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DNA is the Genetic Material Evidence that DNA can transform Bacteria Frederick Griffith was studying Streptoccoccus pneumonia, a bacterium that causes pneumonia in mammals, trying to develop a vaccine against pneumonia. Griffith had two strands (varieties), one pathogenic (disease- causing) and one non-pathogenic (harmless) He killed the pathogenic bacteria using heat and mixed them will the non-pathogenic bacteria and injected the mixture into different mice.
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The Molecular Basis of Inheritance
CHAPTER 16: DNA STRUCTURE AND REPLICATION
You must know• The structure of DNA• The knowledge about DNA gained from the work of Griffith; Avery,
MacLeod, and McCarty; Hershey and Chase; Wilkins and Franklin; and Watson and Crick• Replication is semi-conservative and occurs 5’ to 3’• The roles of DNA polymerase III, DNA polymerase I, ligase,
helicase, primase, and topoisomerase in replication• The general differences between bacterial chromosomes and
eukaryotic chromosomes• How DNA packaging can affect gene expression
DNA is the Genetic Material• Evidence that DNA can transform Bacteria• Frederick Griffith was studying Streptoccoccus pneumonia, a
bacterium that causes pneumonia in mammals, trying to develop a vaccine against pneumonia.
• Griffith had two strands (varieties), one pathogenic (disease-causing) and one non-pathogenic (harmless)
• He killed the pathogenic bacteria using heat and mixed them will the non-pathogenic bacteria and injected the mixture into different mice.
Frederick Griffith Experiment
• S-Strain= pathogenic
• R-Strain= non-pathogenic
• Transformation-a change in genotype and phenotype due to the assimilation of external DNA by a cell.
• What caused this transformation?
Oswald Avery
• Bacteriologist, who spent 14 years identifying the transforming substance. • Focused on 3 main candidates: DNA, RNA,
and proteins. • Avery broke open the heat-killed
pathogenic bacteria and extracted the components.
• Treated the samples with different agents to kill one type of molecule, then tested the samples ability to transform live nonpathogenic bacteria.
• Only when DNA was allowed to remain active did the bacteria transform.
• In 1944, Avery and his colleagues, Maclyn McCarty and Colin MacLeod announced their findings.
• Evidence that Viral DNA can Program Cells• Bacteriphages (phage)- viruses that infect bacteria. • Virus- is little more than DNA enclosed by a protective coat
(protein). • To produce more viruses, a virus must infect a cell and take
over the cell’s metabolic machinery. • Alfred Hershey and Martha Chase performed experiments
showing that DNA is the genetic material of a phage known as T2.
• T2 infects E. coli, turning it into a T2 factory that releases many copies when the cell reptured.
• But which viral component-protein or DNA- was responsible?
Hershey and Chase Experiment• They used radioactive sulfur and
phosphorous to trace the fates of protein and DNA.
• Results: When proteins were labeled (batch 1), radioactivity was found outside the cell.
• When DNA was labeled (batch 2), radioactivity was found inside the cell.
• Bacterial cells with radioactive phage DNA released new phages with some radioactive phosphourus.
• Conclusion: DNA functions as the genetic material of phage T2.
Building a Structural Model of DNA• Each DNA nucleotide monomer consists of a
nitrogenous base (T, A, C or G), the sugar deoxyribose, and a phosphate group.
• The phosphate group of one nucleotide is attached to the sugar of the next, forming a “backbone” of alternating phosphate and sugars from which the bases project.
• The polynicleotide strand has directionality, from the 5’ end (with the phosphate group) to the 3’ end (with the –OH group of the sugar)
• 5’ and 3’ refers to the numbers assigned to the carbons in the sugar ring.
Watson and Crick• Crick was studying the protein structure with a technique called X-ray crystallography.
• Watson saw an X-ray diffraction image of DNA produced by Rosalind Franklin, while visiting Maurice Wilkins lab.
• Watson was familiar with this type of an X-ray diffraction pattern that helical molecules produced, closer examination of the image confirmed that DNA was helical in shape.
• The photo implied that the helix was made of up two strands.
• Watson and Crick began making models of a double helix that conformed to the X-ray measurements.
Franklin & Wilkins – early 1950s
Franklin deduced that DNA was helical and consisted of 2 or 3 chains
James Watson and Francis Crick• In 1953, they showed the world an
elegant double-helix model for the structure of deoxyribonucleic acid. • The (hydrophobic) nitrogenous bases in
the molecule’s interior.• The negatively charged phosphate group
wouldn’t be forced into the interior. • The two sugar-phosphate backbones are
antiparallel
DNA structure
• Sugar-phosphate backbone with rungs of nitrogenous bases
• Strands are antiparallel
1 nucleotide
Nitrogenous base pairing
Nitrogenous Bases:-Purines – Adenine (A) and Guanine (G)-Pyrimidines – Cytosine (C), Thymine (T), Uracil (U)
A Model for DNA replication: the basic concept
Semi-Conservative Replication
A. New strands are composed of 1 strand of parental DNA and 1 strand of newly formed DNA
B. Free-floating nucleotides1. Can be DNA or RNA2. New complimentary DNA strands
are then synthesized by joining together deoxyribonucleotide triphosphates, one at a time, and with the removal of a di-phosphate.
Replication EnzymesEnzyme FunctionHelicase Unzips & unwinds DNA
Topoisomerase Relieves strain of unwound DNASSBs Help hold DNA open and stabilize it
Primase Builds RNA PrimerDNA Polymerase III Builds new DNA strandDNA Polymerase I Replaces RNA primer with DNA
DNA Ligase Joins Okazaki fragments together
https://www.youtube.com/watch?v=OnuspQG0Jd0
1. Helicase unzips DNA (breaks hydrogen bonds) creating a replication bubble at the origin of replication• Multiple origins per chromosome in eukaryotes• Each side of bubble has replication fork• Bubble enlarges as replication proceeds until bubbles meet
2. Primase builds a short primer (RNA chain) of RNA nucleotides (5 – 10 bases)
3. DNA Polymerase III builds the complimentary strand of DNA in the 5’ 3’ direction • Free-floating DNA nucleotides move in to match up with parent
strand, DNA polymerase III moves along and binds them together
4. DNA Polymerase I replaces RNA primer with DNA nucleotides
Leading and Lagging Strands
• New nucleotides must be added on to the 3’ end• Leading strand – Bases easily added as DNA is unzipped
Lagging Strand – has a delay• Section unzips, then strand is built back towards origin• Results in chunks called Okazaki fragments• DNA ligase bonds Okazaki fragments together after primer is replaced
https://www.youtube.com/watch?v=OnuspQG0Jd0
Speed and Accuracy
• ~4000 nucleotides per second• Mismatch repair – repair enzymes fix incorrectly placed
nucleotides• Nucleotide excision repair – enzymes called nucleases cut
out incorrect nucleotides and then gap is filled in with correct nucleotide
telomeres• Lose a small portion of the chromosome every time it is replicated
• Telomeres consist of highly repetitive sequences in order to protect coding genes
• Cancer cells (ex. HeLa) – telomerase is activated – prevents degradation of telomeres and renders cells “immortal”
Interesting article on HeLa cells:http://berkeleysciencereview.com/article/good-bad-hela/
DNA packaging
• Prokaryotes – one circular chromosome associated with very few proteins
• Eukaryotes – linear chromosomes associated with many proteins
• Histones – proteins that associate with DNA to help it coil
• DNA is negatively charged, histones are positively charged
• Chromatin – the packaged DNA and proteins• The more tightly coiled the DNA is, the less accessible it is to
transcription enzymes = coils control gene expression• Euchromatin – very extended and accessible to transcription
– form of most DNA during interphase• Heterochromatin – more condensed (like during mitosis)
and generally not transcribed (Barr bodies are also an example)