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-Amino AcidsoPolar Non-polarAspartic Acid (-) AlanineGlutamic Acid (-) GlycineArginine (+) ValineLysine (+) LeucineHistidine (+) IsoleucineAsparagine (0) ProlineGlutamine (0) PhenylalanineSerine (0) MethionineThreonine (0) TryptophanTyrosine (0) CytesineoBasic is positive, acidic is negativeoUncharged polar is more likely to interact with water'-EnzymesoRNAaseH-Removes Rna primeroPrimase-Makes short RNA primer complimentary to template in ReplicationoDNA polymerase -Reads short RNA Primerelongates-ONLY WORKs 5'-3' (Reads old 3'-5')-Proofreads itself 2 sites: elongation and repair (endonuclease)oRNA polymerase-Does not use primer-Reading rules are the sameoLigase-Joins together fragments (in Okazaki or repair)oSpliciesomes-Finds 5' splice and 3' splice sites (straddles intron)-snRNP comes in and are held together -Breaks intron out -Excised intron is degraded/ snRNPs recycled -DOES NOT RELEASE THE RNA DURINGoHistone acetylase (Histone acetyl transferase)-Acetylates histone (lysine)oHistone deacetylase-Deacetylates histone (lysine)oKinase -Adds phosphate --> becomes negativeoPhosphotase-Removes phosphate groupoGTPase-Part of GAP (GTPase Activating Protein)-Activates hydrolysis in G-proteinoEndonuclease-Used in DNA polymerase proofreading and base repairoChaperones-Assists protein in folding into correct conformationoHelicase -Unzips genes oPhosphodiesterase-Breaks the bond in DNA repairoExcision nuclease-Used in nucleotide excision repairoTopoisomerase-Reduces stress ahead of replication forkoTelomerase-Uses reverse transcriptase-Carries RNA template-Elongates shortened fragments-Controlled in adult cells, active in germ/stem-GGGTTA-TestsoDNA replication is semi-conservative-One strand is conserved as bothoRNAi-Double stranded RNA leads to silencingComes in recognized by Dicer enzyme as foreignSnips into siRNAs, pairs with dicerMatches to complimentary mRNA of interest, removes rest of sequence -No translation of areas, no protein (silencing) -Research silencing to study effects of gene knockdown (dominant negative approach) -PCRoPolymerase chain reactionoUsed to amplify specified DNA sequenceoTake DNA primer, mix with sample, tac-polymerase (good for heat)oHeating (seperation) breaks strandoAnnealing cools down, higher concentration of primers bind to DNA sampleoElongation, polymerase acts on primer and extends sequencesoAfter 3 cycles, you begin to get specific sequence of interestoCycles continue to amplify that sequenceo**Note Primer A should be able to bind to strand 2, B to strand 1 (see drawing)-Gel ElectrophoresisoGel = Ethidum Bromide (EtBr) + agarose (porous)oUsed to compare mix of DNAoSends electric charge which makes DNA move to + terminaloLarger molecules move slower, stay closer to startoUsed to compare sequences, similar sequences with compare with band stripes on gel-Nuclear Run-On AssayoRNA polymerase, DNA, mRNA isolated during transcription (freezing)omRNA tagged, allowed to incubate and extend (no new transcription)oAfter, use cDNA tag (reverse transcriptase) and run blot analysis to quantifyoAllows comparison of mRNA transcription levels between cells, tissues, etc etc-Southern BlottingoDNA analysisoNitrocellulose membrane transfers DNA from geloDetection: Hybridization with radioactive P, compliment to target DNAoIncubate membrane, probe crowds around target DNAoIntensity is the amount of DNA of interest.-Master GeneoInteraction of certain proteins allow "master gene" to tell cells to differentiateoImmature cell interacts with protein to become mature oTest method: transfect with plasmid-Transfect with plasmid: will have gene of interest and antibioctic-You want a gene that is expressed solely in mature cell, not both immature and mature (discard hybrid s )-Stain with antigen to see expression is successful or not-Signal --> Gene activation --> protein interaction --> differention --> mature development-Yeast 2 Hybrid SystemProtein - protein interactionHow mutations affects protein interactions-DNA is genetic materialRadio labeled bacteriophages-S marked protein, P marked DNA-Infected bacterial cells, and isolated P marked bacteria-Because P was incorporated, scientists knew DNA carried genetic information-Replication is bi-directionalOrigin of replication will remain equidistant from the edge of DNA loops-Protein StructureProtein Domains:-Help us to predict structure and function-Similar linear sequences-Aid in adding of complex functions-Complex == more domain variety-Easily shuffled between proteins-Alpha and Beta sheets-Type of secondary structure-Alpha: helicalFormed by hydrogen bonds**hydrophilic (polar peptide backbone hidden in middle)Arranged with alpha carbon in the middle, side chains out sideGap between so small nothing can fit through it-Beta: Anti-parallel or parallel-Primary-This is the linear sequence-Contains all the information for 3D folding-Secondary -Is organization into alpha and beta sheets-Tertiary -Folding: primarily non-covalent bonds-Spatial arrangement of a and beta (as well as connecting Aas)-Determines shape and structure-Assited by chaperone proteins-Di-Sulfide bond ***IS A COVALENT BOND*** b/w two cysteines-Quaternary-Joining together of multiple teritary structures-Affect of heating-Denatures protein to its linear chain-Effects the non-covalent bondsoBond Types-Covalent-Share electrons-Arent affected by heat-Non-Covalent-Ionic-Electrostatic interactionsPartial dipolesWeakened water-Hydrogen bondsH is partial positive due to electronegativityHolds DNA strands together-Van der WaalsHappens but so weak, just interactions cause temporary dipoleoCell Building Blocks-*DNA/RNA-Nucleic Acids-DNA = deoxyribose Sugar2 complimentary strands (anti-parallel double helix)Purines: Adenine, Guanine Pyrimidine: Cytosine, ThymineMatches A-T (2 hydrogen bonds) , C-G (3 hydrogen)Backbone: Phosphate and sugar-Joined by phosphodiester bond (diphosphate removed and remaining 5' phosphate joined to 3' carbon (condensation reaction)-RNA = ribose SugarRNA is very similar, know that it has an extra oxygenRNA; Uracil (U) replaces T and binds to ARNA can be single stranded, and take on several conformations-Information storage and such-*Protein-Amino group bound to alpha carbon bound to caroxyl group (R group characterized by amino acids)-Left terminus is amino group, right hand is carboxl-Each is joined through peptide bond (condensation reactions)

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Pitt BIOSC 1500 - Amino Acids

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