Cytogenetics 2 replication, transcription and translation

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  • Enzymes more than a dozen enzymes & other proteins participate in DNA replication
  • 09/29/12
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  • 09/29/12 Figure: 10.2 Title: Cells synthesize three major types of RNA Caption: RNA consists of a single nucleotide strand whose bases are complementary to the bases within the template strand of the gene. There are three major types of RNA: (a) Messenger RNA (mRNA) carries within its base sequence the information for the amino acid sequence of a protein. (b) Ribosomes contain both ribosomal RNA (rRNA) and proteins. The ribosome is divided into a small and large subunit that join together during protein synthesis. The small subunit binds the mRNA; the large subunit binds tRNA and catalyzes the formation of bonds between amino acids to form a protein. (c) One side of transfer RNA contains an anticodon, which is a sequence of three nucleotides that can form base pairs with a codon in mRNA. Enzymes within the cytoplasm attach a specific amino acid to the opposite side of the tRNA so that it can carry the proper amino acid to the ribosome for incorporation into a new protein.
  • 09/29/12 Figure: 10.3 Title: Genetic information flows from DNA to RNA to protein Caption: Cellular information is stored within the base sequence of DNA. Transcription, the process of RNA synthesis, occurs in the nucleus. During transcription, the nucleotide sequence in a gene specifies the nucleotide sequence in a complementary RNA molecule. For protein-encoding genes, the product is an mRNA molecule that exits from the nucleus and enters the cytoplasm where translation occurs. During translation, the sequence in an mRNA molecule specifies the amino acid sequence in a protein.
  • Table 10-3 The Genetic Code (Codons of mRNA)
  • FIGURE 10-4a Transcription is the synthesis of RNA from instructions in DNA A gene is a segment of a chromosome's DNA. One of the DNA strands will serve as the template for the synthesis of an RNA molecule with bases complementary to the bases in the DNA strand.
  • FIGURE 10-4b Transcription is the synthesis of RNA from instructions in DNA A gene is a segment of a chromosome's DNA. One of the DNA strands will serve as the template for the synthesis of an RNA molecule with bases complementary to the bases in the DNA strand.
  • FIGURE 10-5 RNA transcription in action This colorized electron micrograph shows the progress of RNA transcription in the egg of an African clawed toad. In each treelike structure, the central "trunk" is DNA (blue) and the "branches" are RNA molecules (red). A series of RNA polymerase molecules (too small to be seen in this micrograph) are traveling down the DNA, synthesizing RNA as they go. The beginning of the gene is on the left. The short RNA molecules on the left have just begun to be synthesized; the long RNA molecules on the right are almost finished.
  • FIGURE 10-4c Transcription is the synthesis of RNA from instructions in DNA A gene is a segment of a chromosome's DNA. One of the DNA strands will serve as the template for the synthesis of an RNA molecule with bases complementary to the bases in the DNA strand.
  • FIGURE 10-4d Transcription is the synthesis of RNA from instructions in DNA A gene is a segment of a chromosome's DNA. One of the DNA strands will serve as the template for the synthesis of an RNA molecule with bases complementary to the bases in the DNA strand.
  • 09/29/12 Figure: 10.E4 Title: Eukaryotic genes contain introns and exons Caption: Eukaryotic genes contain introns and exons
  • 09/29/12 Figure: 19-2 part a Title: Viral structure and replication part a Caption: (a) A cross section of the virus that causes AIDS. Inside, genetic material is surrounded by a protein coat and molecules of reverse transcriptase, an enzyme that catalyzes the transcription of DNA from the viral RNA template after the virus enters the host cell. This virus is among those that also have an outer envelope that is formed from the host cell's plasma membrane. Spikes made of glycoprotein (protein and carbohydrate) project from the envelope and help the virus attach to its host cell.
  • 09/29/12 Figure: 19-2 part a Title: Viral structure and replication part a Caption: (a) A cross section of the virus that causes AIDS. Inside, genetic material is surrounded by a protein coat and molecules of reverse transcriptase, an enzyme that catalyzes the transcription of DNA from the viral RNA template after the virus enters the host cell. This virus is among those that also have an outer envelope that is formed from the host cell's plasma membrane. Spikes made of glycoprotein (protein and carbohydrate) project from the envelope and help the virus attach to its host cell.
  • 09/29/12 Figure: 10.6abc Title: Initiation of protein synthesis Caption: Initiation of protein synthesis
  • 09/29/12 Figure: 10.6def Title: Elongation during protein synthesis Caption: Elongation during protein synthesis
  • 09/29/12 Figure: 10.6ghi Title: Termination of protein synthesis Caption: Termination of protein synthesis
  • 09/29/12 Figure: 10.7 Title: Complementary base pairing is critical at each step in decoding genetic information Caption: (a) DNA contains two strands: the template strand is used by RNA polymerase to synthesize an RNA molecule; the other strand, which is complementary to the template strand, is needed for DNA replication. (b) Bases in the template strand of DNA are transcribed into a complementary mRNA. Codons are sequences of three bases that specify an amino or a stop during protein synthesis. (c) Unless it is a stop codon, each mRNA codon forms base pairs with the anticodon of a tRNA molecule that carries a specific amino acid. (d) The ribosome links the amino acids together, forming the protein.
  • Table 10-4 Effects of Mutations in the Hemoglobin Gene
  • Cytogenetics 2 replication, transcription and translation

    1. 1. ‫بسم ا الرحمن‬ ‫الرحيم‬ DNA Replication,Transcription & Translation Dr. SAHAR ABO ELFADL 1
    2. 2. DNA Replication Dr. SAHAR ABO ELFADL 2 2007-2008
    3. 3. Proposed Models of DNA Replication• In the late 1950s, three different mechanisms were proposed for the replication of DNA – Conservative model • Both parental strands stay together after DNA replication – Semi-conservative model • The double-stranded DNA contains one parental and one daughter strand following replication – Dispersive model • Parental and daughter DNA are interspersed in both strands following replication Dr. SAHAR ABO ELFADL 3
    4. 4. Three models for DNA replication The most accepted Dr. SAHAR ABO ELFADL 4
    5. 5. Directionality of DNA PO4 nucleotide• You need to number the carbons! N base – it matters! 5′ CH2 This will be O IMPORTANT!! 4′ ribose 1′ 3′ 2′ OH Dr. SAHAR ABO ELFADL 5
    6. 6. The DNA backbone 5′ PO4• Putting the DNA base 5′ CH2 backbone together O – refer to the 3′ and 5′ ends 4′ C 1′ of the DNA 3′ 2′ O • the last trailing carbon – O P O Sounds trivial, but… O base this will be 5′ CH2 IMPORTANT!! O 4′ 1′ 3′ 2′ OH Dr. SAHAR ABO ELFADL 6 3′
    7. 7. Anti-parallel strands• Nucleotides in DNA backbone are bonded from phosphate to sugar 5′ 3′ between 3′ & 5′ carbons – DNA molecule has “direction” – complementary strand runs in opposite direction Dr. SAHAR ABO ELFADL 7 3′ 5′
    8. 8. Bonding in DNA hydrogen bonds 5′ 3′ covalent phosphodiester bonds 3′ 5′….strong or weak bonds? Dr. SAHAR ABO ELFADL 8How do the bonds fit the mechanism for copying DNA?
    9. 9. Copying DNA• Replication of DNA – base pairing allows each strand to serve as a template for a new strand – new strand is 1/2 parent template & 1/2 new DNA (semi- conservative). Dr. SAHAR ABO ELFADL 9
    10. 10. DNA Replication Let’s meet the team…• Large team of enzymes coordinates replication Dr. SAHAR ABO ELFADL 10
    11. 11. Replication: 1st step • Unwind DNA – helicase enzyme • unwinds part of DNA helix • stabilized by single-stranded binding proteins helicasesingle-stranded binding proteins ABO ELFADL replication fork Dr. SAHAR 11
    12. 12. Replication: 2nd step  Build daughter DNA strand  add new complementary bases  DNA polymerase III But… Where’s the We’re missing ENERGY DNA something! for the bonding!Polymerase III What? Dr. SAHAR ABO ELFADL 12
    13. 13. Energy of Replication Where does energy for bonding usually come from? We come with our own energy! You remember energy ATP! Are there other waysto get energy out of it? And we leave behind a GTP TTP ATP nucleotide! TMP GMP AMP ADP modified nucleotide ELFADL Dr. SAHAR ABO 13
    14. 14. Okazaki Leading & Lagging strandsLimits of DNA polymerase III can only build onto FREE 3′ end of an existing DNA 5′ ents 3′ strand Okaza ki fragm 5′ 3′ 5′ 3′ 5′ 5′ 3′ Lagging strand ligase growing 3′ replication fork 5′ Leading strandLagging strand  3′ 5′ 3′ DNA polymerase III Okazaki fragments joined by ligase Leading strand Dr. SAHAR ABO ELFADL 14  “spot welder” enzyme  continuous synthesis
    15. 15. Replication fork / Replication 5′ bubble 3′ 5′ 3′ DNA polymerase III leading strand 5′ 3′ 3′ 5′ 5′ 5′ 5′ 3′ 3′ lagging strand 3′ 5′ 5′3′ lagging strand leading strand 5′ growing 3′ replication fork 5′5′ growing replication fork 5′ leading strand 3′ lagging strand 3′ 5′ 5′ 5′ Dr. SAHAR ABO ELFADL 15
    16. 16. Starting DNA synthesis: RNA primersLimits of DNA polymerase III can only build onto 3′ end of an existing DNA strand 5′ 3′ 5′ 3′ 5′ 3′ 3′ 5′ growing 3′ primase replication fork DNA polymerase III 5′ RNA 5′RNA primer 3′ built by primase serves as starter sequence for DNA polymerase SAHAR ABO ELFADL Dr. III 16
    17. 17. Replacing RNA primers with DNADNA polymerase I removes sections of RNA DNA polymerase I primer and replaces with 5′ DNA nucleotides 3′ 3′ 5′ ligase growing 3′ replication fork 5′ RNA 5′ 3′But DNA polymerase I stillcan only build onto 3′ end of Dr. SAHAR ABO ELFADLan existing DNA strand 17
    18. 18. Houston, we Chromosome erosion have a problem!All DNA polymerases canonly add to 3′ end of an DNA polymerase Iexisting DNA strand 5′ 3′ 3′ 5′ growing 3′ replication fork DNA polymerase III 5′ RNA 5′Loss of bases at 5′ ends 3′in every replication chromosomes get shorter with each replication Dr. SAHAR ABO ELFADL 18 limit to number of cell divisions?
    19. 19. TelomeresRepeating, non-coding sequences at the endof chromosomes = protective cap 5′ limit to ~50 cell divisions 3′ 3′ 5′ growing 3′ telomerase replication fork 5′ 5′Telomerase TTAAGGG TTAAGGG TTAAGGG enzyme extends telomeres 3′ can add DNA bases at 5′ end different level of activity in different cells Dr. SAHAR ABO ELFADL 19  high in stem cells & cancers -- Why?
    20. 20. Replication fork DNA polymerase III lagging strand DNApolymerase I 3’ Okazaki primase fragments 5’ 5’ ligase SSB 3’ 5’ 3’ helicase DNA polymerase III5’ leading strand 3’ direction of replication Dr. SAHAR ABO ELFADL 20 SSB = single-stranded binding proteins
    21. 21. Fast & accurate!Human cell• copies its 6 billion bases• Completes mitosis in only few hours• remarkably accurate• only ~1 error per 100 million bases• ~30 errors per cell cycle Dr. SAHAR ABO ELFADL 21
    22. 22. NOWLet us see together this video about DNA REPLICATION Dr. SAHAR ABO ELFADL 22
    23. 23. DNA Replication • Origins of replication 1. Replication Forks: hundreds of Y-shaped Forks regions of replicating DNA molecules where new strands are growing. 3’5’ Parental DNA Molecule Replication Fork3’ Dr. SAHAR ABO ELFADL 23 5’
    24. 24. DNA Replication• Origins of replication 2. Replication Bubbles: Bubbles a. Hundreds of replicating bubbles (Eukaryotes). (Eukaryotes) b. Single replication fork (bacteria). Bubbles Bubbles Dr. SAHAR ABO ELFADL 24
    25. 25. DNA Replication• Strand Separation: Separation 1. Helicase: enzyme which catalyze the Helicase unwinding and separation (breaking H- Bonds) of the parental double helix. 2. Single-Strand Binding Proteins: proteins Proteins which attach and help keep the separated strands apart. Dr. SAHAR ABO ELFADL 25
    26. 26. DNA Replication• Priming: 1. RNA primers: before new DNA strands can primers form, there must be small pre-existing primers (RNA) present to start the addition of new nucleotides (DNA Polymerase). Polymerase) 2. Primase: enzyme that polymerizes Primase (synthesizes) the RNA Primer. Dr. SAHAR ABO ELFADL 26
    27. 27. DNA Replication• Synthesis of the new DNA Strands: 1. DNA Polymerase: with a RNA primer in Polymerase place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA strand in the 5’ to 3’ direction. direction5’ 3’ RNA 5’ DNA Polymerase Primer Nucleotide Dr. SAHAR ABO ELFADL 27
    28. 28. DNA Replication 2. Leading Strand: synthesized as a Strand single polymer in the 5’ to 3’ direction. direction5’ 3’ 5’ RNA Nucleotides DNA Polymerase Primer Dr. SAHAR ABO ELFADL 28
    29. 29. DNA Replication 3. Lagging Strand: also synthesized in Strand the 5’ to 3’ direction, but discontinuously direction against overall direction of replication. Leading Strand5 3’’3’ 5’ DNA Polymerase RNA Primer5’ 3’3’ 5’ Lagging Strand Dr. SAHAR ABO ELFADL 29
    30. 30. DNA Replication 4. Okazaki Fragments: series of short Fragments segments on the lagging strand. Okazaki Fragment Okazaki Fragment DNA Polymerase RNA Primer5’ 3’3’ 5’ Lagging Strand Dr. SAHAR ABO ELFADL 30
    31. 31. DNA Replication5. DNA ligase: a linking enzyme that ligase catalyzes the formation of a covalent bond from the 3’ to 5’ end of joining stands.Example: joining two Okazaki fragments together. DNA ligase Okazaki Fragment 1 Okazaki Fragment 25’ 3’3’ Lagging Strand Dr. SAHAR ABO ELFADL 5’ 31
    32. 32. DNATranscription & Translation Dr. SAHAR ABO ELFADL 32 2007-2008
    33. 33. The Link Between DNA and Protein• DNA contains the molecular blueprint of every cell• Proteins are the “molecular workers” of the cell• Proteins control cell shape, function, reproduction, and synthesis of biomolecules• The information in DNA genes must therefore be linked to the proteins that run the cell Dr. SAHAR ABO ELFADL 33
    34. 34. Transcription• Process by which genetic information Translation encoded in DNA is • Process by which copied onto information encoded messenger RNA in mRNA is used to• Occurs in the nucleus assemble a protein at• DNA mRNA a ribosome • Occurs on a Ribosome • mRNA protein Dr. SAHAR ABO ELFADL 34
    35. 35. Three Types of RNAmRNA A A A A U U U U U U U Umessenger G GC G G GG catalytic site Large subunitrRNA 1 2ribosomal Small subunit tRNA docking sites MettRNA Attached amino acidtransfer A anticodon Dr. SAHAR ABO ELFADL 35 G U
    36. 36. Transcription and Translation• DNA directs protein synthesis in a two- step process 1. Information in a DNA gene is copied into mRNA in the process of transcription 2. mRNA, together with tRNA, amino acids, and a ribosome, synthesize a protein in the process of translation Dr. SAHAR ABO ELFADL 36
    37. 37. Information Flow: DNA  RNA  ProteinDr. SAHAR ABO ELFADL 37
    38. 38. The Genetic Code• The base sequence in a DNA gene dictates the sequence and type of amino acids in translation• Bases in mRNA are read by the ribosome in triplets called codons• Each codon specifies a unique amino acid in the genetic code• Each mRNA also has a start and a stop codon Dr. SAHAR ABO ELFADL 38
    39. 39. Dr. SAHAR ABO ELFADL 39
    40. 40. Overview of Transcription• Transcription of a DNA gene into RNA has three stages – Initiation – Elongation – Termination Dr. SAHAR ABO ELFADL 40
    41. 41. Initiation• Initiation phase of transcription 1. DNA molecule is unwound and strands are separated at the beginning of the gene sequence 2. RNA polymerase binds to promoter region at beginning of a gene on template strand Dr. SAHAR ABO ELFADL 41
    42. 42. Dr. SAHAR ABO ELFADL 42
    43. 43. Elongation1. RNA polymerase synthesizes a sequence of RNA nucleotides along DNA template strand2. Bases in newly synthesized RNA strand are complementary to the DNA template strand3. RNA strand peels away from DNA template strand as DNA strands repair and wind up Dr. SAHAR ABO ELFADL 43
    44. 44. Dr. SAHAR ABO ELFADL 44
    45. 45. Elongation• As elongation proceeds, one end of the RNA drifts away from the DNA; RNA polymerase keeps the other end temporarily attached to the DNA template strand Dr. SAHAR ABO ELFADL 45
    46. 46. Dr. SAHAR ABO ELFADL 46
    47. 47. Termination– RNA polymerase reaches a termination sequence and releases completed RNA strand Dr. SAHAR ABO ELFADL 47
    48. 48. Dr. SAHAR ABO ELFADL 48
    49. 49. Dr. SAHAR ABO ELFADL 49
    50. 50. mRNA– The DNA is in the nucleus and the ribosomes are in the cytoplasm– The genes that encode the proteins for a biochemical pathway are not clustered together on the same chromosome Each gene consists of multiple segments of DNA that encode for protein, called exons Exons are interrupted by other segments that are not translated, called introns Dr. SAHAR ABO ELFADL 50
    51. 51. DNA exons introns promoter Transcription from DNA to RNA Initialtranscript s Splicing In tron ut so ped ut it n snnpro d o I pe completed snipmRNA transcript Dr. SAHAR ABO ELFADL 51
    52. 52. mRNA– Transcription of a gene produces a very long RNA strand that contains introns and exons– Enzymes in the nucleus cut out the introns and splice together the exons to make true mRNA– mRNA exits the nucleus through a membrane pore and associates with a ribosome Dr. SAHAR ABO ELFADL 52
    53. 53. Ribosomes• Ribosomes are large complexes of proteins and rRNA Dr. SAHAR ABO ELFADL 53
    54. 54. Ribosomes• Ribosomes are composed of two subunits – Small subunit has binding sites for mRNA and a tRNA – Large subunit has binding sites for two tRNA molecules and catalytic site for peptide bond formation Dr. SAHAR ABO ELFADL 54
    55. 55. Transfer RNAs• Transfer RNAs hook up to and bring amino acids to the ribosome• There is at least one type of tRNA assigned to carry each of the twenty different amino acids• Each tRNA has three exposed bases called an anticodon• The bases of the tRNA anticodon pair with an mRNA codon within a ribosome binding site Dr. SAHAR ABO ELFADL 55
    56. 56. Translation• Ribosomes, tRNA, and mRNA cooperate in protein synthesis, which begins with initiation: 1. The mRNA binds to the small ribosomal subunit 2. The mRNA slides through the subunit until the first AUG (start codon) is exposed in the first tRNA binding site… Dr. SAHAR ABO ELFADL 56
    57. 57. Translation3. The first tRNA carrying methionine (and anticodon UAC) binds to the mRNA start codon completing the initiation complex4. The large ribosomal subunit joins the complex Dr. SAHAR ABO ELFADL 57
    58. 58. Translation:Initiation (1) A tRNA with an attached methionine amino acid binds to a small ribosomal subunit, forming an initiation complex. Dr. SAHAR ABO ELFADL 58
    59. 59. Translation:Initiation (2) The initiation complex binds to end of mRNA and travels down until it encounters an AUG codon in the mRNA. The anticodon of the tRNA in the initiation complex forms base pairs with the AUG codon. Dr. SAHAR ABO ELFADL 59
    60. 60. Translation:Initiation (3) The large ribosomal subunit binds to the small subunit, with the mRNA between the two subunits. The methionine tRNA is in the first tRNA site on the large subunit. Dr. SAHAR ABO ELFADL 60
    61. 61. Translation:Elongation 1 The second tRNA enters the second tRNA site on the large ribosomal subunit. Which tRNA binds depends on the ability of its anticodon (CAA in this example) to base pair with the codon (GUU in this example) in the mRNA. tRNAs with a CAA anticodon carry an attached valine amino acid, which was added to it by enzymes in the cytoplasm. Dr. SAHAR ABO ELFADL 61
    62. 62. Translation:Elongation 2 The "empty" tRNA is released and the ribosome moves down the mRNA, one codon to the right. The tRNA that is attached to the two amino acids is now in the first tRNA binding site and the second tRNA binding site is empty. Dr. SAHAR ABO ELFADL 62
    63. 63. Translation:Elongation 3 The catalytic site on the large subunit catalyzes the formation of a peptide bond linking the amino acids methionine to valine. The two amino acids are now attached to the tRNA in the second binding position. Dr. SAHAR ABO ELFADL 63
    64. 64. Translation:Elongation 4 Another tRNA enters the second tRNA binding site carrying its attached amino acid. The tRNA has an anticodon that pairs with the codon. (Here, the CAU mRNA codon pairs with a GUA tRNA anticodon.) The tRNA molecule carries the amino acid histidine (his). Dr. SAHAR ABO ELFADL 64
    65. 65. Translation:Elongation 5 Binding of tRNAs, & formation of peptide bonds continues. Ribosome reaches STOP codon (UAG). Protein "release factors" signal the ribosome to release the protein. The mRNA is also released and large & small subunits Dr. SAHAR ABO ELFADL separate. 65
    66. 66. Translation:Termination The catalytic site forms a new peptide bond, in this example, between the valine and the histidine. A three-amino acid chain is now attached to the tRNA in the second tRNA binding site. The empty tRNA in the first site is released and the ribosome Dr. SAHAR ABO ELFADL moves one codon to 66 the right.
    67. 67. Complementary Base Pairing gene(a) complementary C etc. T T T A A DNA strand G G G G G template C C C C C A A A C C C C C T G T T DNA strand etc. codons G G G G G G G G G G (b) mRNA C C A A U U U U U U etc. U anticodons C C C C (c) tRNA A A C C U U etc. A C C amino acids Dr. SAHAR ABO ELFADL 67 (d) protein Methionine Glycine Valine etc.
    68. 68. MOVI TIMEDr. SAHAR ABO ELFADL 68
    69. 69. Effects of Mutations on Proteins• Recall that mutations are changes in the base sequence of DNA• Most mutations are categorized as – Substitutions – Deletions – Insertions – Inversions – Translocations Dr. SAHAR ABO ELFADL 69
    70. 70. Effects of Mutations on Proteins• Inversions and translocations – When pieces of DNA are broken apart and reattached in different orientation or location – Not problematic if entire gene is moved – If gene is split in two it will no longer code for a complete, functional protein Dr. SAHAR ABO ELFADL 70
    71. 71. Effects of Mutations on Proteins• Insertions or deletions – Nucleotides are added or subtracted from a gene – Reading frame of RNA codons is changed • THEDOGSAWTHECAT is changed by deletion of the letter “S” to THEDOGAWTHECAT – Resultant protein has very different amino acid sequence; almost always is non- functional Dr. SAHAR ABO ELFADL 71
    72. 72. Effects of Mutations on Proteins• Nucleotide substitutions (point mutations) – An incorrect nucleotide takes the place of a correct one – Protein structure and function is unchanged because many amino acids are encoded by multiple codons – Protein may have amino acid changes that are unimportant to function (neutral mutations) Dr. SAHAR ABO ELFADL 72
    73. 73. Effects of Mutations on Proteins• Effects of nucleotide substitutions – Protein function is changed by an altered amino acid sequence (as in gly val in hemoglobin in sickle cell anemia) – Protein function is destroyed because DNA mutation creates a premature stop codon Dr. SAHAR ABO ELFADL 73
    74. 74. Dr. SAHAR ABO ELFADL 74
    75. 75. Mutations Fuel Evolution• Mutations are heritable changes in the DNA• Approx. 1 in 105-106 eggs or sperm carry a mutation• Most mutations are harmful or neutral• Mutations create new gene sequences and are the ultimate source of genetic variation• Mutant gene sequences that are beneficial may spread through a population and become common Dr. SAHAR ABO ELFADL 75
    76. 76. How Are Genes Regulated?• The human genome contains ~ 30,000 genes• A given cell “expresses” (transcribes) only a small number of genes• Some genes are expressed in all cells• Other genes are expressed only – In certain types of cells – At certain times in an organism’s life – Under specific environmental conditions Dr. SAHAR ABO ELFADL 76
    77. 77. The EndDr. SAHAR ABO ELFADL 77

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