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Gene Expression OverviewBy

Salwa Hassan Teama M.D. N.C.I. Cairo university

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Gene ExpressionGene Expression Eukaryotic CellEukaryotic Cell The Gene StructureThe Gene Structure Protein SynthesisProtein Synthesis Prokaryotes Vs EukaryotesProkaryotes Vs Eukaryotes

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Gene ExpressionGene Expression

Gene expressionGene expression

is the process by which a is the process by which a genes information is converted genes information is converted into the structures and into the structures and functions of a cell by a process functions of a cell by a process of producing a biologically of producing a biologically functional molecule of functional molecule of either either protein or RNA (gene product)protein or RNA (gene product) is made. is made.

GeneGene expressionexpression is assumed to be controlled at is assumed to be controlled at various points in the various points in the sequencesequence leading to protein leading to protein synthesissynthesis. .

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Eukaryotic CellEukaryotic Cell

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Gene StructureGene Structure

Eukaryotic gene Eukaryotic gene structure:structure: Most Most eukaryotic genes in eukaryotic genes in contrast to typical contrast to typical bacterial genes, bacterial genes, the the coding sequence coding sequence (exons)(exons) are interrupted by are interrupted by noncoding DNA noncoding DNA (introns).(introns).

The gene must have The gene must have ( Exon; start signals; stop ( Exon; start signals; stop

signals; regulatory control signals; regulatory control elements).elements).

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Protein SynthesisProtein Synthesis

Protein SynthesisProtein Synthesis is the process in whichis the process in which cellscells buildbuild proteins fromproteins from information in a DNA gene information in a DNA gene in a in a two major stepstwo major steps::

I-Transcription and

II-Translation

Transcription :: synthesis of an RNA synthesis of an RNA (mRNA)(mRNA) that is that is complementary to one of complementary to one of the strands of DNA.the strands of DNA.

Translation Translation :: ribosomes read a ribosomes read a messenger RNA and messenger RNA and make protein according to make protein according to

its instructionits instruction..

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Protein SynthesisProtein Synthesis

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TranscriptionTranscription

http://biology.unm.edu/ccouncil/Biology_124/Images/transcription.gif

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Transcription

RNA polymeraseRNA polymerase copies both the exons and the copies both the exons and the introns. The stretch of DNA that is transcribed into an RNA introns. The stretch of DNA that is transcribed into an RNA molecule is called a molecule is called a transcription unittranscription unit. .

A transcription unit that is translated into protein A transcription unit that is translated into protein containscontains codingcoding sequence that is translated into protein and sequence that is translated into protein and sequencessequences that direct and regulate protein synthesis; that direct and regulate protein synthesis;

Transcription proceeds in the 5' → 3' direction. Transcription proceeds in the 5' → 3' direction. Transcription is divided into 3 phases: Initiation,Transcription is divided into 3 phases: Initiation, ElongationElongation

and Termination.and Termination...

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RNA polymerase;RNA polymerase; eukaryotic nuclei contain eukaryotic nuclei contain three RNA polymerasesthree RNA polymerases . . RNA polymerase IRNA polymerase I is is found in the found in the nucleolusnucleolus; ; the other the other two two polymerasespolymerases are located are located in the in the nucleoplasmnucleoplasm. The . The three nuclear RNA three nuclear RNA polymerase have different polymerase have different roles in transcription. roles in transcription.

Polymerase IPolymerase I makes a large makes a large precursor to the major rRNA precursor to the major rRNA (5.8S,18S and 28S rRNA in (5.8S,18S and 28S rRNA in vertebrates). vertebrates).

Polymerase IIPolymerase II synthesizes synthesizes hnRNAs, which are precursors hnRNAs, which are precursors to mRNAs. It also make to mRNAs. It also make

most small nuclear RNAs most small nuclear RNAs (snRNAs). (snRNAs).

Polymerase IIIPolymerase III makes the makes the precursor to 5SrRNA, the precursor to 5SrRNA, the tRNAs and several other small tRNAs and several other small cellular and viral RNAs.cellular and viral RNAs.

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Initiation

The general transcription The general transcription factors combine with factors combine with

RNA polymeraseRNA polymerase to form a preinitiation to form a preinitiation complex that is complex that is competent to initiate competent to initiate transcription as soon as transcription as soon as nucleotide are available.nucleotide are available.

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Initiation

The enzyme The enzyme RNA polymeraseRNA polymerase recognizes a recognizes a promoter,promoter, which lies upstream of the gene. which lies upstream of the gene. The The polymerase binding causes the polymerase binding causes the unwindingunwinding of the DNA of the DNA double helix. double helix. This is followed by initiation of RNA This is followed by initiation of RNA synthesis at the starting point.synthesis at the starting point.

The The RNA polymeraseRNA polymerase starts building the RNA starts building the RNA chain, it assembles ribonucleotides triphosphates: ATP; chain, it assembles ribonucleotides triphosphates: ATP; GTP; CTP and UTP into a strand of RNA.GTP; CTP and UTP into a strand of RNA.

After the first nucleotide is in place, the polymerase joins After the first nucleotide is in place, the polymerase joins a second nucleotide to the first, forming the initial a second nucleotide to the first, forming the initial phosphodiester bond in the RNA chain. phosphodiester bond in the RNA chain.

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Elongation

RNA polymeraseRNA polymerase directs the sequential binding of directs the sequential binding of riboncleotides to the growing RNA chain in the 5`-3` riboncleotides to the growing RNA chain in the 5`-3` direction.direction.

Each ribonucleotide is inserted into the growing RNA Each ribonucleotide is inserted into the growing RNA strand following the rules of base pairing. This process is strand following the rules of base pairing. This process is repeated till the desired RNA length is repeated till the desired RNA length is synthesized…………………….synthesized…………………….

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TerminationTermination

Other regions at the end of genes; called Other regions at the end of genes; called terminators,terminators, signal termination. These work in conjunction with RNA signal termination. These work in conjunction with RNA polymerase to loosen the association between RNA polymerase to loosen the association between RNA product and DNA template. The result is that the RNA product and DNA template. The result is that the RNA dissociate from RNA polymerase and DNA and so stop dissociate from RNA polymerase and DNA and so stop transcription.transcription.

The product is The product is immature RNA or pre mRNA (Primary immature RNA or pre mRNA (Primary transcript).transcript).

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RNA Processing

Pre-mRNA → mRNAPre-mRNA → mRNA Capping:Capping: Synthesis of the cap. The 5` cap is a 7- methylguanosine Synthesis of the cap. The 5` cap is a 7- methylguanosine

(m7G) . The cap protects the mRNA from being degraded by enzymes; (m7G) . The cap protects the mRNA from being degraded by enzymes; enhancement of mRNA translatability.enhancement of mRNA translatability.

SplicingSplicing:: Step-by-step removal of introns present in the pre-mRNA Step-by-step removal of introns present in the pre-mRNA and joining of the remaining exons. The removal of introns and joining of and joining of the remaining exons. The removal of introns and joining of exons takes place on a special structures called spliceosomes.exons takes place on a special structures called spliceosomes.

Polyadenylation:Polyadenylation: Synthesis of the poly (A) tail involves cleavage Synthesis of the poly (A) tail involves cleavage of its 3' end and then the addition of about 200 of its 3' end and then the addition of about 200 adenine adenine residues to form a residues to form a poly (A)poly (A) tail; This completes the mRNA molecule tail; This completes the mRNA molecule (mature mRNA),(mature mRNA), which is which is now ready for export to the cytosol for protein synthesis.now ready for export to the cytosol for protein synthesis.

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RNA Processing

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RNA SplicingRNA Splicing

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Alternative SplicingAlternative Splicing

Alternative splicing:Alternative splicing: is a very common phenomenon in is a very common phenomenon in higher eukaryotes. It is a way to get more than one protein higher eukaryotes. It is a way to get more than one protein product out of the same gene and a way to control gene product out of the same gene and a way to control gene expression in cells.expression in cells.

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TranslationTranslation

http://www.nature.com/embor/journal/v4/n9/images/embor923-f3.jpg

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TranslationTranslation

Translation is the process by which ribosomes read the Translation is the process by which ribosomes read the genetic message in the mRNA and produce a protein genetic message in the mRNA and produce a protein product according to the message's instruction.product according to the message's instruction.

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Requirement for translationRequirement for translation RibosomesRibosomes tRNA tRNA mRNA templatemRNA template Amino AcidsAmino Acids Initiation factorsInitiation factors Elongation factorsElongation factors Termination factorsTermination factors Aminoacyl tRNA synthetase enzymesAminoacyl tRNA synthetase enzymes Energy sourceEnergy source

TranslationTranslation

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RibosomesRibosomes are the site are the site of of protein biosynthesisprotein biosynthesis using using the mRNA as a template, the the mRNA as a template, the ribosome traverses each ribosome traverses each codon of the mRNA, pairing codon of the mRNA, pairing it with the appropriate amino it with the appropriate amino acid. This is done using acid. This is done using molecules of molecules of transfer RNAtransfer RNA (tRNA) containing a (tRNA) containing a complementary complementary anticodonanticodon on on one end and the appropriate one end and the appropriate amino acid on the other.amino acid on the other.

http://www.molecularexpressions.com/cells/ribosomes/images/ribosomesfigure1.jpghttp://www.molecularexpressions.com/cells/ribosomes/images/ribosomesfigure1.jpg

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tRNAtRNA Act as Act as adaptorsadaptors that can bind an that can bind an

amino acid at one end and interact amino acid at one end and interact with the mRNA at the other.with the mRNA at the other.

mRNAmRNA Source of Source of coding informationcoding information for the for the

protein synthesis system.protein synthesis system. Contains start and stop signals for Contains start and stop signals for

translation.translation. Eukaryotic mRNA is Eukaryotic mRNA is cappedcapped. This is . This is

used as the recognition feature for used as the recognition feature for ribosome binding. ribosome binding.

The site at which protein synthesis The site at which protein synthesis begins on the mRNA is especially begins on the mRNA is especially crucial, since it sets the crucial, since it sets the reading reading frameframe for the whole length of the for the whole length of the messagemessage. . An error of one nucleotide An error of one nucleotide either way at this stage would cause either way at this stage would cause every subsequent codon in the every subsequent codon in the message to be misread, so that a message to be misread, so that a nonfunctional protein would result, nonfunctional protein would result, the rate of initiation thus determines the rate of initiation thus determines the rate at which the protein is the rate at which the protein is synthesizedsynthesized. .

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Amino acidsAmino acids are the monomers are the monomers which arewhich are polymerizedpolymerized to produce to produce proteinsproteins. . The amino acids are The amino acids are loaded ontoloaded onto tRNAtRNA molecules for molecules for use in the process ofuse in the process of translationtranslation..

Initiation factorsInitiation factors help the help the ribosome, initiator tRNA, and other ribosome, initiator tRNA, and other components assemble the at the components assemble the at the correct location on the mRNA and correct location on the mRNA and ensure that protein synthesis ensure that protein synthesis starts in the correct reading starts in the correct reading frameframe ..

Elongation factorsElongation factors are responsible are responsible for moving the ribosome along the for moving the ribosome along the mRNA and maintain the correct mRNA and maintain the correct reading framereading frame. . Facilitate removal Facilitate removal of of ""usedused" " tRNAs and bringing in tRNAs and bringing in ""newnew" " tRNAs.tRNAs.

Termination factorsTermination factors recognize recognize the stop codons and release the stop codons and release proteins and ribosomes.proteins and ribosomes.

Aminoacyl tRNA synthetase Aminoacyl tRNA synthetase enzymes:enzymes: It catalyze the It catalyze the covalent attachment of an covalent attachment of an amino acids to the end of the amino acids to the end of the corresponding tRNA.corresponding tRNA.

Energy source:Energy source: ATP or GTP ATP or GTP which are synthesized in the which are synthesized in the mitochondria.mitochondria.

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Preparatory steps for Preparatory steps for protein synthesisprotein synthesis::

First,First, aminoacyl tRNA aminoacyl tRNA synthetase join amino synthetase join amino acid to their specific acid to their specific

tRNAtRNA . .Second,Second, ribosomes must ribosomes must dissociatedissociate into subunits at into subunits at the end of each round of the end of each round of translationtranslation..

The protein synthesis occur in 3 The protein synthesis occur in 3 phasesphases::

11 - -Accurate and efficient Accurate and efficient initiationinitiation occurs, the ribosomes binds to occurs, the ribosomes binds to the mRNA, and the first amino the mRNA, and the first amino acid attached to its tRNAacid attached to its tRNA..

22 - -Chain Chain elongationelongation,, the the ribosomes adds one amino ribosomes adds one amino acid at a time to the growing acid at a time to the growing polypepyide chainpolypepyide chain . .

33 - -Accurate and efficient Accurate and efficient terminationtermination,, the ribosomes the ribosomes releases the mRNA and the releases the mRNA and the polypeptidepolypeptide..

TranslationTranslation

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TranslationTranslation: Initiation

The initiation phase of The initiation phase of protein synthesis protein synthesis requires over 10 requires over 10 eukaryotic Initiation eukaryotic Initiation Factors (eIFs):Factors (eIFs): Factors Factors are needed to recognize are needed to recognize the cap at the 5` end of the cap at the 5` end of an mRNA and binding to an mRNA and binding to the 40s ribosomal the 40s ribosomal subunit.subunit.

Binding the initiator MetBinding the initiator Met--tRNAiMettRNAiMet (methionyl- tRNA) to the 40S small (methionyl- tRNA) to the 40S small subunit of the ribosomesubunit of the ribosome. .

Scanning to find the start codonScanning to find the start codon by by binding to the 5` cap of the mRNA binding to the 5` cap of the mRNA and scanning downstream until they and scanning downstream until they find the first find the first AUG (initiation codon)AUG (initiation codon)..

The start codon must be located The start codon must be located and positioned correctly in the P and positioned correctly in the P site of the ribosome and the initiator site of the ribosome and the initiator tRNA must be positioned correctly tRNA must be positioned correctly in the same site.in the same site.

Once the mRNA and initiator tRNA Once the mRNA and initiator tRNA are correctly bound, the 60S large are correctly bound, the 60S large subunit binds to form 80 s initiation subunit binds to form 80 s initiation complex with release of the eIF complex with release of the eIF factors.factors.

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The large ribosomal The large ribosomal subunit contains three subunit contains three tRNA binding sites, tRNA binding sites, designated A, P, and E. designated A, P, and E. The The A siteA site binds an binds an aminoacyl-tRNA (a tRNA aminoacyl-tRNA (a tRNA bound to an amino acid); bound to an amino acid); the the P siteP site binds a binds a peptidyl-tRNA (a tRNA peptidyl-tRNA (a tRNA bound to the peptide bound to the peptide being synthesized); and being synthesized); and the the E siteE site binds a free binds a free tRNA before it exits the tRNA before it exits the ribosome.ribosome.

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ElongationElongation

Transfer of proper Transfer of proper aminoacyl-tRNA from aminoacyl-tRNA from cytoplasm to A-site of cytoplasm to A-site of ribosome;ribosome;

Peptide bond formation;Peptide bond formation;

Peptidyl transferase Peptidyl transferase forms a peptide bonds forms a peptide bonds between the amino acid between the amino acid in the P site and the in the P site and the newly arrived aminoacyl newly arrived aminoacyl tRNA in the A site. This tRNA in the A site. This lengthens the peptide by lengthens the peptide by one amino acids.one amino acids.

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ElongationElongation

Translocation;Translocation; translocation of the new translocation of the new peptidyl t-RNA with its peptidyl t-RNA with its mRNA codon in the A site mRNA codon in the A site into the free P site occurs into the free P site occurs Now the A site is free for Now the A site is free for another cycle of another cycle of aminoacyl t-RNA codon aminoacyl t-RNA codon recognition and recognition and elongation. Each elongation. Each translocation events translocation events moves mRNA , one moves mRNA , one codon length through the codon length through the ribosomes. ribosomes.

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TerminationTermination

Translational termination requires specific protein factors Translational termination requires specific protein factors identified as identified as releasing factors, RFsreleasing factors, RFs in E. coli and eRFs in in E. coli and eRFs in eukaryotes.eukaryotes.

The signals for termination are the same in both prokaryotes The signals for termination are the same in both prokaryotes

and eukaryotes. These signals are termination codons and eukaryotes. These signals are termination codons present in the mRNA. There are 3 present in the mRNA. There are 3 termination codons, UAG, termination codons, UAG, UAA and UGA. UAA and UGA.

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TerminationTermination

After multiple cycles of After multiple cycles of elongation and elongation and polymerization of specific polymerization of specific amino acids into protein amino acids into protein molecules, a nonsense molecules, a nonsense codon = termination codon = termination codon of mRNA appear in codon of mRNA appear in the A site. The is the A site. The is recognized as terminal recognized as terminal signal by eukaryotic signal by eukaryotic releasing factors (eRF) releasing factors (eRF) which cause the release which cause the release of the newly synthesized of the newly synthesized protein from the protein from the ribosomal complex. ribosomal complex.

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Protein Synthesis

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Protein Synthesis

www.bseinquiry.gov.uk/report/volume2/fig1_2.htm

Reading the instruction means Reading the instruction means translating the code in the RNA translating the code in the RNA from from basesbases

))building block of DNA and RNAbuilding block of DNA and RNA ( (to to amino acidsamino acids (building block of (building block of proteins)proteins)..

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Prokaryotic vs. EukaryoticProkaryotic vs. Eukaryotic

Eukaryotic DNA is wound around Eukaryotic DNA is wound around histoneshistones to form to form nucleosomesnucleosomes and packaged as and packaged as chromatinchromatin. Chromatin . Chromatin has a strong influence on the accessibility of the DNA to has a strong influence on the accessibility of the DNA to transcription factorstranscription factors and the transcriptional machinery and the transcriptional machinery including including RNA polymerase.RNA polymerase.

Eukaryote genes are not grouped in operons. each Eukaryote genes are not grouped in operons. each eukaryote gene is transcribed separately, with separate eukaryote gene is transcribed separately, with separate transcriptional controls on each gene. transcriptional controls on each gene.

Protein synthesis takes place in the cytoplasm while Protein synthesis takes place in the cytoplasm while transcription and RNA processing take place in the transcription and RNA processing take place in the nucleus.nucleus.

Essentially all humans' genes contain introns. A notable Essentially all humans' genes contain introns. A notable exception is the histone genes which are intronless. exception is the histone genes which are intronless.

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Prokaryotic vs. EukaryoticProkaryotic vs. Eukaryotic

Eukaryotic mRNA is modified through Eukaryotic mRNA is modified through RNA splicingRNA splicing.. Eukaryotic mRNA is generally monogenic Eukaryotic mRNA is generally monogenic

(monocistronic); code for only one polypeptide.(monocistronic); code for only one polypeptide. Eukaryotes have a separate RNA polymerase for each Eukaryotes have a separate RNA polymerase for each

type of RNA. type of RNA. Eukaryotic mRNA contain Eukaryotic mRNA contain no Shine-Dalgarnono Shine-Dalgarno sequence sequence

to show the ribosomes where to start translating. to show the ribosomes where to start translating. Instead, most eukaryotic mRNA have caps at their 5` Instead, most eukaryotic mRNA have caps at their 5` end which directs initiation factors to bind and begin end which directs initiation factors to bind and begin searching for an initiation codon.searching for an initiation codon.

Eukaryotic protein synthesis initiation begins with Eukaryotic protein synthesis initiation begins with methionine not N formyl- methionine.methionine not N formyl- methionine.

In eukaryotes, polysomes are found in the cytoplasm.In eukaryotes, polysomes are found in the cytoplasm.

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Prokaryotic vs. EukaryoticProkaryotic vs. Eukaryotic Bacterial genetics are different.Bacterial genetics are different. Prokaryote genes are grouped in operons. Prokaryote genes are grouped in operons. Prokaryotes have one type of RNA polymerase for all types of Prokaryotes have one type of RNA polymerase for all types of

RNA, RNA, mRNA is not modified mRNA is not modified The existence of introns in prokaryotes is extremely rare. The existence of introns in prokaryotes is extremely rare. To initiate transcription in bacteria, sigma factors bind to RNA To initiate transcription in bacteria, sigma factors bind to RNA

polymerases. RNA polymerases/ sigma factors complex can polymerases. RNA polymerases/ sigma factors complex can then bind to promoter about 40 deoxyribonucleotide bases prior then bind to promoter about 40 deoxyribonucleotide bases prior to the coding region of the gene.to the coding region of the gene.

In prokaryotes, the newly synthesized mRNA is polycistronic In prokaryotes, the newly synthesized mRNA is polycistronic (polygenic) (code for more than one polypeptide chain). (polygenic) (code for more than one polypeptide chain).

In prokaryotes, transcription of a gene and translation of the In prokaryotes, transcription of a gene and translation of the resulting mRNA occur simultaneously. So many polysomes are resulting mRNA occur simultaneously. So many polysomes are found associated with an active gene.found associated with an active gene.

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References & Further Reading Robert F.Weaver. Molecular Biology. Fourth Edition. Page 600. McGraw-Hill International Edition. ISBN 978-0-07-110216-2Robert F.Weaver. Molecular Biology. Fourth Edition. Page 600. McGraw-Hill International Edition. ISBN 978-0-07-110216-2 InnisInnis,David H. ,David H. GelfandGelfand,John J. Sninsky PCR Applications: Protocols for Functional Genomics: ISBN:0123721865 ,John J. Sninsky PCR Applications: Protocols for Functional Genomics: ISBN:0123721865 DanielDaniel H. Farkas. DNA Simplified: The Hitchhiker's Guide to DNA. Washington, DC: AACC Press, 1996, ISBN 0-915274-84-1. H. Farkas. DNA Simplified: The Hitchhiker's Guide to DNA. Washington, DC: AACC Press, 1996, ISBN 0-915274-84-1. William B. Coleman,Gregory J. Tsongalis: William B. Coleman,Gregory J. Tsongalis: MolecularMolecular DiagnosticsDiagnostics: For the : For the ClinicalClinical LaboratorianLaboratorian: : ISBNISBN 1588293564... 1588293564... Robert F. Mueller,Ian D. Young. Robert F. Mueller,Ian D. Young. EmeryEmery''ss ElementsElements of of MedicalMedical GeneticsGenetics: ISBN. : ISBN. 044307125X044307125X DanielDaniel P. P. StitesStites,Abba T. Terr. Basic Human Immunology: ISBN. ,Abba T. Terr. Basic Human Immunology: ISBN. 08385054300838505430

Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology of the cell. ISBN. 9780815341055Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology of the cell. ISBN. 9780815341055

http://www.pubmedcentral.nih.gov/http://www.pubmedcentral.nih.gov/ www.medscape.comwww.medscape.com www.ebi.ac.uk/2can good introduction to bioinformatics and molecular biology www.ebi.ac.uk/2can good introduction to bioinformatics and molecular biology http://www.genomicglossaries.comhttp://www.genomicglossaries.com http://www.gene.ucl.ac.uk/nomenclature/guidelines.html defines the nomenclature for human geneshttp://www.gene.ucl.ac.uk/nomenclature/guidelines.html defines the nomenclature for human genes http://www.accessexcellence.orghttp://www.accessexcellence.org http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Codons.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Codons.html http://www.web-books.com/MoBio/http://www.web-books.com/MoBio/ http://www.expasy.orghttp://www.expasy.org http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPROTSYn.htmlhttp://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPROTSYn.html Cell & Molecular Biology online: http://www.cellbio.com/recommend.htmlCell & Molecular Biology online: http://www.cellbio.com/recommend.html http://www.ornl.gov/sci/techresources/Human_Genome/glossary/glossary.shtml%20http://www.ornl.gov/sci/techresources/Human_Genome/glossary/glossary.shtml%20 http://www.genome.gov/10000715http://www.genome.gov/10000715 http://www.ncbi.nlm.nih.gov/About/primer/mapping.htmlhttp://www.ncbi.nlm.nih.gov/About/primer/mapping.html http://www.lilly.com/research/discovering/targets.html http://www.lilly.com/research/discovering/targets.html http://www.informatics.jax.org/expression.shtml http://www.informatics.jax.org/expression.shtml www.wikipdia.comwww.wikipdia.com http://www.biology.arizona.edu/cell_bio/tutorials/pev/page2.htmlhttp://www.biology.arizona.edu/cell_bio/tutorials/pev/page2.html http://www.genome.ou.edu/protocol_book/protocol_index.htmlhttp://www.genome.ou.edu/protocol_book/protocol_index.html

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