 hides import signals exposes export signal 5 NFAT exits nucleus 0 During mitosis I Lamins are phosphorylated 9 depolymerize and form small stretches I NPC proteins bind nuclear import and export receptors 9 helps with the reassembly and reformation of nucleus at the end of mitosis Translocation into mitochondria o Depends on signal sequences and protein translocators I Differentiates between outer and inner membrane I Proteins can be targeted at membranes at intermembrane space or at matrix 9 complex sorting required 0 Mitochondrial precursor proteins are imported as unfolded polypeptide chains 0 Mitochondrial membrane complexes I TOM Translocase of the Outer Mitochondrial membrane t 914990 9 Required for the import of all nuclearencoded mitochondrial proteins some proteins are encoded in the mitochondria Central proteinconducting channel 25 nm TIM Translocase of the Inner Mitochondrial membrane TIM23 complex transport preproteins into matrix TIM22 complex mediates the integration of carrier proteins integrates membrane proteins into the inner membrane OXA Oxidase Assembly Insertion of mitochondrialencoded protein insertion into the inner membrane Insertion of protein imported into the matrix by TOMTIM into the inner membrane SAM Sorting and Assembly Machinery Specific Assembly of Bbarrel proteins on outer membrane 0 Import steps rPSNNtquot Molecular chaperones of the Hsp70 family keep preproteins unfolded Signal recognition and docking Insertion into TOMTIM Translocation Maturation includes cleavage of signal peptide by peptidase 0 Energy in protein import into mitochondrial matrix space 1 Cytosolic Hsp70 release required hydrolysis by ATP 9 insertion of signal sequence into TOM 9 insertion of polypeptide 9 signal sequence interacts with TIM Signal sequence translocates into matrix 9 requires membrane potential across inner mitochondrial membrane energy used to generate membrane potential to pump protons to the intermembrane space mHsp7O binds to the entering polypeptide requires ATP hydrolysis 9 process pulls protein through the translocation channel prevents protein from sliding back out 0 Protein import from the cytosol into the inner mitochondrial membrane and inner membrane space Signal sequence cleaved after entry of inner membrane proteins into the intermembrane space 9 exposes stop transfer sequencequot which is embedded in membrane protein stays embedded in the inner membrane OR Signal sequence cleaved 9 full translocation of protein into matrix space 9 second signal sequence recognized by OXA complex 9 insertion of protein into inner membrane by the OXA complex OR targeting protein goes to TIM22 complex 9 TIM22 helps form inner membrane protein Peroxisomes o Specialized for carrying out oxidative reactions using molecular oxygen 0 Peroxisomal precursor vesicle buds off from ER with proteins 9 growth occurs by uptake of specific peroxisomal proteins and lipids from cytosol o All proteins imported in a similar way to ER Import signal SKL at Cterminus 23 proteins form the peroxins to translocate peroxisomal proteins Endoplasmic reticulum o Structurally and functionally diverse Ca2 storage uptake and release primary cellular calcium store Detoxification reactions SER Lipid biosynthesis phospholipids cholesterol glycosphingolipids Protein biosynthesis ribosomes 0 Protein translocation 30 or proteins are targeted to the ER signal sequence at Nterminus or internal 5 10 critical HYDROPHOBIC amino acids Cotranslational Ribosomes synthesize translocation signal first Translation and translocation occurs at the same time Posttranslational Synthesized proteins in cytosol are bound to Hsp70 chaperones Proteins are bound to membrane and folded Modifications o Glycosylation o Disulfide bonds 0 Cistrans proline isomerization 0 Stages in targeting of nascent proteins to the ER 1 Nterminus signal sequence is recognized by the Signal Recognition Particle SRP 9 stops translation until SRP binds to SRP receptor SRP binds to SRP receptor on ER membrane SRP helps target the ribosome to the translocon pore ribosomes have to be associated to translocon in order to be associated with ER SRP is released translation resumes ribosome binds to translocon nascent chain enters translocon Nascent polypeptide is translocated in the ER lumen and signal is removed by signal peptidase Signal stays in the membrane but moves away from translocon it is rapidly degraded Sec61 complex aqueous pore in the translocator translocon that allows polypeptide chain to pass through I Usually tightly closed with a plug so that concentrations are constant in the cell 9 regulation allows only proteins to pass through and not other particles I Growing polypeptide chain displaces plug Import of single pass protein I Start and stoptransfer sequences are present in the protein stoptransfer sequence forms transmembrane domain and moves away from the translocon I Continual synthesis of protein moves ribosome away from the membrane I Hsp70 used to prevent folding before completion of translocation Import of multipass proteins I Combinations of start and stoptransfer signals determine the topology of multipass transmembrane proteins Role of Nlinked glycosylation in ER protein folding I Proteins that are not properly folded are recognized by glucose transferases 9 proteins are altered such that only one glucose molecule is present bound to the protein I Calnexin recognizes protein with one sugar folds the proteins properly Protein quality control I Misfolded proteins are exported for degradation and activates the unfolded protein response UPR I No glycans are present in cytosol only in lumen or extracellular space 9 only single sugars I ERAssociated Protein Degradation ERAD Consumes energy when going through cycle 9 chaperones required 9 may affect other proteins 1 Retrotranslocation of misfolded proteins occur 2 Misfolded proteins are marked with ubiquitin 9 signature for proteasome recognition 3 Proteasome degrades the misfolded proteins I UPR is turned on when large accumulation of unfolded proteins are detected Activates genes to increase protein folding capacity of ER Sensors for misfolded proteins on the ER membrane 0 IRE1 I Phosphorylation splices regulatory proteins I Regulated mRNA splicing initiates translation of gene regulatory protein 1 o PERK I Phosphorylation inactivates translation initiation factor 9 reduces amount of proteins entering the ER I Initiates selective translation of gene regulatory protein 2 o ATF6 I Releases regulated proteolysis 9 produces gene regulatory protein 3 Secretion endocytosis and vesicle transport 0 O 0 Specific 9 requires energy Use of different coats in vesicular traffic I Cage structure of coat forces membrane to fuse and bud 9 provides energy for vesicles to bud out and move to targeted regions I ER to golgi anterograde 9 COPII coat Golgi to ER retrograde 9 COPI coat Plasma membrane to lysosome 9 clathrin coat Tethering of a vesicle to a target membrane I TransSNARE complex dependent Present on membrane and on vesicle 9 specific targeted region of fusion Removes water 9 hydrophobic fusion of membranes CYTOSKELETON Internal network of protein filaments Three type of filaments OOOO Microfilaments actin polymer 7 nm Intermediate filaments IF proteins 10 nm Microtubules tubulin polymer 25 nm Maintain cell shape Mechanical strength 9 intermediate filament Cell motility eg immune system sperm 9 good for immune cells not good for cancerous cells metastasizing result in cell migration to other organs Organelle motility Intracellular transport eg vesicular transport Cell polarity eg long cells have different ends Cell division pull chromosomes to daughter cells pinch off cytoplasm in the centre quotPolymersquot of small subunits 0 Not true polymers 9 cytoskeleton are made of noncovalent interactions so that the quotpolymerquot can be disassembled and assembled easily Easy diffusion Rapid reorganization 0 Because movement of filament subunits are required in order to change the direction of movement Multiple protofilaments stacked together allow for thermally stable filaments Actin cytoskeleton o Filaments are concentrated at the cell cortex 0 Used for I Cell shape I Cell movement I Muscle contraction 0 Diameter 7 9 nm 0 Actin monomers globular I Gactinquot I Activated myosin I 43 kDA globular protein I Highly conserved between species 90 amino acid homology 9 essential function I Actin homologues found in bacteria 9 actinlike molecules expression of bacterial filaments in yeast will form filament functions I ATP holds the two lobes of the actin monomer together 0 Actin polymerization I Phases 1 Nucleation lag phase 0 Trimer of Gactin required to provide for the nucleus for polymerization 0 Because dimer of Gactin is not stable 9 can fall apart easily 2 Elongation growth phase 0 Helical polymer is stabilized by multiple contacts 3 Steady state equilibrium phase 0 Growth and loss is the same 9 length does not change much I ATP is hydrolysed to ADP after assembly Rate of subunit addition is faster than hydrolysis 9 appearance of ATP capquot I Treadmilling Net assembly at plus end Net disassembly at minus end Can be observed by uorescently labeling some molecules of Gactin 9 labeled molecules will move from one end to another I Polarity Visualized using myosin heads which use the actin as a track Pointed end of myosin heads show minus end slow growing end barbed end correspond to plus end fast growing end o Actin polymerization measurement methods I Fluorescence enhancement of pyrene conjugates G 9 F actin transition increases uorescence allows researcher to see the polymer action I DNAse inhibition assays Gactin inhibits DNAse but Factin does not I Viscosity measurements Factin is filamentous 9 increases the Viscosity I Spindown assays o Actin arrays Factin will exist in the pellet Gactin remains suspended Compare the amounts in solution I Stress fiber Allows cell to contract Contractile bundle with actin filaments in opposite directions I Cell cortex Gellike network Haphazard arrangement of filaments I Filopodium Fingerlike projections for cells to sense the environment Tight parallel bundles in the same direction 0 Actin binding proteins ABPs I Characteristic actin filaments are determined by ABPs 9 structure of the actin filaments are different depending on the type of ABPs I ABPs include End blocking proteins eg Capz Cross linking proteins eg filamin Filament severing proteins eg Gelsolin Actin filament depolymerizing proteins eg ADF Membrane binding proteins eg ERM I Thymosin 34 Small protein 5 kDa Forms 11 complex with Gactin Maintains a pool of unpolymerised actin 9 binds to Gactin Actin in locked state actinthymosin complex cannot associate with or end of the filament 9 cannot hydrolyse or exchange bound nucleotide Increase in thymosin may promote Factin depolymerisation I Profilin Profilamentous actin profilactin profilin 16 kDa 11 complex with Gactin Binds to the face opposite to nucleotide binding cleft Alters the conformation of Gactin to promote ADPATP exchange 9 generates ATP actin does not stabilize Promotes the formation of filamentous actin 9 removes actin from thymosin Competes with thymosin for binding to Gactin and promotes plus end growth Can be bound to PIP2 on cell membrane 9 ATP bound to profilin bound to PIP2 stays on membrane regulation of actin polymerization allows profilin to be released to compete with thymosin 9 causes Factin assembly I Arp2 3 complex Actin related protein 7protein complex Nucleates actin filament Factor brings Arp2 and Arp3 together to dimerise 9 nucleus forms when one actin binds to dimer Bypasses lag phase 9 actin goes into exponential growth immediately Complex side binding forms branches in the actin filament Regulated by las17 gene which binds and activates Arp2 3 complex which regulates actin formation but no las17 gene does not mean no actin formation I WiskottAldrich Syndrome Protein Xlinked genetic disorder WASP required for the proper function of hematopoietic cells 9 immunodeficiency occurs when defective protein is present due to mutation in genes Activates Arp2 3 complex WASPWIP WASPWASP interactive protein I Capping proteins Plus end capping protein stabilizes plus end 9 binds to plus end and stops growth 0 Growth only allowed at minus end9 slow polymerization 0 Critical concentration of monomers increases by a lot Minus end capping protein is Arp2 3 complex I Actin bundling proteins Crosslink actin filaments into parallel array Two Factin binding sites to allow for crosslink Spacing determines type of structure 0 Eg Villin o Fimbrin 14 nm I Tight packing of parallel bundles prevent myosinII from entering bundle o aactinin 30 nm I Loose packing allows myosinII to enter bundle Actin bundles support membrane projections o Microvillus 9 fingerlike projections to increase surface area for absorption I Actin filament bundle present in microvillus I Narrow space between bundles allow for strength of projections due to movement of liquids around the microvillus I Gel forming proteins Crosslink actin filament into a weblike mesh Have two Factin binding sites Filamin large angle Spectrin red blood cells 2 a and 3 subunits 9 gives RBC mechanical support changes shape of RBC to allow for movement in narrow capillaries without tearing I Severing proteins Increases the number of plus and minus ends 9 allow for faster growth Regulates length of polymer No energy required Binds to two different sites on the actin subunit remains attached to the plus end and serves as cap Gelsolin can be removed Used in platelet activation 9 fast growth of Factin results in clotting I ERM proteins Ezrin Radixin and Moesin Links actin filaments to plasma membrane Cterminal domain binds to side of actin filament N terminal domain binds to cytoplasmic tail of integral membrane proteins 9 links Factin to integral membrane proteins Loss of Merlin moesinezrinradixin related protein leads to neurofibromatosis 9 benign human genetic disease Attachment between actin filament and ECM serve as anchors for cells 9 not whole cell stick to surfaces reduces contact area between cell and surface 0 Actin cytoskeleton regulation I Rho family of GTPase regulate the actin cytoskeleton Act as molecular switches 9 GTP or GDP bound Guanine nucleotide exchange factors GEF o Promotes dissociation of GDP and uptake of GTP 9 make active Guanine nucleotide dissociation inhibitor GDI o Prevents GDP from becoming GTP 9 inhibit activation GTPase activating proteins 0 Accelerates GTPase activity so that GTP becomes GDP I Extracellular signals induce cytoskeletal changes Activates different Rho GTPases 9 arrangements of filaments depend on GTPase type activated which decides subsequent cell activity Rho stress fibers increase Rac lamellipodia increase 9 actin at edges Cdc42 identified using S cerevisiae during genetic screens filopodia increase 9 fine projections 0 Cell polarity I Most cells are polarized so that proteins supposed to be at a particular position will be at the correct location 9 allow for cell function I 2 or more domains I Cells use cytoskeletal filaments to generate and maintain cell polarity I Eg epithelial cells have projection sides facing blood vessels 9 more nutrients I Eg nerve cells I Use of S cerevisiae to analyze cell polarity 9 budding yeast Observe actin patches and cables Most actin patches found in new daughter cell Only when daughter and mother cells are of similar size patches will appear at both ends 9 before division patches move to the centre between the two cells I Haploid yeast cells form shmoo 9 polarization of yeast cells in response to pheromone When a and or cells are put next to each other they secrete pheromones which identifies sides activating signaling pathways 0 Activation of cdc42 9 WASP 9 Arp2 3 complex 9 polymerization of actin to form structures I Tcell polarization When localized signal from target cell to be killed is detected localized actin polymerization occurs in both cells Polymerized actin is used by microtubules as tracks to reorient 9 microtubules used to release factor to kill target Reorganization of cell so as to ensure that the factor released is targeted specifically at target cell and not at cells around the Tcell Phagocytosis 2 possible mechanism 0 Zipper mechanism host cell tries to spread over the bacterium 9 takes up the bacteria 0 Trigger mechanism bacterium injects effector molecules into the host cell 9 activate Rho GTPase actin forms projections ingest bacteria I Listeria monocytogenes 9 intracellular pathogen I Protects itself from other immune cells by going into eukaryotic cells membrane recognized as its ownquot I Uptake by zipper mechanism 9 hemolysin secretion which causes destruction of membrane of phagosome 9 can move around in cell 9 bacterial release and replication Actin polymerization driven movement Express ActA on cell surface not uniform 9 used to polymerise actin to propel itself ActA binds and activates Arp2 3 comples Arp2 3 nucleates new actin filaments Actin polymerization propels listeria Moves directly from cell to cell 9 not exposed to host immune system OR shigella exneri recruits WASP to activate Arp2 3 to drive its movement 0 Microfilament specific poisons Binds to either G or Factin Stabilizes or destabilizes Factin Drives the reaction towards the form to which the toxin binds Phalloidin binds Factin and stabilizes it Latrunculin binds Gactin and stabilizes it o Myosin biggest group of ABP 17 types of myosin All have head neck and tail domains Head domain 9 ATPase also called a motor domain Head domain mediates binding to actin tail domain mediates binding to other structures Head domain is highly conserved as all myosin heads bind to the same Factin structures Tail domain of myosin is not conserved 9 varies between type of binding to specific structures Evidence of motor activity of myosins 1 Myosin heads attached to glass slide 2 Labeled Factin added 3 ATP added Actin filaments move at 4 pm per second 9 myosin serves as molecular motors Plus end motor 9 walks towards the plus end only myosin VI move to the minus end Functions Used to transport vesicles using Factin as tracks Contractions in muscles Attaches to membrane to move filaments Pulls filaments into a new orientation Myosin 11 Two heads two heavy chains four light chains 2 essential light chains ELC 2 regulatory light chains RLC Inactive state 9 tail domain folded on itself Active state 9 phosphorylated RLC assembly into bipolar structures 0 Myosin light chain kinase MLCK phosphorylate the RLC to activate the myosin o Muscles 9 highly specialized contractile machines Skeletal muscle Cells fuse together to form multinucleated structures 9 long cytoplasm Smooth muscle Not multinucleated Cardiac muscle Muscle contraction Motor proteins use energy from ATP to force large movement Rapid contraction Myofibril consist of tiny contractile units sarcomeres Initiated by sudden rise in cytosolic Ca2 signal from nerve terminal 9 T tubules 9 sarcoplasmic reticulum storage for calcium 9 opening of Ca2 channel 9 contraction of myofibril o Ca2 exposes myosin binding sites 9 myosin binding sites on actin originally blocked by tropomyosin relieves blockage by binding troponin c Sarcomere organization Titin 3 MDa muscle protein keeps thick filament at the centre Tropomodulin minus end capping protein Capz plus end capping protein Nebulin binds 200 actin monomers Thick myosin and thin actin filaments slide past one another without shortening o Organelle movement I Melanosomes move along actin cables Gives colour of skin Myosin V associated with surface of melanosomes 9 visualized by antibodies labeled with uorescence that binds to myosin V I mRNA transport Cytoskeleton used to localize RNA as well as proteins 9 more economical to transport RNA than proteins as RNA can be used to make more proteins To determine the fate of sister or daughter cells Eg haploid yeast cells a and a o Ash1 is a repressor of mating switch 9 prevents daughter cell from being able to switch between the mating types 0 Ash1 mRNA is transported to daughter by myosin V 9 translation in daughter cell 0 She2 protein binds to mRNA She3 binds to She2 and She3 binds to myosin V I Acrosome reaction in echinoderm sperm Echinoderm sea urchin 9 thick outer jelly layer covering the mature oocyte protects eggs from the environment Actin polymerization leads to the formation of acrosomal process which pierces the jelly layer and provides passage for the sperm nucleus microvilli like structure for nucleus to be transported for egg fertilization o Contractile ring formed by actin and myosin I Myosin II is required for cytokinesis Loss of myosin 11 results in unicellular mutinucleate cells I Almost all myosin and actin cytoskeleton is at middle of cell where cell is being pinched off 0 Crawling of cells I Majority of eukaryote cells crawl sperms swim prokaryotes swim I Three activities 1 Protrusion o Actin polymerization at plus end protrudes lamellipodium 2 Attachment 0 Focal contact contains integrins 9 cell raised slightly above surface 3 Traction I Three types of protrusion Filopodia 0 Also known as microspikes 0 Similar to microvilli but longer 0 Bundled actin filaments Lamelliopodia o Sheetlike structure 0 Crosslinked mesh of actin filaments o Plane parallel to substrum Pseudopodia o Stubby three dimensional projection 0 Filled with actin filament gel I Model for protrusion at leading edge Arp2 3 complex promotes actin polymerization at the leading edge Net filament disassembly behind the leading edge net filament assembly at the leading edge 9 number of actin molecules is the same recycled Gactin depolymerized Factin behind leading edge and moved to front to form Factin Cofilin action 0 Actin binding protein present in all eukaryotic cells 0 Also called actin depolymerizing factor ADF Binds both G and Factin o Promotes severing and dissociation of ADP actin old actinquot o Helps depolymerize Factin away from the leading edge disassembles older filaments to Gactin 0 Not at leading edge I Adhesion and traction Protruded cell structure adheres to the substratum Adhesion is reversible Uses same molecular machinery as focal contact If protruded lamellipodia fails to stick it is carried backward as ruf e I Polarization and chemotaxis Cell locomotion requires polarization 9 orientation of cell is required to move in specific direction Chemotaxis cell movement along chemical 0 gradient 0 Chemical binds to receptors on cell membrane 0 Reorganization in cytoskeleton occurs to allow for reorientation Both require cytoskeleton Microtubules MT 0 Functions 0 FtsZ Cell movements 9 generate force cilia and agella Vesicle and organelle transport 9 using the MT as tracks Chromosome segregation during mitosis Bacterial homologue of tubulin Also found in chloroplasts 9 reason to suggest that chloroplasts were taken in by eukaryotes Forms protofilaments and rings similar to tubulin Dynamin 9 GTPase which forms ring around endocytic vesicle o Stiff hollow cylinders Long tubular polymer of tubulin subunits 9 a tubulin and B tubulin heterodimer a tubulin binds only GTP 9 catalytically inactive B tubulin binds both GTP and GDP 9 catalytically active y tubulin binds GTP Polarity like microfilaments Headtotail assembly of aB dimers end B tubulin end a tubulin 13 parallel protofilaments Alternating a and B tubulins o Polymerization Similar to actin polymerization Microtubules are very dynamic GTP hydrolysis associated with polymerization Plus ends grow faster than minus end Nucleation growth and equilibrium phase Treadmilling Microtubules nucleation y tubulin ring complex nucleates 9 polymerization of microtubule Single y tubulin ring complex causes polymerization of microtubule from itself Growth at plus end Preferential at plus end GTP is hydrolysed to GDP soon after assembly Rate of subunit addition is faster than hydrolysis 9 GTP Capquot Isolated cilium incubated with tubulin subunits provided with proper conditions show that growth at one end is faster than the other end 0 Fast growth at end 0 Slow growth at end Dynamic instability Fluorescence used to visualize filaments 9 growth of filaments happen in the same medium at captured at different time points to show difference in rates of growth and shrink Individual microtubules grow and shrink at different rates in the cell GTP hydrolysis throughout the cycle occurs 1 Assembly 9 microtubule length increases 2 Catastrophe 9 disassembly starts 3 Rescue 9 start point of growth of microtubule Loss of GTP cap leads to dynamic instability 9 all subunits have GDP only a tubulin have GTP 0 GDP tubulin exposed when GTP cap is lost 0 Rescue occurs when GTP cap is regained 9 rapid growth occurs with the GTPcapped end 0 Microtubule organizing centre MTOC Structure that organizes microtubules 9 also called centrosome Usually found adjacent to the nucleus Microtubules emanate in star like astral conformation 9 nucleating sites y tubulin ring complexes Minus end at the MTOC plus end pointing away towards the cell periphery Pair of centrioles exist in the centrosome matrix Usually found only in animal cells 9 plant cells have no centrioles Hypothesis centrioles are only present in cells that need to move Organize the centrosome matrix percentriolar material 9 not true for plant cells Ensures duplication during each cell cycle responsible for the formation of the mitotic spindles Centrioles are made of microtubules 9 sets of triplets 1 complete and 2 incomplete 9 number of protofilaments of each triplet will not give 39 some protofilaments are shared Centreseeking behavior Single centrosome placed in a square plastic well with tubulin subunits will have MT push against the wall of well 9 equal pushing on all directions will mean that the centrosome is in the middle of the well Microtubule array can find the centre of the cell 0 Cut part of cell that contains MTs but no centrosome o Severed cell fragments will have reorganized MTs with an MTOC lacking centrioles 0 ER and Golgi apparatus rely on MT for integrity I ER and golgi use MT as a track for proper distribution I Golgi fragments while ER collapse to centre when cells are treated with drugs that destroy MT 0 MTspecific poisons I Mitotic spindle is labile 9 easily liable to change I Inhibits formation of mitotic spindle I Antimitotic drugs will kill dividing cells preferentially 9 potential anticancer drug Faster dividing cells will be targeted more compared to slower dividing cells 9 not only cancerous cells will be killed by the drug Actinspecific drugs Phalloidin Binds and stabilizes filaments Latrunculin Binds subunits and prevents their polymerization Cytochalasin Caps filament plus ends Swinholide Severs filaments Microtubulespecific drugs Taxol Binds and stabilizes microtubules Nocodazole Colchicine colcemid Binds subunits and prevents their polymerization Vinblastine vincristine I Taxol Yew tree bark Binds to MT and stabilize them 9 prevents disassembly of MT cells eventually dies without being able to disassemble their MT Arrests cells in mitosis Binds B tubulin I Colichine Meadow saffron Binds irreversibly to B tubulin Prevents polymerization Destabilize polymers I Nocodazole Binds subunits Prevents polymerization o Microtubule Associated Proteins MAPs I 3 classes of MAPs Structural MAPs 0 Promote assembly or disassembly 0 Connect to ME or IE cell polarity or cell shape Motor proteins 0 Slide along MT kinesin plus end and dynein minus end o Specialized directions of travel 0 Membrane traffic Signaling molecules 0 Assembly 0 Monitors if microtubules are assembled properly I Microtubule assembly modulators Katanin severs filaments Unknown caps filaments 9 maybe no cap dynamic property Stathmin binds tubulin dimer y tubulin nucleates filaments Tau MAP1A MAP1B MAPZa MAPZb MAPZc MAP4 Taxol binds to filament wall stabilizes filament I Structural MAPs Abundant in neurons where stabilized MT bundles form core At least one domain which binds have multiple tubulin binding motifs Stabilizes microtubules 9 results in longer less dynamic microtubules Accelerate nucleation Aberrant filamentous assembly of tau 9 hallmark of diseases such as Alzheimer s o Overexpressed MAPZ will result in regularly spaced microtubules longarmed o Shortarmed tau will result in filaments close together I Stathmin Monomer sequester protein 1 mol of stathmin bind two tubulin heterodimers 9 reduces numer of subunits available for polarization after sequestering Help to maintain pool of subunits Phosphorylation of stathmin frees subunits I Kinesin First isolated from squid giant axon 1985 9 transport along microtubules which require a lot of kinesin Motor proteins that move along microtubules Move vesicles and organelles over microtubules 0 Eg cell body to synapse Similar to myosin Several types A tetramer 9 two identical heavy chains two identical light chains Structure 0 Pair of globular heads ATPase force generation 9 conserved sequence 0 A stalk o A tail cthelical which can dimerise binds to cargo 9 sequence not conserved to allow for binding to different cargoes Moves on MTs very processive movement 9 moves for long distance without detaching from MT VS myosin o Kinesin s motor heads act together 0 Microtubule binding releases ADP from head ATP replaces 9 allows kinesin to throw second head forward onto binding site of MT 9 releases ATP from first head 0 In myosin motor heads act independently ADP and phosphate on motor heads binds weakly phosphate released when head binds to actin 9 stored energy released from ATP hydrolysis Kinesin motility assay 1 Purified kinesin attached to glass slide 2 Add labeled MTs and ATP 0 Can observe the movement of microtubules o A single molecule moves at 800 nms under ideal conditions I Dynein Motor protein that power the cilia and the agella of eukaryotic cells Stick out in a clockwise manner to generate force to drive movement Contains an ATPase EM reveals 3 globular attached to a common base by a stalk Minus end motor Several types Carries vesicles I Cilia and agella Swimming organelles o Flagella wavelike motion 0 Cilia a fast power stroke followed by a slow recovery stroke Same structure cilia 9 more per cell and shorter agella 9 few per cell and longer eg sperm Comprised of microtubules 9 2 and associated proteins eg dynein Basal body at the base of cilia and agella MTs radiate from basal body Similar in structure to centrioles Cilium 0 Central core called the axoneme o Surrounded by plasma membrane 0 Axoneme 9 2 I Peripheral doublet I Complete A microtubule 13 I Incomplete B microtubule 10 I central pair of single tubes 13 I Cilia motion Sliding of adjacent MT doublets relative to one another Dyneins probably generate the motion by walking along adjacent MT MT crosslinked so cannot slide past each other Outcome bending I Regular melanosome movement Rapid change in colour Melanosome can change location in cell in response to stimuli Kinesin to cell periphery Dynein to cell centre I Vesicular transport Vesicles transported along MT from cell body dendrite Multiple motor proteins are associated with membrane vesicles 0 Cytosolic dyneins o Cytosolic kinesins o Spindle kinesins KRPs Intermediate filaments 0 00000 Function physical strength 9 not required in every cell IF prominent in cells subject to mechanical stress No polarityquot No nucleotide bound to subunits No motor proteins Mechanism of assembly and disassembly phosphorylation Substantial sequence variation I Fibrous long subunits strong lateral contacts I Central ahelical coiled coil rod highly conserved sequence I Nonhelical globular heads and tails variable Monomer 9 dimer 9 tetramer 9 two tetramers two IF molecules form parallel heterodimer Selfassembles into a tetramer structure Span the entire cytoplasm and are anchored to the plasma membrane enable cells to withstand mechanical stress when cells are stretched Keratin I Type I acidic type II neutralbasic Form heterodimers one basic one acidic Most diverse IF 30 types 20 in epithelial cells 10 specific hair and nails Single cell with multiple forms Used to diagnose carcinomas epithelial cancers Mutation in keratin Epidermolysis bullosa simplex Mutants express a mutant gene encoding a truncated keratin lacking both N and Cterminal domains Defective protein assembles with the normal keratins and thereby disrupts the keratin filament Mutants blister easily 0 Neurofilaments type IV Found in high concentrations along the axons of vertebrate neurons 3 types NFL NFM NFH coassemble NFL plus one of the others NFM and NFH have lengthy Cterminal tail domains Side projections help organize the fibers Neurofilaments appear after completion of growth of the nerve cell MT predominant in growing axons NF in mature axons MT remain necessary for transport function 0 Vimentinlike filaments Can form homo polymers Vimentin is widely distributed fibroblasts etc Desmin 53 kDa is expressed in skeletal cardiac and smooth muscle localized in the Zdisk region Mice lacking desmin have initial normal muscle development adults have a variety of muscle cell abnormalities IF MT MF for ligaments bones and muscle of cell 0 Nuclear lamins type V Lamins A B and C Filamentous proteins lining the inner membrane of the nucleus Longer central rod domain Contain nuclear transport signal Support for nuclear envelope Anchorage for chromosomes and nuclear pores More dynamic 9 phosphorylation for disassembly 0 Cell junctions Intermediate filament link adhesive structures Desmosomes cadherincadherin interaction between cells Hemidesmosome adhesive molecule such as integrin molecule in the cell link to extracellular matrix ECM Actin cytoskeletonlinked adhesive structures I Adherens junction cadherincadherin interaction between cells I Focal adhesions adhesive molecule such as integrin molecules in the cell links to ECM MODEL ORGANISMS Life science research aims O Characterize the molecules of life 9 know what a protein does in what cell and the context Identify genes involved in a given process How these genes and proteins work at a cellular and molecular level How these genes and proteins activities are regulated 9 different molecules in different people results in different responses to a treatment method Methods to characterize gene function 0 O O Phenotype physical or biochemical characteristics of an organism Forward genetics start with a mutant phenotype and subsequently identify the gene mutated 9 induce mutation by UV or by chemicals 1 Decide on process to study 9 study disruption of the process Identify mutant phenotype associated with process Mutagenize the organism Screen mutants for desired phenotype Determine nature of mutation eg recessive dominant determine cloning method Define the gene linkage position of gene with respect to others 7 Put the function in a pathway 9 different mutations occur in sequence 8 Map and clone the genes 9 Sequence the gene predict amino acid sequence based on homology to other genes 10 Determine function of protein Reverse genetics start with a gene of known sequence modify its activity to reveal its function when placed in the context of the organism 914990 9 1 Identify proteins and corresponding genes through molecular biology Genome sequencing Interaction with a protein Altered expression during development Altered expression during disease state Etc 2 Disrupt gene function 3 Analyze phenotype 4 Determine biochemical function of protein Why study model organisms O O O O 0 Ethical reasons cannot test on humans Cost space cost of maintenance 1 test to 1 control Speed reproduction speed Ease of handling smaller organisms are easier to handle Genetics organism with a lot of offspring 9 study gene linkage Requirements for model system 0 O 0 Easy to handle large number of individuals statistical purpose Short generation time Sexual life cycle and genetic cross possible 9 crossing of mutants to determine sequence of gene function required Small genome and easy in cloning gene with known mutations Stable genetic transformation available I After introduction of wild type gene to mutant the ability of the mutant to repair mutation with wild type genes will mean that that is the only mutation and not other mutations are present Model organisms O O O O O O Escherichia coli prokaryote wellcharacterised Saccharomyces cerevisiae Caenorhabditis elegans Drosophila melanogaster Danio rerio zebrafish Mus musculus house mouse Relevance of model 0 O 0 Cell theory cells are the basic units of life 9 processes are more or less conserved Cellular processes are often conserved eg cell division which is highly conserved Some of these processes are found in unicellular organisms Study the problem in a system that is well suited eg cell cycle of yeast is same as that of humans Learn how the process is regulated in a simple system Test if the same mechanism is also utilized in similar processes in other systems Mutations types and causes 0 O Genotype is its entire set of genes and may denote whether an individual carries mutations in one or more genes Phenotype is its function and physical appearance and depends on that individuals genotype Wild type organisms may develop mutations in their DNA sequence thereby altering their genotype and perhaps their phenotype Organisms that develops such mutations are termed mutants Haploid and diploids O Haploid organisms one copy of each chromosome and its phenotype is a consequence of that one copy O O O O Diploid organisms two copies of each chromosome and thus two copies of each gene I Two copies may be the same or different 9 changes may or may not affect function Alleles different forms of each gene Homozygous diploids with identical alleles Heterozygous diploids with different alleles Isolation and analysis of mutants O 0 Procedures used to identify and isolate mutants are termed genetic screensquot Screen used depends on whether the organism is haploid or diploid I Diploid screen used also depends on whether the mutation is dominant or recessive Dominant mutation o Leads to gain of function although they occasionally may lead to a loss of function 0 First filial generation F1 will have half of the offspring mutated Recessive mutation 0 Lead to a loss of function 0 First filial generation F1 will have normal phenotype o Crossing of F1 with F1 will have second filial generation F2 containing 11 mutants I Haploid screen often involve temperaturesensitive mutants with proteins functional at one temperature 9 with one copy of gene some gene functions may be loss with the essential genes present cell therefore behaves differently at different temperatures Yeast as a model organism O 0 000000000 0000 Nonpathogenic don t need to worry about it causing diseases Has all the advantage of bacterial genetics eukaryotic but can carry out all the same experiments as e coli Unicellular eukaryotic Extensive molecular biology tools Stable diploid and haploid states Easy mating of haploid cells to generate diploid cells Efficient transformation by exogenous DNA Efficient homologous recombination Easy to GFP tag proteins Easy to alter gene expression at will Closed mitosis 9 nuclear membrane does not break down human cells have open mitosis 9 nuclear membrane breaks down during mitosis and then forms again Easy to study cell biology Easy to carry out biochemical studies grows in large numbers Genome sequenced Yeast genetics Saccharomyces cerevisiae budding yeast unicellular eukaryote Three tyes of cell Two haploid mating types a and a One diploid mating type produced with mating between the haploid mating types High degree of conservation with higher eukaryotes Cell cycle Signal transduction pathways Secretion Endocytosis Cytoskeleton DNA replication Recombination Etc 0 Advantages of using yeast In isolation of recessive mutations Dominant gene is always expressed when present Dominant mutations o Phenotype detectable in diploids 0 Usually gainoffunctionquot mutation Recessive mutations o Phenotype not detectable in diploids quotcarriersquot 0 Frequently lossof functionquot mutations o Difficult to isolate in diploid organisms Temperature sensitive for function mutations 0 Isolation of mutants 1 2 Cell undergoes mutagenesis Introduce starving conditions so that cell undergoes sporulation Carry out tetrad dissection use a microscope and needle to differentiate and separate the two mating types Mate a mutant with a wild type to check for dominant and recessive mutants If recessive mate mutants with each other Test diploids for gene function secretion If no secretion denotes mutation secretion in a diploid cell shows mutation is dominant step 4 If no secretion mutation is recessive After test for recessive during mating between two mutants if mutation is in different gene secretion will occur 9 wild type dominant allele pairs with mutant recessive allele if mutation is in same gene secretion will not occur 9 recessive mutant alleles pair with each other 0 Putting gene activity in a pathway Staining for protein to see where the protein is accumulated Determine direction of sequence 9 which protein works first using a double mutant o Cloning by complementation 1 Isolate mutants in haploid strain 2 Mutation recessive or dominant Mate to haploid wild type Assess phenotype of diploid I Recessive mutation transform mutant strain with library from wild type yeast screen for wild type phenotype I Dominant mutation transform wild type yeast with library from mutant strain screen for mutant phenotype 0 Transformation in yeast I Yeast plasmids Integrative plasmids for introducing a gene into a yeast chromosome Centrometic plasmids contain a yeast centromere and are low copy 9 suspected toxic genes don t want to over express Episomal plasmids called 2 micron from the 2 micron circles seen in some yeast strains high copy vectors in yeast 20 to 100 copies per cell Easy recovery of plasmids from yeast from library to identify and sequence gene that allow organism to survive I Methods Liacetate 0 Up to 107 transformantsmg DNA 0 Simple easy and cheap Spheroplast 0 104 transformantsmg DNA 0 Have to digest cell wall with zymolyase Electroporation 0 105 transformantsmg DNA 0 Need expensive equipment I Gene disruption Easy to disrupt a gene of known sequence homologous PCR primer designed to disrupt gene of known interest Phenotype can be observed in haploids Easy tagging system as visualization of protein localization can add GFP tag I Expression of proteins in yeast Eukaryotic cells will do modifications to genes 9 cannot use yeast cells to modify human genes as different organisms will modify splice genes differently Prokaryotic cells express all genes 9 no introns I Inducible and constitutive promoters Constitutive promoters are always active 0 ADH1 alcohol dehydrogenase I o PGK 3phosphoglycerate kinase 0 1 each of total yeast mRNA Inducible used when gene expressed is suspected to be toxic 9 induce only after plasmid that can break down toxin is introduced 0 GAL1 GAL10 repressed by glucose induced by galactose o PHOS induced by inorganic phosphate 0 Gene expression increase 10 30 fold I Over expression 1 Purification of protein for biochemical studies obtain large amounts to carry out enzymatic assays 2 Look for phenotype expression of human genes in yeast result in growth at specific conditions due to over expression 0 Growth at high or low temperature 0 Growth in high or low salt When there is overexpressed human genes in yeast phenotype due to 0 Act on the substrate at the wrong time 0 Act on other similar substrates 0 Act on completely different substrate 0 Break a protein complex 0 Etc Over expression studies 0 Use inducible promoters 0 Introduce into cells 0 Turn on the gene and look for phenotype 0 Over expression disruption of protein complexes 9 loss of function will result in observed phenotype 0 Have to bind to the same gene in order for mutants to be rescued 0 Contributions of yeast to life sciences I Cell cycle I Cytokinesis I Check point I Secretion I Endocytosis I Vesicular protein sorting I DNA replication I Signal transduction I Cytoskeleton I Cell polarity I Etc 0 S pombe VS s cerevisiae I S pombe fission yeast as model organism to identify genes essential for cell division 9 more similar to human cells Because s cerevisiae divides by budding 9 no G2 phase Mammalian cells in culture 0 Disadvantages Slow growth 24 h Expensive media Expensive equipment incubator to provide for specific temperature Adherent culture so that mammalian cells can stick on surface to grow on it Most plasmids not maintained plasmid gets lost after cell divides Two copies of most genes Gene families present need to disrupt all genes to observe phenotype 0 Advantages Mammalian cells relevant information Respond to treatment Single type of cells eg liver Many cellular activities can be studied Cells can differentiate All the genes are there mechanisms for control Other model organisms in development 0 Xenopuslaevis O Chick Mouse Development is independent Poor genetics cannot do crossing Available Surgical manipulation required In vitro culture Poor genetics cannot do crossing Good genetics Development is in utero cannot look at development Drosophila Great genetics Great development C elegans Less than 1000 cells Transparent C elegans ideal model organism 0 Small free living soil nematode animal multicellular with different organs Transparent and has no eyes Two sexes O O 0 Self fertilizing hermaphrodites XX and males X0 Single hermaphrodite produces 300 progeny Rapid isolation of mutants 959 cells of which 300 neurons 81 muscle cells Genome size of 97 Mb 6 chromosomes small 000000 0 0 19 000 protein coding genes Easy to maintain large numbers of animals Simple growth media agar plate and bacteria Rapid development and good genetics 3 days between generations Transparent embryo GFP tag will allow to observe where protein goes realtime observation of cells Laser ablation I Use laser to destroy a single cell in early embryo I Identify all cells missing after ablation I Lineage determination I Identify cells which cannot develop normally in absence of descendants of ablated cells evidence of cellcell interactions Lineage of every cell in body is known Phenotypic screens Molecular biology of C elegans I RNAi RNA interference double stranded RNA sequence same as mRNA of gene to be disrupted 9 binds to RNA of particular gene destroys mRNA 9 no protein I Microinjection of antisense RNA I Dominant negative mutants I Mutant interferes with function of normal gene I Methods to knock down gene expression RNAi siRNA small interfering RNA contains two RNA strands sense and antisense shRNA short hairpin RNA same as siRNA except the strands are linked by RNA hairpin siRNP small interfering ribonucleoprotein RISC RNA induced silencing complex Contribution of C elegans to biology I Development of the body plan I Cell lineages and cell differentiation I Formation of the nervous system I Programmed cell death ced genes Zebrafish as a model organism O O 0 00000000 Life cycle well documented Transparent embryo development Vertebrate suitable for some experiments as compared to C elegans and drosophila Prolific produces a lot of offspring Rapid development External fertilization and development Simple system Identifiable stereotyped neurons Available genetics Visualizing GFP tagged proteins Transplantation experiments 0 Over expression by injecting mRNA 0 Rescue of mutants by injecting mRNA 0 Antisense oligo s to modify expression to knock down genes Mammalian development 0 Monkey needs a lot of genes difficult to maintain o Mice Availability of hundreds of singlegene mutations Short generation time Small thousands in a small room Large litter gt 8 Breed readily in captivity Very docile and easy to handle Mammal almost all the same genes as humans with similar sequences Use of mice in experiments 0 Knockout mouse Disruption of gene completely deletion Conditional deletion Possible outcomes Embryonic lethal no offspring deletion of essential gene offspring dies in embryonic stage Still born Dies after birth No phenotype normal possibility of more than one gene redundant disrupted gene or nonimportant disrupted gene 0 Generate NWASPloxploxp Mate female NWASPKOWT and male NWASPKOWT Possible outcomes of crossing homologous wild type homologous knockout heterozygous 9 112 o NWASP KO mouse WASP KO mouse is viable with immune defects NWASP KO mice embryonic lethal 8 10 liter Timed mating Isolate the embryo and determine genotype Embryos die at E11 9 gene deleted is important at this stage not necessarily important at adult stage 0 NWASP conditional knockout knockout gene in specific cells depending on tissue 1 991990 Electroporate 129sv ES cell chromosome in 129sv ES cells from agouti colour mice Select NeoR clones Neo is the selectable marker Check for integration in correct locus Inject into blastocyst of C57BL 6 Get chimeras cells from C57BL 6 and 129sv cell Check for germline transmission ES cells reproductive cells Obtain mice with NWASPlOXPWT genotype transgenic 9 Cross with mice expression p to remove NeoR 9 Self cross NWASPlOXPWT mice to get NWASPloxploxp mice homozygous mice I Generate NWASP OXPIOXPpromotercre enzyme controls deletion of gene 9 promoter expressed tissue specific I Cross NWASPIOXPIOXP with NWASPIOXPWT 4 types of gametes 9 NWASPIOXP Nestin cre NWASPWT Nestincre NWASP10XP NWASPWT to get promotercre mice I NWASPlOXPIOXP Nestincre will result in delection of N WASP in brain I Back calculation if sex of mice is not important 1 in 4 mice will have the genotype if only interested in either sex 1 in 8 mice will have the genotype CELL CYCLE Cell growth and division is the cornerstone of biology millions of cells die every second Lifespan of cells growth of cells will stop at the end of lifespan other than cancerous cells Fusion of two cells egg and sperm 9 adult human 1013 cells Understanding cell cycle can allow us to come up with effective strategies to fight cancer to control cell proliferation Cell theory 0 1838 Schleiden and Schwann I Every living organism is composed of one or more cells I New cells arise only by the division of preexisting cells One exception viruses 9 can proliferate in vitro does not come from viruses divide in living organisms Main jobs of the cell cycle 0 Transmit the genetic information accurately mistakes will lead to the death of the cell 0 Maintain normal ploidy ie diploid in humans eg extra chromosome 21 leads to downs syndrome Purpose of cell division 0 Growth 0 Development 0 Repair 0 Reproduction Under optimal conditions 0 E coli divide every 30 mins 0 Yeast cells divide every 2 hours 0 Human cells divide every 24 hours 0 Adult liver cells do not normally divide cells will only divivde if part of the liver is removed Nerve cells never divide 9 they are removed and differentiated from cell division results in paralysis after injury 0 Red blood cells do not divide 9 no nucleus 0 Before 1950s it is observed that there is lack of chromosomal activity between cell divisions 9 chromosomes that are not condensed cannot be observed 9 resting state interphase Experimental systems for cell cycle studies 0 Genetics to identify mutants I S cerevisiae I Schizosaccharomyces pombe 0 Biochemistry isolation of proteins I Arbacia punctulata sea urchin I Xenopus laevis Mitosis nuclear division 9 condensation of chromosome separation etc Cytokinesis cytoplasmic division G1 Gap 1 S phase DNA synthesis G2 Gap 2 GO resting or differentiated 0 Cycle of duplication and division cell cycle 0 M phase mitosis and cytokinesis 0 Period between two M phases is called interphase Events in interphase 0 Protein and RNA synthesis 9 increase in size otherwise daughter cells will be smaller than parent cell 0 Continuous replication of mitochondria daughters have to have the same organelle o Replication of DNA in the nucleus Sphase 0 Monitoring size readiness to enter Sphase or mitosis G1 and G2 phases checkpoints Restriction point START G1 0 Point at which cell is irreversibly committed to traversing the cell cycle I Mammals restriction point Yeast START 0 Cell cycle proceeds without in uence from environment 9 only stopped by damage 0 Late in G1 0 Cell fusion experiment I Find out essential events of cell division 9 in uence of one nucleus product over the other I Two cells can be made to fuse to form heterokaryons 9 ensure synchronized state of cells all cells in one tube are at G1 or S state etc I S G1 9 G1 nucleus enters S phase immediately S phase nucleus continues DNA replication S phase cytoplasm contains factors that drive a G1 nucleus directly into DNA synthesis I G2 S 9 G2 nucleus stays in G2 S phase nucleus continues DNA replication G2 nucleus have already replicated its DNA there is refractory not responsive to these factors I G1 G2 9 G2 nucleus stays in G2 G1 nucleus enters S according to its own timetable G1 not driven into synthesis cytoplasmic factors for DNA replication that were present in the S phase cell disappear when the cell moves from S to G2 I Factor ensures G2 does not go back to S phase 9 if G2 in uenced by factor in S phase cell will have 4 or more copies of DNA instead of 2 Discovery of cyclin O O 35S methionine to label newly synthesized proteins 9 aliquots pulled out at different times and analyzed for protein content Ribonucleotide reductase used as a loading control to ensure accuracy of results Cyclin was found to increase in the fertilized eggs just before cell division Maturation Promoting Factor MPF discovery 0 O Xenopus egg divides very rapidly every 30 mins multicellular organism obtained in 1 2 days Oocyte grows for months accumulating nutrients Assay for MPF Masui 1971 I Transfer cytoplasm from egg arrested in metaphase 11 into G2arrested oocyte I No need for progesterone in order to make egg leave G2 arrested state MPF activity peaks before each cell division 9 MPF has kinase activity transfers phosphate from ATP to a protein 9 protein kinase MPF contains two parts one of which is cyclin Cell cycle in a cellfree system 0 O 0 Can add or remove things easily Pure cytoplasm from activated xenopus eggs mix with sperm nuclei and ATP energy source Sperm nuclei decondense and then go through repeated cycles of DNA replication and mitosis Cultured mammalian cells 0 Not easy to observe individual cells in intact mammals 0 Use cells from normal or tumors and grow in plastic dishes 9 normal cells will stop dividing after a limited number of division cycles due to shortening telomeres etc o Mutations or expression of viral proteins can lead to immortalized cell lines 0 Cells from tumors are generally immortalized 9 divides in continuous manner M phase 0 Divided into 6 stages 1 Prophase 2 Prometaphase 3 Metaphase 4 Anaphase 5 Telophase 6 Cytokinesis o Separates and segregates the chromosomes 9 separation and segregation must be coordinated separation at the right place must be observed 0 Chromatids and chromosomes I Chromatin DNA associated proteins I Chromatin condense into chromosome I After replication duplicated chromosome when attached are called chromatids 9 sister chromatids daughter chromosomes I Chromosome condensation To prevent entanglement and to allow fit into cells Average human chromosome 130 Mbp45mm Mitotic spindle 10 20 um Need to condense 10 000 fold DNA wrapped around histones 9 nucleosomes reduces length of DNA by 7 fold Further compaction by forming solenoidal fibre Condensin 5 subunit large protein complex 9 uses energy from ATP hydrolysis to drive the coiling of each interphase chromosome one condensing per 10 000 nucleotides Condensation allows for ease of separation while decondensation allows for replication transcription and translation I Cohesins Holds sister chromatids together Multisubunit protein complex Crucial to segregation Broken only late in mitosis o Mitochondria division I Mitochondria is a dynamic organelly I mtDNA replication is not limited to S phase generally nuclear DNA replication is limited to S phase 9 mtDNA replication is independent to nuclear DNA replication I Dynaminlike protein is responsible for fission 9 fission and fusion are dynamic for replication and increasing of mass 0 Nuclear envelope golgi and ER I Golgi stack and ER undergoes extensive fragmentation during mitosis to partition into daughter cells mitosis specific reactions catalyze the process of fragmentation I Vesicles reassemble to form nuclear membrane ER and golgi after daughter cells form I Nuclear membrane remodeling occurs during mitosis 9 higher order eukaryotes undergo open mitosis o Prophase I Inside nucleus Replicated chromosomes condense I Outside the nucleus Mitotic spindle assembles between two centrosome Centrosomes move apart towards the pole Prometaphase I Centrosome at spindle pole I Breakdown of nuclear envelope to allow for capturing of chromosomes at kinetochore by mitotic spindles Yeast no nuclear membrane breakdown spindles in nuclear membrane nucleus migrated to neck area Metaphase I Chromosomes are aligned at the equator of the spindle midway between the spindle poles allow equal share of chromosomes to daughter cells I Kinetochore microtubules attach sister chromatids to opposite poles of the spindle I Checkpoint present Anaphase I Sister chromatids synchronously separate to form two daughter chromosomes and each is pulled slowly toward the spindle pole it faces I Kinetochore microtubules get shorter I Spindle poles move apart 9 initially spindle pole positions are fixed Telophase I Chromosomes arrive at the spindle poles and decondense I A new nuclear envelope reassembles around each set of chromosomes completing the formation of each nuclei and marking the end of mitosis Cytokinesis I A contractile ring made of actin and myosin filaments is assembled 9 late telophase I Cytoplasm is divided by the contractile ring which pinches the cell in two Cytoskeletal machines perform both mitosis and cytokinesis in animal cells I Mitosis by a bipolar mitotic spindle and motor proteins Microtubules of the mitotic spindle capture chromosomes and move them to ends of cells I Cytokinesis by a contractile ring made up of actin and myosin Central spindle used to form contractile ring Mitosis before cytokinesis I Two mechanisms Cell cycle system that activates proteins required for mitosis is thought to inactivate some of the proteins required for cytokinesis 9 cytokinesis occurs when Mcdk is inactivated at the end of mitosis Residual central region of the spindle is required to maintain a functional contractile ring 9 cytokinesis occurs after spindle has separated chromosomes and formed a central spindle o Centriole replication Centrosome pair of centriole and amorphous material 9 centrosome matrix or pericentriolar material matrix contains yTuRC responsible for nucleating microtubules and other proteins Principle MTOC in animal cells centrosome Duplication mechanism unknown Centrosome cycle 1 At G1 phase the pair move apart a few pm At S phase daughter centriole begins to grow At G2 phase growth completed At M phase the two pairs split and move apart Each parental centriole dictates assembly of the other corresponding centriole nepow o Mitotic spindle Made up of microtubule and associated proteins Assembly and function depend on motor proteins kinesin related proteins KRP and dynein Pull the chromosomes toward poles and move the poles apart Three classes of microtubules Astral generate force to separate poles orientate and position spindle moves towards the membrane 9 pulls poles toward membrane to orientate and position spindle Kinetochore attach chromosome to spindle separates sister chromatids Interpolar bipolar shape of spindle forms shape no contact with chromosomes Microtubule instability increases at M phase Dynamic instability Rapid assembly and disassembly of microtubules increases at M phase Due to increase in catastrophes and phosphorylation by Mcdk motor proteins 9 phosphorylation causes dynamic instability MAPs 9 stabilize microtubules ensure catastrophes do not go too high Mitotic spindle assembly and function Random microtubules Interact at overlap zone KRPs crosslink the MT Pushes the centrosome 9 motor proteins try to quotwalkquot on microtubules 9 pushes centrosomes apart to move towards the poles Minus end motors help form the foci plus end motors help form overlap zone and slide MT past each other both act together to form the spindle Length of spindle balance between and end motor protein 9 increasing minus end motor protein Kar3p results in short spindles increasing plus end protein Cin8p results in long spindles o Kinetochore and kinetochore microtubule Prometaphase nuclear envelope breakdown due to phosphorylation of nuclear lamina by Mcdk Microtubules can capture the condensed chromosomes Kinetochore complex protein assembled at the centromere Capture of kinetochores by microtubules Microtubule from centrosome grow towards chromosome Microtubules that attach to a chromosome become stabilized 9 no more catastrophe The microtubule eventually end up attached to kinetochore Opposing forces drive the chromosome to metaphase plate Microtubule with chromosome arm pushes away from pole while kinetochore moves towards pole Experimentally determined by cutting the chromosome arm with laser 0 Chromosomes at metaphase plate Chromosomes attached on both sides bipolar attachment Chromosomes are tugged back and forth Eventually chromosomes assume a position equidistant between the two poles at the metaphase plate They oscillate gently until signal to separate 0 Checkpoints 2 major checkpoints function in mitosis At entry into mitosis G2 M checkpoint At the metaphase to anaphase transition metaphase checkpoint Metaphase checkpoint monitors the attachment of the mitotic spindle to kinetochores and the tension generated by mitotic spindle attachment In the presence of a single unattached kinetochore metaphase checkpoint halts separation of sister chromatids and thereby provides additional time for spindle attachment 9 metaphase checkpoint ensures a high fidelity of chromosome separation and prevents aneuploidy extra chromosomes in one and missing chromosomes in the other during mitosis Unattached kinetochores or kinetochores that are attached wrongly will be released and reattached until all tensions are sensed for the all clear to transit into anaphase o Anaphase promoting complex APC Activated by mitotic cdk complex MPF Marks proteins for degradation 9 ubiquitin ligase Anaphase inhibitor Cyclin in mitotic cdk complex MPF 9 cytokinesis can take place after degradation Allows for sister chromatid separation and segregation o Chromosome separation Separase protease cleaves cohesin destroying the link between sister chromatids Separase is kept inactive by securin APC targets securin for degradation in order to activate separase Cells regulate anaphase entry by delaying securin ubiquitination until all chromosomes have attached to the mitotic spindle After degradation of securin separase cleaves cohesin releasing the cohesin linkage o Anaphase Anaphase A Depends on motor proteins operating at the kinetochore Together with depolymerisation of kinetochore microtubules o ATPdriven motor protein drives both chromosome movement and microtubule disassembly o Microtubule disassembly drives chromosome movement Pull the daughter chromosomes toward to nearest pole Without movement of the spindles Anaphase B Spindle poles move apart Due to elongation and sliding of the overlap microtubules past one another and the outward force exerted by astral microtubules Pulls spindles toward plasma membrane Decreases in overlap of microtubules o Plane of animal division Mitotic spindle specifies location of the contractile ring Contractile ring forms in the plane of the metaphase plate at right angle to long axis of mitotic spindle This ensures that division between two sets of chromosomes Use of the glass bead to show that astral stimulation occurs as division occurs even though there are no chromosomes 0 Asymmetric cell division To produce two cells that differ in size contents or both The mother segregate fate determinantsquot found only in one daughter cell input by the mother cell Spindle has to be moved to make daughter cells of different size Eg stem cells where division occurs to produce one remaining stem cell and the other maturing to become another cell type Spindle rotation to achieve specific pattern of cleavage 9 minus end motors try to move towards the pole but they are immobilized thus causing the spindle pole to come towards them Astral microtubules captured by sites on plasma membrane Ensures that the two daughter cells are still in contact with the same cells that the mother was in contact with 9 cell to cell contact is important 0 Spindle pole body SPB Yeast MTOC is called the SPB On the nuclear membrane Inner and outer plaque 9 microtubules inside and outside the nucleus respectively After duplication moves along nuclear membrane to opposite poles Spindle orientation Kar9 localized to the bud captures EB 1 microtubule capping protein ensuring microtubules are in correct orientation 0 Cytokinesis Cytoplasmic division contractile ring made of actin and myosin In plants No contractile ring semirigid cell wall 9 cannot cause pinching effect Cytokinesis by the formation of new cell wall called cell plate 9 membrane vesicles fuse to form plasma membrane at the centre cell wall forms after 0 Mitosis without cytokinesis Interphase Multiple rounds of nuclear division without cytoplasmic division Eg first 13 nuclear divisions in drosophila embryo to create syncytium cellularisation then occurs coordinated cytokinesis 0 Cells increase in size 0 Organelles are replicated o Centrosome is duplicated 0 DNA is replicated Prokaryote cell division 0 Binary fission O Chromosome circular Single oriC origin of replication Bacteria can divide twice in the time it takes to complete DNA replication 9 replicate each daughter chromosome before completion of the first replication MinC and MinD enzymes in bacteria work together to inhibit cell division I Enzymes oscillates from pole to pole I Therefore middle part of the cell is where time spent is the lowest 9 enzyme for making new cell wall is most active at middle where protein activity is lowers I Min E inhibit MinC D complex at the middle Evolution of chromosome segregation mechanisms 0 O O Bacteria I Daughter chromosomes segregate by means of their replication origin I Separated by the ingrowth of plasma membrane between them Yeasts and diatoms I Nuclear envelope remains intact I Spindle microtubules form inside the nucleus I Nucleated by SPB and associated with the nuclear envelope I A single kinetochore microtubule attaches each chromosome to a pole Animals I Spindle begins to form outside the nucleus I At prometaphase nuclear envelope breaks down to allow chromosomes to capture spindle microtubules which now become kinetochore microtubules Life cycle of higher and some lower eukaryotes o Diploid phase predominates o Haploid phase short 0 Haploid cells specialized for sexual fusion 0 Two types of haploid cells one large nonmotile ovum one small motile spermatozoon 0 Animal cell proliferation by mitosis Meiosis o Chromosomes autosomal and sex chromosomes 0 Diploid organism two similar versions of each chromosome similar but not identical DNA sequence one from each parent 9 homologs o Replicated chromosomes tightly linked and called sister chromatids o Mitosis DNA replication followed by a single cell division Meiosis DNA replication followed by two cell divisions 0 First cell division of meiosis I Replicated chromosomes pair with the replicated homolog forming a structure called a bivalent containing 4 chromatids Pairing mediated by complementary DNA base pair interactions Haploid number of chromosomes diploid amount of DNA 9 2 chromosomes but they are exactly the same 0 Second cell division of meiosis Meiosis I Does not produce haploid cells Each daughter inherit two copies of one homolog Two copies identical except where genetic recombination has taken place Meiosis 11 Produce haploid gametes Each daughter inherit a copy of one homolog Chromosomes fail to separate normally into four haploid cells nondisjunction Some haploids lack a chromosome while others have more than one Eg downs syndrome 3 copies of chromosome 21 after fusion 0 Genome reshuf ing Independent assortment of maternal and paternal homologs during meiosis I 2n genetically different gametes where n is the haploid number 9 human n 23 Crossing over during prophase I exchanges segment of homologous chromosomes and thereby reassorts genes on individual chromosomes On average 2 to 3 crossovers on each pair of chromosomes Recombinations occur between DNA molecules that have sequence homology Enzymatic essential process Important role in the creation of genetic diversity and in the repair of DNA damage Characteristics of genes 0 Types Essential at all conditions 9 lack of which will result in cell death Essential under some condition 9 cells die under certain conditions Nonessential at all conditions 9 possibly due to gene families different or copies of similar gene function present 0 Ts mutant strain Ts for function mutation leads to a protein that is functional at permissive temperature but not at the restrictive temperature 9 due to 1 amino acid change OR mutation in a gene that is only essential under some condition thus the strain becomes temperature sensitive for growth Isolation of tS39 mutants in haploid yeast 1 Mutagenize a culture with chemical or UV mutagen I Titration has to be used 9 cells will die with too much mutagen or over exposure to UV have to find correct concentration dosage where cells can survive 2 Plate them on nutrient plate and incubate at permissive temperature Make replicas and put them at two temperatures 4 Find the difference between the two plates 9 mutations that are temperature sensitive does not allow cells to grow at 36 C but growth occurs at 23 C Cdc VS noncdc mutants o Cdc cell division cycle 0 Cdc mutants all cells will arrest at the point where the protein s function is needed 0 Noncdc mutant expect a diverse range of phenotypes o Ts cdc mutants I After allowing cells to grow at 23 C I Shift cell plates to 36 C I Since ts is confirmed 9 cells will have ts and cdc mutations I At 36 C protein is unable to function I If cells are cdc mutants all cells in the culture will look the same 9 protein is required at one stage of the cell therefore all cells will stop at that point 0 Cdc28 is used throughout the cell cycle 9 mutations of specific points of the protein at particular amino acids lead to arrest at specific stages Libraries 0 To obtain mutated gene I Use GENOMIC library where we want to use yeast mutant to clone yeast gene I Use cDNA library when we want to use yeast mutant to identify human gene Isolation of s cerevisiae cdc genes 0 After addition of plasmid with cdc28 gene if at 35 C the cells in colony are observed to be at various cell cycle stages we isolate the plasmid to find the sequence mutated I Plasmid gene is same as mutated gene 9 complementation occurs and the cells are able to grow at nonpermissive temperatures 0 If no colony is formed it can be concluded that there is no growth at 35 C 9 gene in plasmid is different from mutated gene in chromosome 9 therefore the yeast cells cannot grow and divide S pombe cdc mutants o Cdc 9 arrests cell cycle but the cells keep growing phenotype of super long cells 0 Wee 9 cell cycle accelerates cell becomes small because the cells are not allowed to grow to the right size before division 9 O Mitotic catastrophes 9 over accelerates nucleus gets cut cytokinesis starts before mitosis is completed Checkpoint 9 quotcutquot divides with incomplete spindles or replication cytokinesis in the absence of normal nuclear division Rereplication 9 repeats S phase without an intervening M Isolation of human cdc2 1 2 0000 S pombe cdc2 gene was isolated functionally by transforming a plasmid library of fission yeast genes into a ts cdc2 mutant strain Transform the s pombe cdc2 mutant with a human cDNA library and look for genes which complemented the mutant Human cdc2 had 63 identity with s pombe cdc2 Human cdc2 is functionally equivalent to yeast cdc2 Human cdc2 s pombe cdc2 s cerevisiae cdc 28 Mechanism controlling the onset of mitosis is conserved from yeast to humans human gene is able to perform the same function as yeast Cell cycle regulatory proteins 0 O Consists of a serine threonine protein kinase cyclin dependent kinase 9 kinase but requires cyclin to function associated with a cyclin I Kinase adds phosphate group on other proteins on their serine or threonine Activity of cyclincdk is in uenced by phosphorylation of the cdk subunit the binding of inhibitory proteins and proteolysis of cyclin The kinase associates with different cyclins at different phase of the cell cycle MPF 9 cdk cyclin Regulation of cell cycle regulatory proteins 0 O O O Cyclical changes in cyclins Phosphorylation of cdk subunits inhibitory and activation I Activation of cdkcyclin by phosphorylation Without cyclin cdk cannot function as a kinase Cdk when bound to cyclin is only partially active Phosphorylation of cde by CAK cdk activating kinase results in full activation I Inhibitory phosphorylation Active cyclincdk is turned off when the wee1 kinase phosphorylates two closely spaced sites 9 wee1 delays entry into next cell cycle stage removal of these phosphates by cdc25 phosphatase results in activation of cyclincdk Binding of inhibitory subunits I Inhibition of cyclincdk by CKI Binding of cdk inhibitory proteins CKIs regulates cyclincdk complexes P27 27 refers to molecular weight binds to cdk cyclin and distorts the active site cdk and also blocks the ATP binding site on cdk Proteolysis of cyclin subunit I Core of cell cycle control system Cdk associates with different cyclins at different phases of cell cycle 9 cyclins are destroyed by proteolysis Noneconomicalquot to be destroying proteins via proteolysis 100 inactivation of protein 9 cell cycle is so important that protein has to be present only at certain times in order for a successful cycle I APC together with other proteins act as ubiquitin ligase After one cell cycle stage is completed the cyclin at that stage has to be degraded in order to ensure cell is able to proceed Inactive ARC is activated by cdc20 9 activated APC adds polyubiquitin chain on cyclin 9 results in degradation of cyclin in proteasome I SCF is constitutively active always active Recognizes phosphorylated substrate SCF together with Fbox protein forms active SCF complex Polyubiquitin chain is added to for example phosphorylated CKI resulting in degradation of CKI in proteasome Regulation of chromosome duplication 0 Licensing factorquot I Current models of the DNA synthesis regulation propose a licensing factorquot which allows each replication origin to be used only ONCE within a cell cycle Regulation of the initiation of DNA replication 0 ORC origin recognition complex binds to the replication origins o S cdk triggers S phase by phosphorylating cdc6 o S cdk prevents rereplication by triggering the destruction of cdc6 9 ensures that cdc6 cannot come back and start synthesis again Control of cell cycle 0 Checkpoints ensure that each phase of cell cycle is completed successfully prior to starting next phase of cell cycle I Maintains the order of unrelated events I Signaling if something goes wrong DNA damage Incomplete replication Incomplete establishment of mitotic apparatus I Usually not essential o If any phase is not successfully completed cdk present is stopped to arrest cell at that stage to allow time for repair 0 Three components of checkpoints I Generate the signals I Transduce the signals checkpoint proteinsquot I Receive the signal 0 Essential processes of the cell cycle are controlled O O O I DNA replication I Mitosis I Cytokinesis Quality control of the cell cycle I START checkpoint Cell size Nutrients Growth factors DNA damage I GZM checkpoint All DNA replicated Environment favourable I Metaphase to anaphase transition All chromosomes attached to spindle Checkpoint mutants I Can repair damage but unable to sense the problem 9 cells cannot arrest the stage leading to inviable microcolonies I The other type of mutant able to detect the problem but cannot repair 9 checkpoint arrested mutants stay as single cells DNA replication checkpoint in mammalian cells I Hydroxyurea blocks DNA synthesis I Caffeine interfere with checkpoint signaling I Cells go into suicidal mitosis passing on incomplete genome Control of cell division and growth 0 0 Size depends on total cell mass cell number cell divison and cell death and cell size cell growth Determinants I Mitogens stimulate cell division PDGF EGF promotes cell division TGFB inhibits cell division I Growth factors stimulate cell growth I Survival factors promote cell survival Potential mechanisms for coordinating cell growth and division I Extracellular factor cell division in yeast cell growth and division in animal cells Growth factor cell growth in animal cells Mitogen cell division in animal cells Cells compete for extracellular signal proteins I Densitydependent inhibition due to nutrients I Not contact dependent inhibition where cells stop dividing after they touch each other Role of inhibitory factor in maintaining size I Belgian blue I Myostatin Cell size and ploidy I Salamander Increase ploidy 0 Same organism size 0 Increase in cell size 0 Decrease in cell number Cancer 0 Uncontrollable destructive reproduction of cells in a multicellular organism 0 Genetic disease of inherited and somatic mutations Multistep process 0 Mutated genes I Oncogenes I Tumor suppressors o Mechanisms controlling cell cycle entry I Oncogenes and tumor suppressors I Mutation in Ras can lead to a constitutively active Ras 9 mutation in gene such that it is always active even without the mitogen I Mutation in Rb retinoblastoma I Mutation in Brcal breast cancer Apoptosis o Programmed cell death not present in cancerous cells 0 Part of normal development 0 Decreases the number of somatic cells 0 Opposite of mitosis 0 EXPERIMENTAL STUFFS Flow cytometry 0 Method used to determine the type of cells in a tube 0 Separates cells based on their DNA content and the proteins on the cell surface Cell fractionation o Centrifugation I Low speed yields whole cells nuclei and cytoskeletons in pellet I Medium speed yields mitochondria lyzosomes and peroxisomes in pellet I High speed yields microsomes and small vesicles in pellet I Very high speeds yield ribosomes viruses and large macromolecules Antibodies 0 Used extensively in research 0 Seeks antigens 0 Animals make antibodies against the epitopes part of antigen that is recognized by immune system 0 If antigen has 3 epitopes a cell will make an antibody against an epitope I Polyclonal 9 collection of immunoglobulin molecules that react against a specific antigen each identifying a different epitope I Monoclonal 9 collection of molecules that bind to the same antigen Agarose gel electrophoresis O O Easiest way to resolve DNA fragments based on size Simpler than resolving proteins since nucleic acids are negatively charged Useful for resolving DNA 500 to 25 000 bp Migration is affected by I Conformation DNA topology Speed of migration relaxed lt linear lt supercoiled 9 differences mainly due to the structural differences of the molecules I Pore size of gel I Voltage gradient I Salt concentration of buffer Pulsedfield gel electrophoresis 0 DNA molecules larger than 25 kb are poorly resolved by standard gel electrophoresis 9 longer molecules can be resolved by periodically changing the direction of the electric field Compares the difference in the amount of time required for a molecule to change direction in an electric field I Larger fragments require more time to change direction Simplest form field inversion 10 to 2000 kb Have to ensure that the DNA fragments do not shear into small pieces during handling 9 manipulations are done on agarose blocks cells are embedded in gel and digested with enzymes to release the DNA Radioactivity O 32P used to label nucleotides 9 in order to label on DNA P closest to the nucleotide is labeled trinucleotides Blotting techniques 0 0 Principles of hybridization I Process whereby two complementary nucleic acid strands form a double helix during an annealing period due to base pairing I Powerful techniques for identifying specific nucleic acid sequences I Cooling denatured DNA slowly allows singlestranded DNA to hybridize 9 rapid cooling does not allow hybridization SOUTHERN blotting I By EM Southern 1975 I Used to analyze DNA fragments I Needs a DNA acid probe against gene of interest Complementary to the sequence of interest 0 gt 20 nucleotides in length Very sequence specific Sensitive 9 depending on the detection method I Applications I Steps 1 2 3 4 Number of copies of a gene in a DNA sample Digest DNA template into smaller fragments Treat with RNAse Resolve DNA fragments on an agarose gel Transfer DNA fragments onto a membrane support 9 immobilizes DNA fragments membrane carried a semipermanent reproduction of the banding pattern of the gel Hybridization analysis carried out on DNA 9 bands with sequence similarity to a labeled probe can be identified 0 NORTHERN blotting I Analyze RNA fragments I Labeled probe used to identify RNA molecules I Application Presence of RNA species 0 In specific cell types 0 At different points in development Presence of abnormal RNA species 0 Deletions insertions and splicing Loss of RNA transcripts in disease state Purify RNA molecules from tissue cell sample I General steps 1 3 RNA is resolved against agarose gel under denaturing conditions 9 RNA is S S and therefore prone to secondary structure formation keeps RNA denatured so that RNA will separate based on size and not structure Transfer RNA to nylon or nitrocellulose membrane by capillary transfer Identify RNA sequence of interest by labeled DNA or RNA I When used to check for presence of transcript Dot or slot plot is used 0 RNA is loaded using an apparatus which has either a slot or dotlike aperture 0 RNA on the membrane is probed with a labelled probe Loading control is used eg actin because it is expressed in all cells 0 To ensure that presence of no band is no mistake 9 with presence of control but no band it can be certain that that RNA is missing Applications 0 Effect of a treatment on transcription 0 WESTERN blotting Analyze protein Need antibody against protein of interest Dideoxy Sanger sequencing old method 0 ddNTP used missing OH at C3 9 unable to add anymore nucleotides onto ddNTP o Invitro DNA synthesis by DNA polymerase 9 DNA polymerase incorporates dNTP onto a growing DNA chain if ddNTP is incorporated chain synthesis terminates 4 concurrent reactions one for each nucleotide Small amounts of each ddNTP in each mixture of nucleotides 9 randomly incorporated ddNTP are labeled with a radioactive isotope Reactions are run on polyacrylamide gel 9 separates DNA fragments according to size resolution of 1 nt 9 dried gel is exposed to Xray sequence is read from the bottom up Automated DNA sequencing new method 0 Replaces Sanger method 9 radioactive labeling is hazardous manual sequencing is laborious and time consuming 0 Used for genome sequencing projects 0 Use of uorescent ddNTPs Library Each ddNTP is a different colour Reaction is run using machines which use capillary or traditional gel electrophoresis Colour is noted by laser as fragments passes through DNA sequence printout is generated 0 Collection of clones each of which contains a different fragment of genomic DNA and which together include all sequences in the genome OR a collection of cloned DNA sequences whose location and identity can be established by mapping the genome of a particular organism When the whole genome of an organism is used as the starting point for cloning it is known as a shotgun clonequot 0 GENOMIC library ALL DNA is represented 9 coding noncoding etc All DNA is represented in EQUAL PROPORTION Complexity of the library is the number of independent DNA clones and is defined by genome equivalents GE CE 1 when number of independent clones genome sizeaverage size insert 0 Human genomic DNA library of 40 kb average insert size contains 75 000 independent clones 1fold library To have a high chance of recovering a specific gene the GE should be higher than 1 Isolation of genomic DNA 1 Lyse the cell or break open the cell 9 eg grind a piece of tissue along in a mortar with a pestle 2 Break down and emulsify cell membrane fat and proteins using solution containing proteinase K and detergent SDS sodium dodecyl sulfate 3 Precipitate the DNA by adding ethanol 9 ethanol causes watersoluble DNA to settle out of solution leaving all remaining cellular components 4 Spool the DNA only an inoculating loop Construction 1 Isolate genomic DNA from organism 2 Digest with enzymes 0 Do a partial digest to get the right size fragments I Plasmids up to 15 kb I Lambda phage up to 25 kb I Cosmid up to 45 kb BAC and YAC o The larger the size of appropriate fragments the lower the number of clones required 3 Prepare vector 4 Ligate 5 Introduce into appropriate host 6 Screen Number of clones to be screened can be determined N N ln1 P ln1F o P probability of getting the clone 0 F fragment sizegenome size 0 cDNA library Genes which are EXPRESSED exons are represented 9 sequence information that ends up in mRNA introns tRNA promoter etc are not present Sequences are present PROPORTIONAL to the level of gene expression cDNA library made from different tissues will include different clones mRNA expressed from proteincoding genes is purified from a specific tissue or developmental stage then reverse transcribed into its complementary DNA 9 cDNA only contains the exons of the expressed proteincoding genes and is cloned in a suitable vector Isolation of mRNA RNA consists rRNA tRNA and mRNA 9 mRNA is the most useful because it codes for proteins 1 Total RNA is denatured to expose polyA tail 9 characteristic that separates the mRNA from the rest 2 PolyA tailcontaining RNA is then bound to oligodT 9 sequence of many Ts 3 Unbound RNA is washed off 4 PolyA is eluted by removing salt from solution 9 destabilizes dTrA hybrid I Construction 1 2 3 4 S 7 8 9 Isolate mRNA Synthesis of 1St strand cDNA using reverse transcriptase Remove mRNA Synthesize 2rld strand cDNA using DNA polymerase Methylate cDNA with EcoRI methylase o In case gene has restriction sites 9 prevents cutting of cDNA accidentally by restriction enzymes Add linkers adaptors o Linkers restriction sites 0 Adaptors linkers that have already been cut Digest with enzymes Prepare vector Ligate cDNA to vector 10 Introduce into competent cells 0 Expression VS nonexpression library I Requires 0 Probes I Types Right promotor o Inducible can be regulated or constitutive always on Tagged or untagged proteins 0 Eg GFP CMyc His Flag Directional cloning o Promotor 9 ATG 9 gene 0 1 in 3 chance of being in the right frame Nondirectional cloning o Fragment with same restriction sites at both ends 0 1 in 6 chance of being in the right frame Previously cloned genes 0 Eg use a gene from one organism to look for the same gene from another organism Synthetic oligonucleotides 0 Eg use an oligonucleotide derived from cDNA to screen for the genomic copy of the gene Degenerate oligonucleotides 0 Eg based on partial amino acid sequence synthesize a degenerate oligonucleotide Antibodies raised against protein of interest I Visualizing probes Radioactivity o Incorporate base with labeled 32P 33P or 35S o Radioactive decay event exposes a film 0 Hazardous Nonradioactive O O 0 Light exposes film when enzymes react with substrate I Small recognition molecule incorporated into nucleic acid probe I Enzyme is conjugated to protein molecule which recognizes small labeled molecule BiotinAvidin 9 detect using enzymatic activity of HRP DigoxigeninUTPAntibody Enzymes crosslinking 9 direct coupling of enzyme to gene least preferred I Uniformly labeled probes 0 DNA probes 1 2 3 Nick translation Random priming method Label using PCR 0 RNA probes 1 oesAw Clone fragment into vectors with eg T7 promoter Cut vector with restriction enzymes Add T7 polymerase Add 4 NTPs with one labeled Result is several copies of insert with uniform lengths Protein probes O 0 Library screening 35S methionine or other radioactively labeled amino acid 9 incorporated during translation Radioactively labeled crosslinkers used Antibodies against proteins can be labeled directly or labeled secondary antibodies can be used I Eliminated unwanted clones I Hybridization screening DNA from a library is bound to a membrane then exposed to a probe that should base pair to the sequence of interest 0 Replica of master plate is made on nitrocellulose filter 9 filter treated 0 Compare developed film to master plate to identify colonies containing gene of interest I Expression vectors used Proteins are produced 9 detect the protein of interest to screen for gene of interest Immunological detection o Overlay nitrocellulose filter 9 lyse and process to mark with radioactive antibodies When a protein of interest is not captured on film it does not mean that the gene is not present 9 is just means that the gene is not expressed Visualizing cells 0 Terms I Lenses Objective 0 Collects a cone of light rays to create an image Condenser o Focuses a cone of light rays onto each point of the specimen I Resolution Depends on the width of the cone of illumination and therefore on both the condenser and objective lens Resolution 061 Ajn sine o A wavelength oflight used 053 pm assumed for white light 0 9 half the angular width of the cone of rays collected by the objective lens from a typical point in this specimen since max width 180 sinO has a max value of 1 o n refractive index of the medium usually air or oil separating the objective and condenser lens I Numerical aperture Is a function of the light collecting ability of the lenses n sine The higher the numerical aperture the greater the resolution and the brighter the image 9 obtained at the expense of very short working distances and very small depth of field 0 For dry lens it cannot be more than 1 but for oilimmersion lens it can be as high as 14 0 Types I Light microscopy Brightfield microscopy 0 Simplest of all microscopy techniques 0 Low contrast 0 Low resolution Phasecontrastmicroscopy Nomarski differentialinterferencecontrast DIC microscopy 0 Allows visualization of the 3D structure detail of the cells Darkfield microscopy 0 Only re ected light goes through the lens I Fluorescence microscopy Source of light and visualized light is of different wavelength 3 parts 0 First barrier filter lets through only blue light with wavelength of 450 490 nm 0 Beamsplitting mirror re ects light below 510 nm but transmits light above 510 nm 0 Second barrier filter cuts out unwanted uorescent signals passing the specific green uorescein emission between 520 and 560 nm I Confocal uorescence microscope Makes use of laser light for visualization with dichromatic mirror concentrated at the pinhole I Transmission and scanning electron microscope TEM and SEM Specimen contrast provided for by electron scattering 0 Fluorescence probes eg GFPtagged proteins 0 Indirect immunocytochemistry primary antibody recognizes antigen 9 secondary antibodies are markercoupled and recognizes the primary antibody 0 Fluorescence resonance energy transfer FRET I 2 proteins one which absorbs violet and emits blue light another absorbs blue light and emits green light I Occurs when proteins are in close contact 9 protein interaction green light detected I Acceptor excitation wavelengths cannot overlap donor excitation wavelengths 9 otherwise transfer will be observed whether or not interaction between proteins are present 0 Photo activation used to trigger emission of uorescence in selected region 0 Fluorescence recovery after photobleaching FRAP I Photobleach region of cell to study protein movement 9 loss of uorescence will be recovered because protein moves on the cell I Diffusion constant of proteins in membrane can be calculated from bleaching of GFP proteins using computer controlled lasers I Movement of proteins results in recovery of GFP uorescence but not to original level 9 bleached proteins will NEVER regain uorescence I If proteins are stationary uorescence will not be recovered I Mobility of the proteins allow other proteins to come into place and uoresce Studying cell cycle 0 Cells exposed to 3H thymidine I Radioactivity only in cells that divide 9 base is incorporated during replication I 3Hthymidine is an analog of thymidine 0 Cells exposed BrdU and probed with uorescent antiBrdU antibodies 9 visualization of dividing cells Fluorescence activated cell sorter o Allows for synchronization of cells 0 Fluorescence antibodies label cell type cell phase 0 G2 and M phase are sorted together 9 G2 has same amount of DNA as M GFP tagging of proteins to observe protein movement 0 DNA fragment encoding YFG fused with DNA encoding GFP 0 Fusion can be either at C or Nterminal Purification of proteins using column chromatography 0 Usually multiple columns are used before purification o Ionexchange chromatography 9 ionic interaction use of negatively charged column to isolate positively charged proteins 0 Gelfiltration chromatography 9 size of protein 0 Affinity chromatography 9 needs an antibody specific fast BIOTECHNOLOGY Uses 0 Helps meet our basic needs Food clothing shelter health safety Production of improved plants animals and other organisms Used in maintaining a good environment Genetic manipulations to get new organisms or new products from organisms Ancient biotechnology 0 Meat scarce 9 easier to get meat from reared animals 9 capture small animals and rear them 0 Breeding and nurturing techniques Food supplies often seasonal 9 select for plants that produce more grains than their wild counterparts 10 000 years ago farming Focused on having food and other human needs Developments in agriculture and animal farming Useful plants brought from the wile select for good breeders Use genetics to improve quality and quantity Increased food require ability to store and preserve I Techniques include cheese rennetquot enzyme from stomach lining of calves I New purified from genetically engineered organism I Genetically modified food Modern biotechnology o Manipulation of genetic material within organisms O O O O 0 000000 O O Commonly referred to as genetic engineeringquot 9 make new DNA constructs and express in different organisms Microscopy and advanced computer technology used Indepth knowledge of gene function and expression 9 active sites to work on different substrates change in expression Use of biotech to produce new life forms new tools etc Good knowledge of animal and plant genetics Made possible by recombinant DNA technology 9 precise sequencespecific for constructs Used in food preparation and preservation Bread baking produce C02 causes dough to rise Fermented products from sugar beer wine etc species of fungi Some are useful eg s cerevisiae Some may cause diseases eg candida albicans Wine and beer 0 O Fungus extreme heat and cold affect production of grapes Use genetics to improve crossing of breeds in an attempt to get hybrid cannot control the transfer of unwanted genes Identify and transplant genes that make native grapes resistant Genetic engineering transfer specific traits without changing other traits eg avor Genetic engineering faster than it takes to grow hybrids 9 multiple crosses required before desired plant is obtained Beer 3rd most popular drink made from malted barley water and hops Improve the barley stock immunity to a virus resistance to fungal root rot 9 conferring resistance to obtain a larger viable number Bioethanol and biofuel O O 0 Use yeast and other microorganisms to produce bioethanol 1St generation corn wheat barley sugar cane sweet sorghum 9 result in price increases of foods as farmers become more interested in making ethanol than food 2nd generation lignocellulose biomass wood straw grass etc cannot be digested Convert to glucose and xylose S cerevisiae efficient conversion of glucose to ethanol but poor xylose metabolism Pichia stipites able to metabolize xylose efficiently Transfer genes from pichia to s cerevisiae 9 able to metabolize both and convert to ethanol well 3rd generation biofuel microalgae and biomass 9 uses light directly bioethanol required multiple conversions Antibiotics O O Penicillin discovered by Alexander Fleming 1928 to combat bacterial infections Widespread use from the 1940s WWII First drug from microbes used to improve human health Increase production by chemical synthesis or transfer the biosynthetic pathway into other organisms that are easier to grow 0 Use genetic engineering to make new antibiotics modify enzyme to give slightly new antibiotics 0 Eg erythromycin has similar activity to penicillin I Used in patients with allergy to penicillin I Erythromycin from the fermentation of Saccharopolyspora erythraea hard to scale up I Transfer the genes responsible to e coli 9 which grows easily in labs Vaccination o E Ienner 1796 inoculated a boy with cowpox material to immunize against smallpox infection 9 cross immunity 0 Attenuated weakened whole agent vaccines 9 still can multiply I Long term immunity 9 full virus there therefore body can make antibodies for all proteins belonging to virus I May mutate back to virulent strain rare Inactivated killed vaccines I Not as long lasting I Safe 9 no chance of it surviving and coming back Subunit vaccines 9 protection against whole virus bacteria by creating immunity against a part of the virus bacteria I Uses fragments antigens from virus or bacteria I Produced by recombinant methods I Safe Antibodies O Polyclonal and monoclonal 9 recognizes many epitopes and recognizes one epitope of a protein respectively Specific recognition Diagnostic use I Direct tests detect antigens I Indirect tests detect antibodies Therapeutic use I Chemotherapy is toxic to normal and cancerous cells thus side effects Magic bullet I A drug that works only on cancerous cells I Add a toxin or radioisotope to an antibody specific to cancer cells I The specificity of antibodyantigen reaction kills only cancer cells I Identify antibody which recognize cancer cells 9 antigen that is specific to cancer cells I Enzymelinked immunosorbent assay ELISA Direct ELISA o Antibody adsorbed to well 0 Patient sample added 0 Complementary antigen binds to antibody o Enzymelinked antibody specific for test antigen is added and binds to antigen forming sandwich o Enzyme s substrate is added reaction produces a product that causes a visible colour change Indirect ELISA o Antigen adsorbed to well 0 Patient antiserum is added complementary antibody binds to antigen 0 Enzymelinked antiHISG is added and binds to bound antibody 0 Enzyme s substrate is added reaction produces a product that causes a visible colour change Humanized antibodies 0 Active immunization expose to immunogen 0 Passive immunization transfer antibody 0 Monoclonal antibodies mAb from mouse 9 mAb short half life in humans as they are detected as foreign 0 Need to make mAb less immunogenic in humans 9 humanize 0 Need to know antigen recognizing region of mAb 9 top part of the Y structure of antibodies In vitro generation of antibodies 0 scFv singlechain variable fragment 9 light and heavy chains linked together by a linker I A fusion protein of the variable regions of the heavy VH and light VL chains of immunoglobulins connected with a short linker peptide of 10 to about 25 aa 0 Can be converted into intact immunoglobulins by cloning the variable region into plasmids Combinatorial library library of different constructs 0 Contains many different random combinations of nucleotides 0 In vitro library where binding site is random Lactose intolerance o Hypolactasia Inability to metabolize lactose glucose galactose Due to lack of lactase Abdominal pain bloating atulence diarrhea etc Decreased lactase activity 5 in northern Europe gt90 in Asian countries Lactose free milk produce the calcium needed 0 Lactose supplement purified from fungi aspergillus oryzae 9 converts lactose to glucose and galactose Diabetes 0 Type I I Auto immune disorder I Body produces antibodies that kills insulinproducing cells I Most ofter occurring in children and young adults 0 O O O O I Treated with daily insulin injections I Accounts for 5 10 of diabetes Type II I Due to body s inability to make enough or properly use insulin I Most common form of the disease I Accounts of90 95 of diabetes I Nearing epidemic proportions due to an increased af uence Purified insulin from bovine pancreases Treated dogs whose pancreas had been removed 9 induced diabetes Availability of an effective treatment insulin injections first patient treated in 1922 Insulin I From bovine cow 9 foreign I Patients immune systems produce antibodies against it I Clone the human gene and express 9 e coli humulin I 2 peptides purified and mixed I Yeast two peptides synthesized and secreted with perfect three dimensional structure I Stem cells to treat type I diabetes 9 hopefully body will secrete insulin I Insulinsecreting pancreaticlike cells from mouse embryonic stem cells 1 Establish ES cells 2 Determine the conditions for differentiation invitro 3 Inject differentiated cells into diabetic animal Establish the techniques in mice Toxin for medical and aesthetic treatment 0 O Botulinum toxin botox produced by the bacterium clostridium botulinum the most powerful neurotoxin ever discovered 7 subtypes designated with letters A through G 1g enough to kill 1 million people 9 LDso 3 ngkg inhalation Injecting overactive muscles with minute quantities of botulinum toxin type A will result in decreased muscle activity by blocking the release of acetylcholine from the neuron Thus effectively weakening the muscle for a period of three to four months I Purify from c botulinum 9 danger of contamination I Therefore Clone the gene Express in e coli Purify and inject I Safer 9 ensures that only one toxin is produced Restriction fragment length polymorphism RFLP O 0 Restriction enzymes cut DNA at specific sites Many individuals have variations in their DNA sequences 9 if variations occur frequently within a population the location is said to be polymorphic 0 Use of a DNA probe which hybridizes to the DNA in the region of interest 9 polymorphisms allow chromosomes to be tracked I Southern blot I Digest DNA I Run agarose gel I Transfer to membrane I Add labeled probe I Wash I Expose to xray film 0 Genetic testing use of RFLP analysis to get this data 9 results can be very reliable 1 in 1 billion chance of a false positive I Carrier screening I Preimplantation genetic diagnosis IVF I Prenatal diagnostic testing I Newborn screening I Presymptomatic testing for adultonset disorders diseases I Confirm diagnosis I Foreignidentitytesting Genetic engineering of organism 0 Identify the process to improve Identify the organism OrgZ with the most ideal characteristics Identify the steps to improve Identify the source genes Clone the genes Construct appropriate vector 9 promoter appropriateness etc 0 Introduce into the OrgZ Probes based on partial protein sequences 0 Identify protein 9 identify purity 9 identify amino acid sequence 9 identify DNA sequence 0 Choose least degenerate 20base region 9 create pool of primers that have all the possible combinations 9 one will bind to the DNA perfectly others will not work so well Subtracted cDNA library 0 Eg Tcells have only one mRNA that is Tcell specific 1 Create first strand cDNA of Tcells from mRNA Remove RNA by alkali Anneal excess RNA from Bcell with Tcell cDNA Pass through column which retains D S DNA Because Tcell specific cDNA stays single stranded that strand of cDNA will be left Differential hybridization 0 Eg differentiate between activated and resting cells to find out the difference in gene expression Functional screening 0 Mutants of other organisms used to screen for homologues 0 Expression library in a suitable vector is used I Promoter I Transcription terminator ensures that transcription stops after the gene OOOOO wewv O I Multiple cloning site I Two markers I Two origin of replication Transform library into mutant and look for clones which rescue the defects in the mutant Look for human genes that can take over function of yeast genes 9 compensates for defect in yeast genes Eg human cdc2 was cloned using yeast mutant Screening for biological activity 0 If cloned gene is expressed to give a functional protein clones showing a new biological activity can be identified Transform library and look for new biological activity Eg clones expressing an enzyme can be detected using chromogenic substrates like e coli and XGal Eg say s pombe can degrade toxic chemical which s cerevisiae cant 9 transform s pombe library into s cerevisiae and look for clones that can degrade the toxic chemical Divide colonies into sublibraries instead of doing individual assays on all 9 test sublibraries for biological activity Eg I Screening for proteins which make up gas vesicles 9 allow oating of bacterium I Protease positive clone 9 able to break down milk which causes halo in milk agar plate I Genes that degrade starch 9 clear zone after iodine is added Mosquitoes O O O 0 Cause more human suffering than any other organism Spread many diseases I Dengue I Elephantiasis I Malaria One strain of the disease hemorrhagic dengue fever is often deadly Range of the mosquito that carries disease is limited by temperatures 9 disease is limited to tropics but global warming is changing that Nepal and Bhutan saw their first cases in recent years 9 people not previously exposed to mosquitoborne diseases are now at risk Chemical and biological control I Use of pesticides to reduce pests to an acceptable or tolerable lecel Insecticides 9 DDT dichlorodiphenyltrichloroethane formerly used throughout the world but is now banned in most developing countries 9 causes pollution and its metabolites are not good for humans Total elimination is rarely possible I Use of any living agent to suppress pest populations Predators Parasitoids Bacteria Fungi Nematodes o Mosquitocidal toxins Entomopathogenic species of Bacilli B sphaericus and B thuringiensis produce one or more toxins that kill mosquito larvae even at pMolar range Advantages Many different genes with varying potency Species specificity Different modes of action Safe for animals and the environment Do not affect nonpest insects Disadvantages Rapid sedimentation from the larval feeding zone 9 gram positive bacteria that produces spores which sinks heavy UVlight sensitive Narrow host range 9 kills only one or few species TOO species specific Cloning and expression of the toxin Purify the toxin Sequence the toxin Design degenerate primers Screen a library Choice of organism o Persist in the larval feeding zone I Eg free oating bacteria like Caulobacter cresentus Asticcacaulis excentricus Ancylobacter aquaticus Cyanobacteria 0 Express high levels of toxin 0 Selection of promoters and vectors 0 Sterile insect technique Principle make insects sterile mainly using radiation Successfully used in tsetse control Research underway for malaria vectors including anopheles arabiensis Determine optimal dose range 9 amount of mosquitoes to release to maintain the mosquito populations Needs release of large number of sterile males 101 9 males do not spread diseases Effectiveness is dependent on population structure and dynamics 9 effective when the target populations are small and the target area is isolated crossborder issues o Transgenic mosquitoes I Principle genetically transforming mosquitoes to be less efficient vectors or nontransmitters of malaria O O Labreared mosquitoes genetically modified Release into the wild Transgenic strain breed with the wild mosquitoes introduce disease resistance into the wild mosquitoes Number of disease carrying nonresistant mosquitoes reduced or replaced I Size of the targeted wild mosquito population I Fitness of the genetically engineered mosquitoes Caterpillar pests Lepidoptera moths and butter ies are the second most pest insect Out numbered only by the beetles Adult moths and butter ies are usually beneficial insects Caterpillars however are defoliators or miners or succulent plant tissues Genetically modified cotton I Cotton primarily grown in dry tropical and subtropical climates at temperatures between 11 C and 25 C I Cotton insects are the principle cause of yield losses 9 losses almost 15 of world annual production I Bacillus thuringiensis or Bt is a grampositive soil dwelling bacterium commonly used as a biological pesticide I Bt is closely related to B cereus a soil bacterium and B anthracis 9 three organisms differ mainly in their plasmids I Bt crop Identify the genes encoding the toxin Prepare vector with Bt toxin gene under plant promoter Transfer gene with promoter to Agrobacterium tumefaciens gram negative bacteria Use Agrobacterium to transfer gene cassette to plants Select for plants with gene cassette Cultivate I Advantages Level of toxin expression can be very high thus delivering sufficient dosage to the pest Toxin expression is contained within the plant system hence only those insects that feed on the crop perish Toxin expression can be modulated by using tissue specific promoters eg expression in the leaves only and replaces the use of synthetic pesticides in the environment Latter observation has been documented worldwide Biological control gone wrong 0 0 Sugar plantations in Puerto Rico were protected from beetles by cane toad Australia introduced 102 cane toads in 1935 to control insect pests of sugar cane cane beetle and greyback cane beetle Study of feeding habits were done and release of 62 000 more cane toads were done in 1937 Failure did not control the insects and the cane toad itself became a pest Insect pests were later controlled using insecticides and other suitable management practices Golden rice O 0000 Children growing in poor countried suffer from vitamin A deficiency More than 100 million children suffer from the problem Causes blindness and death Not easy to deliver vitamin pills Improved vitamin A content in widely consumed crops an attractive alternative Rice is a staple diet in many of the poor countries but is vitamin A deficient Production of vitamin A I Isopentenyldiphosphate 9 geranylgeranyl diphosphate 9 phytoene synthase phytoene 9 phytoene desaturase 9 Ecarotene desaturase lycopene 9 lycopenebeta cyclase Bcarotene vitamin A precursor I Rice is missing enzymes phytoene synthase phytoene desaturase Ecarotene desaturase and lycopenebeta cyclase while other enzymes responsible for making vitamin A is present in rice I 3 genes in plasmid construct is introduced Daffodil gene phytoene synthase Single bacterial gene which performs functions of phytoene desaturase and Ecarotene desaturase Another daffodil gene lycopenebetacyclase I Plasmids were introduced via agrobacteria Atlantic salmon O 0 Found in northern Atlantic Ocean Anadromous fish 9 fresh and salt water phase born in fresh water migrates to salt water where greatest feeding and growth occurs then migrates back to fresh water to breed Take growth hormone gene from Chinook salmon Growth hormone is the principle stimulator of skeletal growth and plays a key role in lipid mobilization protein synthesis and feeding behavior Pharming Use of genetic engineering to insert genes that code for useful pharmaceuticals into host animals or plants Express with signal sequencequot eg express in milk or eggs to allow for easy purification so that killing of the organism is not required to purify products Pharmaceutical products produced in large quantity I Cow Human growth hormone Collagen Fibrinogen Lactoferrin Antithrombin 111 Alpha1 antitrypsin CD137 41BB MAb antibody Malaria vaccine Spider silk protein I Chickens Recombinant proteins Human poly and monoclonal antibodies Interferon alpha and single chain antibodies Bioremediation 0 0000000 Use of microorganism metabolism to remove pollutants Insitu onsite or exsitu treat at a plant far away Prokaryotes survive in very harsh environments Identify microorganisms from polluted areas able to survive Cultivate them and screen for ideal organism OR genetically engineer microorganism OR both Uses I Oil spillage petroleum I Industrial pollutions cadmium sulfur chromium copper etc cleaning of industrial areas Gene therapy 0 0 Proposed in 1972 use of DNA as a pharmaceutical agent to treat diseases Use DNA that encodes a functional gene in order to replace a mutated gene DNA that encodes a therapeutic protein is packaged within a vector usually a virus 9 virus infects and integrates the gene 9 gene is expressed by the cell 9 normal protein treats the patients diseases Over 1700 clinical trials have been conducted 9 successful treatment of patients with Xlinked SCID severe combined immunodeficiency Therapeutic cloning O O Somatic cell nuclear transfer or research cloning Objections in humans due to ethical reasons etc 0 Insert somatic nucleus into egg with removed nucleus 9 activate eggs 9 cell division of egg occurs 9 formation of embryo 9 kill embryo and remove the inner cell mass from the blastocyst to form personalized ES cells 0 Personalized ES cells have the same genetic material and background as the patient 9 will not be rejected 0 Strategies for delivery I Direct delivery Therapeutic gene packaged into delivery vehicle such as retrovirus Retrovirus injected into patient s target organ I Cellbased delivery Therapeutic gene packaged into delivery vehicle and introduced to invitro differentiated stem cells Genetically modified cells are introduced into the pa ent Test tube meat 0 Hamburger made from cow s stem cells MISCELLANOUS All cells express its information by transcription of DNA into RNA then translation until proteins making use of mRNA tRNA and rRNAs in the process Proteins consists linear peptides folded into 3D forms with reactive sites Thus proteins bind with high specificity to other molecules They also act as enzymes to catalyse reactions to make or break covalent bonds Prokaryotes greatest biochemical diversity no distinct nuclear compartment small size 1 2 pm live as independent individuals or in loosely organized communites typically rod or spherical shaped tough cell wall some have agella which propels them through media some are photosynthetic Eukaryotes big genome size measured in nucleotide pair in haploid genome cell size increases with genome size Modern eukaryotic cells evolved from symbiosis eg mitochondria double membrane with own DNA generally accepted that anaerobic eukaryote engulfed an aerobic bacteria chloroplasts has double membrane with own DNA as well Tree of life used to classify based on outward appearance therefore not possible for microorganisms Biochemical tests genome analysis and rRNA sequences can be used to classify microorganisms Prokaryotes are broken into bacteria and archaea 9 archaea resembles eukaryotes in terms of handling genetic information transcription and translation Packaging of E coli chromosome 9 prokaryote before the nucleusquot 46 x 106 base pairs 17 mm DNA in cell 2 pm Single circular chromosome DNA is supercoiled HU protein HU and topoisomeraseI Chromosome conformation capture 3C 9 technique used to determine the position of loops in interphase Method 0 Cells are treated with formaldehyde to crosslink DNADNA and DNAprotein 0 DNA is digested with restriction nucleases 0 Free ends generated are ligated with DNA ligase o Crosslinks are revered and polymerase chain reaction PCR is used to amplify and determine the junctions Results show that DNA in human chromosomes are organized into loops of different lengths 9 a typical loop is 50 000 to 200 000 nt pairs genes in the loop regions are highly expressed Embryo divide and give rise to all cell types adult somatic cells cannot give all cell types stem cells can differentiate into all different cell types In order to generate the various cell types invitro we need to identify genes that are expressed and how to turn them on Induce Pluripotent stem cells iPS 9 forces the adult somatic cells to become pluripotent Genomic reprogramming 9 reprograms adult cells to give rise to a complete organism reprogramming in embryo results in epigenetic modifications being reset Cells Basic unit of life smallest living unit Very small 05 to 20 um Light microscope Robert Hooker 1665 Electron microscope Ernst Ruska 1930s The cytosol is reducing 9 proteins can form disulfide bonds outside of the cytosol but they remain as sulfhydryl SH groups within the cytosol Cell theory history 1950s DNA was the carrier of genetic information 1960s identification of cytoskeleton molecules cell shape 1970 Rao and Johnson cell fusion experiment 1970 Hartwell Culotti and Reid yeast mutants s cerevisiae 1971 Masui and Markert discovery of MPF 1987 Nurse identification of human CDC2


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