The Molecular Basis of Inheritance

Transcription

1 LEURE PRESENIONS For MPBELL BIOLOY, NINH EDIION Jane B. Reece, Lisa. Urry, Michael L. ain, Steven. Wasserman, Peter V. Minorsky, Robert B. Jackson hapter 6 he Molecular Basis of Inheritance Lectures by Erin Barley Kathleen Fitzpatrick Overview: Life s Operating Instructions In 953, James Watson and Francis rick introduced an elegant double-helical for the structure of deoxyribonucleic acid, or, the substance of inheritance, is the most celebrated molecule of our time Hereditary information is encoded in and reproduced in all cells of the body his program directs the development of biochemical, anatomical, physiological, and (to some extent) behavioral traits Figure 6. oncept 6.: is the genetic material Early in the 0th century, the identification of the molecules of inheritance loomed as a major challenge to biologists he Search for the enetic Material: Scientific Inquiry When. H. Morgan s group showed that genes are located on chromosomes, the two components of chromosomes and protein became candidates for the genetic material he key factor in determining the genetic material was choosing appropriate experimental organisms he role of in heredity was first discovered by studying bacteria and the viruses that infect them Evidence hat an ransform Bacteria he discovery of the genetic role of began with research by Frederick riffith in 98 riffith worked with two strains of a bacterium, one pathogenic and one harmless

2 When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic He called this phenomenon transformation, now defined as a change in genotype and phenotype due to assimilation of foreign Figure 6. EXPERIMEN Living S cells (control) Living R cells (control) Heat-killed S cells (control) Mixture of heat-killed S cells and living R cells RESULS Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells Evidence hat Viral an Program ells In 944, Oswald very, Maclyn Mcarty, and olin MacLeod announced that the transforming substance was heir conclusion was based on experimental evidence that only worked in transforming harmless bacteria into pathogenic bacteria Many biologists remained skeptical, mainly because little was known about More evidence for as the genetic material came from studies of viruses that infect bacteria Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research nimation: Phage Reproductive ycle Figure 6.3 Bacterial cell Phage head ail sheath ail fiber 00 nm In 95, lfred Hershey and Martha hase performed experiments showing that is the genetic material of a phage known as o determine this, they designed an experiment showing that only one of the two components of ( or protein) enters an E. coli cell during infection hey concluded that the injected of the phage provides the genetic information nimation: Hershey-hase Experiment

3 Figure 6.4- Figure 6.4- EXPERIMEN Phage protein EXPERIMEN Phage protein Empty protein shell Bacterial cell Bacterial cell Batch : sulfur ( 35 S) Batch : sulfur ( 35 S) Phage Batch : phosphorus ( 3 P) Batch : phosphorus ( 3 P) Figure EXPERIMEN Phage Bacterial cell protein Empty protein shell Radioactivity (phage protein) in liquid dditional Evidence hat Is the enetic Material Batch : sulfur ( 35 S) Batch : phosphorus ( 3 P) Phage entrifuge Pellet (bacterial cells and contents) It was known that is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group In 950, Erwin hargaff reported that composition varies from one species to the next his evidence of diversity made a more credible candidate for the genetic material entrifuge Pellet Radioactivity (phage ) in pellet nimation: and RN Structure Figure 6.5 Sugar phosphate backbone end Nitrogenous bases wo findings became known as hargaff s rules he base composition of varies between species In any species the number of and bases are equal and the number of and bases are equal he basis for these rules was not understood until the discovery of the double helix hymine () denine () ytosine () Phosphate uanine () Sugar (deoxyribose) Nitrogenous base nucleotide end 3

4 Figure 6.6 Building a Structural Model of : Scientific Inquiry fter was accepted as the genetic material, the challenge was to determine how its structure accounts for its role in heredity Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure Franklin produced a picture of the molecule using this technique (a) Rosalind Franklin (b) Franklin s X-ray diffraction photograph of Figure 6.6a Figure 6.6b (b) Franklin s X-ray diffraction photograph of (a) Rosalind Franklin Figure 6.7 Franklin s X-ray crystallographic images of enabled Watson to deduce that was helical he X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous bases he pattern in the photo suggested that the molecule was made up of two s, forming a double helix nm end 3.4 nm end 0.34 nm Hydrogen bond end end (a) Key features of structure (b) Partial chemical structure (c) Space-filling nimation: Double Helix 4

5 end Figure 6.7a Figure 6.7b Hydrogen bond end 3.4 nm nm 0.34 nm end end (a) Key features of structure (b) Partial chemical structure (c) Space-filling Watson and rick built s of a double helix to conform to the X-rays and chemistry of Franklin had concluded that there were two outer sugar-phosphate backbones, with the nitrogenous bases paired in the molecule s interior Watson built a in which the backbones were antiparallel (their subunits run in opposite directions) t first, Watson and rick thought the bases paired like with like ( with, and so on), but such pairings did not result in a uniform width Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray data Figure 6.UN0 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data Watson and rick reasoned that the pairing was more specific, dictated by the base structures hey determined that adenine () paired only with thymine (), and guanine () paired only with cytosine () he Watson-rick explains hargaff s rules: in any organism the amount of =, and the amount of = 5

6 Figure 6.8 Sugar denine () Sugar hymine () oncept 6.: Many proteins work together in and repair he relationship between structure and function is manifest in the double helix Watson and rick noted that the specific base pairing suggested a possible copying mechanism for genetic material Sugar Sugar uanine () ytosine () Figure 6.9- he Basic Principle: Base Pairing to a emplate Strand Since the two s of are complementary, each acts as a template for building a new in In, the parent molecule unwinds, and two new daughter s are built based on base-pairing rules (a) Parent molecule nimation: Replication Overview Figure 6.9- Figure (a) Parent molecule (b) Separation of s (a) Parent molecule (b) Separation of s (c) Daughter molecules, each consisting of one parental and one new 6

7 Watson and rick s semiconservative of predicts that when a double helix replicates, each daughter molecule will have one old (derived or conserved from the parent molecule) and one newly made ompeting s were the conservative (the two parent s rejoin) and the dispersive (each is a mix of old and new) Figure 6.0 (a) onservative (b) Semiconservative (c) Dispersive Parent cell First Second Experiments by Matthew Meselson and Franklin Stahl supported the semiconservative hey labeled the nucleotides of the old s with a heavy isotope of nitrogen, while any new nucleotides were labeled with a lighter isotope he first produced a band of hybrid, eliminating the conservative second produced both light and hybrid, eliminating the dispersive and supporting the semiconservative Figure 6. EXPERIMEN Bacteria cultured in medium with 5 N (heavy isotope) RESULS 3 sample 4 sample centrifuged centrifuged after first after second Bacteria transferred to medium with 4 N (lighter isotope) Less dense More dense ONLUSION Predictions: First Second Figure 6.a EXPERIMEN Bacteria cultured in medium with 5 N (heavy isotope) Bacteria transferred to medium with 4 N (lighter isotope) onservative Semiconservative Dispersive RESULS 3 sample 4 centrifuged after first sample centrifuged after second Less dense More dense 7

8 Figure 6.b ONLUSION Predictions: First Second Replication: loser Look he copying of is remarkable in its speed and accuracy More than a dozen enzymes and other proteins participate in onservative Semiconservative Dispersive Figure 6. (a) Origin of in an E. coli cell Origin of (b) Origins of in a eukaryotic cell Parental (template) Origin of Daughter (new) Doubleed molecule Replication fork Parental (template) Replication bubble Bubble Daughter (new) Replication fork wo daughter molecules wo daughter molecules 0.5 µm Replication begins at particular sites called origins of, where the two s are separated, opening up a bubble eukaryotic chromosome may have hundreds or even thousands of origins of Replication proceeds in both directions from each origin, until the entire molecule is copied Double-ed molecule nimation: Origins of Replication 0.5 µm etting Started Figure 6.a Figure 6.b (a) Origin of in an E. coli cell Origin of (b) Origins of in a eukaryotic cell Double-ed Origin of molecule Parental (template) Daughter (new) Doubleed molecule Replication bubble Parental (template) Daughter (new) Replication fork Bubble Replication fork wo daughter molecules wo daughter molecules 0.5 µm 0.5 µm 8

9 Figure 6.c Figure 6.d 0.5 µm 0.5 µm Figure 6.3 Primase t the end of each bubble is a fork, a Y-shaped region where new s are elongating Helicases are enzymes that untwist the double helix at the forks Single- binding proteins bind to and stabilize single-ed opoisomerase corrects overwinding ahead of forks by breaking, swiveling, and rejoining s opoisomerase Helicase Single- binding proteins RN primer polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the end he initial nucleotide is a short RN primer n enzyme called primase can start an RN chain from scratch and adds RN nucleotides one at a time using the parental as a template he primer is short (5 0 nucleotides long), and the end serves as the starting point for the new 9

10 Synthesizing a New Strand Enzymes called polymerases catalyze the elongation of new at a fork Most polymerases require a primer and a template he rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells Each nucleotide that is added to a growing is a nucleoside triphosphate dp supplies adenine to and is similar to the P of energy metabolism he difference is in their sugars: dp has deoxyribose while P has ribose s each monomer of dp joins the, it loses two phosphate groups as a molecule of pyrophosphate Figure 6.4 New emplate ntiparallel Elongation Sugar Phosphate OH Base polymerase he antiparallel structure of the double helix affects polymerases add nucleotides only to the free end of a growing ; therefore, a new can elongate only in the to direction P P P OH Nucleoside triphosphate P P i OH Pyrophosphate P i Figure 6.5 Overview Origin of long one template of, the polymerase synthesizes a leading continuously, moving toward the fork Primer Overall directions of Origin of RN primer Sliding clamp Parental pol III nimation: Strand 0

11 Figure 6.5a Figure 6.5b Origin of Overview Origin of RN primer Sliding clamp Primer Overall directions of Parental pol III Figure 6.6 Overview o elongate the other new, called the lagging, polymerase must work in the direction away from the fork he lagging is synthesized as a series of segments called fragments, which are joined together by ligase emplate RN primer for fragment RN primer for fragment fragment fragment Origin of Overall directions of nimation: Strand Overall direction of Figure 6.6a Figure 6.6b- emplate Overview Origin of Overall directions of

12 Figure 6.6b- Figure 6.6b-3 emplate RN primer for fragment emplate RN primer for fragment fragment Figure 6.6b-4 Figure 6.6b-5 emplate RN primer for fragment emplate RN primer for fragment RN primer for fragment fragment fragment RN primer for fragment fragment fragment Figure 6.6b-6 Figure 6.7 emplate RN primer for fragment Overview Origin of RN primer for fragment fragment fragment Parental pol III Primer Primase pol III 4 Overall directions of pol I ligase 3 Overall direction of

13 Figure 6.7a Overview Origin of Figure 6.7b Overview Origin of Overall directions of Overall directions of Parental pol III Primer Primase Primer pol III pol I ligase 4 3 he Replication omplex he proteins that participate in form a large complex, a machine he machine may be stationary during the process Recent studies support a in which polymerase molecules reel in parental and extrude newly made daughter molecules Figure 6.8 Parental onnecting protein pol III pol III Helicase template nimation: Replication Review Proofreading and Repairing polymerases proofread newly made, replacing any incorrect nucleotides In mismatch repair of, repair enzymes correct errors in base pairing can be damaged by exposure to harmful chemical or physical agents such as cigarette smoke and X-rays; it can also undergo spontaneous changes In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of Figure 6.9 Nuclease polymerase ligase 3

14 Evolutionary Significance of ltered Nucleotides Error rate after proofreading repair is low but not zero Sequence changes may become permanent and can be passed on to the next generation hese changes (mutations) are the source of the genetic variation upon which natural selection operates Replicating the Ends of Molecules Limitations of polymerase create problems for the linear of eukaryotic chromosomes he usual machinery provides no way to complete the ends, so repeated rounds of produce shorter molecules with uneven ends his is not a problem for prokaryotes, most of which have circular chromosomes Figure 6.0 Ends of parental s Last fragment RN primer Next-to-last fragment Figure 6.0a Ends of parental s Parental Removal of primers and replacement with where a end is available Last fragment RN primer Next-to-last fragment New leading New lagging Second round of Parental Removal of primers and replacement with where a end is available Further rounds of Shorter and shorter daughter molecules Figure 6.0b New leading New lagging Second round of Further rounds of Eukaryotic chromosomal molecules have special nucleotide sequences at their ends called telomeres elomeres do not prevent the shortening of molecules, but they do postpone the erosion of genes near the ends of molecules It has been proposed that the shortening of telomeres is connected to aging Shorter and shorter daughter molecules 4

15 Figure 6. If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce n enzyme called telomerase catalyzes the lengthening of telomeres in germ cells µm Figure 6.UN03 he shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions here is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist pol III synthesizes leading continuously Parental Helicase pol III starts synthesis at end of primer, continues in direction synthesized in short fragments, later joined by ligase Origin of Primase synthesizes a short RN primer pol I replaces the RN primer with nucleotides Figure 6.UN04 Figure 6.UN05 5

16 Figure 6.UN06 Figure 6.UN07 6