1. Genetics: From Genes to Genomes
Powerpoint to accompany
Genetics: From Genes to Genomes
Third Edition
Hartwell ● Hood ● Goldberg ● Reynolds ● Silver ● Veres
Chapter 6
Prepared by Malcolm D. Schug
University of North Carolina Greensboro
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2. DNA
How the Molecule of Heredity Carries, Replicates, and Recombines Information
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3. Outline of Chapter 6
How investigators pinpointed DNA as the genetic material
The elegant Watson-Crick model of DNA structure
How DNA structure provides for the storage of genetic information
How DNA structure gives rise to the semiconservative model of molecular replication
How DNA structure promotes the recombination of genetic information
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4. Chemical characterization localizes DNA in the chromosomes.
1869 – Friedrich Meischer extracted a weakly acidic, phosphorous rich material from nuclei of human white blood cells which he named nuclein.
DNA – deoxyribonucleic acid
Deoxyribose – a sugar; acidic
Four subunits belonging to class of compounds called nucleotides linked together by phosphodiester bonds
Chromosomes are composed of DNA.
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5. The chemical composition of DNA
Figure 6.2
Fig. 6.2
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6. Are genes composed of DNA or protein?
Only four different subunits make up DNA.
Chromosomes contain less DNA than protein by weight.
Protein
20 different subunits – greater potential variety of combinations
Chromosomes contain more protein than DNA by weight.
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7. Bacterial transformation implicates DNA as the substance of genes.
1928 – Frederick Griffith – experiments with smooth (S), virulent strain Streptococcus pneumoniae, and rough (R), nonvirulent strain
Bacterial transformation demonstrates transfer of genetic material.
1944 – Oswald Avery, Colin MacLeod, and Maclyn McCarty determined that DNA is the transformation material.
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8. Griffith experiment Fig. 6.3a Figure 6.3 a
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9. Griffith experiment Fig. 6.3 b Figure 6.3 b
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10. Avery, MacLeod, McCarty Experiment
Figure 6.4 a
Fig. 6.4 a
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11. Avery, MacLeod, McCarty experiment
Figure 6.4 c
Fig. 6.4 c
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12. Hershey and Chase experiments
1952 – Alfred Hershey and Martha Chase provide convincing evidence that DNA is genetic material.
Waring blender experiment using T2 bacteriophage and bacteria
Radioactive labels 32P for DNA and 35S for protein
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13. Hershey and Chase Waring blender experiment
Figure 6.5 a,b
Fig. 6.5 a,b
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14. Hershey and Chase Waring blender experiment
Figure 6.5 c
Fig. 6.5 c
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15. The Watson-Crick Model: DNA is a double helix.
1951 – James Watson learns about x-ray diffraction pattern projected by DNA
Knowledge of the chemical structure of nucleotides (deoxyribose sugar, phosphate, and nitrogenous base)
Erwin Chargaff’s experiments demonstrate that ratios of A and T are 1:1, and G and C are 1:1.
1953 – James Watson and Francis Crick propose their double helix model of DNA structure.
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16. X-ray diffraction patterns produced by DNA fibers – Rosalind Franklin and Maurice Wilkins
Figure 6.6
Fig. 6.6
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17. DNA’s chemical constituents
3. Four nitrogenous bases Purines
1. Deoxyribose sugar.
Figure 6.7
Fig. 6.7a
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18. DNA’s chemical constituents
Assembly into a nucleotide
Figure 6.7
Fig. 6.7b
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19. DNA’s chemical constituents
Figure 6.7 c
Fig. 6.7c
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20. Chargaff’s ratios
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21. Complementary base pairing by formation of hydrogen bonds explain Chargaff’s ratios.
Figure 6.8
Fig. 6.8
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22. DNA is double helix
Strands are antiparallel with a sugar-phosphate backbone on outside and pairs of bases in the middle.
Two strands wrap around each other every 30 Angstroms, once every 10 base pairs.
Two chains are held together by hydrogen bonds between A-T and G-C base pairs.
Figure 6.9
Fig. 6.9
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23. Structurally, purines (A and G) pair best with pyrimadines (T and C).
Thus, A pairs with T and G pairs with C, also explaining Chargaff’s ratios.
Figure 6.9 d
Fig. 6.9 d
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24. Double helix may assume alternative forms.
Figure 6.10
Fig. 6.10
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25. Some DNA molecules are circular instead of linear.
1. Prokaryotes
2. Mitochondria
3. Chloroplasts
4. Viruses
Some viruses carry single-stranded DNA.
1. bacteriophages
Some viruses carry RNA.
1. e.g., AIDS
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26. Four requirements for DNA to be genetic material
Must carry information
Cracking the genetic code
Must replicate
DNA replication
Must allow for information to change
Mutation
Must govern the expression of the phenotype
Gene function
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27. DNA stores information in the sequence of its bases.
Much of DNA’s sequence-specific information is accessible only when the double helix is unwound.
Proteins read the DNA sequence of nucleotides as the DNA helix unwinds. Proteins can either bind to a DNA sequence, or initiate the copying of it.
Human genome is believed to be 250 million nucleotides long. Four possible nucleotides. Thus 4250,000,000 possible sequences in the human genome.
An average single coding gene sequence might be about 10,000 bases long. Thus, 410,000 possibilities for an average gene.
Some genetic information is accessible even in intact, double-stranded DNA molecules.
Some proteins recognize the base sequence of DNA without unwinding it.
One example is a restriction enzyme.
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28. Some viruses use RNA as the repository of genetic information.
Figure 6.13
Fig. 6.13
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29. Complementary base pairing produces semiconservative replication.
DNA replication: Copying genetic information for transmission to the next generation
Complementary base pairing produces semiconservative replication.
Double helix unwinds
Each strand acts as template
Complementary base pairing ensures that T signals addition of A on new strand, and G signals addition of C.
Two daughter helices produced after replication
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30. Figure 6.14
Fig. 6.14
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31. Semiconservative replication – Watson and Crick model
Experimental proof of semiconservative replication – three possible models
Semiconservative replication – Watson and Crick model
Conservative replication: The parental double helix remains intact; both strands of the daughter double helix are newly synthesized.
Dispersive replication: At completion, both strands of both double helices contain both original and newly synthesized material.
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32. Figure 6.15
Fig. 6.15
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33. Meselson-Stahl experiments confirm semiconservative replication.
Figure 6.16
Fig. 6.16
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34. The mechanism of DNA replication
Arthur Kornbuerg, a nobel prize winner and other biochemists deduced steps of replication.
Initiation
Proteins bind to DNA and open up double helix.
Prepare DNA for complementary base pairing
Elongation
Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA.
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35. Figure 6.17
Fig. 6.18a
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36. Figure 6.17
Fig. 6.18b
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37. Enzymes involved in replication
Pol III – produces new stands of complementary DNA
Pol I – fills in gaps between newly synthesized Okazaki segments
DNA helicase – unwinds double helix
Single-stranded binding proteins – keep helix open
Primase – creates RNA primers to initiate synthesis
Ligase – welds together Okazaki fragments
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38. Replication is bidirectional.
Replication forks move in opposite directions.
In linear chromosomes, telomeres ensure the maintenance and accurate replication of chromosome ends
In circular chromosomes, such as E. coli, there is only one origin of replication.
In circular chromosomes, unwinding and replication causes supercoiling, which may impede replication.
Topoisomerase – enzyme that relaxes supercoils by nicking strands
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39. The bidirectional replication of a circular chromosome
Figure 6.18 amb
Fig. 6.19a-b
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40. Figure 6.18 c - f
Fig. 6.19c-f
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41. Cells must ensure accuracy of genetic information.
Redunancy
Basis for repair of errors that occur during replication or during storage
Enzymes repair chemical damage to DNA.
Errors during replication are rare.
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42. Recombination reshuffles the information content of DNA.
During recombination, DNA molecules break and rejoin.
Meselson and Weigle - Experimental evidence from viral DNA and radioactive isotopes
Coinfected E. coli with light and heavy strains of virus after allowing time for recombination
Separated on a CsCl density gradient
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43. Meselson and Weigle demonstrate recombination occurs by breakage and rejoining of DNA.
Figure 6.19
Fig. 6.20
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44. Heteroduplexes mark the spot of recombination.
Products of recombination are always in exact register; not a single base pair is lost or gained.
Two strands do not break and rejoin at the same location; often they are hundreds of base pairs apart.
Region between break points is called heteroduplex.
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45. Heteroduplex region Fig .6.21a-b Figure 6.20 a,b
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46. In heterozygotes, mismatches within heteroduplexes must be repaired.
Gene conversion – a deviation from expected 2:2 segregation of alleles due to mismatch repair.
Studied most extensively in yeast where tetrad analysis makes possible to follow products of meiosis
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47. Gene conversion in yeast Mismatch leads to 3:1 ratio of a:A
Gene conversion in yeast Mismatch leads to 3:1 ratio of a:A. Ratio of B:b and C:c which lie outside of heteroduplex are both 2:2, as expected.
Insert figure 6.21c
Redrawn here
Figure 6.20 c
Fig c
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48. Double stranded break model of meiotic recombination
Homologs physically break, exchange parts, and rejoin.
Breakage and repair create reciprocal products of recombination.
Recombination events can occur anywhere along the DNA molecule.
Precision in the exchange prevents mutations from occurring during the process.
Gene conversion can give rise to unequal yield of two different alleles. 50% of gene conversions are associated with crossing over of adjacent chromosomal regions, and 50% of gene conversions are not associated with crossing over.
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49. Double stranded break formation Spo11 protein breaks one chromatid on both strands.
Figure 6.22 step 1
Fig step 1
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50. Resection 5’ ends on each side of break are degraded to produce two 3’ single stranded tails.
Figure 6.22 step 2
Fig step 2
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51. First strand invasion RecA binds 3’ tail and double helix allowing invasion and migration.
Figure 6.22 step 3
Fig step 3
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52. Formation of Holliday junctions New DNA synthesis forms two X structures called Holliday junctions.
Figure 6.22 step 4
Fig step 4
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53. Branch migration Both invading strands zip up and migrate while newly created heteroduplex molecules rewind behind.
Figure 6.22 step 5
Fig step 5
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54. The Holliday intermediate Interlocked nonsister chromatids disengage
The Holliday intermediate Interlocked nonsister chromatids disengage. Two resolutions are possible.
Figure 6.22 step 6
Fig step 6
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55. Alternative resolutions Endonuclease cuts Holliday intermediate
Figure 6.22 step 7
Fig step 7
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56. Probability of crossover occurring Resolution of Holliday junction in same plan results in noncrossover chromatids. Resolution in different planes results in crossover.
Insert figure 6.23 Step 8
Redrawn here
Figure 6.22 step 8
Fig step 8
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57. Essential Concepts DNA is the nearly universal genetic material.
The Watson-Crick model shows that DNA is a double helix composed of two antiparallel strands of nucleotides: each nucleotide consists of one of four nitrogenous bases (A,T,C, or G), a deoxyribose sugar, and a phosphate. An A pairs with a T and a G pairs with a C.
DNA carries information in the sequence of its bases, which may follow one another in any order.
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