The Molecular Basis of Inheritance

Transcription

1 6 he Molecular Basis of Inheritance MPBELL BIOLOY ENH EDIION Reece Urry ain Wasserman Minorsky Jackson Life s Operating Instructions In 953, James Watson and Francis rick introduced an elegant double-helical for the structure of deoxyribonucleic acid, or DN Hereditary information is encoded in DN and reproduced in all cells of the body his DN program directs the development of biochemical, anatomical, physiological, and (to some extent) behavioral traits Lecture Presentation by Nicole unbridge and Kathleen Fitzpatrick Figure 6. What is the structure of DN? oncept 6.: DN is the genetic material Early in the 0th century, the identification of the molecules of inheritance loomed as a major challenge to biologists DN is copied during DN replication, and cells can repair their DN 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 DN and protein became candidates for the genetic material he role of DN in heredity was first discovered by studying bacteria and the viruses that infect them Evidence hat DN an ransform Bacteria he discovery of the genetic role of DN began with research by Frederick riffith in 98 riffith worked with two strains of a bacterium, one pathogenic and one harmless 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 DN

2 Figure 6. an a genetic trait be transferred between different bacterial strains? Experiment Living S cells (pathogenic control) Results Living R cells (nonpathogenic control) Heat-killed S cells (nonpathogenic control) Mixture of heatkilled S cells and living R cells Mouse dies Mouse healthy Mouse healthy Mouse dies In 944, Oswald very, Maclyn Mcarty, and olin MacLeod announced that the transforming substance was DN Many biologists remained skeptical, mainly because little was known about DN Living S cells Evidence hat Viral DN an Program ells Figure 6.3 virus infecting a bacterial cell More evidence for DN 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 virus is DN (sometimes RN) enclosed by a protective coat, often simply protein Phage head DN ail sheath ail fiber enetic material Bacterial cell 00 nm In 95, lfred Hershey and Martha hase showed that DN is the genetic material of a phage known as hey designed an experiment showing that only one of the two components of (DN or protein) enters an E. coli cell during infection hey concluded that the injected DN of the phage provides the genetic information Figure 6.4 Experiment Batch : Radioactive sulfur ( 35 S) in phage protein Labeled phages gitation frees outside infect cells. phage parts from cells. Radioactive protein entrifuge Batch : Radioactive phosphorus ( 3 P) in phage DN Radioactive DN 3 entrifuged cells form a pellet. 4 Radioactivity (phage protein) found in liquid Pellet entrifuge Pellet 4 Radioactivity (phage DN) found in pellet

3 dditional Evidence hat DN Is the enetic Material It was known that DN is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group Figure 6.5 Sugar phosphate backbone end Nitrogenous bases hymine () denine () In 950, Erwin hargaff reported that DN composition varies from one species to the next ytosine () his evidence of diversity made DN a more credible candidate for the genetic material Phosphate end Sugar DN (deoxyribose) nucleotide Nitrogenous base uanine () wo findings became known as hargaff s rules he base composition of DN 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 Building a Structural Model of DN: Scientific Inquiry fter DN 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 DN molecule using this technique Figure 6.6 (a) Rosalind Franklin (b) Franklin s X-ray diffraction photograph of DN Franklin s X-ray crystallographic images of DN enabled Watson to deduce that DN 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 DN molecule was made up of two s, forming a double helix 3

4 Figure 6.7 nm 3.4 nm end Hydrogen bond end Watson and rick built s of a double helix to conform to the X-rays and chemistry of DN Franklin had concluded that there were two outer sugar-phosphate backbones, with the nitrogenous bases paired in the molecule s interior 0.34 nm end end Watson built a in which the backbones were antiparallel (their subunits run in opposite directions) (a) Key features of DN structure (b) Partial chemical structure (c) Space-filling Figure 6.UN0 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 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data Figure 6.8 Watson and rick reasoned that the pairing was more specific, dictated by the base structures Sugar hey determined that adenine () paired only with thymine (), and guanine () paired only with cytosine () denine () Sugar hymine () he Watson-rick explains hargaff s rules: in any organism the amount of, and the amount of Sugar Sugar uanine () ytosine () 4

5 oncept 6.: Many proteins work together in DN replication 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 he Basic Principle: Base Pairing to a emplate Strand Since the two s of DN are complementary, each acts as a template for building a new in replication In DN replication, the parent molecule unwinds, and two new daughter s are built based on base-pairing rules Figure 6.9- Figure 6.9- (a) Parental molecule (a) Parental molecule (b) Separation of parental s into templates Figure Watson and rick s semiconservative of replication 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 (a) Parental molecule (b) Separation of parental s into templates (c) Formation of new s complementary to template s ompeting s were the conservative (the two parent s rejoin) and the dispersive (each is a mix of old and new) 5

6 Figure 6.0 (a) onservative (b) Semiconservative (c) Dispersive Parent cell First Second replication replication 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 replication produced a band of hybrid DN, eliminating the conservative second replication produced both light and hybrid DN, eliminating the dispersive and supporting the semiconservative Figure 6. Experiment Bacteria cultured Bacteria transferred in medium with 5 N to medium with 4 N (heavy isotope) (lighter isotope) Results 3 DN sample centrifuged after first replication DN sample centrifuged after second replication onclusion Predictions: First replication Second replication 4 Less dense More dense DN Replication: loser Look he copying of DN is remarkable in its speed and accuracy More than a dozen enzymes and other proteins participate in DN replication onservative Semiconservative Dispersive etting Started Replication begins at particular sites called origins, where the two DN s are separated, opening up a replication bubble eukaryotic chromosome may have hundreds or even thousands of origins Replication proceeds in both directions from each origin, until the entire molecule is copied Figure 6. (a) Origin in an E. coli cell Origin of replication Bacterial chromosome Doubleed DN molecule wo daughter DN molecules Parental (template) Daughter (new) Replication bubble Replication fork (b) Origins in a eukaryotic cell Origin of replication Eukaryotic chromosome Double-ed DN molecule Bubble Parental (template) Daughter (new) Replication fork wo daughter DN molecules 0.5 µm 0.5 µm 6

7 Figure 6.3 Primase t the end of each replication bubble is a replication fork, a Y-shaped region where new DN s are elongating Helicases are enzymes that untwist the double helix at the replication forks opoisomerase Replication fork RN primer Single- binding proteins bind to and stabilize single-ed DN opoisomerase corrects overwinding ahead of replication forks by breaking, swiveling, and rejoining DN s Helicase Single- binding proteins DN polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to an existing 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 DN as a template he primer is short (5 0 nucleotides long), and the end serves as the starting point for the new DN Synthesizing a New DN Strand Enzymes called DN polymerases catalyze the elongation of new DN at a replication fork Most DN polymerases require a primer and a DN 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 DN is a nucleoside triphosphate dp supplies adenine to DN 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 DN, it loses two phosphate groups as a molecule of pyrophosphate 7

8 Figure 6.4 New emplate ntiparallel Elongation Sugar Phosphate Base he antiparallel structure of the double helix affects replication OH Nucleotide DN polymerase OH P P i Pyrophosphate DN polymerases add nucleotides only to the free end of a growing ; therefore, a new DN can elongate only in the to direction P i Figure 6.5 Leading Overview Origin Lagging Parental DN Lagging DN pol III starts to synthesize leading. Primer Overall directions Leading Origin RN primer Sliding clamp DN pol III ontinuous elongation in the to direction long one template of DN, the DN polymerase synthesizes a leading continuously, moving toward the replication fork o elongate the other new, called the lagging, DN polymerase must work in the direction away from the replication fork he lagging is synthesized as a series of segments called Okazaki fragments, which are joined together by DN ligase Figure 6.6 emplate Leading 3 Lagging Overview Origin Primase makes RN primer. Origin of replication RN primer for fragment DN pol III detaches. Overall directions Okazaki fragment Lagging Leading DN pol III makes Okazaki fragment. RN primer for fragment Okazaki fragment DN pol III makes Okazaki fragment. DN pol I replaces RN with DN. DN ligase forms bonds between DN fragments. Overall direction Figure 6.7 Single- binding proteins Helicase Parental DN Leading template Leading DN pol III Primer Primase 5 DN pol III 4 Leading Lagging Lagging Lagging template Overview Origin of replication Overall directions 3 Leading Lagging DN pol I DN ligase 8

9 able 6. he DN Replication omplex he proteins that participate in DN replication form a large complex, a DN replication machine he DN replication machine may be stationary during the replication process Recent studies support a in which DN polymerase molecules reel in parental DN and extrude newly made daughter DN molecules Figure 6.8 Leading template BioFlix: DN Replication DN pol III Parental DN onnecting protein DN pol III Helicase Leading Lagging template Lagging Proofreading and Repairing DN Figure 6.9- DN polymerases proofread newly made DN, replacing any incorrect nucleotides In mismatch repair of DN, repair enzymes correct errors in base pairing Nuclease DN 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 DN 9

10 Figure 6.9- Figure Nuclease Nuclease DN polymerase DN polymerase DN ligase Evolutionary Significance of ltered DN 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 DN Molecules Limitations of DN polymerase create problems for the linear DN of eukaryotic chromosomes he usual replication machinery provides no way to complete the ends, so repeated rounds of replication produce shorter DN molecules with uneven ends his is not a problem for prokaryotes, most of which have circular chromosomes Figure 6.0 Ends of parental DN s Lagging Parental Last fragment RN primer New leading New lagging Leading Lagging Next-to-last fragment Removal of primers and replacement with DN where a end is available Second round Further rounds Shorter and shorter daughter molecules Figure 6.0a Ends of parental DN s Lagging Parental Last fragment RN primer Leading Lagging Next-to-last fragment Removal of primers and replacement with DN where a end is available 0

11 Figure 6.0b New leading New lagging Second round Further rounds Shorter and shorter daughter molecules Eukaryotic chromosomal DN molecules have special nucleotide sequences at their ends called telomeres elomeres do not prevent the shortening of DN molecules, but they do postpone the erosion of genes near the ends of DN molecules It has been proposed that the shortening of telomeres is connected to aging 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 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 oncept 6.3: chromosome consists of a DN molecule packed together with proteins he bacterial chromosome is a double-ed, circular DN molecule associated with a small amount of protein Eukaryotic chromosomes have linear DN molecules associated with a large amount of protein In a bacterium, the DN is supercoiled and found in a region of the cell called the nucleoid

12 Figure 6. In the eukaryotic cell, DN is precisely combined with proteins in a complex called chromatin hromosomes fit into the nucleus through an elaborate, multilevel system of packing DN double helix ( nm in diameter) Histones DN, the Histones double helix Nucleosome (0 nm in diameter) Histone tail H Nucleosomes, or beads on a string (0-nm fiber) 30-nm fiber 30-nm fiber Loops Scaffold 300-nm fiber hromatid (700 nm) Looped Replicated chromosome domains (,400 nm) (300-nm fiber) Metaphase chromosome nimation: DN Packing Video: artoon and Stick Model of a Nucleosomal Particle Figure 6.3 hromatin undergoes changes in packing during the cell cycle t interphase, some chromatin is organized into a 0-nm fiber, but much is compacted into a 30-nm fiber, through folding and looping Interphase chromosomes occupy specific restricted regions in the nucleus and the fibers of different chromosomes do not become entangled 5 µm

13 Most chromatin is loosely packed in the nucleus during interphase and condenses prior to mitosis Loosely packed chromatin is called euchromatin During interphase a few regions of chromatin (centromeres and telomeres) are highly condensed into heterochromatin Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions Histones can undergo chemical modifications that result in changes in chromatin organization 3