1 The Genetic Code of Genes and Genomes

1. 1 The Genetic Code of Genes and Genomes

2. DNA: Molecule of Heredity Inherited traits are affected by genes that are transmitted from parents to offspring in reproduction Genes are composed of the chemical deoxyribonucleic acid = DNA

3. DNA: Molecule of Heredity DNA was discovered by Friedrich Miescher in 1869 In 1920s microscopic studies with special stains showed that DNA is present in chromosomes In 1944 Avery, McLeod and McCarty provided the first evidence that DNA is the genetic material

4. 4 Griffith's experiment demonstrating bacterial transformation

5. Fig. 1.3

6. DNA Structure: Double Helix In 1953 Watson and Crick proposed the three dimensional structure of DNA Molecular structure of DNA is a double-stranded helix comprised of a linear sequence of paired subunits = nucleotides Each nucleotide contains any one of four bases = adenine, thymine, guanine and cytosine Pairing between nucleotides of the double helix is complementary: adenine pairs with thymine guanine pairs with cytosine

7. DNA Structure: Double Helix DNA backbone forms right-handed helix Each DNA strand has polarity = directionality The paired strands are oriented in opposite directions = antiparallel

8. Central Dogma

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10. Mutations Mutation refers to any heritable change in a gene The change may be: substitution of one base pair in DNA for a different base pair; deletion or addition of base pairs Any mutation that causes the insertion of an incorrect amino acid in a protein can impair its function

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13. 13 Genome Size The genetic complement of a cell or virus constitutes its genome In eukaryotes, this term is commonly used to refer to one complete haploid set of chromosomes, such as that found in a sperm or egg The C-value = the DNA content of the haploid genome The units of length of nucleic acids in which genome sizes are expressed : � kilobase (kb) 103 base pairs � megabase (Mb) 106 base pairs

14. 14 Genome Size Viral genomes are typically in the range 100�1000 kb: Bacteriophage MS2, one of the smallest viruses, has only four genes in a single stranded RNA molecule of about 4000 nucleotides (4kb) Bacterial genomes are larger, typically in the range 1�10 Mb: The chromosome of Escherichia coli is a circular DNA molecule of 4600 kb. Eukaryotic genomes are typically in the range 100�1000 Mb: The genome of a fruit fly, Drosophila melanogaster is 180 Mb Among eukaryotes, genome size often differs tremendously, even among closely related species

15. 15 The C-value Paradox Genome size among species of protozoa differ by 5800-fold, among arthropods by 250-fold, fish 350-fold, algae 5000-fold, and angiosperms 1000-fold. The C-value paradox: Among eukaryotes, there is no consistent relationship between the C-value and the metabolic, developmental, or behavioral complexity of the organism The reason for the discrepancy is that in higher organisms, much of the DNA has functions other than coding for the amino acid sequence of proteins

16. 16 DNA: Chemical Composition DNA is a linear polymer of four deoxyribonucleotides Nucleotides composed of 2'- deoxyribose (a five-carbon sugar), phosphoric acid, and the four nitrogen-containing bases denoted A, T, G and C

17. 17 DNA: Chemical Composition Two of the bases, A and G, have a double-ring structure; these are called purines The other two bases, T and C, have a single-ring structure; these are called pyrimidines

18. 18 Fig. 6.4

19. 19 DNA Structure The duplex molecule of DNA consists of two polynucleotide chains twisted around one another to form a right-handed helix in which the bases form hydrogen bonds Adenine pairs with thymine; guanine with cytosine A hydrogen bond is a weak bond The stacking of the base pairs on top of one another also contribute to holding the strands together The paired bases are planar, parallel to one another, and perpendicular to the long axis of the double helix.

20. 20 DNA Structure The backbone of each polynucleotide strand consists of deoxyribose sugars alternating with phosphate groups that link 5 ' carbon of one sugar to the 3' carbon of the next sugar in line The two polynucleotide strands of the double helix run in opposite directions The paired strands are said to be antiparallel

21. 21 Fig. 6.5

22. 22 DNA Replication Watson-Crick model of DNA replication: Hydrogen bonds between DNA bases break to allow strand separation Each DNA strand is a template for the synthesis of a new strand Template (parental) strand determines the sequence of bases in the new strand (daughter)= complementary base pairing rules

23. 23 Fig. 6.9

24. 24 Autoradiogram of the intact replicating circular chromosome of E. coli shows that DNA synthesis is bidirectional Replication starts from a single site called origin of replication (OR) The region in which parental strands are separating and new strands are being synthesized is called a replication fork Circular DNA Replication

25. 25 Replication of Linear DNA The linear DNA duplex in a eukaryotic chromosome also replicates bidirectionally Replication is initiated at many sites along the DNA Multiple initiation is a means of reducing the total replication time

26. 26 DNA Synthesis One strand of the newly made DNA is synthesized continuously = leading strand The other, lagging strand is made in small precursor fragments = Okazaki fragments The size of Okazaki fragments is 1000�2000 base pairs in prokaryotic cells and 100�200 base pairs in eukaryotic cells.

27. 27 Fig. 6.22

28. 28 Fig. 6.15

29. 29 Nucleic Acid Hybridization DNA denaturation: Two DNA strands can be separated by heat without breaking phosphodiester bonds DNA renaturation = hybridization: Two single strands that are complementary or nearly complementary in sequence can come together to form a different double helix Single strands of DNA can also hybridize complementary sequences of RNA

30. 30 Fig. 6.24

31. 31 Restriction Enzymes Restriction enzymes cleave duplex DNA at particular nucleotide sequences The nucleotide sequence recognized for cleavage by a restriction enzyme is called the restriction site of the enzyme In virtually all cases, the restriction site of a restriction enzyme reads the same on both strands A DNA sequence with this type of symmetry is called a palindrome

32. 32 Fig. 6.26

33. 33 Southern Blot Analysis DNA fragments on a gel can often be visualized by staining with ethidium bromide, a dye which binds DNA Particular DNA fragments can be isolated by cutting out the small region of the gel that contains the fragment and removing the DNA from the gel. Specific DNA fragments are identified by hybridization with a probe = a radioactive fragment of DNA or RNA Southern blot analysis is used to detect very small amounts of DNA or to identify a particular DNA band by DNA-DNA or DNA-RNA hybridization

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35. 35 Polymerase Chain Reaction Polymerase Chain Reaction (PCR) makes possible the amplification of a particular DNA fragment Oligonucleotide primers that are complementary to the ends of the target sequence are used in repeated round of denaturation, annealing, and DNA replication The number of copies of the target sequence doubles in each round of replication, eventually overwhelming any other sequences that may be present

36. 36 Polymerase Chain Reaction Special DNA polymerase is used in PCR = Taq polymerase isolated from bacterial thermophiles which can withstand high temperature used in procedure PCR accomplishes the rapid production of large amounts of target DNA which can then be identified and analyzed

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38. 38 Fig. 6.28

39. 39 DNA Sequence Analysis DNA sequence analysis determines the order of bases in DNA The dideoxy sequencing method employs DNA synthesis in the presence of small amounts of fluorescently labeled nucleotides that contain the sugar dideoxyribose instead of deoxyribose

40. 40 DNA Sequencing: Dideoxy Method Modified sugars cause chain termination because it lacks the 3�-OH group, which is essential for attachment of the next nucleotide in a growing DNA strand The products of DNA synthesis are then separated by electrophoresis. In principle, the sequence can be read directly from the gel

41. 41 DNA Sequencing: Dideoxy Method Each band on the gel is one base longer than the previous band Each didyoxynucleotide is labeled by different fluorescent dye G, black; A, green; T, red; C, purple As each band comes off the bottom of the gel, the fluorescent dye that it contains is excited by laser light, and the color of the fluorescence is read automatically by a photocell and recorded in a computer

42. 42 Fig. 6.31