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16 the molecular basis of inheritance

This document provides an overview of the discovery of DNA as the genetic material. It discusses early evidence from experiments transforming bacteria and showing that viral DNA enters bacterial cells. It also describes Rosalind Franklin's X-ray crystallography work that provided insights into DNA's structure and allowed Watson and Crick to deduce the double helix model with paired nitrogenous bases. Their model resolved the structure and showed how DNA could store and replicate the genetic information required for inheritance.

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LECTURE PRESENTATIONS For CAMPBELL BIOLOGY, NINTH EDITION Jane B. Reece, Lisa A. Urry, Michael L. Cain, Steven A. Wasserman, Peter V. Minorsky, Robert B. Jackson Chapter 16 The Molecular Basis of Inheritance Lectures by Erin Barley Kathleen Fitzpatrick © 2011 Pearson Education, Inc.
Overview: Life’s Operating Instructions • In 1953, James Watson and Francis Crick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA • DNA, the substance of inheritance, is the most celebrated molecule of our time • Hereditary information is encoded in DNA and reproduced in all cells of the body • This DNA program directs the development of biochemical, anatomical, physiological, and (to some extent) behavioral traits © 2011 Pearson Education, Inc.
Figure 16.1
DNA is the genetic material • Early in the 20th century, the identification of the molecules of inheritance loomed as a major challenge to biologists © 2011 Pearson Education, Inc.
The Search for the Genetic Material: Scientific Inquiry • When T. H. Morgan’s group showed that genes are located on chromosomes, the two components of chromosomes—DNA and protein—became candidates for the genetic material • The key factor in determining the genetic material was choosing appropriate experimental organisms • The role of DNA in heredity was first discovered by studying bacteria and the viruses that infect them © 2011 Pearson Education, Inc.
Evidence That DNA Can Transform Bacteria • The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928 • Griffith 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 DNA © 2011 Pearson Education, Inc.
Figure 16.2 EXPERIMENT Mixture of Heat-killed heat-killed Living S cells Living R cells S cells S cells and (control) (control) (control) living R cells RESULTS Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells
• In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA • Their conclusion was based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteria • Many biologists remained skeptical, mainly because little was known about DNA © 2011 Pearson Education, Inc.
Evidence That Viral DNA Can Program Cells • More evidence for DNA 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 © 2011 Pearson Education, Inc.
Figure 16.3 Phage head Tail sheath Tail fiber DNA 100 nm Bacterial cell
• In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2 • To determine this, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection • They concluded that the injected DNA of the phage provides the genetic information © 2011 Pearson Education, Inc.
Figure 16.4-3 EXPERIMENT Radioactive Empty protein protein shell Radioactivity Phage (phage protein) in liquid Bacterial cell Batch 1: Radioactive DNA sulfur Phage (35S) DNA Centrifuge Radioactive Pellet (bacterial DNA cells and contents) Batch 2: Radioactive phosphorus (32P) Centrifuge Radioactivity Pellet (phage DNA) in pellet
Additional Evidence That DNA Is the Genetic Material • It was known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group • In 1950, Erwin Chargaff reported that DNA composition varies from one species to the next • This evidence of diversity made DNA a more credible candidate for the genetic material © 2011 Pearson Education, Inc.
• Two findings became known as Chargaff’s rules – The base composition of DNA varies between species – But, in any species the number of A and T bases are equal and the number of G and C bases are equal • The basis for these rules was not understood until the discovery of the double helix © 2011 Pearson Education, Inc.
Figure 16.5 Sugar–phosphate Nitrogenous bases backbone 5′ end Thymine (T) Adenine (A) Cytosine (C) Phosphate Guanine (G) Sugar (deoxyribose) DNA Nitrogenous base nucleotide 3′ end
Building a Structural Model of DNA: Scientific Inquiry • After DNA 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 DNA molecule using this technique © 2011 Pearson Education, Inc.
Figure 16.6 (a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA
• Franklin’s X-ray crystallographic images of DNA enabled Watson to deduce that DNA was helical • The X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous bases • The pattern in the photo suggested that the DNA molecule was made up of two strands, forming a double helix © 2011 Pearson Education, Inc.
Figure 16.7 G 5′ end C C G Hydrogen bond 3′ end G C G C T A 3.4 nm T A G C G C C G A T 1 nm C G T A C G G C C G A T A T 3′ end A T 0.34 nm T A 5′ end (a) Key features of (b) Partial chemical structure (c) Space-filling DNA structure model
• Watson and Crick built models of a double helix to conform to the X-rays and chemistry of DNA • Franklin had concluded that there were two outer sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior • Watson built a model in which the backbones were antiparallel (their subunits run in opposite directions) © 2011 Pearson Education, Inc.
• At first, Watson and Crick thought the bases paired like with like (A with A, 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 © 2011 Pearson Education, Inc.
Figure 16.UN01 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data
• Watson and Crick reasoned that the pairing was more specific, dictated by the base structures • They determined that adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C) • The Watson-Crick model explains Chargaff’s rules: in any organism the amount of A = T, and the amount of G = C © 2011 Pearson Education, Inc.
Figure 16.8 Sugar Sugar Adenine (A) Thymine (T) Sugar Sugar Guanine (G) Cytosine (C)
Many proteins work together in DNA replication and repair • The relationship between structure and function is manifest in the double helix • Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material © 2011 Pearson Education, Inc.
The Basic Principle: Base Pairing to a Template Strand • Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication • In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules © 2011 Pearson Education, Inc.
Figure 16.9-3 A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C (a) Parent molecule (b) Separation of (c) “Daughter” DNA molecules, strands each consisting of one parental strand and one new strand
• Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand • Competing models were the conservative model (the two parent strands rejoin) and the dispersive model (each strand is a mix of old and new) © 2011 Pearson Education, Inc.
Figure 16.10 Parent First Second cell replication replication (a) Conservative model (b) Semiconservative model (c) Dispersive model
• Experiments by Matthew Meselson and Franklin Stahl supported the semiconservative model • They labeled the nucleotides of the old strands with a heavy isotope of nitrogen, while any new nucleotides were labeled with a lighter isotope • The first replication produced a band of hybrid DNA, eliminating the conservative model • A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model © 2011 Pearson Education, Inc.
Figure 16.11 EXPERIMENT 1 Bacteria 2 Bacteria cultured in transferred to medium with medium with 15 N (heavy 14 N (lighter isotope) isotope) RESULTS 3 DNA sample 4 DNA sample Less centrifuged centrifuged dense after first after second replication replication More dense CONCLUSION Predictions: First replication Second replication Conservative model Semiconservative model Dispersive model
Getting Started • Replication begins at particular sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble” • A bacterial chromosome has 1 origin of replication • A eukaryotic chromosome may have hundreds or even thousands of origins of replication • Replication proceeds in both directions from each origin, until the entire molecule is copied © 2011 Pearson Education, Inc.
Figure 16.12 (a) Origin of replication in an E. coli cell (b) Origins of replication in a eukaryotic cell Origin of Double-stranded Parental (template) strand Origin of replication DNA molecule replication Daughter (new) strand Parental (template) Daughter (new) strand strand Replication Double- fork stranded DNA molecule Replication bubble Bubble Replication fork Two daughter DNA molecules Two daughter DNA molecules 0.25 µm 0.5 µm
• At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating • Helicases are enzymes that untwist the double helix at the replication forks • Single-strand binding proteins bind to and stabilize single-stranded DNA • Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands © 2011 Pearson Education, Inc.
• DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3′ end • The initial nucleotide strand is a short RNA primer • An enzyme called primase can start an RNA chain from scratch and adds RNA nucleotides one at a time using the parental DNA as a template • The primer is short (5–10 nucleotides long), and the 3′ end serves as the starting point for the new DNA strand © 2011 Pearson Education, Inc.
Figure 16.13 Primase 3′ Topoisomerase 5′ RNA 3′ primer 5′ 3′ Helicase 5′ Single-strand binding proteins
Synthesizing a New DNA Strand • Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork • Most DNA polymerases require a primer and a DNA template strand • The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells © 2011 Pearson Education, Inc.
• Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate • dATP supplies adenine to DNA and is similar to the ATP of energy metabolism • The difference is in their sugars: dATP has deoxyribose while ATP has ribose • As each monomer of dATP, dGTP, dCTP, or dTTP joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate © 2011 Pearson Education, Inc.
Figure 16.14 New strand Template strand 5′ 3′ 5′ 3′ Sugar A T A T Phosphate Base C G C G G C G C DNA OH polymerase 3′ A T A T P P Pi OH P P C Pyrophosphate 3′ C OH Nucleoside 2Pi triphosphate 5′ 5′
Antiparallel Elongation • The antiparallel structure of the double helix affects replication • DNA polymerases add nucleotides only to the free 3′ end of a growing strand; therefore, a new DNA strand can elongate only in the 5′ to 3′ direction • Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork © 2011 Pearson Education, Inc.
Figure 16.15 Overview Leading Origin of replication Lagging strand strand Primer Lagging Leading strand strand Origin of Overall directions replication of replication 3′ 5′ 5′ RNA primer 3′ 3′ Sliding clamp DNA pol III Parental DNA 5′ 3′ 5′ 5′ 3′ 3′ 5′
• To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork • The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase © 2011 Pearson Education, Inc.
Figure 16.16a Overview Leading Origin of replication Lagging strand strand Lagging strand 2 1 Leading strand Overall directions of replication
LE 16-15_6 Primase joins RNA nucleotides into a primer. 3′ 5′ 5′ 3′ Template strand DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 3′ 5′ RNA primer 3′ 5′ After reaching the next RNA primer (not shown), DNA pol III Okazaki falls off. fragment 3′ 3′ 5′ 5′ After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 5′ 3′ 3′ 5′ DNA pol I replaces the RNA with DNA, adding to the 3′ end 5′ of fragment 2. 3′ 3′ 5′ DNA ligase forms a bond between the newest The lagging DNA and the adjacent DNA strand in the region of fragment 1. is now complete. 5′ 3′ 3′ 5′ Overall direction of replication
Figure 16.17 Overview Leading Origin of replication Lagging strand strand Leading Lagging strand strand Overall directions Leading strand of replication 5′ DNA pol III 3′ Primer Primase 3′ 5′ 3′ Parental DNA pol III Lagging strand DNA 5′ 4 DNA pol I DNA ligase 3′5′ 3 2 1 3′ 5′
The DNA Replication Complex • The proteins that participate in DNA replication form a large complex, a “DNA replication machine” • The DNA replication machine may be stationary during the replication process • Recent studies support a model in which DNA polymerase molecules “reel in” parental DNA and “extrude” newly made daughter DNA molecules • Primase may act as a molecular brake, coordinating primer placement and the rates of replication on the leading and lagging strands © 2011 Pearson Education, Inc.
Figure 16.18 DNA pol III Parental DNA Leading strand 5′ 5′ 3′ 3′ 3′ 3′ 5′ 5′ Connecting Helicase protein 3′ 5′ Lagging DNA strand 3′ Lagging strand template pol III 5′
Proofreading and Repairing DNA • DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides • In mismatch repair of DNA, repair enzymes correct errors in base pairing • DNA 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 DNA © 2011 Pearson Education, Inc.
Figure 16.19 5′ 3′ A thymine dimer distorts the DNA molecule. 3′ 5′ Nuclease A nuclease enzyme cuts the damaged DNA strand 5′ at two points and the 3′ damaged section is removed. 3′ 5′ DNA polymerase Repair synthesis by a DNA polymerase 5′ 3′ fills in the missing nucleotides. 3′ 5′ DNA ligase DNA ligase seals the 5′ 3′ free end of the new DNA to the old DNA, making the strand complete. 3′ 5′
Evolutionary Significance of Altered DNA 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 • These changes (mutations) are the source of the genetic variation upon which natural selection operates © 2011 Pearson Education, Inc.
Replicating the Ends of DNA Molecules • Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes • The usual replication machinery provides no way to complete the 5′ ends, so repeated rounds of replication produce shorter DNA molecules with uneven ends • This is not a problem for prokaryotes, most of which have circular chromosomes © 2011 Pearson Education, Inc.
LE 16-18 5′ End of parental Leading strand DNA strands Lagging strand 3′ Last fragment Previous fragment RNA primer Lagging strand 5′ 3′ Primer removed but Removal of primers and cannot be replaced replacement with DNA with DNA because where a 3′ end is available no 3′ end available for DNA polymerase 5′ 3′ Second round of replication 5′ New leading strand 3′ New leading strand 5′ 3′ Further rounds of replication Shorter and shorter daughter molecules
• Eukaryotic chromosomal DNA molecules have special nucleotide sequences at their ends called telomeres • Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules • It has been proposed that the shortening of telomeres is connected to aging © 2011 Pearson Education, Inc.
Figure 16.21 1 µm
• If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce • An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells • The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions • There is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist © 2011 Pearson Education, Inc.
• Chromatin, a complex of DNA and protein, is found in the nucleus of eukaryotic cells • Chromosomes fit into the nucleus through an elaborate, multilevel system of packing © 2011 Pearson Education, Inc.
• Chromatin undergoes changes in packing during the cell cycle • At interphase, some chromatin is organized into a 10-nm fiber, but much is compacted into a 30-nm fiber, through folding and looping • Though interphase chromosomes are not highly condensed, they still occupy specific restricted regions in the nucleus © 2011 Pearson Education, Inc.
Figure 16.22a Nucleosome (10 nm in diameter) DNA double helix (2 nm in diameter) H1 Histone Histones tail Nucleosomes, or “beads on DNA, the double helix Histones a string” (10-nm fiber)
Figure 16.22b Chromatid (700 nm) 30-nm fiber Loops Scaffold 300-nm fiber 30-nm fiber Replicated chromosome (1,400 nm) Looped domains (300-nm fiber) Metaphase chromosome
Figure 16.22f Loops Scaffold
Figure 16.22g Chromatid (700 nm)
• 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 © 2011 Pearson Education, Inc.