chapter 15: molecular basis of inheritance. dna is the genetic material early in the 20th century,...
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Chapter 15: Molecular Basis of Inheritance
DNA is the genetic material
• Early in the 20th century, the identification of the molecules of inheritance loomed as a major challenge to biologists
The Search for the Genetic Material
• 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
DNA is Genetic Material
• 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
Living S cells (control)
Living R cells (control)
Heat-killed S cells (control)
Mixture of heat-killed S cells and living R cells
Mouse diesMouse dies Mouse healthy Mouse healthy
Living S cells
RESULTS
EXPERIMENT
Frederick Griffith (1928)- Gave mice the bacterium that causes pneumonia (2 strains)
- Smooth strain killed the mice (DEADLY)- Rough strain did not kill the mice (HARMLESS)- Killed smooth strain with heat, did not kill the mice
- Mixed the harmless strain and the heat-treated deadly strain- He found that the harmless rough strain, TRANSFORMED into
a deadly form.- He discovered the TRANSFORMING principle which is now
DNA
Oswald Avery(1944)- Wanted to test if protein was the transforming factor
- Treated Griffith’s mixture of heat-treated deadly strain and live harmless strain with protein-destroying enzymes.
- The bacterial colonies grown from the mixture were still transformed
- Then treated the mixture with DNA destroying enzymes.- The bacterial colonies failed to transform.- CONCLUSION: DNA IS THE GENETIC MATERIAL OF THE
CELL
Additional Evidence
• Viruses: small infectious agent that can only reproduce in living cells
• More evidence for DNA as the genetic material came from studies of bacteriophages
Bacteriophages- viruses that infect bacteria
Fig. 16-3
Bacterial cell
Phage head
Tail sheath
Tail fiber
DNA
100
nm
Alfred Hershey and Martha Chase(1952)
- Conducted experiments using viruses to be sure DNA was the transforming factor, not protein.
- A virus is DNA wrapped in a protein. They reproduce by infecting a living cell with genetic materal. A virus that infects bacteria is a bacteriophage.
- Concluded that the phage’s DNA entered the bacterial cell during infection but the proteins did not.
Fig. 16-4-3
EXPERIMENT
Phage
DNA
Bacterial cell
Radioactive protein
Radioactive DNA
Batch 1: radioactive sulfur (35S)
Batch 2: radioactive phosphorus (32P)
Empty protein shell
Phage DNA
Centrifuge
Centrifuge
Pellet
Pellet (bacterial cells and contents)
Radioactivity (phage protein) in liquid
Radioactivity (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
• Chargaff’s rules state that in any species there is an equal number of A and T bases, and an equal number of G and C bases
Sugar–phosphate backbone
5’ end
Nitrogenous
bases
Thymine (T)
Adenine (A)
Cytosine (C)
Guanine (G)
DNA nucleotide
Sugar (deoxyribose)
3’ end
Phosphate
Building a Structural Model of DNA\
• After most biologists became convinced that DNA was the genetic material, the challenge was to determine how its structure accounts for its role
• 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
• 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 width suggested that the DNA molecule was made up of two strands, forming a double helix– 2 strands, that are twisted
NucleotidesMonomers of DNA- nucleotides
Made up of:
1.Pentose Sugar
2.Phosphate group
3.Nitrogenous Base
Nucleotides join together• Joined together by covalent bonds that connect sugar of
one nucleotide to the phosphate group of the next
• The repeating pattern of sugar-phosphate is called a sugar-phosphate “backbone”
Fig. 16-7a
Hydrogen bond 3’ end
5’ end
3.4 nm
0.34 nm
3’ end
5’ end
(b) Partial chemical structure(a) Key features of DNA structure
1 nm
Fig. 16-7b
(c) Space-filling 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 antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior
Antiparallel- run side by side in opposite directions
• 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
• 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
Purine:
Adenine and Guanine
2 fused rings
Pyrimidine:
Thymine and Cytosine
1 ring
Fig. 16-UN1
Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: width consistent with X-ray data
Fig. 16-8
Cytosine (C)
Adenine (A) Thymine (T)
Guanine (G)
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
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
How does DNA act as a template?
Fig. 16-9-1
A T
GC
T A
TA
G C
(a) Parent molecule
Fig. 16-9-2
A T
GC
T A
TA
G C
A T
GC
T A
TA
G C
(a) Parent molecule (b) Separation of strands
Fig. 16-9-3
A T
GC
T A
TA
G C
(a) Parent molecule
A T
GC
T A
TA
G C
(c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand
(b) Separation of strands
A T
GC
T A
TA
G C
A T
GC
T A
TA
G C
• 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)
Fig. 16-10Parent cell
First replication
Second replication
(a) Conservative model
(b) Semiconserva- tive model
(c) Dispersive model
DNA Replication
• The copying of DNA is remarkable in its speed and accuracy
• More than a dozen enzymes and other proteins participate in DNA replication
Getting Started
1. Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble”
• 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
Fig. 16-12Origin of replication Parental (template) strand
Daughter (new) strand
Replication fork
Replication bubble
Two daughter DNA molecules
(a) Origins of replication in E. coli
Origin of replication Double-stranded DNA molecule
Parental (template) strandDaughter (new) strand
Bubble Replication fork
Two daughter DNA molecules
(b) Origins of replication in eukaryotes
0.5 µm
0.25 µm
Double-strandedDNA molecule
Fig. 16-12b
0.25 µm
Origin of replication Double-stranded DNA molecule
Parental (template) strandDaughter (new) strand
Bubble Replication fork
Two daughter DNA molecules
(b) Origins of replication in eukaryotes
Fig. 16-13
Topoisomerase
Helicase
PrimaseSingle-strand binding proteins
RNA primer
5’5’
5’ 3’
3’
3’
2. 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 protein binds to and stabilizes single-stranded DNA until it can be used as a template
• Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands
Enzymes/Proteins Involved in the beginning….
http://www.youtube.com/watch?v=EYGrElVyHnU
Antiparallel Elongation• The antiparallel
structure of the double helix (two strands oriented in opposite directions) 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’to3’ direction
Synthesizing a New DNA Strand
• Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork (adding new nucleotides)
• The rate of elongation is about per second in bacteria and 50 per second in human cells500 nucleotides
• Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork
• DNA polymerases can only add nucleotides to a FREE 3’ end
• THERE IS NO FREE 3’ end on lagging strand
• Uses a short “primer” to initiate synthesis
• The initial nucleotide strand is a short RNA primer- used on lagging strand
• 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
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Fig. 16-13
Topoisomerase
Helicase
PrimaseSingle-strand binding proteins
RNA primer
5’5’
5’ 3’
3’
3’
• 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
Table 16-1
Fig. 16-17
OverviewOrigin of replication
Leading strand
Leading strand
Lagging strand
Lagging strandOverall directions
of replication
Leading strand
Lagging strand
Helicase
Parental DNA
DNA pol III
Primer Primase
DNA ligase
DNA pol III
DNA pol I
Single-strand binding protein
5
3
5
5
5
5
3
3
3
313 2
4
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 chemicals, radioactive emissions, X-rays, UV light, and certain molecules (in cigarette smoke for example)
• In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA
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Fig. 16-18
Nuclease
DNA polymerase
DNA ligase
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
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Fig. 16-19Ends of parental DNA strands
Leading strand
Lagging strand
Lagging strand
Last fragment Previous fragment
Parental strand
RNA primer
Removal of primers and replacement with DNA where a 3 end is available
Second round of replication
New leading strand
New lagging strand
Further rounds of replication
Shorter and shorter daughter molecules
5
3
3
3
3
3
5
5
5
5
http://www.youtube.com/watch?v=AJNoTmWsE0s http://www.youtube.com/watch?
v=x1zw6uRxKYU
• Eukaryotic chromosomal DNA molecules have at their ends nucleotide sequences 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
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• 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
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
• 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
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
• The bacterial chromosome is a double-stranded, circular DNA molecule associated with a small amount of protein
• Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein
• In a bacterium, the DNA is “supercoiled” and found in a region of the cell called the nucleoid
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
• Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells
• Histones are proteins that are responsible for the first level of DNA packing in chromatin
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 16-21a
DNA double helix (2 nm in diameter)
Nucleosome(10 nm in diameter)
Histones Histone tailH1
DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)
Fig. 16-21b
30-nm fiber
Chromatid (700 nm)
Loops Scaffold
300-nm fiber
Replicated chromosome (1,400 nm)
30-nm fiber Looped domains (300-nm fiber)
Metaphase chromosome