Chapter 9. Topics - Genetics - Flow of Genetics - Regulation - Mutation - Recombination

Transcription

1 Chapter 9 Topics - Genetics - Flow of Genetics - Regulation - Mutation - Recombination 1

2 Genetics Genome Chromosome Gene Protein Genotype Phenotype 2

3 Terms and concepts gene Fundamental unit of heredity DNA segment which codes for protein or RNA clone population of cells that are genetically identical genome all genes present in a cell or virus haploid one set of genes (eg., bacteria) diploid two sets of genes (eg., humans) genotype specific set of genes an organism possesses phenotype set of observable characteristics 3

4 The sum total of genetic material of a cell is referred to as the genome. Fig. 9.2 The general location and forms of the genome 4

5 DNA and Chromosomes 5

6 The Organization of DNA in Cells Chromosomes neatly packaged DNA molecule. organization differs in prokaryotic and eukaryotic cell types 6

7 Chromosome Prokaryotic Histonelike proteins condense DNA Eukaryotic Histone proteins condense DNA Subdivided into basic informational packets called genes 7

8 Plasmids usually small, closed circular DNA molecules exist and replicate independently of chromosome not required for growth and reproduction may carry genes that confer selective advantage (e.g., drug resistance) 8

9 Genes Three categories Structural Regulatory Encode for functional/regulatory RNAs Genotype Specific set of genes an organism possesses Phenotype Set of observable characteristics 9

10 Flow of Genetics NA replication (DNA => DNA; RNA => RNA) Gene Expression (DNA =>RNA=>Protein) Replication Transcription Translation Post-translational modification 10

11 The Central Dogma 11

12 Representation of the flow of genetic information. Fig. 9.9 Summary of the flow of genetic information in cell. 12

13 DNA Structure - Fig. 9.3 An Escherichia coli cell disrupted to release its DNA molecule. Replication - today 13

14 Structure Nucleotide Phosphate Deoxyribose sugar Nitrogenous base Double stranded helix Antiparallel arrangement 14

15 DNA as Genetic Material established by several critical experiments Fred Griffith (1928) Oswald T. Avery, C. M. MacLeod, and M. J. McCarty (1944) Alfred D. Hershey and Martha Chase (1952) 15

16 Griffith s Experiment 16

17 Griffith s Experiment: Transforming principle 17

18 Structure of DNA Rosalind Franklin "for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material" X-ray analysis of DNA structure Francis Harry Compton Crick James Dewey Watson Maurice Hugh Frederick Wilkins 18

19 DNA Structure nitrogenous bases A, T, G, C pentose sugar deoxyribose chain of nucleotides linked by phosphodiester bonds usually a double helix, composed of two complementary strands base pairing rules A with T G with C 19

20 Nucleic Acid Structure 20

21 Nitrogenous bases Purines Adenine Guanine Pyrimidines Thymine Cytosine 21

22 DNA Structure 22

23 DNA Structure two polynucleotide chains are anti-parallel 23

24 RNA Structure nitrogenous bases A, G, C, U (instead of T) pentose sugar ribose usually consists of single strand of nucleotides linked by phosphodiester bonds can coil back on itself forms hairpin-shaped structures with complementary base pairing and helical organization base pairing rules A with U G with C 24

25 Prokaryotic chromosome usually exists as closed circular, supercoiled molecule associated with basic proteins relaxed circle supercoiled DNA 25

26 Eukaryotic DNA linear molecules associated with histones coiled into repeating units called nucleosomes 26

27 Eukaryotic Chromosomes A chromosome is formed from a single DNA molecule that contains many genes. A chromosomal DNA molecule contains three specific nucleotide sequences which are required for replication: a DNA replication origin; a centromere to attach the DNA to the mitotic spindle.; a telomere located at each end of the linear chromosome. 27

28 Purines and pyrimidines pair (A-T or G-C) and the sugars (backbone) are linked by a phosphate. Fig. 9.4 Three views of DNA structure 28

29 Replication Nature: Semiconservative Involved Enzymes (in Primosome & Replisome) Leading strand Lagging strand Okazaki fragments 29

30 Semiconservative New strands are synthesized in 5 to 3 direction 30

31 T A C G A C T A A T A G G C G C A T C C A C G A T C 3' 5' Minus ( ) strand of DNA

32 5' 3' Plus (+) A T G C T G A T T A T C C G C G T A G G T G C T A G strand of DNA T A C G A C T A A T A G G C G C A T C C A C G A T C 3' 5' Minus ( ) strand of DNA

33 5' 3' A T G C T G A T T A T C C G C G T A G G T G C T A G T A C G A C T A A T A G G C G C A T C C A C G A T C 3' 5' A U G C U G A U U A U C C G C G U A G G U G C U A G Plus (+) strand of DNA Minus ( ) strand of DNA 5' 3' mrna

34 Semiconservative replication of DNA synthesizes a new strand of DNA from a template strand. Fig. 9.5 Simplified steps to show the semiconservative replication of DNA 34

35 Enzymes Helicase DNA polymerase III Primase DNA polymerase I Ligase Gyrase 35

36 The function of important enzymes involved in DNA replication. Table 9.1 Some enzymes involved in DNA replication 36

37 Leading strand DnaA,DnaB proteins at Origin or Replication (circular genetic element) or RNA primer (linear genetic element) initiate the 5 to 3 synthesis of DNA in a continuous manner 37

38 Lagging strand Because the direction of synthesis (5 -> 3 ) is opposite to fork movement, the lagging strand is synthesized in form of multiple DNA (Okazaki) fragments Primer synthesis performed by RNA polymerase (Primase), DNA synthesis performed by DNA polymerase III RNA primer removal and fill-in with DNA by DNA polymerase I Okazaki fragments are ligated together by DNA ligase to form one continuous strand 38

39 The steps associated with the DNA replication process. Fig. 9.6 The bacterial replicon: a model for DNA Synthesis 39

40 Patterns of DNA synthesis in procaryotes bidirectional from a single origin of replication replicon portion of the genome that contains an origin and is replicated as a unit 2 forks move in opposite directions 40

41 Mechanism of DNA Replication in bacteria DNA polymerase III synthesizes DNA keep strands separated synthesized by primase unwind strands and relieve tension caused by unwinding 41 Quinolone Target

42 Some amazing facts 30 proteins required to replicate E. coli chromosome occurs with great fidelity error frequency = 10-9 or per base pair replicated due to proofreading activity of DNA polymerases III and I occurs very rapidly 750 to 1,000 base pairs/second in procaryotes base pairs/second in eucaryotes 42

43 Helicase separates the double-stranded molecule 5' 3'

44 3' 5' Helicase separates the double-stranded molecule Synthesis of the leading strand proceeds continuously. 5' 3'

45 3' 5' Helicase separates the double-stranded molecule 5' 3' RNA primer 1. Primase synthesizes an RNA primer Synthesis of the leading strand proceeds continuously. Synthesis of the lagging strand is discontinuous; synthesis is reinitiated periodically, generating a series of fragments that are later joined.

46 3' 5' Helicase separates the double-stranded molecule 5' 3' RNA primer 1. Primase synthesizes an RNA primer Synthesis of the leading strand proceeds continuously. Synthesis of the lagging strand is discontinuous; synthesis is reinitiated periodically, generating a series of fragments that are later joined. 2. DNA polymerase adds nucleotides onto the 3' end of the fragment

47 3' 5' Helicase separates the double-stranded molecule 5' 3' RNA primer 1. Primase synthesizes an RNA primer Synthesis of the leading strand proceeds continuously. Synthesis of the lagging strand is discontinuous; synthesis is reinitiated periodically, generating a series of fragments that are later joined. 2. DNA polymerase adds nucleotides onto the 3' end of the fragment 3. DNA polymerase replaces the RNA primer with deoxynucleotides

48 3' 5' Helicase separates the double-stranded molecule 5' 3' RNA primer 1. Primase synthesizes an RNA primer Synthesis of the leading strand proceeds continuously. Synthesis of the lagging strand is discontinuous; synthesis is reinitiated periodically, generating a series of fragments that are later joined. 2. DNA polymerase adds nucleotides onto the 3' end of the fragment 3. DNA polymerase replaces the RNA primer with deoxynucleotides 4. DNA ligase seals the gaps between adjacent fragments 3' 5'

49 Replication processes from other biological systems (some Grampositive bacterial genomes, plasmids, viruses) involve a rolling cycle. 5 3 Fig. 9.8 Simplified model of rolling circle DNA Replication 49

50 RNA Transcription Message RNA (mrna) Transfer RNA (trna) Ribosomal RNA (rrna) Codons (nucleotide triplett) 50

51 (a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.* Cycle 1 DNA Sample Heat to 94 C *For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA. Polymerase Chain Reaction 51

52 (a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.* Cycle 1 DNA Sample Heat to 94 C Denaturation Strands separate Cycle 1 *For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA. 52

53 (a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.* Cycle 1 DNA Sample Heat to 94 C Denaturation Strands separate Priming Oligonucleotide primers attach at ends of strands to promote replication of amplicons 50 to 65 C Amplicons Primer Primer Cycle 1 *For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA. 53

54 (a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.* Cycle 1 DNA Sample Heat to 94 C Denaturation Strands separate Priming Oligonucleotide primers attach at Amplicons ends of strands to promote replication of amplicons Extension DNA polymerase synthesizes complementary strand Primer 50 to 65 C Primer 72 C Polymerase Cycle 1 *For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA. 54

55 (a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.* Cycle 1 DNA Sample Heat to 94 C Denaturation Strands separate Priming Oligonucleotide primers attach at Amplicons ends of strands to promote replication of amplicons Extension DNA polymerase synthesizes complementary strand Cycle 2 Denaturation New strand Primer 2 copies Original strands Heat to 94 C 50 to 65 C Primer 72 C Polymerase Cycle 1 New strand *For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA. Cycle 2 55

56 (a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.* Cycle 1 DNA Sample Heat to 94 C Denaturation Strands separate Priming Oligonucleotide primers attach at Amplicons ends of strands to promote replication of amplicons Extension DNA polymerase synthesizes complementary strand Cycle 2 Denaturation New strand Priming Primer 2 copies Original strands Heat to 94 C 72 C 50 to 65 C Primer 72 C Polymerase Cycle 1 New strand *For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA. Cycle 2 56

57 (a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.* Cycle 1 DNA Sample Heat to 94 C Denaturation Strands separate Priming Oligonucleotide primers attach at Amplicons ends of strands to promote replication of amplicons Extension DNA polymerase synthesizes complementary strand Cycle 2 Denaturation New strand Priming Extension Primer 2 copies Original strands Heat to 94 C 72 C 50 to 65 C 4 copies Primer 72 C Polymerase Cycle 1 New strand *For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA. Cycle 2 57

58 (a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.* Cycle 1 DNA Sample Heat to 94 C Denaturation Strands separate Priming Oligonucleotide primers attach at Amplicons ends of strands to promote replication of amplicons Extension DNA polymerase synthesizes complementary strand Cycle 2 Denaturation New strand Priming Extension Primer 2 copies Original strands Heat to 94 C 72 C 50 to 65 C 4 copies Primer 72 C Polymerase Cycle 1 New strand *For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA. Cycle 2 (b) A view of the process after 6 cycles, with 64 copies of amplified DNA. Continuing this process for 20 to 40 cycles can produce millions of identical DNA molecules. Cycles 3, 4,... repeat same steps 1* fragment 2 copies 4 copies 8 copies 16 copies 32 copies 64 copies 58