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Molecular Biology (BIO1004/2001) Kathleen Lobban

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1 Molecular Biology (BIO1004/2001) Kathleen Lobban

2 MODULE CONTENT Structure of DNA Structure of DNA DNA Replication DNA Replication Transcription and Protein synthesis Transcription and Protein synthesis Molecular Biology Techniques Molecular Biology Techniques Recombinant DNA technology Recombinant DNA technology Gene Expression and Regulation in Prokaryotes and Eukaryotes Gene Expression and Regulation in Prokaryotes and Eukaryotes

3 ASSESSMENT PROCEDURES Test 1 ( Week of October 12, 2009 )15% Test 1 ( Week of October 12, 2009 )15% Test 2 ( Week of November 16, 2009 ) 15% Test 2 ( Week of November 16, 2009 ) 15% Practicals10% Practicals10% Project ( Week of October 26, 2009 ) 10% Project ( Week of October 26, 2009 ) 10% Final Exam ( Week of October 12, 2009 )50% Final Exam ( Week of October 12, 2009 )50%

4 UNIT 1 Structure of DNA

5 DNA Deoxyribonucleic acid Deoxyribonucleic acid Belongs to the class of macromolecules called nucleic acids. Belongs to the class of macromolecules called nucleic acids. The other nucleic acid is RNA The other nucleic acid is RNA (ribonucleic acid) (ribonucleic acid)

6 Composition of DNA DNA is a polymer. DNA is a polymer. The monomer units of DNA are nucleotides The monomer units of DNA are nucleotides Therefore the polymer is known as a "polynucleotide." Therefore the polymer is known as a "polynucleotide." Each nucleotide consists of Each nucleotide consists of a 5-carbon sugar (deoxyribose) a 5-carbon sugar (deoxyribose) a nitrogen-containing base a nitrogen-containing base a phosphate group. a phosphate group.

7 Composition of DNA

8 Nitrogenous Bases

9 The Nucleotides BaseNucleosideNucleotide (dNTP) Adenineadenosineadenosine triphosphate ATP / dATP Guanineguanosineguanosine triphosphate GTP / dGTP GTP / dGTP Cytosinecytidinecytidine triphosphate CTP / dCTP CTP / dCTP Uraciluridineuridine triphosphate UTP /dUTP UTP /dUTP Thyminethymidinethymidine triphosphate TTP / dTTP

10 Other Functions of Nucleotides They carry chemical in their bonds which can be easily released for use by the cell eg ATP and GTP They carry chemical in their bonds which can be easily released for use by the cell eg ATP and GTP They combine with other group to form coenzymes: eg coenzyme A They combine with other group to form coenzymes: eg coenzyme A They are used as specific signaling molecules within cells, eg. Cyclic AMP (cAMP) serves to signal switching on of the lac operon in prokaryotes. They are used as specific signaling molecules within cells, eg. Cyclic AMP (cAMP) serves to signal switching on of the lac operon in prokaryotes.

11 Composition of DNA Base Pairs Adenine forms 2 hydrogen bonds with Thymine on the opposite strand Adenine forms 2 hydrogen bonds with Thymine on the opposite strand Adenine (A) will only bond with Thymine (T) Adenine (A) will only bond with Thymine (T) Guanine forms 3 hydrogen bonds with Cytosine on the opposite strand. Guanine forms 3 hydrogen bonds with Cytosine on the opposite strand. Guanine (G) will only bond with Cytosine (C). Guanine (G) will only bond with Cytosine (C).

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14 DNA molecule First described by James D. Watson and Francis Crick in First described by James D. Watson and Francis Crick in In a DNA molecule, the two strands are not parallel, but intertwined with each other. In a DNA molecule, the two strands are not parallel, but intertwined with each other. The two strands form a "double helix" structure The two strands form a "double helix" structure

15 DNA molecule The DNA backbone is an alternating sugar-phosphate sequence. The DNA backbone is an alternating sugar-phosphate sequence. The deoxyribose sugars are joined at both the 3'- and 5'-carbon to phosphate groups by phosphodiester bonds. The deoxyribose sugars are joined at both the 3'- and 5'-carbon to phosphate groups by phosphodiester bonds.

16 DNA molecule Chain has a direction (polarity) Chain has a direction (polarity) 5΄ end - phosphate 5΄ end - phosphate 3΄ end - hydroxyl 3΄ end - hydroxyl

17 DNA molecule The two polynucleotide chains run in opposite directions - antiparallel

18 The DNA Double Helix Two DNA strands form a right-handed Two DNA strands form a right-handed helical spiral The sugar-phosphate backbones of the two DNA strands wind around the helix axis like the railing of a spiral staircase The sugar-phosphate backbones of the two DNA strands wind around the helix axis like the railing of a spiral staircase The bases of the individual nucleotides are on the inside of the helix, stacked on top of each other like the steps of a spiral staircase The bases of the individual nucleotides are on the inside of the helix, stacked on top of each other like the steps of a spiral staircase

19 DNA molecule The sugar-phosphate backbone of DNA is polar, and therefore hydrophilic. The sugar-phosphate backbone of DNA is polar, and therefore hydrophilic. The bases, are relatively non-polar and therefore hydrophobic. The bases, are relatively non-polar and therefore hydrophobic. These hydrostatic forces have a very stabilizing effect on the overall structure of the DNA double helix These hydrostatic forces have a very stabilizing effect on the overall structure of the DNA double helix Therefore there is a strong pressure holding the two strands of DNA together. Therefore there is a strong pressure holding the two strands of DNA together.

20 The DNA Double Helix The helix makes a turn every 3.4 nm The helix makes a turn every 3.4 nm The distance between two neighboring base pairs is 0.34 nm. The distance between two neighboring base pairs is 0.34 nm. Hence, there are about 10 pairs per turn. Hence, there are about 10 pairs per turn. The intertwined strands make two grooves of different widths The intertwined strands make two grooves of different widths The major groove The major groove facilitate binding with specific proteins. The minor groove The minor groove

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23 Watson and Crick’s Model of DNA In 1928 Frederick Griffith was working on a project that enabled others to point out that DNA was the molecule of inheritance. In 1928 Frederick Griffith was working on a project that enabled others to point out that DNA was the molecule of inheritance. The experiment involved mice and bacteria that cause pneumonia - a virulent and a non-virulent kind. The experiment involved mice and bacteria that cause pneumonia - a virulent and a non-virulent kind. He injected the virulent into a mouse and the mouse died. He injected the virulent into a mouse and the mouse died. Next he injected the non-virulent into a mouse and the mouse lived. Next he injected the non-virulent into a mouse and the mouse lived. After this, he heated up the virulent strain to kill it and then injected it into a mouse. The mouse lived. After this, he heated up the virulent strain to kill it and then injected it into a mouse. The mouse lived. Last he injected non-virulent pneumonia and virulent pneumonia, that had been heated and killed, into a mouse. This mouse died. Last he injected non-virulent pneumonia and virulent pneumonia, that had been heated and killed, into a mouse. This mouse died.

24 Watson and Crick’s Model of DNA Why? Griffith thought that the killed virulent bacteria had passed on a characteristic to the non-virulent one to make it virulent - TRANSFORMATION. He thought that this characteristic was in the “inheritance molecule”. Why? Griffith thought that the killed virulent bacteria had passed on a characteristic to the non-virulent one to make it virulent - TRANSFORMATION. He thought that this characteristic was in the “inheritance molecule”.

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26 Watson and Crick’s Model of DNA In 1942 Oswald Avery continued with Griffith’s experiment to see what the inheritance molecule was. continued with Griffith’s experiment to see what the inheritance molecule was. He destroyed the lipids, ribonucleic acids, carbohydrates, and proteins of the virulent pneumonia. Transformation still occurred after this. He destroyed the lipids, ribonucleic acids, carbohydrates, and proteins of the virulent pneumonia. Transformation still occurred after this. Next he destroyed the deoxyribonucleic acid. Transformation did not occur. Avery had found the inheritance molecule, DNA! Next he destroyed the deoxyribonucleic acid. Transformation did not occur. Avery had found the inheritance molecule, DNA!

27 Watson and Crick’s Model of DNA 1940’s Erwin Chargaff noticed a pattern in the amounts of the four bases: adenine, guanine, cytosine, and thymine. noticed a pattern in the amounts of the four bases: adenine, guanine, cytosine, and thymine. In samples of DNA from different organisms : In samples of DNA from different organisms : the amount of adenine = to the amount of thymine the amount of adenine = to the amount of thymine the amount of guanine = to the amount of cytosine. the amount of guanine = to the amount of cytosine. This discovery later became Chargaff’s Rule. This discovery later became Chargaff’s Rule.

28 Watson and Crick’s Model of DNA Rosalind Franklin and Maurice Wilkins Decided to try to make a crystal of the DNA molecule. They obtained an x-ray pattern. Decided to try to make a crystal of the DNA molecule. They obtained an x-ray pattern. The pattern appeared to contain rungs, like those on a ladder between to strands that are side by side. It also showed by an “X” shape that DNA had a helix shape. The pattern appeared to contain rungs, like those on a ladder between to strands that are side by side. It also showed by an “X” shape that DNA had a helix shape.

29 Watson and Crick’s Model of DNA In 1953 James Watson and Francis Crick In 1953 James Watson and Francis Crick saw Franklin and Wilkin's picture of the X-ray and saw Franklin and Wilkin's picture of the X-ray and made an accurate model - a double helix with little rungs connecting the two strands made an accurate model - a double helix with little rungs connecting the two strands But how to bond the bases together ? But how to bond the bases together ? How to solve the problem of the sizes of the bases? How to solve the problem of the sizes of the bases? Adenine and Guanine were purines having two carbon- nitrogen rings in their structures. Adenine and Guanine were purines having two carbon- nitrogen rings in their structures. Thymine and Cytosine were pyrimidines having one carbon- nitrogen ring in its structure. Thymine and Cytosine were pyrimidines having one carbon- nitrogen ring in its structure. If purines and the pyrimidines were together, then DNA would look wobly and crooked. If purines and the pyrimidines were together, then DNA would look wobly and crooked. If they paired Thymine with Adenine and Guanine with Cytosine, DNA would look uniform (Chargaff's rule) If they paired Thymine with Adenine and Guanine with Cytosine, DNA would look uniform (Chargaff's rule) Each side is a complete compliment of the other. Each side is a complete compliment of the other.

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31 Watson and Crick Model of DNA

32 Remembering Rosalind By using the picture of the crystallized DNA, Watson and Crick were able to put together the model of DNA. Watson and Crick used information from Avery, Chargaff, Griffith, and others. They simply pieced together the puzzle. The Nobel Prize was awarded to Watson, Crick, and Maurice Wilkins. Rosalind Franklin did not receive the prize because she had died of cancer by this time. Maurice Wilkins was able to share the prize with Watson and Crick, though, because of his work with Franklin. Her accomplishment should never be forgotten.

33 Forms of DNA DNA exists in many possible conformations. DNA exists in many possible conformations. only A-DNA, B-DNA, and Z-DNA have been observed in cells. only A-DNA, B-DNA, and Z-DNA have been observed in cells. conformation depends on: conformation depends on: the DNA sequence the DNA sequence the amount and direction of supercoiling the amount and direction of supercoiling chemical modifications of the bases chemical modifications of the bases solution conditions (eg concentration of metal ions, salt concentration and level of hydration ). solution conditions (eg concentration of metal ions, salt concentration and level of hydration ).

34 B -Form of DNA Most common under the conditions found in cells. Most common under the conditions found in cells. Has a major and minor groove Has a major and minor groove Has 10 base pairs per turn Has 10 base pairs per turn One turn spans 3.4 nm One turn spans 3.4 nm

35 A -Form of DNA With higher salt concentrations or with alcohol added, the DNA structure may change to A form With higher salt concentrations or with alcohol added, the DNA structure may change to A form A wider right-handed spiral A wider right-handed spiral A shallow, wider minor groove A shallow, wider minor groove A narrower, deeper major groove A narrower, deeper major groove Has 11 bases per turn Has 11 bases per turn One turns spans 2.3 nm One turns spans 2.3 nm

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37 Z -Form of DNA The DNA molecule with alternating G-C sequences in alcohol or high salt solution tends to have such structure The DNA molecule with alternating G-C sequences in alcohol or high salt solution tends to have such structure Bases seem to zigzag Bases seem to zigzag The strands turn about the helical axis in a left- handed spiral. The strands turn about the helical axis in a left- handed spiral. Has a narrow deep groove Has a narrow deep groove Has 12 bases per turn. Has 12 bases per turn. One turn spans 4.6 nm One turn spans 4.6 nm

38 A – DNAB-DNAZ-DNA

39 Chromosomes Chromosomes – highly coiled condensed packages of DNA Chromosomes – highly coiled condensed packages of DNA Present in both eukaryotes and prokaryotes Present in both eukaryotes and prokaryotes Eukaryotes – linear Eukaryotes – linear Prokaryotes – most closed circular Prokaryotes – most closed circular The human genome has 3,000,000,000 base pairs packed into 23 chromosomes!

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41 Organization of Eukaryotic Genomes DNA is packed into chromosomes with the help of proteins - histones. DNA is packed into chromosomes with the help of proteins - histones. The histones associate with DNA forming structures called nucleosomes. The histones associate with DNA forming structures called nucleosomes. Each nucleosome complex consists of a beadlike structure with 146 base pairs of DNA wrapped around a disc-shaped core of eight histone molecules. Each nucleosome complex consists of a beadlike structure with 146 base pairs of DNA wrapped around a disc-shaped core of eight histone molecules.

42 Nucleosome

43 Chromosomes Nucleosomes are 11nm in diameter. Nucleosomes are 11nm in diameter. The packed nucleosome state occurs when a ninth histone called H1, associates with the linker DNA packing adjacent nucleosomes together forming a 30nm diameter thread. The packed nucleosome state occurs when a ninth histone called H1, associates with the linker DNA packing adjacent nucleosomes together forming a 30nm diameter thread. In the extended chromosome, these 30nm diameter threads form large coiled loops held together by a set of non-histone scaffolding proteins. In the extended chromosome, these 30nm diameter threads form large coiled loops held together by a set of non-histone scaffolding proteins.

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45 Eukaryotic Genomes organized into multiple chromosomes - varying in size and numbers depending on the species: organized into multiple chromosomes - varying in size and numbers depending on the species: Human cells haploid 23 Human cells haploid 23 Fruit flies haploid 4 Fruit flies haploid 4 Yeast haploid 16 Yeast haploid 16 Cats haploid 19 Cats haploid 19 Dogs haploid 39 Dogs haploid 39

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47 Bacterial Genome Organization Most have covalently closed, circular chromosomes and plasmids Most have covalently closed, circular chromosomes and plasmids Not all bacteria have a single circular chromosome: Not all bacteria have a single circular chromosome: some bacteria have multiple circular chromosomes some bacteria have multiple circular chromosomes many bacteria have linear chromosomes and linear plasmids. many bacteria have linear chromosomes and linear plasmids.

48 Bacterial Genome The genetic material of a bacterium lies in the cytoplasm and is not surrounded by a nuclear envelope. The genetic material of a bacterium lies in the cytoplasm and is not surrounded by a nuclear envelope. In most species it is contained in a single circular DNA molecule. In most species it is contained in a single circular DNA molecule. If stretched out to its full length, this molecule would be 1000 times longer than the cell itself. If stretched out to its full length, this molecule would be 1000 times longer than the cell itself. Unlike eukaryotic chromosomes, the bacterial DNA has little proteins associated with it. Unlike eukaryotic chromosomes, the bacterial DNA has little proteins associated with it.

49 Plasmids In addition to the genomic DNA, most bacteria have a small amount of genetic information present as one or more plasmids – extrachromosomal circular DNA. In addition to the genomic DNA, most bacteria have a small amount of genetic information present as one or more plasmids – extrachromosomal circular DNA. Plasmids can replicate independently of the genomic DNA or become integrated into it. Plasmids can replicate independently of the genomic DNA or become integrated into it. Bacterial plasmids frequently have genes that code for: Bacterial plasmids frequently have genes that code for: catabolic enzymes catabolic enzymes genetic exchange genetic exchange resistance to antibiotic. resistance to antibiotic.

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52 Viral Genomes virus genomes may contain their genetic information encoded in either DNA or RNA virus genomes may contain their genetic information encoded in either DNA or RNA

53 Viral Genomes Many of the DNA viruses of eukaryotes closely resemble their host cells in terms of the biology of their genomes: Many of the DNA viruses of eukaryotes closely resemble their host cells in terms of the biology of their genomes: Some DNA virus genomes are complexed with cellular histones to form a chromatin-like structure inside the virus particle. Some DNA virus genomes are complexed with cellular histones to form a chromatin-like structure inside the virus particle.

54 Viral Genomes Genome may be : Genome may be : Circular or linear Circular or linear segmented – (two or more separate molecules of nucleic acid) segmented – (two or more separate molecules of nucleic acid) Single-stranded Single-stranded Double-stranded Double-stranded Double-stranded with regions of single-strandedness Double-stranded with regions of single-strandedness DNA viruses DNA viruses RNA viruses RNA viruses Positive sense Positive sense Negative sense Negative sense Ambisense Ambisense Both DNA and RNA (at different stages in the life cycle) Both DNA and RNA (at different stages in the life cycle) Reverse transcribing viruses Reverse transcribing viruses

55 Viral Genomes I: dsDNA viruses I: dsDNA viruses (e.g. Herpesviruses) (e.g. Herpesviruses) II: ssDNA viruses (+)sense DNA II: ssDNA viruses (+)sense DNA (e.g. Parvoviruses) (e.g. Parvoviruses) III: dsRNA viruses III: dsRNA viruses (e.g. Reoviruses - Rotavirus) (e.g. Reoviruses - Rotavirus) IV: (+)ssRNA viruses IV: (+)ssRNA viruses (+)sense RNA (e.g. Picornaviruses -Enterovirus) (+)sense RNA (e.g. Picornaviruses -Enterovirus) V: (-)ssRNA viruses V: (-)ssRNA viruses (-)sense RNA (e.g. Orthomyxoviruses – Influenza A, B, C) (-)sense RNA (e.g. Orthomyxoviruses – Influenza A, B, C) VI: ssRNA-RT viruses VI: ssRNA-RT viruses (+)sense RNA with DNA intermediate in life-cycle (e.g. Retroviruses – HIV, lukemia and tumor viruses) (+)sense RNA with DNA intermediate in life-cycle (e.g. Retroviruses – HIV, lukemia and tumor viruses) VII: dsDNA-RT viruses VII: dsDNA-RT viruses (e.g. Hepadnaviruses – hepatitis B) (e.g. Hepadnaviruses – hepatitis B)

56 Genes Unit of inheritance Unit of inheritance A sequence of nucleotides which provide a cell with the instructions for the synthesis of a specific polypeptide or a type of RNA A sequence of nucleotides which provide a cell with the instructions for the synthesis of a specific polypeptide or a type of RNA Genes determine traits Genes determine traits Most are 1,000 to 4,000 nucleotides long (may be shorter or significantly longer) Most are 1,000 to 4,000 nucleotides long (may be shorter or significantly longer)

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58 Genes

59 Genes Most eukaryotic genes contain non coding regions - introns Most eukaryotic genes contain non coding regions - introns

60 Genes, Viruses and Cancer Cancer is a disease in which cells escape the restraints on normal cell growth. Cancer is a disease in which cells escape the restraints on normal cell growth. Cancer is an inheritable disease (at least from cell to daughter cells). Cancer is an inheritable disease (at least from cell to daughter cells). Once a cell has become cancerous, all of its descendant cells are cancerous. Once a cell has become cancerous, all of its descendant cells are cancerous. Gross chromosomal abnormalities are often visible in cancerous cells. Gross chromosomal abnormalities are often visible in cancerous cells. Most carcinogens (cancer-generating factors) are also mutagens (mutation-generating factors). Most carcinogens (cancer-generating factors) are also mutagens (mutation-generating factors). Oncogenes are genes resembling normal genes but in which something has gone wrong, resulting in a cancer. Oncogenes are genes resembling normal genes but in which something has gone wrong, resulting in a cancer.

61 Genes, Viruses and Cancer Viruses seem able to cause cancer in three ways: Viruses seem able to cause cancer in three ways: Presence of the viral DNA may disrupt normal host DNA functions. Presence of the viral DNA may disrupt normal host DNA functions. Viral proteins needed for virus replication may also affect normal host gene regulation. Viral proteins needed for virus replication may also affect normal host gene regulation. Since most cancer-causing viruses are retroviruses, the virus may serve as a vector for oncogene insertion. Since most cancer-causing viruses are retroviruses, the virus may serve as a vector for oncogene insertion.

62 Images’ source structure.jpg&imgrefurl=http://themedicalbiochemistrypage.org/dna.html&usg=__rj_0WyKjg3pd93RGtskLC_qn- Jg=&h=828&w=500&sz=90&hl=en&start=82&tbnid=ONMf9ibvdHh0sM:&tbnh=144&tbnw=87&prev=/images%3F q%3Ddna%2Bstructure%26gbv%3D2%26ndsp%3D18%26hl%3Den%26sa%3DN%26start%3D72 structure.jpg&imgrefurl=http://themedicalbiochemistrypage.org/dna.html&usg=__rj_0WyKjg3pd93RGtskLC_qn- Jg=&h=828&w=500&sz=90&hl=en&start=82&tbnid=ONMf9ibvdHh0sM:&tbnh=144&tbnw=87&prev=/images%3F q%3Ddna%2Bstructure%26gbv%3D2%26ndsp%3D18%26hl%3Den%26sa%3DN%26start%3D72 structure.jpg&imgrefurl=http://themedicalbiochemistrypage.org/dna.html&usg=__rj_0WyKjg3pd93RGtskLC_qn- Jg=&h=828&w=500&sz=90&hl=en&start=82&tbnid=ONMf9ibvdHh0sM:&tbnh=144&tbnw=87&prev=/images%3F q%3Ddna%2Bstructure%26gbv%3D2%26ndsp%3D18%26hl%3Den%26sa%3DN%26start%3D72 structure.jpg&imgrefurl=http://themedicalbiochemistrypage.org/dna.html&usg=__rj_0WyKjg3pd93RGtskLC_qn- Jg=&h=828&w=500&sz=90&hl=en&start=82&tbnid=ONMf9ibvdHh0sM:&tbnh=144&tbnw=87&prev=/images%3F q%3Ddna%2Bstructure%26gbv%3D2%26ndsp%3D18%26hl%3Den%26sa%3DN%26start%3D72 table : table : History of DNA History of DNA psf.org/Chr16/Chr16.home.html&usg=__zOSiNtQbR9DdY2ZBsog2FGR1Ag8=&h=284&w=280&sz=51&hl=en&sta rt=6&um=1&tbnid=zYTbfjqc9ubbgM:&tbnh=114&tbnw=112&prev=/images%3Fq%3Drosalind%2Bfranklin%26hl% 3Den%26sa%3DG%26um%3D1 psf.org/Chr16/Chr16.home.html&usg=__zOSiNtQbR9DdY2ZBsog2FGR1Ag8=&h=284&w=280&sz=51&hl=en&sta rt=6&um=1&tbnid=zYTbfjqc9ubbgM:&tbnh=114&tbnw=112&prev=/images%3Fq%3Drosalind%2Bfranklin%26hl% 3Den%26sa%3DG%26um%3D1 psf.org/Chr16/Chr16.home.html&usg=__zOSiNtQbR9DdY2ZBsog2FGR1Ag8=&h=284&w=280&sz=51&hl=en&sta rt=6&um=1&tbnid=zYTbfjqc9ubbgM:&tbnh=114&tbnw=112&prev=/images%3Fq%3Drosalind%2Bfranklin%26hl% 3Den%26sa%3DG%26um%3D1 psf.org/Chr16/Chr16.home.html&usg=__zOSiNtQbR9DdY2ZBsog2FGR1Ag8=&h=284&w=280&sz=51&hl=en&sta rt=6&um=1&tbnid=zYTbfjqc9ubbgM:&tbnh=114&tbnw=112&prev=/images%3Fq%3Drosalind%2Bfranklin%26hl% 3Den%26sa%3DG%26um%3D line/lifecycle/images/ jpg line/lifecycle/images/ jpg line/lifecycle/images/ jpg line/lifecycle/images/ jpg


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