3
Termsandconcepts• gene– Fundamentalunitofheredity– DNAsegmentwhichcodesforproteinorRNA
• clone– populationofcellsthataregeneticallyidentical
• genome– allgenespresentinacellorvirus• haploid–onesetofgenes(eg.,bacteria)• diploid–twosetsofgenes(eg.,humans)
• genotype– specificsetofgenesanorganismpossesses
• phenotype– setofobservablecharacteristics
4
The sum total of genetic material of a cell is referred to as thegenome.
Fig. 9.2 The general location and forms of the genome
6
TheOrganizationofDNAinCells• Chromosomes–neatlypackagedDNAmolecule.
• organizationdiffersinprokaryoticandeukaryoticcelltypes
7
Chromosome
• Prokaryotic– Histonelike proteins condense DNA
• Eukaryotic– Histone proteins condense DNA
• Subdivided into basic “informationalpackets” called genes
8
Plasmids• usuallysmall,closedcircularDNAmolecules• existandreplicateindependentlyofchromosome• notrequiredforgrowthandreproduction• maycarrygenesthatconferselectiveadvantage(e.g.,drugresistance)
9
Genes
• Three categories– Structural– Regulatory– Encode for functional/regulatory RNAs
• Genotype– Specific set of genes an organism possesses
• Phenotype– Set of observable characteristics
10
Flow of Genetics
• NA replication (DNA => DNA; RNA => RNA)
• Gene Expression (DNA =>RNA=>Protein)– Replication– Transcription– Translation– Post-translational modification
12
Representation of the flow of genetic information.
Fig. 9.9 Summary of the flow of genetic information in cell.
13
DNA
• Structure - √
• Replication - today
Fig. 9.3 An Escherichia coli cell disrupted to release its DNA molecule.
14
Structure
• Nucleotide– Phosphate– Deoxyribose sugar– Nitrogenous base
• Double stranded helix– Antiparallel arrangement
15
DNAasGeneticMaterial• establishedbyseveralcriticalexperiments– FredGriffith(1928)– OswaldT.Avery,C.M.MacLeod,andM.J.McCarty(1944)– AlfredD.HersheyandMarthaChase(1952)
18
"fortheirdiscoveriesconcerningthemolecularstructureofnucleicacidsanditssignificanceforinformationtransferinlivingmaterial"
MauriceHughFrederickWilkins
JamesDeweyWatson
FrancisHarryComptonCrick
Structure of DNA
X-ray analysis of DNA structure
Rosalind Franklin
19
DNAStructure• nitrogenousbases– A,T,G,C
• pentosesugar– deoxyribose
• chainofnucleotideslinkedbyphosphodiesterbonds
• usuallyadoublehelix,composedoftwocomplementarystrands– basepairingrules• AwithT• GwithC
24
RNA Structure
• nitrogenous bases– A, G, C, U (instead of T)
• pentose sugar– ribose
• usually consists of single strand of nucleotides linkedby phosphodiester bonds– can coil back on itself
• forms hairpin-shaped structures with complementarybase pairing and helical organization
• base pairing rules– A with U– G with C
25
Prokaryotic chromosome
• usually exists asclosed circular,supercoiledmoleculeassociated withbasic proteins
relaxed circle
supercoiled DNA
26
EukaryoticDNA• linearmolecules• associatedwithhistones• coiledintorepeatingunitscallednucleosomes
27
EukaryoticChromosomes
A chromosome is formed from asingle DNA molecule thatcontains many genes.
A chromosomal DNA moleculecontains three specificnucleotide sequences which arerequired for replication: a DNAreplication origin;a centromere to attach the DNAto the mitotic spindle.;a telomere located at each endof the linear chromosome.
28
Purines and pyrimidines pair (A-T or G-C) and the sugars(backbone) are linked by a phosphate.
Fig. 9.4 Three views of DNA structure
29
Replication
• Nature: Semiconservative• Involved Enzymes (in Primosome & Replisome)
• Leading strand• Lagging strand
– Okazaki fragments
Plus (+)strandof DNA
5' 3'
Minus (–)strandof DNA
3' 5'
T A C G A C T A A T A G G C G C A T C C A C G A T C
A T G C T G A T T A T C C G C G T A G G T G C T A G
A T G C T G A T T A T C C G C G T A G G T G C T A G
A U G C U G A U U A U C C G C G U A G G U G C U A G
Plus (+)strandof DNA
Minus (–)strandof DNA
5' 3'
3' 5'
5' 3' mRNA
T A C G A C T A A T A G G C G C A T C C A C G A T C
34
Semiconservative replication of DNA synthesizes a new strand of DNA from atemplate strand.
Fig. 9.5 Simplified steps to show the semiconservative replication of DNA
36
The function of important enzymes involved in DNA replication.
Table 9.1 Some enzymes involved in DNA replication
37
Leading strand
• DnaA,DnaB proteins at Origin or Replication(circular genetic element) or
• RNA primer (linear genetic element) initiate the 5’ to3’ synthesis of DNA in a continuous manner
38
Lagging strand
• Because the direction of synthesis (5’ -> 3’) isopposite to fork movement, the lagging strand issynthesized in form of multiple DNA (Okazaki)fragments
• Primer synthesis performed by RNA polymerase (Primase),• DNA synthesis performed by DNA polymerase III• RNA primer removal and fill-in with DNA by DNA polymerase I
• Okazaki fragments are ligated together by DNAligase to form one continuous strand
39
The steps associated with the DNA replication process.
Fig. 9.6 Thebacterialreplicon: amodel for DNASynthesis
40
PatternsofDNAsynthesis…• inprocaryotes– bidirectionalfromasingleoriginofreplication– replicon• portionofthegenomethatcontainsanoriginandisreplicatedasaunit
2 forks movein oppositedirections
41
MechanismofDNAReplication in bacteria
unwind strands and relievetension caused by unwinding
keep strands separated
synthesizedby primase
DNA polymerase III synthesizes DNA
Quinolone Target
42
Someamazingfacts• ≥30proteinsrequiredtoreplicateE.colichromosome• occurswithgreatfidelity– errorfrequency=10-9or10-10perbasepairreplicated– duetoproofreadingactivityofDNApolymerasesIIIandI
• occursveryrapidly– 750to1,000basepairs/secondinprocaryotes– 50-100basepairs/secondineucaryotes
5'3'
Synthesis of the leadingstrand proceeds continuously.
3'5'
Helicase separatesthe double-strandedmolecule
5'3'
Synthesis of the leadingstrand proceeds continuously.
Synthesis of the lagging strand isdiscontinuous; synthesis is reinitiatedperiodically, generating a series offragments that are later joined.
1. Primase synthesizes an RNA primer
3'5'
Helicase separatesthe double-strandedmolecule
RNA primer
5'3'
Synthesis of the leadingstrand proceeds continuously.
Synthesis of the lagging strand isdiscontinuous; synthesis is reinitiatedperiodically, generating a series offragments that are later joined.
1. Primase synthesizes an RNA primer
3'5'
Helicase separatesthe double-strandedmolecule
2. DNA polymerase adds nucleotides onto the 3' end of the fragment
RNA primer
5'3'
3'5'
Synthesis of the leadingstrand proceeds continuously.
Synthesis of the lagging strand isdiscontinuous; synthesis is reinitiatedperiodically, generating a series offragments that are later joined.
1. Primase synthesizes an RNA primer
3. DNA polymerase replaces the RNA primer with deoxynucleotides
Helicase separatesthe double-strandedmolecule
2. DNA polymerase adds nucleotides onto the 3' end of the fragment
RNA primer
5'3'
3'5'
Synthesis of the leadingstrand proceeds continuously.
Synthesis of the lagging strand isdiscontinuous; synthesis is reinitiatedperiodically, generating a series offragments that are later joined.
1. Primase synthesizes an RNA primer
3. DNA polymerase replaces the RNA primer with deoxynucleotides
3'5'
Helicase separatesthe double-strandedmolecule
2. DNA polymerase adds nucleotides onto the 3' end of the fragment
RNA primer
4. DNA ligase seals the gaps between adjacent fragments
49
Replication processes from other biological systems (some Gram-positive bacterial genomes, plasmids, viruses) involve a rolling cycle.
Fig. 9.8 Simplified model of rolling circle DNA Replication
5’3’
50
RNA
• Transcription– Message RNA (mRNA)– Transfer RNA (tRNA)– Ribosomal RNA (rRNA)
• Codons (nucleotide triplett)
51
(a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.*
*For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA.
3′DNA SampleDNA SampleCycle 1
Heat to 94°C3′5′
5′
Polymerase Chain Reaction
52
(a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.*
*For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA.
3′DNA SampleDNA SampleCycle 1
Heat to 94°CDenaturationDenaturation
Cycle1
Strandsseparate
3′
3′
3′ 5′
5′5′
5′
53
(a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.*
*For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA.
3′DNA SampleDNA SampleCycle 1
Heat to 94°C
50° to 65°C
DenaturationDenaturation
Oligonucleotideprimers attach atends of strands topromote replicationof amplicons
PrimerPrimer Cycle1
Strandsseparate
Amplicons
PrimingPriming
3′
3′
3′
3′
3′
3′ 5′
5′
5′5′
5′
5′
5′3′ 5′
54
(a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.*
*For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA.
3′DNA SampleDNA SampleCycle 1
Heat to 94°C
50° to 65°C
DenaturationDenaturation
Oligonucleotideprimers attach atends of strands topromote replicationof amplicons
PrimerPrimer
ExtensionExtensionDNA polymerasesynthesizescomplementary strand
Cycle1
Strandsseparate
72°C
Amplicons
PrimingPriming
3′
3′
3′
3′3′
3′
3′
3′
3′
3′
5′
5′
5′
5′5′
5′
5′
5′
5′3′ 5′
5′
Polymerase5′
55
(a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.*
*For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA.
3′DNA SampleDNA SampleCycle 1
DenaturationDenaturation New strandOriginalstrands
Heatto
94°C
Cycle2
New strand
Cycle 22 copies
Heat to 94°C
50° to 65°C
DenaturationDenaturation
Oligonucleotideprimers attach atends of strands topromote replicationof amplicons
PrimerPrimer
ExtensionExtensionDNA polymerasesynthesizescomplementary strand
Cycle1
Strandsseparate
72°C
Amplicons
PrimingPriming
3′
3′
3′
3′3′
3′
3′
3′
3′
3′
5′
5′
5′
5′5′
5′
5′
5′
5′3′ 5′
5′
Polymerase5′
56
(a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.*
*For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA.
3′DNA SampleDNA SampleCycle 1
DenaturationDenaturation
PrimingPriming
New strandOriginalstrands
Heatto
94°C
Cycle2
New strand
Cycle 22 copies
72°C
Heat to 94°C
50° to 65°C
DenaturationDenaturation
Oligonucleotideprimers attach atends of strands topromote replicationof amplicons
PrimerPrimer
ExtensionExtensionDNA polymerasesynthesizescomplementary strand
Cycle1
Strandsseparate
72°C
Amplicons
PrimingPriming
3′
3′
3′
3′3′
3′
3′
3′
3′
3′
5′
5′
5′
5′5′
5′
5′
5′
5′3′ 5′
5′
Polymerase5′
57
(a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.*
*For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA.
3′DNA SampleDNA SampleCycle 1
DenaturationDenaturation
PrimingPriming
New strandOriginalstrands
Heatto
94°C
Cycle2
New strand
ExtensionExtension
Cycle 22 copies
4 copies
72°C
Heat to 94°C
50° to 65°C
DenaturationDenaturation
Oligonucleotideprimers attach atends of strands topromote replicationof amplicons
PrimerPrimer
ExtensionExtensionDNA polymerasesynthesizescomplementary strand
Cycle1
Strandsseparate
72°C
Amplicons
PrimingPriming
3′
3′
3′
3′3′
3′
3′
3′
3′
3′
5′
5′
5′
5′5′
5′
5′
5′
5′3′ 5′
5′
Polymerase5′
58
(a) In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis.*
(b) A view of the process after 6 cycles, with 64 copies of amplified DNA. Continuing this process for 20 to 40 cycles can produce millions of identical DNA molecules.
*For simplicity's sake, we have omitted the elongation of the complete original parent strand during the first cycles. Ultimately, templates that correspond only to the smaller fragments dominate and become the primary population of replicated DNA.
3′DNA SampleDNA SampleCycle 1
DenaturationDenaturation
PrimingPriming
New strandOriginalstrands
Heatto
94°C
Cycle2
Cycles 3, 4, . . . repeat same steps
New strand
ExtensionExtension
Cycle 22 copies
4 copies
72°C
Heat to 94°C
50° to 65°C
DenaturationDenaturation
Oligonucleotideprimers attach atends of strands topromote replicationof amplicons
PrimerPrimer
ExtensionExtensionDNA polymerasesynthesizescomplementary strand
Cycle1
Strandsseparate
72°C
Amplicons
PrimingPriming
1* fragment2 copies
4 copies8 copies
16 copies32 copies64 copies
3′
3′
3′
3′3′
3′
3′
3′
3′
3′
5′
5′
5′
5′5′
5′
5′
5′
5′3′ 5′
5′
Polymerase5′