The Genetic Material
The Genetic MaterialLecture 1Brooker Chapters 3, 9 & 10BIO 184Dr. Tom PeavyHOW DO WE KNOWDNAIS THEGENETIC MATERIAL?1
What are the requirements of“Genetic Material”?Evidence that Genes Reside within Chromosomes 1667- Anton van Leeuwenhoek (microscopy)– Hypothesis: spermatozoa (“sperm animals”) enter theegg to achieve fertilization– Homunculus (spermists vs ovists)2
Late 1800’s – microscopy studies– egg and sperm nuclei unite and contribute equally(e.g. frogs, sea urchins)– dyes used to stain the nucleus andobserved long, threadlike bodies Chromosomes (“colored bodies)– Mitosis described(nucleus is equallypartitioned into daughter cells)– Sex Determination( and chromosomes) Homologous Chromosomes: The pair ofchromosomes in a diploid individual that have thesame overall genetic content.– One member of each homologous pair ofchromosomes is inherited from each parent.3
Chromosome theory of Inheritance(Sutton and Boveri 1902) Chromosomes are in pairs and genes, or theiralleles, are located on chromosomes Homologous chromosomes separate duringmeiosis so that alleles are segregated Meiotic products have one of each homologouschromosome but not both Fertilization restores the pairs of chromosomesChromosomes Approximately 40% DNA and 60% protein4
Evidence for DNA as GeneticMaterial Used simple experimental organisms tostudy question– Bacteria with single circular chromosomewithout a nucleus (prokaryotes)– Bacteriophage (“bacteria eaters”)Frederick Griffith Experiments In 1928, Griffith studied the bacterium Streptococcuspneumoniae S. pneumoniae comes in two strains– S Smooth (strain IIIS) Secretes a polysaccharide capsule (evades immunesystem) Produce smooth colonies on solid media– R Rough (strain IIR) Unable to secrete a capsule Produce colonies with a rough appearance5
Figure 9.2dead smoothlive roughdead smoothlive smooth6
The Experiments of Avery, MacLeod &McCarty realized that Griffith’s observations could be usedto identify the genetic material or “transformingprinciple” In essence, the formation of the capsule is guidedby the bacteria’s genetic material– Transformed bacteria acquired information to make thecapsule– Variation exists in ability to make capsule– The information required to create a capsule isreplicated and transmitted from mother to daughtercellsThe Experiments of Avery, MacLeod &McCarty At the time of their experimentation in the 1940s, it was knownthat DNA, RNA, proteins and carbohydrates are the majorconstituents of living cells Prepared cell extracts from smooth cells (IIIS) and added torough cells (IIR) for transformation in culture medium Only the DNA enriched extract was able to convert rough cellsinto smooth cells7
Figure 9.3Method Allow sufficient time for theDNA extract to be taken up bythe rough cells Add antibody that aggregatesrough bacteria (not transformed) Gentle centrifugation (removesaggregated rough bacteria) Plate remaining cells insupernatant and incubateovernight (i.e. smooth cells willgrow, if any)8
Still.More Verification was NeededHershey and Chase Experiment (1952) Studied thebacteriophage T2– It is relatively simplesince its composed ofonly twomacromoleculesInside thecapsid DNA and proteinMade upof proteinFigure 9.49
Figure 9.5Life cycle of theT2 bacteriophageFigure 9.5Life cycle of theT2 bacteriophage10
Hypothesis– Only the genetic material of the phage isinjected into the bacterium Isotope labeling will reveal if it is DNA or proteinMethod– Used radioisotopes to distinguish DNA from proteins 32P labels DNA specifically 35S labels protein specifically– The two different Radioactively-labeled phages wereused to infect non-radioactive Escherichia coli cellsseparately– After allowing sufficient time for infection to proceed,the residual phage particles were sheared off the cells Phage ghosts and E. coli cells were separated– Radioactivity was monitored using a scintillationcounter11
Total isotope in supernatant (%)RESULTS100Extracellular 35S8080%Blending removes 80%of 35S from E. coli cells.6040Extracellular 32P35%Most of the 32P (65%)remains with intactE. coli cells.200012345678Agitation time in blender (min)Data from A. D. Hershey and Martha Chase (1952) Independent Functions of Viral Protein and Nucleic Acid in Growth ofBacteriophage. Journal of General Physiology 36, 39–56.RNA can alsoserve as thegeneticmaterial inmany viruses12
THE STRUCTURE OFDNA AND RNANucleotides The nucleotide is the repeating structural unit ofDNA and RNA It has three components– A phosphate group– A pentose sugar– A nitrogenous base13
Repeating unit comprised of: phoshate group pentose sugar nitrogenous base Nucleotides are covalently linked together byphosphodiester bonds– A phosphate connects the 5’ carbon of one nucleotide tothe 3’ carbon of another Therefore the strand has directionality– 5’ to 3’ The phosphates and sugar molecules form thebackbone of the nucleic acid strand– The bases project from the backbone14
removed duringpolymerizationA, G, C or TOO PA, G, C or OHDeoxyribose(a) Repeating unit ofdeoxyribonucleicacid ��OHOHRibose(b) Repeating unit ofribonucleic acid (RNA)15
Discovery of the Structure of DNA In 1953, James Watson and Francis Crickdiscovered the double helical structure of DNA The scientific framework for their breakthrough wasprovided primarily by:– Rosalind Franklin (X-ray diffraction)– Erwin Chargaff (chemical composition)Rosalind Franklin She used X-raydiffraction to study wetfibers of DNA The diffraction patternshe obtained suggestedseveral structuralfeatures of DNA– Helical– More than one strand– 10 base pairs percomplete turnThe diffraction pattern is interpreted(using mathematical theory)This can ultimately provideinformation concerning thestructure of the molecule16
Erwin Chargaff’s Experiment Chargaff pioneered many of the biochemicaltechniques for the isolation, purification andmeasurement of nucleic acids from living cells It was already known then that DNA contained thefour bases: A, G, C and T Chargaff analyzed the the base composition ofDNA in different species to see if there was apattern17
Chargaff’s rulePercent of adenine percent of thymine (A T)Percent of cytosine percent of guanine (C G)A G T C(or purines pyrimidines)Modeling the structure of DNAbased on data observations(Watson, Crick and Maurice Wilkinswere awarded the Nobel Prize in1962 for their discoveries) Barrington Brown/Photo Researchers(a) Watson and Crick Hulton Archive by Getty Images(b) Original model of the DNA double helix18
The DNA Double Helix General structural features– Two strands are twisted together around acommon axis– There are 10 bases per complete twist– The two strands are antiparallel One runs in the 5’ to 3’ direction and the other 3’ to 5’– The helix is primarily right-handed in the B form As it spirals away from you, the helix turns in aclockwise directionThe DNA Double Helix General structural features– The double-bonded structure is stabilized by 1. Hydrogen bonding between complementary bases– A bonded to T by two hydrogen bonds– C bonded to G by three hydrogen bonds 2. Base stacking– Within the DNA, the bases are oriented so that the flattenedregions are facing each other19
20
The DNA Double Helix General structural features– There are two asymmetrical grooves on theoutside of the helix 1. Major groove 2. Minor groove Certain proteins can bind within these grooves– They can thus interact with a particular sequence of basesCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or ve Laguna Design/Photo Researchers(a) Ball-and-stick model of DNAFigure 9.17(b) Space-filling model of DNA9-4821
DNA Can Form Alternative Types ofDouble Helices The DNA double helix can form differenttypes of secondary structure However, under certain in vitro conditions,A-DNA and Z-DNA double helices can formA-DNA The predominant form found in living cells isB-DNARight-handed helix11 bp per turnOccurs under conditions of low humidityLittle evidence to suggest that it is biologically importantZ-DNA Left-handed helix12 bp per turnIts formation is favored by Alternating purine/pyrimidine sequences, at high saltconcentrations (e.g. GCGCGCGCGC)Evidence from yeast suggests that it may play a role intranscription and recombinationCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display9-5022
GA- DNAB- DNAGZ- DNA(note the tilt of the bases for A- and Z-type DNA)DNA Can Form a Triple Helix synthetic DNA oligomers (short pieces)were found to complex to double strandedDNA forming a triplex found to occur in nature during someinstances of recombination and also duringtelomerase activity (extension of DNA ends)23
RNA Structure The primary structure of an RNA strand is muchlike that of a DNA strand RNA strands are typically several hundred toseveral thousand nucleotides in length In RNA synthesis, only one of the two strands ofDNA is used as a template Although usually single-stranded, RNA moleculescan form short double-stranded regions– This secondary structure is due to complementary basepairing A to U and C to G– This allows short regions to form a double helix RNA double helices typically– Are right-handed (11-12 base pairs per turn) Different types of RNA secondary structures arepossible24
Complementary regionsHeld together byhydrogen bondsFigure 9.23Noncomplementary regionsAlso calledhair-pinHave bases projecting awayfrom double stranded regionsMolecule containssingle- and doublestranded regionsThese spontaneouslyinteract to producethis 3-D structureFigure 9.24 the tertiary structure of tRNAphe(transfer RNA carrying the amino acid phenylalanine)25
GENOME/ CHROMOSOMEORGANIZATIONProkaryotic vs. EukaryoticWhat are the essential differences?How would this impact chromosomeorganization?26
The main function of the genetic material is to storethe information required to produce an organism– The DNA molecule does that through its base sequence DNA sequences are necessary for––––1.2.3.4.Synthesis of RNA and cellular proteinsReplication of chromosomesProper segregation of chromosomesCompaction of chromosomes So they can fit within living cellsViral Genomes The genome can be– DNA or RNA– Single-stranded or double-stranded– Circular or linear Viral genomes vary in size from a few thousand tomore than a hundred thousand nucleotides27
During an infection process, mature viral particlesneed to be assembledSingle-stranded RNA moleculeViruses with asimple structuremay self-assemble Genetic materialand capsid proteinsspontaneously bindto each otherExample: Tobaccomosaic virusCapsidproteinCapsid composed of 2,130identical protein subunits Complex viruses, such as T2 bacteriophages,undergo a process called directed assembly– Virus assembly requires proteins that are not part of themature virus itself The noncapsid proteins usually have two mainfunctions– 1. Carry out the assembly process Scaffolding proteins that are not part of the mature virus– 2. Act as proteases that cleave viral capsid proteins This yields smaller capsid proteins that assemble correctly28
BACTERIAL CHROMOSOMES The bacterial chromosome is found in a region of the cellcalled the nucleoid (not enclosed in membrane) Bacterial chromosomal DNA is usually a circular moleculethat is a few million nucleotides in length– Escherichia coli 4.6 million base pairs– Haemophilus influenzae 1.8 million base pairs A typical bacterial chromosome contains a few thousanddifferent genes– Structural gene sequences (encoding proteins) accountfor the majority of bacterial DNA– The nontranscribed DNA between adjacent genes aretermed intergenic regions To fit within the bacterial cell, the chromosomalDNA must be compacted about a 1000-fold– This involves the formation of loop domainsThe looped structure compactsthe chromosome about 10-foldFigure 10.529
DNA supercoiling is a second important way tocompact the bacterial chromosomeSupercoiling within loopscreates a more compact DNAFigure 10.6 The control of supercoiling in bacteria isaccomplished by two main enzymes– 1. DNA gyrase (also termed DNA topoisomerase II) Introduces negative supercoils using energy from ATP It can also relax positive supercoils when they occur– 2. DNA topoisomerase I Relaxes negative supercoils The competing action of these two enzymesgoverns the overall supercoiling of bacterial DNA30
How Gyrase WorksUpper jawsDNA wraps aroundthe A subunits in aright-handed direction.DNA binds tothe lower jaws.Lower jawsUpper jawsclamp onto DNA.DNA held in lower jawsis cut. DNA held in upperjaws is released and passesdownward through the openingin the cut DNA. This processuses 2 ATP molecules.DNAA subunitsB subunits(a) Molecular mechanism of DNA gyrase functionCircularDNAmolecule2 negativesupercoilsDNA gyrase2 ATPCut DNA is ligated backtogether, and the DNA isreleased from DNA gyrase.1210 bpper turnPlates preventing DNAends from rotating freely345(a)360 right-handedturn (overwinding)360 left-handedturn (underwinding)Both overwinding andunderwinding caninduce supercoilingFewer turnsor1112.5 bpper turn(not astablestructure)2310 bpper turnplus 1negativesupercoil4(b)More turnsor24538.3 bpper turn(not astablestructure)234510 bpper turnplus 1positivesupercoil134256(c)These two DNA conformations donot occur in living cells(d)(e)These three DNA conformationsare topoisomers of each other31
The chromosomal DNA in bacteria is negativelysupercoiled– In E. coli, there is one negative supercoil per 40 turns ofthe double helix Negative supercoiling has two major effects– 1. Helps in the compaction of the chromosome– 2. Creates tension that may be released by DNAstrand separation Two main classes of drugs inhibit gyrase and otherbacterial topoisomerases– 1. Quinolones– 2. Coumarins(note: these do not inhibit eukaryotic topoisomerases)Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.Area mosomeFigure 10.8This enhancesDNA replicationand transcription10-1732
EUKARYOTIC CHROMOSOMES Eukaryotic genomes vary substantially insize– The difference in the size of the genome is notbecause of extra genes Rather, the accumulation of repetitive DNAsequences–These do not encode proteinsVariation in Eukaryotic Genome SizeHas a genome that is morethan twice as large as that ofFigure 10.1033
Eukaryotic Chromatin Compaction-Problem If stretched end to end, a single set of humanchromosomes will be over 1 meter long- but cell’snucleus is only 2 to 4 µm in diameter!!! How does the cell achieve such a degree ofchromatin compaction?First Level Chromatin organized as repeating unitsNucleosomes Double-stranded DNA wrapped around an octamer of histone proteins Connected nucleosomes resembles “beads on a string”– seven-fold reduction of DNA length34
Histone proteins are basic– They contain many positively-charged amino acids Lysine and arginine– These bind with the phosphates along the DNA backbone There are five types of histones– H2A, H2B, H3 and H4 are the core histones Two of each make up the octamer– H1 is the linker histone Binds to linker DNA Also binds to nucleosomes– But not as tightly as are the core histonesNucleosome core particle(b) Molecular model for nucleosome structure35
Second level: Nucleosomes associate with each otherto form a more compact structure termed the 30 nm fiberHistone H1 plays a role in this compaction(non-histone proteins also play a role)The 30 nm fiber shortens the total length ofDNA another seven-foldThese two events compact the DNA7x7 49 ( 50 fold compaction)Further Compaction of the Chromosome A third level of compaction involves interactionbetween the 30 nm fiber and the nuclear tregions (SARs)MARs are anchoredto the nuclearmatrix, thus creatingradial loops36
The nuclear matrix is composed of two parts: Nuclear lamina (Fibers that line the inner nuclear membrane) Internal matrix proteins (Connected to nuclear lamina andfills interior of nucleus)Protein boundto internalnuclear taphase chromosomes2532646Z41635Z425Interphase3213Z52 mEach of seven types of chicken chromosomes is labeled a differentcolor. Each occupies a specific territory during interphase.37
Heterochromatin vs Euchromatin The compaction level of interphase chromosomesis not completely uniform– Euchromatin Less condensed regions of chromosomes Transcriptionally active Regions where 30 nm fiber forms radial loop domains– Heterochromatin Tightly compacted regions of chromosomes Transcriptionally inactive (in general) Radial loop domains compacted even furtherFigure 10.20 There are two types of heterochromatin– Constitutive heterochromatin Regions that are always heterochromatic Permanently inactive with regard to transcription– Facultative heterochromatin Regions that can interconvert between euchromatin andheterochromatin38
Figure 10.21Compaction levelin euchromatinDuring interphasemost chromosomalregions areeuchromaticCompaction levelin heterochromatinFigure 10.2139
Condensin plays a critical role in condensationDuring interphase,condensin is in thecytoplasm300 nm radial loops — euchromatin700 nm — heterochromatinCondensinCondesin binds tochromosomes andcompacts theradial someG1, S, and G2 phasesCondesin travelsinto the nucleusStart of M phaseCohesin plays a critical role in sister chromatid alignmentCohesinCohesin atcentromereis degraded.Cohesins alongchromosome arms arereleasedCentromereregionCohesinremains atcentromere.ChromatidEnd of S phaseG2 phase(decondensed sisterchromatids, armsare cohered)Beginning ofprophase(condensedsisterchromatids,arms are cohered)Middle ofprophase(condensedsisterchromatids,arms are free)Anaphase(condensed sisterchromatids haveseparated)40
Data from A. D. Hershey and Martha Chase (1952) Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage. Journal of General Physiology 36, 39–56. Extracellular 35S Extracellular 32P RESULTS R
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Le genou de Lucy. Odile Jacob. 1999. Coppens Y. Pré-textes. L’homme préhistorique en morceaux. Eds Odile Jacob. 2011. Costentin J., Delaveau P. Café, thé, chocolat, les bons effets sur le cerveau et pour le corps. Editions Odile Jacob. 2010. 3 Crawford M., Marsh D. The driving force : food in human evolution and the future.
