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Search for Genetic Search for Genetic MaterialMaterial
Looking for a molecule that could Looking for a molecule that could be specific and show great be specific and show great variationvariation
Molecule needs to be abundantMolecule needs to be abundant Needs to be able to be copied Needs to be able to be copied
preciselyprecisely What is your guess based on these What is your guess based on these
requirements?requirements?
Evidence of Genetic Evidence of Genetic MaterialMaterial
Griffith looking for vaccine against Griffith looking for vaccine against Streptococcus pneumoniaeStreptococcus pneumoniae
2 strains: S-smooth colonies; R-2 strains: S-smooth colonies; R-roughrough
S are encapsulated with S are encapsulated with polysaccharide coatpolysaccharide coat
Alternative phenotypes (S and R) Alternative phenotypes (S and R) are inheritedare inherited
Griffith ExperimentGriffith Experiment
Injected live S strain into mice: mice died of Injected live S strain into mice: mice died of pneumonia (S is pathogenic)pneumonia (S is pathogenic)
Injected live R strain into mice: mice healthy Injected live R strain into mice: mice healthy (R is nonpathogenic)(R is nonpathogenic)
Mice injected with heat killed S: mice Mice injected with heat killed S: mice healthyhealthy
Mice injected with heat killed S mixed w live Mice injected with heat killed S mixed w live R cells: mice diedR cells: mice died
Blood samples from dead mice contained Blood samples from dead mice contained live S cells: R cell acquired from dead S cells live S cells: R cell acquired from dead S cells ability to make coats TRANSFORMATIONability to make coats TRANSFORMATION
ImplicationsImplications
Transformation: assimilation of Transformation: assimilation of external genetic material by a cellexternal genetic material by a cell
Not a protein-heat denatures Not a protein-heat denatures proteins but heat did not destroy proteins but heat did not destroy the transforming ability of the the transforming ability of the genetic material in the heat killed S genetic material in the heat killed S cellscells
Later Avery, McCarty, and MacLeod Later Avery, McCarty, and MacLeod discovered transforming agent was discovered transforming agent was DNADNA
Outer layer doesn’t stain-pathenogenicOuter layer doesn’t stain-pathenogenicno outer coat-coat does stain-no outer coat-coat does stain-
nonpathenogenicnonpathenogenic
Gram negative Gram positive
Evidence of Viral DNAEvidence of Viral DNA Bacteriophage (phage): virus that Bacteriophage (phage): virus that
infects bacteriainfects bacteria Alfred Hershey & Martha Chase Alfred Hershey & Martha Chase
DNA genetic material of phage T2DNA genetic material of phage T2 Virus was DNA and a protein coatVirus was DNA and a protein coat Protein tagging: T2 and E. coli Protein tagging: T2 and E. coli
were grown were grown DNA tagging: T2 and E. coli were DNA tagging: T2 and E. coli were
grown in media w 32 Pgrown in media w 32 P
Hershey and ChaseHershey and Chase
Protein labeled infected E. coliProtein labeled infected E. coli DNA labeled infected separate E. coliDNA labeled infected separate E. coli Mixtures were agitated to break loose Mixtures were agitated to break loose
phage coats from bacteriaphage coats from bacteria Mixtures were centrifuged; cells in the Mixtures were centrifuged; cells in the
pellet; viruses in the supernatantpellet; viruses in the supernatant S labeled in supernatantS labeled in supernatant P labeled in the pelletP labeled in the pellet Bacteria P labeled released viruses w PBacteria P labeled released viruses w P
Conclusions Hershey & Conclusions Hershey & ChaseChase
Viral proteins remain outside the Viral proteins remain outside the host cellhost cell
Viral DNA injected into host cellViral DNA injected into host cell Injected DNA molecules cause cells Injected DNA molecules cause cells
to produce additional viruses w to produce additional viruses w more viral DNA and proteinsmore viral DNA and proteins
Nuclei acids not proteins are Nuclei acids not proteins are hereditary materialhereditary material
Chargaff’s ExperimentChargaff’s Experiment Analyzed DNA of different organismsAnalyzed DNA of different organisms DNA composition is species specific: DNA composition is species specific:
amount and ratios of nitrogenous amount and ratios of nitrogenous bases vary from one species to bases vary from one species to anotheranother
Adenine residues equaled number of Adenine residues equaled number of thymines; cytosines equaled number thymines; cytosines equaled number of guaninesof guanines
Chargaff’s rules A=T; G=CChargaff’s rules A=T; G=C This molecular diversity supports This molecular diversity supports
DNA as hereditary materialDNA as hereditary material
Circumstantial Evidence for Circumstantial Evidence for DNADNA
Eukaryotic cell doubles DNA Eukaryotic cell doubles DNA content prior to mitosiscontent prior to mitosis
During mitosis, the doubled DNA is During mitosis, the doubled DNA is equally divided btwn 2 daughter equally divided btwn 2 daughter cellscells
Organism’s diploid cells have 2x Organism’s diploid cells have 2x DNA as haploid gametesDNA as haploid gametes
Watson, Crick, & FranklinWatson, Crick, & Franklin
Working on 3D structureWorking on 3D structure Wilkins fed Watson and Crick Wilkins fed Watson and Crick
Franklin’s X ray of DNA crystalFranklin’s X ray of DNA crystal Watson and Crick deduced:Watson and Crick deduced: Helix w uniform width of 2 nmHelix w uniform width of 2 nm Purine and pyrimidine bases Purine and pyrimidine bases
stacked .34 nm apartstacked .34 nm apart Helix makes 1 full turn 3.4 nmHelix makes 1 full turn 3.4 nm There are 10 layers of bases in ea There are 10 layers of bases in ea
turnturn
DNA StructureDNA Structure
Tried sugar phosphate chains on Tried sugar phosphate chains on inside no goinside no go
On outside, hydrophobic On outside, hydrophobic interactions of nitrogenous bases interactions of nitrogenous bases pushed them to insidepushed them to inside
Ladder twisted into a spiralLadder twisted into a spiral 2 sugar phosphate backbones of 2 sugar phosphate backbones of
the helix are antiparallel; they run the helix are antiparallel; they run in opposite directionsin opposite directions
DNA rungsDNA rungs Pair of nitrogenous basesPair of nitrogenous bases Purine must pair w pyrimidines to Purine must pair w pyrimidines to
get .34 nmget .34 nm W Chargaff, A purine + T pyrimidineW Chargaff, A purine + T pyrimidine G purine + C pyrimidineG purine + C pyrimidine Suggests mechanisms for DNA Suggests mechanisms for DNA
replicationreplication Sequences of bases highly variable Sequences of bases highly variable
allowing specificity for genetic codingallowing specificity for genetic coding Hydrogen bonds and van der waals Hydrogen bonds and van der waals
stabilize DNAstabilize DNA
DNA ReplicationDNA Replication Watson & Crick proposed genes on Watson & Crick proposed genes on
original DNA strand are copied by original DNA strand are copied by specific pairing of complementary specific pairing of complementary bases, creating a complementary bases, creating a complementary strandstrand
Complementary strand can funtion as Complementary strand can funtion as template to produce a copy of original template to produce a copy of original strandstrand
2 strands separate each acts as 2 strands separate each acts as template for complementary strandtemplate for complementary strand
Enzymes link nucleotides together at Enzymes link nucleotides together at sugar-phosphate groupssugar-phosphate groups
Meselson and StahlMeselson and Stahl
3 hypotheses3 hypotheses Conservative: parental double helix remain Conservative: parental double helix remain
intact and second DNA molecule entirely intact and second DNA molecule entirely new moleculenew molecule
Semiconservative: each DNA molecules Semiconservative: each DNA molecules should be composed of one original & one should be composed of one original & one new strandnew strand
Dispersive: both strands of newly produced Dispersive: both strands of newly produced DNA molecules should contain mix of old DNA molecules should contain mix of old and new DNAand new DNA
Meselson & Stahl Meselson & Stahl ExperimentExperiment
Grew E coli on medium w 15N (heavy Grew E coli on medium w 15N (heavy nitrogen)nitrogen)
Transferred to medium w 14NTransferred to medium w 14N 11stst generation DNA extracted from E coli after generation DNA extracted from E coli after
on generation of growth in light mediumon generation of growth in light medium 22ndnd generation DNA extracted from E coli after generation DNA extracted from E coli after
2 replications in light medium2 replications in light medium Isolated DNA was mixed w CsCl & centrifugedIsolated DNA was mixed w CsCl & centrifuged Centrifugal force created CsCl gradient w Centrifugal force created CsCl gradient w
>conc at bottom; DNA moved to place >conc at bottom; DNA moved to place density matched density of CsCldensity matched density of CsCl
Results Meselson & StahlResults Meselson & Stahl
Parents: 1 distinct band / tubeParents: 1 distinct band / tube 11stst generation 1 distinct band near center generation 1 distinct band near center 22ndnd generation 2 bands one near center generation 2 bands one near center
other lightother light
Conclusions: Meselson & Conclusions: Meselson & StahlStahl
11stst generation all hybrid: generation all hybrid: semiconservative modelsemiconservative model
11stst generation eliminated conservative, generation eliminated conservative, but not dispersivebut not dispersive
22ndnd generation eliminated dispersive; generation eliminated dispersive; only one band would have occurred if only one band would have occurred if dispersive replicationdispersive replication
DNA ReplicationDNA Replication
Helical molecule must untwist (helicase) Helical molecule must untwist (helicase) while it copies its two antiparallel strands while it copies its two antiparallel strands simultaneouslysimultaneously
Requires 2 dozen enzymes and other Requires 2 dozen enzymes and other proteinsproteins
Prokaryotes: 500 nucleotides/secProkaryotes: 500 nucleotides/sec Few hours to copy 6 billion bases of single Few hours to copy 6 billion bases of single
human cellhuman cell Accurate: 1 in a billion nucleotides is Accurate: 1 in a billion nucleotides is
incorrectly pairedincorrectly paired
Origins of ReplicationOrigins of Replication DNA replication begins at sites called DNA replication begins at sites called
origins of replication that have a specific origins of replication that have a specific sequence of nucleotides sequence of nucleotides
Specific proteins required to initiate Specific proteins required to initiate replication bind to originreplication bind to origin
DNA double helix opens at origin and DNA double helix opens at origin and replication forks spread in both directions replication forks spread in both directions away from point form replication bubbleaway from point form replication bubble
Prokaryotes one origin; eukaryotes Prokaryotes one origin; eukaryotes thousandsthousands
Elongating a new strandElongating a new strand Helicases are enzymes which catalyze Helicases are enzymes which catalyze
unwinding of parental double helixunwinding of parental double helix Single strand binding proteins keep Single strand binding proteins keep
strands apart and stabilize the unwound strands apart and stabilize the unwound DNA until new strand can be synthesizedDNA until new strand can be synthesized
DNA polymerases catalyze synthesis of a DNA polymerases catalyze synthesis of a new DNA strandnew DNA strand
New nucleotides align on template of oldNew nucleotides align on template of old DNA polymerase links nucleotides to DNA polymerase links nucleotides to
growing strand; only grow from 5’ to 3’ growing strand; only grow from 5’ to 3’ only add to 3’only add to 3’
Replication is endergonicReplication is endergonic Requires energyRequires energy Nucleoside triphosphate is sourceNucleoside triphosphate is source Covalently linked to 5’ carbon of pentoseCovalently linked to 5’ carbon of pentose Nucleoside triphosphate lose 2 Nucleoside triphosphate lose 2
phosphates form covalent linkages to the phosphates form covalent linkages to the growing chaingrowing chain
Hydrolysis of phosphate bond drives Hydrolysis of phosphate bond drives synthesis of DNAsynthesis of DNA
AntiparallelAntiparallel Continuous synthesis of both DNA strands Continuous synthesis of both DNA strands
is not possible due to the antiparallel is not possible due to the antiparallel constructionconstruction
Can only elongate from 5’ to 3’ Can only elongate from 5’ to 3’ Continuous synthesis occurs on the leading Continuous synthesis occurs on the leading
strand which is 5’ to 3’strand which is 5’ to 3’ The lagging strand (complementary strand) The lagging strand (complementary strand)
has discontinuous synthesishas discontinuous synthesis Lagging strand produced as a number of Lagging strand produced as a number of
short segments called Okazaki fragmentsshort segments called Okazaki fragments
Okazaki FragmentsOkazaki Fragments
Synthesized in 5’ to 3’ directionSynthesized in 5’ to 3’ direction Fragments are 1000-2000 nucleotides in Fragments are 1000-2000 nucleotides in
length in bacteria and 100-200 length in bacteria and 100-200 nucleotides long in eukaryotesnucleotides long in eukaryotes
Fragments are ligated by DNA ligase, Fragments are ligated by DNA ligase, linking enzyme that catalyzes formation linking enzyme that catalyzes formation of a covalent bond between the 3’ end of of a covalent bond between the 3’ end of each new fragment and the 5’ end of the each new fragment and the 5’ end of the growing chaingrowing chain
primerprimer Primer is a short RNA segment that is Primer is a short RNA segment that is
complementary to DNA segment & that is complementary to DNA segment & that is necessary to begin DNA replicationnecessary to begin DNA replication
Primers are polymerized by an enzyme called Primers are polymerized by an enzyme called primaseprimase
Portion of parental DNA serves as template for Portion of parental DNA serves as template for primer w a base sequence that is about 10 primer w a base sequence that is about 10 nucleotides long in eukaryotesnucleotides long in eukaryotes
Primer formation must precede DNA replication, Primer formation must precede DNA replication, DNA polymerase only add nucleotides to a DNA polymerase only add nucleotides to a polynucleotide that is already correctly base-polynucleotide that is already correctly base-paired w complementary strandpaired w complementary strand
primersprimers
Only one is needed for leading strandOnly one is needed for leading strand Thousands are needed for lagging strandThousands are needed for lagging strand RNA primer must initiate the synthesis of RNA primer must initiate the synthesis of
each Okazaki fragmenteach Okazaki fragment Fragments are ligated in 2 steps to produce a Fragments are ligated in 2 steps to produce a
continuous DNA strandcontinuous DNA strand DNA polymerase removes the RNA primer DNA polymerase removes the RNA primer
and replaces it w DNA; DNA ligase catalyzes and replaces it w DNA; DNA ligase catalyzes linkagelinkage
Between 3’ end of each fragment & 5’ of Between 3’ end of each fragment & 5’ of chainchain
Enzymes repair damageEnzymes repair damage Initial pairing errors occur at a frequency of 1 Initial pairing errors occur at a frequency of 1
in 10Kin 10K DNA can be repaired as it is being synthesized: DNA can be repaired as it is being synthesized:
mismatch repair DNA polymerase proofreads mismatch repair DNA polymerase proofreads each newly added nucleotide against its each newly added nucleotide against its template; if incorrect removes and replaces it template; if incorrect removes and replaces it (eukaryotes have proteins too to proofread)(eukaryotes have proteins too to proofread)
Excision repair: accidental changes in DNA can Excision repair: accidental changes in DNA can result from exposure; 50 different DNA repair result from exposure; 50 different DNA repair enzymes; one excises and gap filled by base-enzymes; one excises and gap filled by base-pairing by DNA polymerase and DNA ligasepairing by DNA polymerase and DNA ligase
Repair SignificanceRepair Significance The importance of proper function of repair The importance of proper function of repair
enzymes is clear from the inherited enzymes is clear from the inherited disorder xeroderma pigmentosum.disorder xeroderma pigmentosum.– These individuals are hypersensitive to sunlight.These individuals are hypersensitive to sunlight.– In particular, ultraviolet light can produce In particular, ultraviolet light can produce
thymine dimers between adjacent thymine thymine dimers between adjacent thymine nucleotides.nucleotides.
– This buckles the DNA double helix and This buckles the DNA double helix and interferes with DNA replication.interferes with DNA replication.
– In individuals with this disorder, mutations in In individuals with this disorder, mutations in their skin cells are left uncorrected and cause their skin cells are left uncorrected and cause skin cancer.skin cancer.
Telomere replicationTelomere replication
Limitations in the DNA polymerase Limitations in the DNA polymerase create problems for the linear DNA create problems for the linear DNA of eukaryotic chromosomes.of eukaryotic chromosomes.
The usual replication machinery The usual replication machinery provides no way to complete the 5’ provides no way to complete the 5’ ends of daughter DNA strands.ends of daughter DNA strands.– Repeated rounds of replication produce Repeated rounds of replication produce
shorter and shorter DNA moleculesshorter and shorter DNA molecules
The ends of eukaryotic chromosomal The ends of eukaryotic chromosomal DNA molecules, the DNA molecules, the telomerestelomeres, have , have special nucleotide sequences.special nucleotide sequences.– In human telomeres, this sequence is In human telomeres, this sequence is
typically TTAGGG, repeated between 100 typically TTAGGG, repeated between 100 and 1,000 times.and 1,000 times.
Telomeres protect genes from being Telomeres protect genes from being eroded through multiple rounds of DNA eroded through multiple rounds of DNA replication.replication.
Eukaryotic cells have evolved a Eukaryotic cells have evolved a mechanism to restore shortened mechanism to restore shortened telomeres.telomeres.
TelomeraseTelomerase uses a short molecule of uses a short molecule of RNA as a template to extend the 3’ RNA as a template to extend the 3’ end of the telomere.end of the telomere.– There is now room for There is now room for
primase and DNA primase and DNA polymerase to extend polymerase to extend the 5’ end.the 5’ end.
– It does not repair the It does not repair the 3’-end “overhang,”3’-end “overhang,”but it does lengthenbut it does lengthenthe telomere.the telomere.
TelomeraseTelomerase Telomerase is not present in most cells of Telomerase is not present in most cells of
multicellular organisms.multicellular organisms. Therefore, the DNA of dividing somatic cells and Therefore, the DNA of dividing somatic cells and
cultured cells does tend to become shorter.cultured cells does tend to become shorter. Thus, telomere length may be a limiting factor in Thus, telomere length may be a limiting factor in
the life span of certain tissues and the organism.the life span of certain tissues and the organism. Telomerase is present in germ-line cells, Telomerase is present in germ-line cells,
ensuring that zygotes have long telomeres.ensuring that zygotes have long telomeres. Active telomerase is also found in cancerous Active telomerase is also found in cancerous
somatic cells.somatic cells.– This overcomes the progressive shortening This overcomes the progressive shortening
that would eventually lead to self-destruction that would eventually lead to self-destruction of the cancer.of the cancer.