Nucleic acids

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Nucleic Acids
( The Alphabet of the life )
◘ Eukaryotic Chromosomes
• In non-dividing eukaryotic cells, the
chromosomal material called chromatin.
• Chromatin has been isolated and analyzed.
• It consists of fibers that contain protein and DNA
in approximately equal masses, plus a small
amount of RNA.
• The DNA in the chromatin is not "bare" ( Free) but is very tightly
associated with proteins called histones, which package and order the
DNA into structural units called nucleosomes that resemble beads on a
string .

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♦ The Histones :
• The histones present in chromatin are of five major types, designated
H1, H2A, H2B, H3 and H4, fall into two main groups:
The first groups of histones form the structural core of the individual
nucleosome "beads".
- Two molecules each of H2A, H2B, H3 and H4 associate to form the
nucleosome core, around which a segment of the DNA double helix is
wound forming a negatively super-twisted helix.
- Each nucleosome contains about 140 base pairs of DNA.
- Histone H1, of which there are several related species that form the
second group of histones, is not found in the nucleosome core but
appears to bind to the spacer DNA chain between the nucleosome beads
(the linker DNA).
Approximately 60 base pairs of spacer between core particles.-
- H1 appears that to aid the packing of nucleosomes into a more compact
structure called the chromatin fiber.
- During cell division, the chromatin condenses further to form the
chromosomes that are visible in the light microscope.

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◘ Deoxynucleic acid ( DNA ) and its components :
◘ DNA Overview :
• Cells contain a structure called the nucleus.
• DNA looks like a winding staircase.
• Chromosomes contain tightly wound and folded DNA .
• Within the nucleus DNA is found as chromosomes .
♦ Chemical structure of Nucleic Acids :
• DNA and RNA are chain-like molecule .
• The Monomeric unit of DNA is called Deoxyribonucleic acid .
• The Monomeric unit of RNA is called Ribonucleic acid .

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• In the DNA molecule , each sugar is attached to one of four types of
molecule called Bases ( Purines & Pyrimidines )
♦ DNA Structure :
• DNA is made up of molecules called nucleotides .
• Each nucleotide contains ( Phosphate group & Sugar & Nitrogen Base ).

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♦ Structure of Nucleotide :
♣ Types of Bases :
1- Purines
• Two of the bases , A & G have a double-
ring structure these are called purines
• M.F. : ( C5N4H4 )
2- Pyrimidines
• T , C , and U have a single-ring structure; these are called pyrimidines.
• M.F. : ( C4N2H4 )
• T found in DNA only
• U found in RNA only

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• Example :
• Guanosine + Phosphoric acid
Nucleotide ( Guanosine 5'- MonoPhosphate )
• Nucleotides are phosphoric esters of nucleosides called Ribonucleosides
5' monophosphate ( if the sugar ribose ) , or deoxyribonucleosides 5'
monophosphate ( if the sugar deoxyribose ) .

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♦ Importance of Nucleotides :
• They are activated precursors of DNA & RNA building blocks.
( without these cpds neither DNA nor RNA can produced )
• Nucleotide derivatives are activated intermediates in many
biosynthesis.
• Nucleotides are energy rich cpds ( ATP ) & ( AMP ).
• Nucleotides are Structural components of enzyme cofactor.
( Adenine are component of 3 major coenzymes " NAD , FAD, Co A. " )
• Nucleotides are Metabolic regulators .
♣ Function :
• N.A. biosynthesis .
• Protein biosynthesis .
• Energy production and transduction.
• Regulatory cascades .
• Signal transduction .
♦ Nucleotide linking :
• Nucleotides are linked via Phosphodiester bond .
• The 3' OH of one Nucleotide is linked to the 5' phosphate gp of the next Nucleotide .

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♦ Base pairing :
♠ Chargaff's Rule :
• The amount of Adenine = The amount of Thymine
• The amount of Guanine = The amount of Cytosine
• Always pairing a purine and a pyrimidine yields a
constant width .
• Chargaff's Rule = DNA composition = A + G = T + C
♠ Watson and Crick Model :
• James Watson and Francis Crick a rounded with Nobel Prize for
(discovering) DNA structure (1953).
• Two helical polynucleotide chain are right handed coiled around a common axis .
• The helical structure repeats after 10 residues on
each chain .
- complete turn contain 10 base pairs ( 0.34 * 10 =
3.4 nm ) .
- one turn of the helix is 3.4 nm.
- X – rays diffraction studies indicated that
distances between two paired nucleotides is 3.4 A0
( 0.34 nm )
• Two chains run in opposite direction .
• Sugar phosphate are outside , base pair are inside .
• Nanometer = Angstrom * 0.1

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♦ The Double helix of DNA has these features (characteristics ):
 It contains two polynucleotide strands wound around each other.
 The backbone of each consists of alternating deoxyribose and phosphate
groups.
 The phosphate group bonded to the 5' carbon atom of one deoxyribose is
covalently bonded to the 3' carbon of the next.
 The two strands are "anti-parallel"; that is, one strand runs 5′ to 3′ while the
other runs 3′ to 5′.
 The purine or pyrimidine attached to each deoxyribose projects in toward the
axis of the helix.
 Each base forms hydrogen bonds with the one directly opposite it,
forming base pairs (also called nucleotide pairs).
- A with T: the purine adenine (A) always
pairs with the pyrimidine thymine (T)
- C with G: the pyrimidine cytosine (C)
always pairs with the purine guanine (G)
(Two H-bond between A & T; Three H-
bond between C & G)
 The diameter of the helix is 20 Å.
 Complete turn contain 10 base pairs ( 0.34 * 10 = 3.4
nm ) .
 The distances between two paired nucleotides is 3.4 A0
( 0.34 nm )
 The path taken by the two backbones forms a major (wider) groove and a
minor (narrower) groove .
 The molecule is stabilized by
- Larg numbers of H-bond between bases
- Hydrophobic bonding
 This structure of DNA was worked out by Francis Crick and James D.
Watson in 1953.

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◘ Denaturation and Renaturation
• Heating double stranded DNA can overcome the hydrogen bonds
holding it together and cause the strands to separate resulting in
denaturation of the DNA.
• When cooled relatively weak hydrogen bonds between bases can reform
and the DNA renatures.
• DNA with a high guanine and cytosine content has relatively more
hydrogen bonds between strands.
• This is because for every GC base pair 3 hydrogen bonds are made while
for AT base pairs only 2 bonds are made.
• Thus higher GC content is reflected in higher melting or denaturation
temperature.

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• Comparison of melting temp can be used to determine GC content of the
organism genome .
• To do this , it is necessary to be able to detect whether DNA is melted or
not .
• The loss of helical structure in DNA is called denaturation.
• Tm ( Melting temperature ): is the temperature at which half of the DNA
is melted .
◘ GC Content Of Some Genomes
% GCOrganism
39.7 %Homo sapiens
42.4 %Sheep
42.0 %Hen
43.3 %Turtle
41.2 %Salmon
35.0 %Sea urchin
51.7 %E. coli
50.0 %Staphylococcus aureus
55.8 %Phage 
48.0 %Phage T7

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◘ Determination GC content :
• Absorbance at 260 nm of DNA in solution provides a means of
determining how much is single standard .
• Single standard DNA absorbs 260 nm ultraviolet light more strongly
than double stranded DNA does although both absorb at this wavelength .
• Thus, increasing absorbance at 260 nm during heating indicates
increasing concentration of single stranded DNA .
◘ The Central Dogma of Molecular Biology :
• The Watson Crick termed the central dogma of molecular genetics, which states
that genetic information flows from DNA to RNA to Protein .
• This is the same for bacteria to humans
• DNA is the genetic instruction or gene
DNA  RNA is called Transcription
RNA chain is called a transcript
RNA  Protein is called Translation

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• Central dogma comprises 3 steps :
1- Replication
2-Transcription
3- Translation
Prokaryotic DNA synthesis :◘
• The enzymes involved in the DNA replication process are template directed
polymerases that can synthesize the complementary sequence of each strand
with extra ordinary fidelity.
• DNA-dependent DNA polymerase requires in addition to a DNA template , all
four deoxy-ribinucleoside 5' – tri-phosphates ( ATP , GTP, TTP , and CTP ) as
precursors of the nucleotide units of DNA as well as Mg2+
.
• Separation of the 2 complementary DNA strands
• In order for the two strands of parental double helical DNA to be replicated,
they must first separate ( or Melt ) at least in a small region because the
polymerase use only single strand DNA template .
• When the two strands of the DNA double helix are separated, each can serve as
template for the replication of the next complementary strands.
• This produces two daughter molecules, each of which contains two DNA
strands with an anti-parallel orientation.
• In prokaryotic organisms, DNA replication begins at a single, unique
nucleotide sequence, a site called the origin of replication.

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◘ Prokaryotic DNA Replication :
‫بعذ‬ ‫دي‬ ‫انخطوات‬ ‫نكم‬ ‫اختصار‬ ‫وفي‬ ‫ساليذ‬ ‫عهي‬ ‫مش‬ ‫فقط‬ ‫فيذيو‬ ‫عهي‬ ‫اال‬ ‫ماتشرحش‬ ‫دا‬ ‫انشرح‬
‫عهيه‬ ‫سؤال‬ ‫جه‬ ‫نو‬ ‫عارفينه‬ ‫نكون‬ ‫عشان‬ ‫انشرح‬.
• E. coli has a single origin of replication on its one chromosome,
as do most prokaryotes .
-This sequence of base pairs is recognized by certain proteins that
bind to this site.
• An enzyme called helicase unwinds the DNA by breaking the
hydrogen bonds between the nitrogenous base pairs.
• As the DNA opens up, Y-shaped structures called replication forks are formed.
• Two replication forks are formed at the origin of replication and these get extended bi-
directionally as replication proceeds.
• Single-strand binding proteins coat the single strands of DNA near the replication fork to
prevent the single-stranded DNA from winding back into a double helix.
• The next important enzyme is DNA polymerase III, also known as DNA pol III, which adds
nucleotides one by one to the growing DNA chain.
• The addition of nucleotides requires energy; this energy is obtained from the nucleotides that
have three phosphates attached to them.
• In addition to ATP, there are also TTP, CTP, and GTP. Each of these is made up of the
corresponding nucleotide with three phosphates attached.
• When the bond between the phosphates is broken, the energy released is used to form the
phosphodiester bond between the incoming nucleotide and the existing chain.
• DNA polymerase is able to add nucleotides only in the 5′ to 3′ direction (a new DNA strand
can be only extended in this direction).
• It requires a free 3′-OH group (located on the sugar) to which it can add the next nucleotide by
forming a phosphodiester bond between the 3′-OH end and the 5′ phosphate of the next
nucleotide.
• This essentially means that it cannot add nucleotides if a free 3′-OH group is not available.
• Then how does it add the first nucleotide? The problem is solved with the help of
a primer that provides the free 3′-OH end.
• Another enzyme, RNA primase, synthesizes an RNA primer that is about five to ten
nucleotides long and complementary to the DNA.
- RNA primase does not require a free 3′-OH group.

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• DNA polymerase can now extend this RNA primer, adding nucleotides one by one that are
complementary to the template strand.
• DNA polymerase can only extend in the 5′ to 3′ direction, which poses a slight problem at the
replication fork.
• As we know, the DNA double helix is anti-parallel; that is, one strand is in the 5′ to 3′
direction and the other is oriented in the 3′ to 5′ direction.
• One strand, which is complementary to the 3′ to 5′ parental DNA strand, is synthesized
continuously towards the replication fork because the polymerase can add nucleotides in this
direction.
• This continuously synthesized strand is known as the leading strand.
• The other strand, complementary to the 5′ to 3′ parental DNA, is extended away from the
replication fork, in small fragments known as Okazaki fragments, each requiring a primer to
start the synthesis.
• The strand with the Okazaki fragments is known as the lagging strand.
• The leading strand can be extended by one primer alone, whereas the lagging strand needs a
new primer for each of the short Okazaki fragments.
• The overall direction of the lagging strand will be 3′ to 5′, and that of the leading strand 5′ to
3′.
• Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication
fork as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and
then resealing it.
• As synthesis proceeds, the RNA primers are replaced by DNA pol I, which breaks down the
RNA and fills the gaps with DNA nucleotides.
• The nicks that remain between the newly synthesized DNA (that replaced the RNA primer)
and the previously synthesized DNA are sealed by the enzyme DNA ligase that catalyzes the
formation of phosphodiester linkage between the 3′-OH end of one nucleotide and the 5′
phosphate end of the other fragment.
• Once the chromosome has been completely replicated, the two DNA copies move into two
different cells during cell division.

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◘ ♦ ◘ The process of DNA replication can be summarized as follows:
1. DNA unwinds at the origin of replication.
2. Helicase opens up the DNA-forming replication forks; these are extended in both
directions.
3. Single-strand binding proteins coat the DNA around the replication fork to prevent
rewinding of the DNA.
4. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling
(over-winding).
5. Primase synthesizes RNA primers complementary to the DNA strand.
6. DNA polymerase III starts adding nucleotides to the 3′-OH (sugar) end of the primer.
7. Elongation of both the lagging and the leading strand continues.
8. RNA primers are removed and gaps are filled with DNA by DNA pol I.
9. The gaps between the DNA fragments are sealed by DNA ligase.
◘ Prokaryotic DNA Replication: Enzymes and Their Function
Specific FunctionEnzyme/protein
Exonuclease activity removes RNA primer and replaces with newly
synthesized DNA
DNA pol I
Repair functionDNA pol II
Main enzyme that adds nucleotides in the 5′-3′ directionDNA pol IIl
Opens the DNA helix by breaking hydrogen bonds between the
nitrogenous bases
Helicase
Seals the gaps between the Okazaki fragments to create one continuous
DNA strand
Ligase
Synthesizes RNA primers needed to start replicationPrimase
Helps relieve the stress on DNA when unwinding by causing breaks and
then resealing the DNA
Topoisomerase
Binds to single-stranded DNA to avoid DNA rewinding back.Single-strand
binding proteins
(SSB)
♣ Topoisomerase types :
◘ Topoisomerase l : Produce single stranded breaks in one of the DNA backbones, allows one
of the phosphodiester bonds to rotate freely around the other relieving tortional stress .
◘ Topoisomerase ll : Produce double stranded breals in the DNA backbone at the same tome.
-This is necessary to untangle replicated chromosomes .

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• RNA is a complimentary copy from DNA template except Uracil is
instead of thymine.
• RNA is synthesized by RNA polymerase.
• DNA dependant RNA polymerase requires, in addition to a DNA
template, all four ribonucleotide 5' – triphosphate (ATP, GTP, UTP and
CTP) as a precursors of the nucleotide units of RNA as well as Mg2+
.
• The protein is also binds one zn2+
.
• RNA elongates on RNA strand by adding ribonucleotide unites to the 3'-
Hydoxyl end (OH).
-Building RNA in the 5' 3' direction.
• The 3'-OH group acts as a nucleophile, attacking the phosphate of the
incoming ribonucleoside triphosphate and releasing pyrophosphate.
• The overall reaction is
(NMP)n +NTP (NMP)n+1 + PPi

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♦ Coding Strand and Template Strand :
♣ Coding Strand ( Un-transcipted DNA Strand ) :
• Is the strand whose base sequence specifies the amino acid sequence of
the encoded protein therefore also is termed as sense strand ( non-template
strand or positive strand ).
♣ Template Strand (Transcipted DNA Strand ) :
• Is the strand from which the RNA is actually transcribed is also termed
anti-sense strand or non-coding strand or negative strand.
◘ Prokaryotic RNA polymerase structure :
FunctionsSubunits
Determine the DNA to be transcipted
Catalyze polymerizationβ
Bind and spin DNA template'β
Recognize the promoter and facilitate initiation .
Releases after nucleotides of RNA are linked together .
σ
2ββ'σ 2ββ' + σ

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◘ Steps of RNA synthesize :
1- Initiation.
2- Elongation.
3- Termination.
1- Initiation
• In contrast to DNA polymerase, RNA polymerase doesn't need a primer
to begin synthesis.
• The transcription is initiated by the bonding of RNA polymerase to a
specific region of DNA double helix.
• This site is called promoter site or promoter region.
• This region is recognized by Sigma factor σ subunit of RNA polymerase.
• When RNA polymerase recognizes this region, it binds to it leading to a
local unwinding ( separation ) of the promoter region into two single
strand .

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a- DNA strand that is transcipted into mRNA and called template
strand or antisense strand .
b- The other strand is coding strand or sense strand that contain gene
to be translated (This strand not transcipted, not used).
♦ Direction of Transcription :
• RNA polymerase will read the information sequence on DNA
template from 3' 5' direction , so RNA is synthesized antiparallel
to DNA template i.e. from 5' 3' direction .
2- Elongation :
• Once RNA polymerase recognizes promoter region, it begins to
synthesize a transcript (copy) of DNA template.
• RNA Polymerase recognize the TTGACA region and slides to the
TATAAT region, then opens the DNA duplex.
• The unwound region is about 17±1 bp.

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• The -35 region of TTGACA sequence is the recognition site and the binding site
of RNA-pol.
• The -10 region of TATAAT is the region at which a stable complex of DNA and
RNA-pol. is formed.
♦ DNA polymerase Holoenzyme:
•The release of the σ subunit causes the conformational change of the core enzyme.
•The core enzyme slides on the DNA template toward the 3' end.

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3- Termination :
• Process of elongation of RNA continues
until reach what is called: termination
region which is recognized by rho factor
(ρ) resulting in release of the enzyme and
the synthesized RNA .
♦ Difference between Replication and Transcription:
TranscriptionReplication
New RNA is formedNew DNA is formed
DNA –RNA hybrid complexDNA-DNA hybrid complex
RNA polymerase enzyme.DNA polymerase enzyme.
Primer not requiredPrimer is required
Ribonucleotides usedDeoxyrinonucleotides used
Very small portion of genome
transcribed
Entire genome is copied.
No proofreading.Proofreading
Information is transferred from
gene to protein
Genetic information is Inherited.
Base pair : A-U,G-C, T-ABase pair : A-T,G-C
♦ Difference between DNA & RNA Polymerase:
RNA
Polymerase
DNA
Polymerase
No primer
required
Primer required
Does not posses
endo &
exonuclease
activity
Posses endo &
exonuclease
activity
No proofreadingProof reading
done

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3- Translation :
♦ Genetic code :
• A code used to convert the language of mRNA to Amino acids .
♦ Codons: 3 Nucleotides on mRNA
each codon ( codes ) for a specific
amino acid .
♦ Anti Codon: 3 Nucleotides on
tRNA that matches up with a
complimentary mRNA codon .

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•The start codon is the first codon of a messenger RNA (mRNA) transcript
translated by a ribosome.
- The start codon always codes for methionine in eukaryotes and a modified Met
(fMet) in prokaryotes.
- The most common start codon is AUG.
• The stop codon (or termination codon) is a nucleotide triplet within messenger
RNA that signals a termination of translation into proteins.
♦ Sets of three bases must be employed to encode the standard amino acids used by
living cells to build proteins, which would allow a maximum of 43
= 64 amino
acids.
-We have 4 bases: A, U, C, G .
-The code has 3 bases.
-So, 43
= 64 amino acids .
-3 codes represent stop codons, and 61 codon translated to amino acids .

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3-Translation :
♦ process in 3 steps :
1- Initiation.
2- Elongation.
3- Termination.
1- Initiation.
• The direction of the protein synthesized
N-terminal C-terminal
• The direction of template mRNA 5' 3' end.
♦ Initiation Process : ( Four steps )
1- Separation Between 50S and 30S subunit.
2- Positioning mRNA on the 30S subunit.
3- Registering fMet-tRNAimet on the P site.
4- Associating the 50S subunit.
♦ Formation of initiation complex in bacteria:
• The complex form in 3 steps at the expense of the hydrolysis of GTP to
GDP and Pi.
• IF-1, IF-3, and IF-3 are initiation factors.
• P designates the peptidyl site.
• A the aminoacyl site.
• E the exit site.

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• IF-3 and IF-1 bind to the 30S subunit, making separation between 50S
and 30S subunit.
• The mRNA then binds to 30S subunit.
• The complex of the GTP bound IF-2 and the fMet-tRNA enters the P
site.
• GTP is hydrolyzed to GDP and Pi.
• All three Initiation Factors depart from this complex.
• The 50 subunit combines with this complex.
2-Elongation:
♦ Elongation Step in Bacteria :
• Three step in each cycle
1-Positioning an aminoacyl tRNA in the A site ( Entrance ) .
2-Forming a peptide bond ( peptide bond formation ) .
3-Translocation of the ribosomes to the next codon ( Translocation ) .
♣ Elongation Factors ( EF ) are required .

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1- Step I ( Entrance ):
• An AA-tRNA occupies the empty A site.
• Registration of AA-tRNA consume one GTP.
• The entrance of AA-tRNA needs to activate EF-Tu.
.
2- Step II ( Peptide bond formation )
• Occurs at the A site.
• The formylmethionyl group is transferred to -NH2 of the AA-tRNA at
the A site by a peptidyl transferase .

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3-Step III ( Translocation ) :
• EF-G is tranlocase .
• GTP bound EF-G provides the energy to move the ribosome one codon
toward the 3' end on mRNA .
• After the translocation, the uncharged tRNA is released from the E-site.

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♦ Termination of protein synthesis in bacteria :
• Termination occurs in response to a termination codon in the A-site
(UAG).
• Prokaryotes have 3 release factors : RF-1, RF-2, and RF-3.
• RF-1 & RF-2 : Recognizing the termination codons.
• RF-3 : GTP hydrolysis and coordinating RF-1/RF-2.
• This leads to hydrolysis of the ester linkage between the nascent
polypeptide and the tRNA in the P site and release of the completed
polypeptide.
• Finally, the mRNA, deacylated tRNA, and release factor, leave the
ribosomes and the ribosome dissociates into its 30S and 50S subunits.
The lec. Is Done ☺ Q.A

Nucleic acids

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