Genetics Notecards[1]

8/3/2019 Genetics Notecards[1]

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DNA:What r the Basic Properties of DNA (2):

Difference in Bases of RNA vs DNA

Differentiate between Pyrimidines vs.

Purines

1. Replication/Transcription/Translation

2. Transforms a genome throughincorporation

TAGC (U instead of for RNA)

1. TA/ U are pyrimidines ( single rings thatmake 2 bonds) *U is substituted for T inRNA

2. GC ( makes 3 bonds)

3. Bases are hydrophobic and are theinterior of the strand

DNADefine Exo vs Endo nuclease

What is DNA ligase

Name the causes of Denaturation

Differentiate between Nucleotide vsNucleoside

- Exonuclease: cleave the last nucleotideresidue

- Endonuclease cleave bonds in theinterior of the strand

- DNA ligase: enzyme that forms DNA 5-3bonds

(high temp, extreme pH etc)

May anneal and become single strandedrandom coils

Nucleotide : The Bases TAGC/U + phosphate(activated)

Nucleoside : base + pentose ring sugar


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Chromosomal OrganizationDescribe the Hierachical Structure of aChromosome:

- Oligonucleotide

- Polynucleotides

- Introns

- Exons

What is a Histone what are its parts?

How is H1 different?

Do prokaryotic cells have histones?

- Oligonucleotide (short polymers to 30nucleotides)

- Polynucleotides (long nucleotidepolymers)

- Introns : pieces of mRNA that are splicedout (not coded) *rare in prokaryotic cells

- Exons : splicing that remains

Histone : functions to stabilize and condenseDNA. Made of 8 proteins that form a diskaround which DNA wraps two ea of : (H2A,H2B, H3, H4). H1 is a stabilizer of the lastDNA turn. It wraPs around the outside of disk +DNA.

- prokaryotic cells do not have histones

Chromosomal Organization contd.Describe the following:

- Nucleosome

- Chromatosome:

- nucleofilament

- Supercoils

- Chromatin

Differentiate betweenEuchromatin/HeteroChromatin

What is in a Chromosome?

- Nucleosome = histone +DNA (no H1)*onlye 1 3/4 turns of DNA

- Chromatosome : histone disk +DNA +H1 (2 full turns of DNA)

- Nucleofilament : long beaded string of chromatosomes + linker DNA

- Supercoils : DNA ladders wrappedaround eachother- Chromatin : packed supercoils +

histonesEu = dispersed , hetero = condensed; Duringdivision becomes condensed.Chromosome= looped condensed DNA of centromere + telomere


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DNA structure:DNA conformations

- B-DNA- A-DNA- Z-DNA

What is DNA Bending:Explain Cruciforms

Triple-Strands;. In what groove?What are Hoogsteen Pairs, function,example?

Quadruplex DNA: 4 strands; seen when andwhere?

crazy Z = L-handed- B-DNA: normal (Watson/Crick)- A-DNA (DNA-RNA hybrid during

transcription)- Z-DNA (Left handed coil, like during

transcription)

DNA bending : *bending occurs when enzymesbind and due to photochemical damageCruciforms : intrachain H-bonds occur, theseform a function in control of replication andtranscription.Triple-Strands :3 rd strand H-bonded in major groove forming Hoogsteen Pairs . form inregions with many purine bases. Function incontrol of transcription etc.. confers stability ontelomeres .( Eg.termination of fetal Hbtranscription)Quadruplex : seen at telomeres duringrecombination

Special DNA Sequences:Be able to explain each, maybe give anexample.

Inverted repeats

Mirror repeats

Direct repeats

What is a Structural Gene? How is itdifferent from the rest of the code?

What is DNA replication vs DNAtranscription?

Inverted repeats : palindromeread the samefront to back and vice versa

Mirror repeats : same sequence towards eachother

Direct repeats : sequences that continuallyrepeat

Structural Gene : eukaryotic DNA that actuallycodes for protein , the rest take part inregulation and development

Replication = making daughter DNA strands Transcription= de-coding the message (makingmRNA)

DNA replication

Topoisomerases:- What is it?- What are the types?

What is an antibiotic used to treat UTIs andwhy?

- Gyrase

Topoisomerase :Enzymes that wind and unwind DNA byCatalyzing breaking and rejoining of dna strand.

- Type I : relieves supercoil by breakingone strand *also contains ligase

- Type II : breaks both strands and reseals*requires ATP

- Type III (gyrases): act to relievesupercoiling at transcription bubbleNote: not a Helicase. Helicase is responsible foropening the replication bubble.Nalidixic Acid : inhibits bacterial gyrase (usefulfor acidic environment of urethra)

- Gyrase is the bacterial analog totopoisomerase


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Prokaryotic DNA synthesis:What are the requirements for DNAreplication?What is Primer/Primase?

DNA polymerase what does it do and how?

- Does is have exoncuclease activity?High or Low?NOTE:

adds a 5 carbon of free nucleotide tothe 3-OH attached to templatestrand.

Synthesis from 5-3 means that thenew strand is being made from itsfree 5(C next to O-- ) end towardsthe 3 end (OH on 3 rd C)

Template, primer, nucleosides, DNApolymerasePrimer = short segments of RNA synthesizedby primase enzyme to provide free OH groupfor nucleotides.

DNA Polymerase = synthesize DNApolynucleotides by polymerizing nucleotidesreleasing PPi

*require primer +template*add nucleotides only to 3 end , thereforesynthesizing in the 5 3 direction*proof-reads through exonuclease activity (3-5removal)*high fidelity

Prokaryotic DNA REPLICATION contd.

Is a template always necessary for DNAreplication?

Is a double-strand important?

How is RNA primer removed?

What is semi conservative replication ?

Yes

NOTE: DNA must be in double-stranded formbefore replication, even in organisms that carry single-stranded DNA ( its just how the machineworks)

Polymerases also have 5-3 exonucleaseactivity that removes RNA primer.

Each new helix has one old and one new strand

Steps in DNA replication:*the following words must be included inyour story:

Replication ForkHelicaseGyraseSSBRNA primase/primerDNA polymerase I and IIILeading (5-3)/Lagging strand(3-5)

*opposite of templateOkazaki Fragments (are synthesized inwhich direction?)DNA ligase

1. Replication Fork is created throughbreaking of H-bonds by Helicases

2. Gyrases reduce the number of supercoils behind the fork, SSB (single-stranded binding proteins) prevent thetwo strands from re-annealing

3. RNA primase lays down a primer on theleading strand and many shortsequences on the lagging

4. DNA pol III starts forming the leadingstrand from the free 5 end, pol I fills inthe free 5 ends created by the primerson the lagging strand (the pieces arecalled okazaki fragments (5-3 pieces)

5. Primers are removed, gaps are filled byDNA polymerase and segments are

joined by DNA ligase


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Prokaryotic DNA polymerases

What is the function of polymerases?

What are the differences between types of polymerases? I/II/III

What is a klenow fragment?

Function: add nucleotides to the end of agrowing DNA chain, and repair (3-5exonuclease)

Pol I/III DNA synthesis

Pol I synthesizes the lagging strand I (3-5exonuclease ) , removes primer (also 5-3exonuclease)Pol II - synthesizes ribosomes?Pol III - synthesizes the leading strand

Klenow Fragment : 70kDa fragment (portion)of Pol I enzyme contains the polymerization and3-5 exonuclease activity, as opposed to thesmaller fragment for 5-3 exonuclease activity.

More on Prokaryotic DNA synthesis:

Define Processivity :- Which of the polymerases has thehighest?

- What is the Sliding Clamp?

What is a replisome and where does itoccur?

The ability of an enzyme to remain on itssubstrate- POL III has higher efficiency than either

Pol I or Pol II- Sliding clamp is an associated protein

that confers stability to Pol III conferringits processivity.

A replisome is the replication complexconsisting of primosome: primases, lipases,helicases etc.. as well as the DNA strand,polymerases etc.. it occurs at the ORI or origin

*there might be more words in this section weneed to know, diff variations on replisome

Prokaryotic vs Eukaryotic Replication:

What are the 4 major differences?

What is a replicon?

1. Size of the DNA of eukaryotes is muchlarger

2. Eukaryotic cells pack DNA , chromatin3. Eukaryotes replication fork movement is

much slower .4. Prokaryotes have one ORi for replication

(circular DNA), eukaryotic have manyand replication is bidirectional .

Replicon: Segments between the various ORIon a DNA. DNA can contain up to 200 ORI.


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When does replication occur ineukaryotic cells?

When are histones synthesized and why?

What controls the cell-cycle?

How do G1 and G2 differ in pro vseukaryotes?

DNA replication occurs during the S-phasesynthesis between the two dormant G1 andG2 phases.

Note: histones are also synthesized during s- phase so that new DNA contains both parental

and new histones.Cyclins are a substrate for enzymes calledcyclin dependant kinases (Cdksusuallyassociated with tumourous growth). That controlthe cell-cycle through catalyzing microtubuleformation and chromatin remodeling etc..

Note: G1 and G2 are very short in prokaryotesallowing for faster division. Ie. Bacteria growslike the dickens.

DNA polymerases- Eukaryotic:What is a polymerase?

- Differentiate between Polymerasesigma vs Pol alpha?

What is PCNA- proliferating cell nuclearantigen?

- Antigen: helper

RPA- replication protein A:

What are the major differences betweenprokaryotic and eukaryotic polymerases?

Polymerase link together DNA nucleosides.

Sigma - high processivity, 3-5 exonuclease,leading strandAlpha - slow processivity, synthesizes laggingstrand diassembles nucleosome prior to DNAreplication

PCNA = proliferating cell nuclear antigen (likeprokaryotic sliding clamp)

RPA : Analogous to SSB in prokaryotes, disallowsthe strands to anneal

?? looping of lagging strands allow both alphaand sigma to elongate and proceed in thedirection of fork movement??

TelomerasesWhy cant DNA be fully replicated with areplisome?

What does telomerase do? How?

How do telomerases contribute to aging?

How does telomerase differ in relation totumors?

Bc the lagging strand cannot be synthesized atthe end where the last primer is removed (gap= 8-12 nucleotides)

Telomerase : reverse transcriptase uses an RNAtemplate for DNA synthesis.

Contains RNA and is therefore templateand enzyme

Lacks exonuclease activity and are errorprone

Retroviruses do the same

Telomerase activity decreases with age andtherefore is related to cell senescence.*telomere length is inversely proportional toage.

Tumors have upregulated telomerase activityconferring immortality prone to errors intranscription


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Name a modern day antibiotic and how itrelates to reverse transcriptases: think HIV.

DNA Recombination: name the 3 types

Give an example of site-specificrecombination:

What is a transposon?

AZT : dideoxythymidine, is an analogue of dTand incorporates into viral genome inhibitingreverse transcriptase and cutting short viralDNA synthesis*used in the treatment of AIDS.

1. Meiosis : homologous2. Site-specific ( insertion bybacteriophage into genome)

3. Transposition: jumping DNAEg. When e.coli bacteriphageinserts into a hostgenome at specific sites.

Genes that insert and are removed seeminglyon their own. jumping gene Usually carriedon plasmids

RNA vs DNA

How do DNA and RNA bases differ?

What is the common structure of RNA?

What are the Types of RNA (9 types): whatare their respective percentages?

RNA contains Uracil instead of THymidineAlso has post-translational modification such as

methylated bases.

RNA is single stranded with hairpin pairingsthat confer stability (60% of strand)

mRNA 1%, tRNA 15%, rRNA 80%,small nuclear RNA ( snRNA ), scRNA (smallcytoplasmic), mtRNA (mitochondrial), RNAi(interface plural)RNP (ribonucleoprotein particles, Ribozymes,

mRNA:What is the general structure for eukaryoticmRNA?What is the function of the cap?

what is polycistronic mRNA? Is it morecommon in pro or eukaryotes?

What is a cistron?

What protein has the shortest of all themRNA half-lives? Why?

Name two exceptions to short mRNA half-lives.

5-5 cap is followed by a nontranslated leadersequence, startcodon, AA instructions, stop,nontranslated tailing sequence (usually poly A).

- The cap confers stability and protects

Polystronic mRNA = mRNA that encode formore than one protein, uncommon ineukaryotes?cistron = DNA segment that codes for onepolypeptide.

Histone mRNAs are the shortest. Lack poly Atail.

1. Unfertilized eggs (inactive and readyfor fast synthesis

2. Hb mRNA , reticulocyte mRNA does notturnover (no nucleus remember).


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Name two mRNA modifications that extendtheir half-life. What is the primarydeterminate of mRNA stability?

tRNA:

what are the two functions of tRNA?

rRNA:where is it synthesized?Made up how how many parts what are theycalled?

- Inverted methylated base ( 5-5phosphate link cap )

- Poly A tail . Primary determinate of mRNA stability. Histones lack this tail,thus the short life. The longer the tail, thelonger the life I guess.

tRNA : T shaped AA carriers1. Activate amino acids (acceptor stem)2. Recognize codons in mRNA (anticodon

loop)rRNA : ribosome subunits

Synthesized: nucleolus2 parts: Large and SMall

Explain what each of the following is in onesentence:

snRNA

scRNAmtRNARNPRNAiRibozymes

Does RNA turnover? Why or why not?

How long is mRNA half-life?tRNA half life?

What catalyzes this degredation?

snRNA : (small nuclear)recognizes introns onmRNAscRNA (small cytoplasmic): selects proteins for

transportmtRNA : its own exclusive with one tRNA for ea.AARNP : function in RNA processing (splicing etc..)RNAi : control cell phenotype duringdevelopmentRibozymes : RNA that act as enzymes eg.RNase P that cleaves pre-tRNA to mature tRNA

Yes. Bc RNA is not repaired like DNA, RNA isdegraded and newly synthesized.

mRNA Half-life = minutes to hours , tRNA=5 days

RNA is destroyed by ribonucleases,exonucleases, endonucleases.

RNA synthesis:List the steps to RNA synthesis:

Know the definitions of the following:pribnow boxPrimary transcriptribonuclease

What are the types of post-transcriptional

modification?

How does transcription ensureunidirectionality?

How is a promoter determined weak orstrong?

1. RNA polymerase searches for promotersite on DNA .(TATAAT box, or Pribnowbox )

2. attaches promoter/ Releases subunitsigma

3. RNA polymerase creates RNA strand theprimary transcript .

4. Post transcriptional modification createsmature mRNA that is then shuttled

outside nucleus.a. Removal of external/internal

nucleotides by ribonucleases.(degradation)

b. Base modificationc. Addition of nucleotides

The TATAAT box does not have symmetricalbases in the complimentary strand.


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Whether it is complimented by enhancers orrepressors.

RNA polymerase

Can RNA polymerase synthesizein 3-5direction?

Does RNA polymerase require a primer?

Which is more accurate DNA or RNApolymerase?

PROKARYOTIC RNA POL .What is a holoenzyme? Core enzyme?

Which RNA subunit recognizes the promoterregion?

No RNA polymerase, like DNA polymerase canonly make 3-5 linkages.

No.

DNA polymerase. RNA polymerase does nothave exonuclease activity and therefore cannotproofread.

Holoenzyme is the prokaryotic RNA subunitcomplex (2A, B, B, omega, sigma). Coreenzyme = all subunits minus sigma(recall, sigma is released upon binding topromoter region)

Sigma acts to recognize the DNA promoter

EUKARYOTIC RNA POL

What antibiotic can distinguish between proand eukaryotic RNA pol?

What is used to detect eukaryotic RNA pol?- 4 types: pol I, pol II, pol II, and

mitochondrial.

What is the primary purpose of RNA pol I?

What is a nuclear organizer?

Rifampicin . Used to treat TB binds to B subunitin prokaryotic RNA pol.

A mushroom toxin: amanitin

RNA pol I: 50 % of all RNA. synthesizes onlythe transcript for rRNA subunits within thenucleolus at the nuclear organizer

Nuclear organizer : The region within thenucleolus containing DNA that codes for rRNA.


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EUKARYOTIC RNA POL contd.

Which RNA pol does the main RNAtranscription?Which RNA pols are sensitive to amantin(the eukaryotic RNA pol detector)?

What is the role of RNA pol III?

Describe ribonuclease P.

RNA pol II transcribes structural genes (pre-mRNA), 20-40% of all RNA.

I no, II/III yes, mitochondrial*. No (*butsensitive to bacterial antibiotic rifampicin).

RNA pol III transcribes tRNA, and small 5srRNA. Makes up 10% of RNA activity

Ribonuclease P is a ribozyme that trims the 5end of the primary tRNA transcript.

Splicing:Name the process for intron removal.

What is the role of snurps in splicing?

Describe alternative splicing:

Splicing. Note: exons are what are splicedtogether and kept as the primary transcript.

snRNPs: Snurps are small nuclearribonucleoproteins. They (U1RNA,

U2RNA)discriminate splice sites and removeintrons.

A splicing is alternative when the same primarymRNA transcript is spliced differently coding forslightly different proteins. Eg. Tropomyosin.

Translation:

What is a codon?Are codons necessarily specific?

What is the sequence for start codon?

What are the three stop codons?

Is an AA necessarily in the synthesizedprotein if its codon exists in the transcript?

mRNA = code, tRNA = activate AAs, rRNAtranslates code.

Nucleotide base triplet that codes for an AA.

No. however some are more specific. Eg. Met,the tRNA initiator, has only 1 codon.

The start codon is AUG. however the startcomplex begins first with the TATAAT box.

UAA, UAG, UGA. Note: they all begin with U.

No. modification of the transcript and eventuallythe mature protein can remove AAs duringprocessing.


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What are the 3 characteristics of thegenetic code.

mRNA conventions:- Usually write code from 5-3, the

same direction it is translated.

Proteins are written by convention fromamino terminus to carboxyl

Where is an anticodon triplet found?

What is the wobble effect ?

1. Degenerate (AAs have more than onecodon)

2. Universal (both pro and eukaryotes usethe same nucleotide triplets)

3. Continous (codons are not separated byspacers)

On tRNA anticodon loop. to recognize specificAAs. Many codons for each AA.

The ability of an anticodon to pair with morethan one codon. Eg. A single tRNA can recognizeall 4 Ala codons.

What is the siginificance of inosine (I)?

Why doesnt the wobble effect occur at the3 end?

How are amino acids activated?

Note: aminoacyle tRNA synthetase arehighly selective. proofread

Inosine is a modified base often found in the 5(last)position of an anticodon that allows for less

stringent base pairing.Eg. UorI, Gor I, A or I etc

3 end is first base read in the codon. Highconservation of this codon is necessary becauseof stop codons. (recall all stop codons start withC or U)

Activation of AA requires aminoacylation byaminoacyle tRNA synthetase . Each AA has itsown specific enzyme for activation. 2 reactions:

1. Activation of AA with ATP 2. Transfer of aminoacyl group to tRNA

Initiator tRNA, pro vs eukaryotes:

Protein synthesis is initiated by theformation of? Terminated by?*non-formylated Met exists inside theprotein and is not an initiator in prokaryotes

Name the leader sequence small subunitrRNA binds to on the mRNA transcript?

What is the anticodon on met-tRNA?

How many sites are in rRNA? Name them.

In which direction is mRNA read? Proteinformed?

Prokarytoes: fMet-tRNA fmet (formyl Met)Eukaryotes: met-tRNA

Termination = stop codon. No associated AA.

Pro: Shine-Delgarno sequence, Eu: Kozaksequence

UAC. Recall that AUG is the start codon,therefore the initator AA will have the anticodonto the start sequence.

1. A (aminoacyl/entrance site)2. P (peptidyl transferase binds 2 AAs)3. E (exit site)

Read: 5-3, formed: amino to carboxyl. N to Cterminus.


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Common antibiotics that inhibit proteinsynthesis:Briefly describe mechanism and commonuse.

1. STREPTOMYOCIN

2. TETRACYCLINES (group)

3. PUROMYCIN*

4. ERYTHROMYCIN

5. CYCLOHEXIMIDE*

6. RICIN

Note: I have included clinical use for mypersonal reference

1. First line antibiotic for TB. Preventbinding of fmet-tRNA to rRNA smallsubunit in prokaryotes. Mutations canconfer resistance (ie. MDR-TB)

2. Cholera, rosacea, acne. Inhibitsaminoacyl-tRNA binding to mRNA-ribosome complex.

3. Used only in research, toxic to both proand eukaryotes. Is an aminoacyl-tRNAanalog that irreversibly binds to A-site.Stops elongation.

4. Used in case of penicillin resistance (2 ndline). Inhibits peptidyl transferase inprokaryotes

5. Used only in research. Inhibits peptidyltransferase in eukaryotes only.

6. Lethal to humans. Castor bean toxin.

Removes adenine from rRNAAmino Acids:Describe Structural vs. dynamic proteins.

What are 2 specific characteristics of AAs inliving organisms?

What is the general structure of AAs?

What is a pH function of AAs?- Which AA is the exception?

Structural: matrix for bone and CT = form.Dynamic = movement. So everything else.(enzymes, transport, regulation etc)

1. made of -amino acids (contain central alphacarbon).2. AAs are L-chiral. That is, have a left handedchiral center.

-C has 4 attachements:- Carboxyl group (COOH/ COO-)- Amino group (NH2/ NH3+)

- H atom- R side chainAAs can act as buffers due to ability to hold andrelease protons (H+s)Glycine: has 2 Hs (R-group is an H)

Peptide Bond:Define a peptide bond.

What does peptide formation release?

Characteristics of peptide bond:

Differentiate between protein vs. peptidevs. polypeptide

Peptide bond: primary amino group catalyzed toprimary carboxyl group of another AA.

Reaction releases H2O through release of H-group on -C.

- Very strong (50% double bond character)- In one plane field (planar)

- Rotation does not occur around carbonyl-Nitrogen however bonds around -C canrotate during folding.

Protein: >50 polymersPeptide:


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Chemical and charge properties of amino acids and proteins:

Formation of some AAs are derivations of other AAs. Give 2 examples.

Why are disulfide bridges important?

What are two group/side chain functions of AAs and why?

Cystine: derived from two cysteines. (derivedthrough thiol oxidation forming a S-S bond)

Glutathion: derived from 3 amino acids. Glu-Cys-Gly. Functions as electron donor(reduces) to

repair oxidative damage.*S-S bridge is used to stabilize protein structureand in Redox reactions. (as in cystine andglutathione)

1. Ionizable groups influence proteinproperties

2. Hydrophobic/non-polar sidechains, foundon the surface of proteins, critical forprotein folding.

Protein structure: structure determines function

Differentiate primary vs secondary vstertiary and quarternary structure.

How does hydrophobicity confer stability toa protein?

Primary : its amino acid sequence

Secondary : local folding of the amino acidsequence; -helix and -pleated sheets, turns,and random coils.Tertiary: 3-D structure of the protein.bonds:electrostatic (salt bridges btw and + AAchains) , hydrophobic, H-bond (between AAs),disulfide.Quarternary: association of discretepolypeptide units functioning as a multi-unitprotein. Formed by homomultimers (of the samepolypeptide) and heteromultimers

Hydrophobic chains prevent interactions withwater. The more hydrophobic chains, the morestable the protein is.

Describe an -helix/ -pleated sheets

Globular vs Fibrillar proteins?

-helix: H-bond Right handed helix, side chainproject outwards

-pleated sheets: two chains aligned in parallel.Side chains project about and below.

-turn, reverses the direction of the polypeptidechain to form compact proteins

Random coildrandom functioning binding sites

Globular: tertiary structure resembling globesFibrillar: structure resembles rods


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What are the advantages of quarternarystructure?

What types of interactions initiate proteinfolding?

What is a proteins native structure?

Stability, genetically economical (same gnemany proteins), efficiency (no transportnecessary) , cooperativity (facilitation)

Primary structure contains properties to

spontaneously fold but is limited to short range.Facilitation by other proteins called chaperonesto create domains.

Final conformation is called native structure

Chaperones:

When do chaperones first bind to theprotein?

What happens each time a protein crosses amembrane?

What are functions that require proteinchaperones?

Why are chaperones called cellular heatshock proteins?

They bind as the protein is synthesized toprevent aggregation/precipitation.

They become unfolded.

Refolding of proteins after crossing membranesor with temperature increases. Facilitatetransport into mit. And through ER. Proteinassembly.

Cells cultured at higher temperatures havegreater percentages of chaperones bc morerefolding is needed to retain native proteinstructure.

Denaturation:

Always causes loss of function

Caused by?

pH, ionic strength, temp, (solvent, denaturantssuch as urea, detergents, guanidine)*weaken AA interactions.

Denaturation of improper folding triggersenxymatic breakdown and phagocytosis of protein.


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Explain the molecular reasoning fordevelopment of Alzheimers andCreutfeldt-Jakob disease.

Alzheimers : proteins form abnormally due toincorrect cleavage of extracellulartransmembrane portions of . These formspontaneous aggregates called amyloid whichare not phagocytized.

Creutzfeld-Jakob (CJD ): fibrous amyloidplaques develop in the brain. Thought to be of prion origin causing insolubility of protein.Characterized by causing nervous systemdysfunction such as ataxia, paralysis as wella sdementia.

*prion protein normally has 3 a-helix and 2 b-strands, altered protein exhibits transformationof 2 a-helixes into b-strands which then formfibers.

Blood proteins:

2 types of blood proteins:

Define serum:

Proteins from blood serum can be used todiagnose disease through separation basedon net charges, what is this process called?

Name some of the major gel peaks:

1. Functional role within blood: highconcentration (eg. RBC)

2. Carried by blood function outside it*disease may be caused by increase or decreasein the amt of ea. Of these in the blood

Serum = blood plasma without clotting factors(no fibrinogen, rbcs?, or platelets?)

Electrophoresis: separating charged particleswithin a gel, usually done at ph 8.6, proteinsmove towards (+) or (_) poles. Gel is thenscanned to show protein peaks.

Albumin, alpha/beta globins, gamma globins

What is the most abundant blood plasmaprotein?What is its function?

What is the clinical sign forhypoalbuminemia?

Name some examples of disorders thatcause hypoalbuminemia:

Albumin makes up almost 50%Function: to maintain osmotic pressure and totransport molecules such as bilirubin, FAs, andhormones.

Edema bc oncotic pressure out of the tissues isnot great enough.

Liver dysfunction (Hepatitis C, alcoholism, etc..),Kwashiorker disease (protein malnutrition inchildren), hypoproteinemia from renal and bowldiseases.


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Common gel protein electrophoresispatterns:

- Normal- Immediate response- Delayed response

- Hypogammaglobinemia- Hepatic cirrhosis- Monoclonal gammopathy- Nephritic- Protein-losing

- Normal- Immediate response : stress or

inflammation- Delayed response : infection, increase

in immunoglobulins

- Hypogammaglobinemia :immunosuppressive diseases- Hepatic cirrhosis : reduction in albumin

increase in gamma (to retain osmolarity)- Monoclonal gammopathy : gamma

myelomas- Nephritic : inflammation + low synthesis

of small proteins that are lost at kidney- Protein-losing : loss of albumin and

gamma (stress and nephritic issues)

Define the following:- Antibody, antigen, epitope, hapten

Describe the structure of IgG molecules interms on constant and variable regions.

How does the composition of thehypervariable region account for antibodyspecificity?

What is the role of the constant region?

What do disulfide bonds do?

What is an immunoglobulin fold?

Epitope: specific portion of antigen to which theantibody bindsHapten: a small molecule which by itself doesnot produce an immune response

Ig:2 heavy chains: constant region (Ch and Cl) arehighly conserved sequences2 Light chains: variable region epitope bindingside (Vh and Vl) responsible for specificity

- Hypervariable region: multiple N-terminalloop part of light chain that aids indiversity of epitope binding.

*2 regions connected through S-S bonds thatstabilize the domains

A structural motif seen in Ig and elsewhereDefine the Hinge region of Ig.

Describe the general biological function of Ig:

After antigen binds to Ig what happens?

What is the molecular mechanism of multiple myeloma?

Area btw Ch1 and Ch2 connected through an S-S bond can swivel to accommodateconformations

IG function:- To activate the complement-binding

site system. An 11 protein cascadesystem that eventually cause cell lysis.

- Initiates a conformational changecascade within the Ig itself to triggercascade.

- Amplification of the signal also occurs (B-cell system?)

Activated B-cell experiences oncogenetranslation or limitless promotion (loss of stopcodon) and over produces antibody, thereforeattacking various organ systems.


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What determines the different classes of immunoglobulins?

Describe the functions and different classes:

- IgG:o What does it look like?o what are the cleavages it

undergoes?

5 main classes:- The H chain classes are differentiated

into IgG, iGA, IgM, IgE, IgD.- Two isotypes of L chain- kappa and

lambda.

IgG = most abundant, 75%- Looks like : Y- Gamma variations in light chain- Undergoes enzymatic cleavage into

functional subunits- Papain:3 fragments = 2 antigen binding

fragments, Fc- complement fragmentcontrol Ig catabolism.

- Pepsin: 1 function unit, and 1proteolyzed Fc region. Can bind 2antigens.

- IgA:o What does it look like?o

What are the two majorsubtypes?o Where is it normally found?

- IgM:o What other cell type is it

associated with?o What does it look like?

IgA:- Looks like: Y or 2Ys joined by a J chain

- Serves as early defense mechanismprevent pathogen adherence- IgA binds and then phagocytosis occurs- Normally found in locations with

environmental contact (tears, mucous,saliva,)

IgM:- First antibody made by B-cell and is

presented on its surface (first linedefense)

- Primary antibody in a fetus- Can be found as a Y monomer or 5Y

pentamero Note: although there are 10 active

sites only 5 actually work.

- IgEo Primarily found where?o What disease process is it

associated with?o What does it look like?

- igDo associated with what other

cell?o

IgE:- Primarily in immune surveillance of

mucosal membranes: intestine/lung- Induces release of histamines allergic

reaction- Anaphalaxis associated- Exists only as a Y monomer

IgD:- Also present on cell surface of B-cell- Exists only as a monomer- Less than 1% of total Igs


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Hemoglobin and myoglobin:- briefly define the following:

Globular vs fibrillar proteins:

Globin fold motif:

Hemoglobin vs Myoglobin

Heme

Globular vs fibrillar proteins: globular =sphere like and soluble, fibrillar= looks like fiber,insoluble

Globin fold motif : 8 -helices, highly

conserved throughout evolution although AAseries conservation is very low.

Hemoglobin vs Myoglobin : Hb = tetramerico2 binding transport protein, Mg= monomericmuscle protein that binds a single o2

Heme: iron- porphyrin common to both Hb andMg, found in other various metalloproteins suchas peroxides etc..

what is the sickle cell anemia mutation?

General Structure of heme:

What is the significance of the His residue inheme?

Primary sequence point mutation change in Hbchanging glu to Val in one helice.Creates a hydrophobic pathch promoting

aggregation and thus changes in RBCmembrane.

4 pyrrole rings and a fe2+Resides in the hydrophobic core of Hb and mg.

2 axial positions coordinated by His residue- when bound protected from oxidation- *genetic mutations can compromise this

state- Oxidized Fe cannot transport O2

how are Hb and mg related?

What are the chain types that hb consistsof?

How does the expressions of these changeover time?

Hb:

- Basically 4 myoglobins that posseshydrophobic patches that assistquarternary structure

- Typically composed of o 2 chainso 2 chains

Expression of ea of these chains changes overdevelopment into 5 different subtypes

- Fetal is predominantly - Adult has both and


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O2 binding and release by Hb and Mg:Describe the difference btw:

- Tense state:

- Relaxed state:

Describe binding Cooperativity of Hb:

What is the shape of the myoglobindissociation curve and why?

Tense state : si deoxy state, ferrous ion standsout of the plane.

Relaxed state :oxygen binds and ferrous ionmoves to center of porphyrin ring. Energetically

more favorable state.As ea. O2 binds, conformation changes so thato2 binding becomes more and more favorable.Conversely, as o2 is dropped off in tissues, o2affinity decreases as ea. O2 is removed. ( curveis sigmoidal )*binding of 4 th O2 requires no E!!

*Myoglobin does not experience cooperativityand thus has a linear dissociation curve

What is BPG/DPG?

How is it relevant clinically?

How does it contribute to fetaldevelopment?

2,3, diphoshoglycerate is produced in high

quantities in RBCs, has a high affinity fordeoxygenated state of Hb. Bind subunitstogether. Thus, lowers Hb o2 affinity.(considered a metabolic byproduct and o2 drop-off molecule)

Clinical:- Hypoxia and high altitude adaptation.- oxygen trap of anticoagulant acid-

citrate-dextrose decreases DPG synthesis- Fetal Hb does not bind DPG very well

shifting dissociation curve to the left sothat o2 can be taken from the mother.

Describe the Bohr effect:

What are the 2 ways Co2 is transported?

Why do people pass out when theyhyperventilate for too long?

When Hb is in R state, certain factors contributeto decreasing o2 affinity of Hb.

- Increase in pH, DPG, metabolites, low o2concentration, high CO2, high temp

1. Can be chemically bound to Hb2. Can be coupled to H+ transport through

carbonic anhydrase present in RBCs.(releases HCO3- and H+ from CO2 andH2O)

Blow off too much CO2 cause a Left shift to Hb

droping off less O2 to brain tissues andultimately fainting.


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Give an example of a triplet expansiondisease:

What is a hemoglobinopathy?

Describe molecular basis of

hemoglobinopathy categories:

Fragile X: primary defect in initation orelongation of transcription.

- Conditions of abnormal Hb that result inanemia

- Unstable Hb structure: mutations of

AA residues at helix-formation sites.Precipitate as Heinz Bodies- Increased or decreases O2 affinity:- Increases heme oxidation:

Mathemoglobin ; oxidation of ferrousion, higher than 10% show cyanosis.

- Imbalance of chain synthesis or- Changes in chain properties:

thalaseemias

- thalaseemia:- Homo vs hetero zygous

- thalaseemia: fetal -globin continuessynthesis after birth.

- Heterozygous: some adult Hb do not

require treatment- Homozygous: severely anemic during 1 st

year of life requiring blood transfusions.(Cooleys disease)

o This can result in Fe overload anddeath btw ages of 15-25 yrs.

-thalassemia:

Barts Hb?

Review HbS: sickle cell (val glut)

Give an example of an acquired Hb opathy:

-thalassemia:- 2 copies of the gene ( and ) on ea.

Chromosome, several levels of deficiency.

- 2/4 defective = mild anemia- = Hb H disease with sever hemolytic

anemia- 4/4 = hydrops fatalis, no Hb at all.

Barts Hb:- Synthesis of 4 gamma chains does form

tetramers however exhibits too high of an affinity for O2 and is useless.

Carbonmonoxide poisoning:-CO increases affinity for O2 to HB slowlysuffocating the individual. High O2 pressure(hyperbaric chambers) are used to treat this.(carboxy-Hb)


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Fibrillar Proteins:

Collagen type I has 4 AAs that make itunique. What are they?

Obviously these AAs include

posttranslational modifications. What 3enzymes are responsible for these?

4 components are required for theseenzymes to work. What are they?

In all collagens what AAs occur every 3 rdresidue?

AAs: Gly, Pro, HydroxyPro, HydroxyLys

1. Lysyl hydroxylase (lys to OH-Lys)

2. Prolyl-4-hydroxylase (Pro to OH-pro)3. Prolyl-3-hydroxylase (4-OH-Pro to 3-OH-Pro)

Components:1. Fe2+, O2, -ketoglutarate, ascorbic acid

Gly, and Pro

What are the structural consequences of this unique sequence?

What special helix is made? What are itscharacteristics?

Which AA varies in the collagen structure indifferent species and why?

*PS> this is why UV causes wrinkles.

1. Gly- the smallest AA fits into small holesin the core of the structure

2. Pro- is a helix breaker, does not allowregular alpha-helices ( polyproline typeII helix )

3. OH groups react further to formcrosslinks and eventually the 3 braidedstrand of Collagen.

polyproline type II helix :- L-handed- Ea. Peptide bond is perpendicular to the

axis- 3 member super helices formed by Gly

(this upser helix is R-handed)Hyp residues allow for interchain H-bonds,higher degree of Hyp = more stability andresistance to degredation with temperature.

Varies by species.Collagen has 2 major structuralarrangements, what are they?

How are mesh-works formed? Describe themechanism.

What region of collagen is considered of globular character, what are they called,what happens to them?

Random for structural tissue strengthMeshwork for filtering

Covalent cross-links for meshes. Some Lysare converted to allysine by enzyme lysylamino oxidase (copper dependent ) which thenspontaneously reacts with non-modified Lys of another collagen chain.

Collagen fiber termini form globular ends calledtelopeptides , usually extracellularly cleaved inorder to form poly-fibers.


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Describe the steps in the biosynthesisof collagen.*collagen is formed from fibroblastsExplain how ea one comes about and what

defines it.

1. Preprocollagen2. Procollagen3. Tropocollagen4. Collagen

5. Translation of 3--chains occur onribosomes in RER. To formpreprocollagen (pre-refers to signalpeptide)

6. Signal peptidases cleave signal peptideforming procollagen

7. Hydroxylation modifications occur in ERlumen by 3 enzymes, the glycosylation8. Intra and inter chain bond formations

begin to occur and triple helix is formedinside RER, procollagen then sent to golgifor end glycosylation

9. Procollagen is excreted via exocytosis10. Telopeptides are cleaved by procollagen

peptidadese = tropocollagen11. Fibers self-assembleinto fibrils and fibers

in quarter staggered configurationsand stabilized by cross-links.

3 major categories of Collagen types: namethem.

What are the three major diseasesassociated with collagen? Explain theirgeneral mechanism.

Fibrillar (I-II, V), network-forming (Type IV),Fibrillar associated (IX, XII) etc..

1. Scurvy : ascorbic acidic deficiency(collagen enzymes dont work right).Usually osteoporosis, fragile capillaries(bruising), loss of teeth and bleedinggums.

2. Osteogenesis imperfect : Debilitatinggenetic disorder. Unstable procollagenhelices (lack of Gy residues), brittlebones

3. Ehler-Danlos Syndrome : group of disorders referring to weak CT.hyperextensibility, impaired jointmechanics, etc..

What is Elastin? why is it special?

Talk about elastins general structure:

Aging does what to elastin? What aboutscarring?

Elastin provides elasticity to tissues throughcoils that are relatively distensible , insolublein water .

They are critical to arteries and vessels - thinkAORTA.

No secondary structure, unordered coils withmobile AA residues. Modified lys residues(allysine) are found. Catabolised by lysyleamino oxidase . (also seen in collagen cross-links)

These react to form heterocyclic structure of desmosine an isodesmosine to form cross-links.

With age, more cross-links form diminishingelastins distensability properties. Scarring =
http://img161.imageshack.us/img161/882/untitled1wk3.jpghttp://images.google.com/imgres?imgurl=http://www.medical-look.com/diseases_images/scurvy.jpg&imgrefurl=http://www.medical-look.com/Nutritional_supplement/Scurvy.html&usg=__9wvQRS9ClJWOaCFjfK1lMYvCfYE=&h=284&w=222&sz=15&hl=en&start=45&sig2=5hlXKkih3SI_yfgzMxgv9w&um=1&tbnid=L0mAyaImnBqQvM:&tbnh=114&tbnw=89&prev=/images?q=scurvy&ndsp=18&hl=en&rls=com.microsoft:en-us&sa=N&start=36&um=1&ei=5JxcS-nrBZ6ONdvj6IIP


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same thing.

Intracellular fibrillar proteins contribute tocytoskeleton: name them. (3)

Microfilaments are found mostly where?What are they?

Microtubules : what is there generalstructure, what are they made of?

Cells use microtubules for which generalprocesses?

1. Microtubules2. Microfilaments3. Associated filament binding proteins

(Eg. Microtubule associated proteinssuch as kinesin, dynein)

Microfilaments are found in muscle:- Actin : polymerization of globular (G-

actin) units- Myosin : motor protein, produces

movement through ATP dependentbinding to Actin.

Microtubules:- Hollow tubes made from polymerized

alpha and beta-tubulin monomers.

Reinforce cytoskeleton , constructcilia/flagella , separate chromosomes duringmitosis . * trafficking

Define (+) and (-) microtubule ends andtheir general properties.

What are Kinesins and Dyneins, why arethey cool?

(+) = fast-growing end. Most likely seenoriented towards cell periphery which modulate.Anterograde trafficking.(-) = retrograde vesicular trafficking (thinkneurons)

Microtubules provide cellular train tracks forATP dependent transport of organelles/vesicles.

Kinesins: move vesicles to (+) end: anterograde*has two heads that bind to microtubule, andtail for transportDyneins: move to (-) end: retrograde*eg. Endocytosed molecules.
http://images.google.com/imgres?imgurl=http://www.wiley.com/legacy/college/boyer/0470003790/structure/kinesin/kinesin.gif&imgrefurl=http://www.wiley.com/legacy/college/boyer/0470003790/structure/kinesin/kinesin_intro.htm&usg=__TPavZeh-Vbp7MeESorglHT8s1Ug=&h=337&w=300&sz=12&hl=en&start=8&sig2=yFzqve8fcp-ORQIPTk7utw&um=1&tbnid=7tjmtUABwS2IQM:&tbnh=119&tbnw=106&prev=/images?q=kinesin&hl=en&rls=com.microsoft:en-us&sa=N&um=1&ei=CaNcS9KxBZ2UMtHUkZEP


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Intermediate Filaments:what are they?What are their general categories?

What are the two types of keratin?What is the general structure of keratin?

Why can girls straighten and curl their hair?(boys can too.. you just probably shouldnt.)

- are named such bc of their size relative tomicrotubules and microfilaments.- they are exclusively structural- 2 general categories: Keratins, Lamins (note:lamins were not discussed in our book)

Soft Keratins = internal structuresHard Keratins = hair, skin, claws

-helix dimer that forms coiled-coils (recall,collagen is a triple helix of helices). dimers areassembled into multimers stabilized by disulfidebridges.

- modified by reducing agents that breakdisulfide bonds (such as temp/straighteners/curling irons)- re-shaped by oxidizing agents (like water/rain!)

If youre in medical school you better know

the difference between an enzyme and asubstrate .

Function of enzymes?

Enzyme attributes:

Enzyme Domains:

What is a zymogen?

Nuff said.

Remember enzymes are proteins that speed up/ Slow down reactions. (otherwise takes years)

Attributes:Specific (regulate), reversible/irreversible,competitive/non-competitive .Domains:

- Prodomain- Catalytic- Substrate Binding- Transmembrane

Zymogen: inactive proform of an enzyme

General Regulators of enzyme activity: (4)

Specific eg. In lecture were given for each of these and are included briefly above.

1. Substrate availability (amount andlocation)

- Eg. Alzeihmers: overproduction of specific cleavage enzyme which disallowssubstrate for other enzyme. excesscleavage and amyloid precipitate

2. Enzme availability ( synthesis)- Cyclooxygenase turn arachidonic acid

into prostaglandins (inflammation).Downregulation of this enzyme (aspirin)decreases inflammation

3. Enzyme activation (non-zymogen)- Activation of trypsinogen by

enterokinase. See acute pancreatitis.4. enzyme inhibitors , endogenous vs

exogenous.- Endogenous tend to be non-specific.


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Define the following:

1. oxidoreductases:

2. transferases:

3. hydrolases:4. isomerases:

5. lyases:

6. ligases:

1. oxidoreductases : transfer hydrogen oroxygen from one substrate to another (e.g.oxidases)2. transferases: transfer a specific group (forexample a phosphate) from one substrate toanother (e.g. kinases).

3. hydrolases : catalyze hydrolysis of asubstrate (e.g. digestive enzymes)4. isomerases: change the molecular form of asubstrate5 . lyases: remove or add a group to a substratein a non-hydrolyticalway (e.g. carboxylases)6. ligases: join two molecules through theformation of bonds between carbonand O, S, N(e.g. citric acid synthetase)

What are Proteases? What do they do?

Name the 4 major classes of proteases. There is an example of ea.

Give some examples of proteases functions:

Proteases: hydrolases that cleave peptidebonds

1. Serine proteases ( trypsin)2. Aspartyle proteases ( HIV)3. Metalloproteases ( matrix)4. Cystein proteases ( caspases)

-maturation of viral proteins (HIV proteases)- food digestion (pepsin, trypsin etc..)- intracellular protein degradation(macrophages)-Orchestrating biochemical cascades (eg.ACE)-Apotosis- activation of complementECM remodeling


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Course objective: illustrate proteasesignificance in CT components.

3 examples:1. Physiological ECM remodeling2. Pathological remodeling

3. Cancer

Metalloproteases: what do they do?

Eg. Of Ehlers-Danlos syndrome is a mutationin the ADAMTS family of metalloproteinases.

1. Physiological ECM remodeling :breaking and remodeling of tissues (eg. Endochondralossification)

2. Pathological remodeling :

tissues broken down duringdisease (eg. Arthritis)3. Cancer: cells use proteases to

invade tissues

Metalloproteases : use Zn to coordinateand activate H2O molecule, mediateturnover of matrix molecules. (Development,Maintenance, Reproduction)

Endongenous inhibitor of metalloproteinases:eg. -2 macroglobulins (produced by theliver)

and selective inhibitors TIMPS?

Drug Development: methods, pitfalls, safety- Selectivity- Tissue distribution of target enzyme- Reversible vs. irreversible.

Post translational modifications:

Signal Peptides: describe where they are andtheir sequence.

Describe the steps for which signal peptidesare involved.

1. Signal peptide2. Signal recognition particle3. Docking protein4. Translocon5. Signal peptidadeses

Signal peptide : 15-30 AA at aminoterminus . Contains hydrophobic AAs, is +charged and contains cleavage site forsignal peptidase .

1. Signal peptide emerges from atranslating sequence

2. Signal recognition particle bindsand halts synthesis

3. Ribosome moves to ER membraneand binds to docking protein ,

4. translocon opens a hole and insertspeptide+ribosome into ER lumen

5. after termination signalpeptidadses cleave signal peptide.


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Describe the formation of insulin.

Do protein modification occurposttranslationally or posttranscriptionally?

Formation of insulin is similar to collagen

Preproinsulin still contains signal peptideAfter cleavage become proinsulin

Proinsulin folds forming disulfide bonds

Transported to golgi and packaged intosecretory vesicles

Then proteolytically modified by proteasescleaving specific residues and formingmature active insulin

Posttranslationally .

What is Phosphorylation?

Distinguish between a Kinase andphosphatase.

What is glycosylation?

What is a Proteoglycan?

Aggrecan?

When and why does cartilage fluoresce?

Phosphorylation is a covalent alterationwhich may activate or inactivate an enzymeKinases : add phosphate groupPhosphatases: remove phosphate groups.Glycosylation : addition of carbohydrates toproteins to change stability, solubility, andphysical bulk, may also act as recognitionpeptides/targeting, function modification.Proteoglycans : contain protein andcarbohydrates (similar to glycoprotein) butprimarily carbohydrateAggrecan : is a large negatively chargedparticle to which H2O binds, single proteincore with extensive sugar chains. Primarycomponent of cartilage.

Fluoresces with age due to AdvancedGlycation End (AGE) Products, or cross-linkformation.


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