Chapter 11 : DNA ReplicationOutline:* Semiconservative Replicationo Meselson-Stahl Experiment* DNA polymerases and DNA elongation* Molecular model of DNA replicationo Initiation of Replicationo Semidiscontinuous DNA replicationo Rolling circle replication* Replication of telomeres in eukaryotesDNA replication underlies the process of inheritance at all levels(cellular, organismal, population). DNA replication occurs as preludeto cell division ( S phase of cell cycle in eukaryotes). DNA in allorganisms is the end point in a continuous series of replications goingback to the origin of life, almost 4 billion yrs ago. DNA replication isbased on complementarity of DNA molecules and on ability ofproteins to form specific interactions with specific sequences of DNA.Semiconservative Replication* Watson and Crick model of DNA suggested that each strand couldserve as template for synthesis of new strand. Their model is calledsemiconservative DNA replication* Two other models based on template-based synthesis were alsoproposed by others (Fig 11.1):
o Conservative model: parental strands rejoin after they are used astemplates, resulting in two DNA moleucles, one made of two parentalstrands, and the other made entirely of newly synthesized DNA.o Dispersive model: parental DNA cleaved into DNA segments thatact as templates for the synthesis of new DNA and then somehowsegments reassemble into double stranded DNA made of parental andprogeny DNA which are interspersed.o All three models made different predictions about the nature ofDNA after one and two rounds of replication (Fig 11.1).Meselson-Stahl Experiment* Meselson and Stahl (1958) used a heavy isotope of nitrogen (15N)and equilibrium density gradient centrifucation to show that DNAreplicated in semiconservative manner in E. coli (Fig11.2).o grew E. coli for many generations in medium containing 15NH4Cl(15N is a heavier isotope than 14N). This resulted in DNA containing15N instead of 14N. 15N DNA can be seperated from 14N DNA byultracentifugation in a CsCl gradient.o 15N-labeled bacteria were then transferred to medium containing14N and allowed to grow for several generations, and sampled aftereach replication cycle.o After one generation in 14N, all the DNA had a density intermediatefrom 15N-DNA and 14N-DNA, just as predicted by thesemiconservative and dispersive models.+ this result ruled out the conservative model because it predicted thatthere should be two bands (one containing light DNA and the otherheavy DNA).o To distinguish between the semiconservative model and thedispersive model, E.coli were grown for another generation. Twobands were observed, as expected by the semiconservative model. Thedispersive model predicted that there should only be one band,therefore it was also ruled out. The results were all consistent with thesemiconservative model.o* Semiconservative DNA replication also occurs in eukaryotes (seeharlequin chromosomes in Fig 11.3).DNA polymerases and DNA elongation
* In 1955, Arthur Korberg identified the first DNA polymerase (DNAPol I). Initially it was thought to be the main DNA replication enzyme,but mutant E.coli defective in the gene encoding for DNA pol Idivided normally, indicating that there must be other enzymesinvolved.* Five DNA polymerases have now been identified in E. coli. DNAPol II, IV, and V are involved in DNA repair. DNA pol I and III areinvolved in DNA replication.* All DNA polymerases catalyze the polymerization of nucleotideprecursors (dNTPs) into a DNA chain . The reaction is shown in Fig11.4 and has three main features:1. DNA pols catalyze the formation of a phosphodiester bond betweenthe 3-OH group of the deoxyribose on the last nucleotide in the chainand the 5-phosphate of the incoming nucleotide. The energy issupplied by the hydrolysis of the two phosphates from the dNTP. AllDNA polymerases require a primer (i.e they can not add the firstnucleotide ).2. DNA polymerases require a template. The particular nucleotideadded depends on correct complementary base pairing with thetemplate. DNA pols are fast. In E. coli, DNA pol I and II canpolymerize ~ 850 nt per sec. In humans, its a lot slower (60-90 nt/sec).3. All DNA polymerases synthesize DNA in the 5 to 3 direction.* DNA pol I and II also have exonuclease activity.o DNA pol I and III have 3-> 5 exonuclease activity. This is aproofreading mechanism. DNA pols add an incorrect base with afrequency of 10-6. When an incorrect base is added, the enzymedetects that it made a mistake, and uses its 3 to 5 exonuclease activityto move back and remove the incorrect base. With proofreading, theerror rate drops to 10-9.o DNA pol I also has 5 -> 3 exonuclease activity. This allows it toremove DNA or RNA from the 5 end of a moleecule. This is essentialduring DNA replication of the lagging strand.Model of DNA Replication in E. coli* The bare-bone mechanics of DNA replication is similar in allorganisms. However, we will only focus on DNA replication in E.coli, where it is best understood. Along the way, significant
differences between prokaryotic and eukaryotic DNA replication willbe highlighted.* Basic research into the mechanisms of DNA replication in E. coli(as well as transcription and translation) has led to the identificationand cloning of dozens of genes involved in these processes (Table11.1). The creative use of these gene products has given us atremendous power to manipulate genes and genomes according to ourwill.Initiation of Replication* Initiation of replication starts at a DNA sequence called thereplicator, which includes the origin of replication (OriC) (AT-rich)where DNA is denatured into single strands to form a replicationbubble. At either end of a bubble there is a replication fork, whereDNA synthesis occurs, using each separated strand as a template.o Circular genomes of prokaryotes contain a single origin ofreplication.o In eukaryotes, linear chromosomes contain many origins ofreplication (allows faster replication).o Synthesis proceeds bidirectionaly at replication fork. Eventually,replicated double helices join each other, producing two daughtermolecules (Fig 11.9)(sister chromatids, in eukaryotes)* Initiation of replication starts with the binding of an initiator proteinwhich denatures the oriC and then recruits a DNA helicase (one foreach strand) which untwists the DNA in both directions (energycomes from hydrolysis of ATP) (Fig 11.5).* Next, each helicase recruits a DNA primase to form a primosome.DNA primase makes the necessary RNA primers ( 5-10 nts) neededby DNA polymerase III.* The next step involves the assembly of the rest of the proteinsinvolved in DNA replication. These proteins associate to form areplisome. There is a replisome at each replication fork.
Semidiscontinuous DNA replication* The replication steps are identical at each replication fork, so wefocus on just one. The entire process is shown in Fig 11.6.* After the helicase unwinds the DNA, the single stranded DNA isprevented from reannealing by binding to single-strand DNA-bindingproteins (SSBs) (about 200 /rep fork).* DNA pol III dimer (part of replisome) now initiates polymerizationby adding dNTPs to the RNA primer on each of the strands. Becausestrands in double helix are in antiparallel configuration, and DNApolymerases add dNTPs in 5 to 3 direction, the two strands aresynthesized differently:o Leading strand synthesized continuously; only one primer required;DNA pol III moves in same direction as replication fork.o Lagging strand synthesized discontinuously as Okazaki fragments,which are later ligated by DNA ligase. Each Okazaki fragmentrequires a primer. DNA pol III moves in opposite direction toreplication fork.
* In Lagging-strand synthesis, DNA Pol III ends polymerization whenit encounters double stranded DNA ahead (from previous Okazakifragment). It dissociates from the DNA, leaving a gap in one strand.This gap is recognized as damaged DNA and is repaired by DNA PolI.* DNA Pol I removes primers and fills in gaps (has 5-3 exonucleaseactivity).This image has been resized. Click this bar to view the full image. The original image is sized 720x540and weights 49KB.
* DNA ligase joins 3 end of one Okazaki fragment to 5 end ofdownstream Okazaki fragment (Fig 11.7).* As helicase unwinds DNA ahead of replication fork, positivesupercoils form elsewhere in the molecule. For replication fork tomove, the helix must rotate (estimated at 50 revolutions/sec). Theproblem of supercoiling is solved by the action of topoisomerases(specifically a Gyrase) which introduce negative supercoils tocounteract positive supercoils intoduce by helicases.Rolling circle replication* For many viral DNAs and some plasmids (e.g. F plasmid in E. coli),rolling circle replication has been demonstrated.This image has been resized. Click this bar to view the full image. The original image is sized 690x499and weights 79KB.
* Synthesis usually continues beyond a single chromosomal unit. Thisresults in many head-to-tail copies of the plasmid, which is then cutand rejoined into new circular molecules.Replication of telomeres in eukaryotes* There are special problems associated with replication of the ends oflinear chromosomes (called telomeres). Recall that DNA polymerasesonly add nucleotides to the 3 end of a growing chain. When the linearchromosomes of eukaryotes replicate, the resulting daughtermolecules will each have an RNA primer left over at the 5end (Fig11.14). This RNA primer is removed, leaving a single stranded DNAsegment. If not fixed, this single-stranded DNA region will getdegraded, and the linear chromosomes will get shorter with eachround of DNA replication.* In most eukaryotes, an enzyme called telomerase, maintains the endsof chromosomes by adding telomere repeats to chromosome ends. Themechanism is shown below (and in Fig 11.5).
* Telomerase is a ribonucleoprotein (has RNA molecule as part of itsstructure) which adds tandem repeats to the 3 end of chromosomesusing an RNA molecule as a template. After is has added manytelomeric repeats and has left, a new DNA molecule is made startingfrom a new RNA primer, which is again is removed, but by this timethe chromosme has already been extended.* The absence of telomerase activity in cells is correlated withsenescence of cells (i.e. die after certain number of cell divisions).Conversely, enhanced telomerase activity correlated with cellimmortalily (i.e. cells divide indefinately).o cells with short telomerse undergo fewer doublings than ones withlong telomerase.o fibroblasts form individuals with progeria (rare disease characterizedby premature aging) have short telomeres.o most somatic cells have no active telomerase (divide only 20-60times)o sperm cells, stem cells and unicellular eukaryotes (essentiallyimmortal ) have active telomerase and stable telomeres.
o cancer cells, which are also essentially immortal, have activetelomerase (promising target for drug design)o Elimination of telomerase activity in somatic cells may be a cellularsenescence mechanism that protects multicellular organisms fromcancer
Chapter 13 : Transcription* Outlineo Genes and RNAo Properties of RNAo Classes of RNAo Making functional transcripts+ RNA polymerases+ Initiation+ Elongation+ Terminationo RNA processing in eukaryotesGenes and RNABiological information flow from DNA to protein requires an RNAintermediate. RNA is produced by a process that copies the nucleotidesequence in DNA to produce a transcript. This process is calledtranscription.
Properties of RNA1. Single stranded, but can undergo intramolecular base-pairing- forms variety of 3D structures specified by sequence.2. Ribose sugar (not deoxyribose)3. Uracyl in place of thymineClasses of RNA* There are a variety of different RNAs that can be classified into twoclasses.o 1. Informational RNAs (e.g. messenger RNA)+ intermediate which is later translated into protein.+ most genes encode mRNAo 2. Functional RNAs
+ never translated+ diverse roles in cell+ main classes of functional RNAs play critical roles in various stepsin the information processing of DNA to protein:# rRNA - components of ribosome# tRNA - bring amino acids to mRNA during translation# snRNA (small nucleolar RNAs) - involved in splicing of introns# scRNAs (small cytoplasmic RNAs) - protein trafficking* All DNA and RNA function is based on two key elements:o 1. Complementary bases in single stranded nucleotide chains can H-bond to form double stranded structures.o 2. Specific sequences can be recognized by specific nucleic-acidbinding proteins.Making functional transcripts* Transcription uses one DNA strand as templateo Strands of double helix must be separated, so that one of thesestrands (template strand) can serve as template to direct the synthesisof transcript.* Either strand along the chromosome can serve as template, but for agiven gene, its always the same strand.* RNA polymerase catalyzes the synthesis of RNA using DNAtemplate (Fig 13.1).o RNA grows in 5 to 3 direction, and the template is read in the 3 to5 direction.o sequence of RNA is complementary to template strand (noncodingstrand), but the same as nontemplate strand (coding strand) except Treplaced with U.* A typical prokaryotic gene has the folowing features:
RNA Polymerases* Prokaryotes have only one RNA Polymerase but eukaryotes have 3:1. RNA Pol I: transcribes rRNA genes2. RNA Pol II: transcribes protein encoding genes3. RNA Pol III: transcribes other functional RNAs (tRNAs, snoRNAsetc...)* In eukaryotes, transription takes place in nucleus.* In prokaryotes, transcription and translation are coupled.* Transcription involves 3 distinct stages: initiation, elongation, andtermination.Initiation* In E. coli, transcription requires a complex of RNA polymerase andthe sigma factor (s) which binds to a promoter. The RNA polymerasecore enzyme (4 has four subunits, two a, one b and one b) complexedwith the sigma factor is known as the holoenzyme. Once transcriptionis initiated, the sigma factor dissociates.* promoter = DNA sequence to which RNA Pol binds to initiatetranscription.o note that by convention, gene is labelled the same way as RNAtranscript. So promoter is at 5 end of gene (Fig 13.3).* RNA pol + sigma factor scans DNA for promoter sequence, bindsDNA at the promoter sequence (- 10 region and -35 region), unwindsit, and begins synthesis of a transcript at transcription initiation site.Promoter sequences are not transcribed. NOTE: RNA pol does notneed a primer to initiate RNA synthesis not does it need a helicase.o there are consensus sequences for all promoters in E. coli. Aconsensus sequence is the sequence found most frequently at eachposition. E.g consensus sequence at -10 position is 5-TATAAT-3o The more similar the promoter sequence is to the consensus, thehigher the rate of transcription.o It is the sigma factor that binds the promoter. Different sigma factorsbind different promoters.* What is described above is the minimum required for transcriptioninitiation. In chapter 19 we will study how genes are regulated inprokaryotes in more detail.
Elongation* RNA pol moves along DNA, maintaining transcription "bubble" toexpose template strand, and catalyzes the 3 elongation of transcript.o energy for reaction derived from splitting high-energy triphosphatesinto monophosphates.o rate of transcription is about 30-50 nt/secTermination* Results from different mechanisms signalled by terminationsequences at 3 end of a gene. Two mechanisms known:o Rho-independent termination+ involves formation of hairpin loop (Fig 13.5) in nascent transcriptcausing RNA strand and RNA Pol to be released from DNA template.o Rho-dependent termination+ Rho is a protein that binds RNA terminator sequence and then usesenergy from ATP hydrolysis to separate transcript from RNApolymerase.+ Rho-dependent terminators lack hairpin loop.* The transcript that is made is called mRNA and in prokaryotes it hasthe following structure (note that its always larger than what is neededto encode the polypeptide):
* Prokaryotes have coupled transcription and translation, so that evenas mRNA is being transcribed, ribosomes attach to 5 end and begintranslation.* Many mRNAs in prokaryotes are polycistronic, i.e. they can encodemore than one polypeptide (E.g. lac operon)RNA processing in Eukaryotes* Transcription in eukaryotes is more complicated, in that there aremore regulatory sequences involved, and there is a sequentialassembly of many different transcription factors at the promoterbefore RNA polymerase binds and initiates trancription. We will studytranscription in eukaryotes in more detail in chapter 20.* In this chapter, we will focus on processing of the initial transcript .* In eukaryotes, the initial product of transcription (primary transcript)is processed in several ways before transport to cytosol. Inprokaryotes, there is no such processing.* Processing performed by RNA binding proteins.This image has been resized. Click this bar to view the full image. The original image is sized 672x462and weights 62KB.
* Processing involves (Fig 13.9 and Fig 13.11):o 1. addition of 5 cap+ Guanyltransferase adds 7-methylguanosine using a 5-to-5triphosphate linkage+ protects transcript aginst degradation by exonucleases.+ also important for binding of ribosome during translationo 2. addition poly(A+) tail+ transcript cleaved 20 bases downstream of AAUAAA sequence near3 end by an endonuclease, then 50-250 adenine nucleotides added byPoly(A) polymerase.+ Poly(A+) tail required for efficient transport out of the nucleus intocytoplasm. Once in cytoplasm, polyA tail also protects against earlydegradation by exonulceases.o 3. RNA splicing to remove introns (Fig 13.13)+ the GU-AG rule+ spliceosome
Chapter 10: DNA as a genetic materialSearch for genetic material* Scientists reasoned early on that whatever the genetic materialturned out to be, it had to have 3 important characteristics:1. Store information (about structure, function, development,reproduction)2. Replicate accurately (so progeny can receive information fromparents)3. Capable of change. Without mutation there is no variation andadaptation. Evolution does not occur.Griffith’s transformation experiment[/b]* In 1928, Frederick Griffith was working with two different strains ofStreptococcus pneumoniae (causes pneumonia).1. S strain: forms smooth colonies; highly infectious because it formsa capsule which allows bacteria to evade immune system of host(virulent).2. R strain: forms rough colonies; harmless because it lacks a capsule,therefore gets detected by immune system early and effectively(avirulent).* Griffith injected mice with the different strains and checked forvirulence. The experiment was as follows:* The experiment showed that bacteria need to be alive and to have apolysaccharide capsule to be infectious and kill the host. Moreimportantly, it also showed that bacteria could uptake genetic materialfrom their surroundings. Griffith called this material the transformingprinciple, which he believed was protein. The real importance of
Griffith’s experiment is that it provided the experimental basis forfurther experiments on the chemical nature of the transformingprinciple.Transformation experiments of Avery, MacLeod and McCarty* In the 1930’s and 40’s these researchers followed up on Griffithsexperiments, by fractionating the heat killed cell extract into proteins,and nucleic acids. Only the nucleic acid fraction was capable ofcausing transformation of a rough strain into a virulent S strain. Thisindicated that proteins were not the transforming principle, and socould not be the genetic material.* Since there are two types of nucleic acids, DNA and RNA, a furtherfractionation and transformation experiment was carried out as shownbelow:* These results strongly suggested that DNA was the genetic material.However, too many scientists were still too predisposed to thinkingthat proteins were the genetic material. Further proof was necessary.Hershey-Chase experiment with labeled phage* In 1953, Alfred Hershey and Martha Chase finally proved to thesatisfaction of most scientists that DNA was the genetic material.* Hershey and Chase studied bacteriophage T2, which was known tobe made entirely of protein and DNA. T2 phage replicates by invadingE. coli, taking over its replication machinery to make progeny phage.The host cell then lyses and releases progeny phage capable ofinfecting new cells.
* Their experiment started with the radioactive-labeling of either DNA(with 32P, not found in proteins) or proteins (with 35S, not found inDNA) in phage as shown below:* These labeled phage were then used to show that DNA was thegenetic material in T2 phage as in the experiment shown below:* They reasoned that since it was DNA and not protein that enteredthe cell, DNA must be genetic material, which is responsisble for thefunction and reproduction of phage.* It is now known that all organisms have DNA as their geneticmaterial. However, in some viruses the genetic material is made ofRNA (e.g. HIV, phage Qb, and tobacco mosaic virus).Composition and structure of nucleic acids* By the very early 1950s, nucleic acids (DNA and RNA) werealready known to be polymers composed of nucleotides linked
together in long chains.* Nucleotides are made of 3 chemical groups:o Phosphateo Sugar (deoxyribose in DNA, ribose in RNA)(Fig 10.6).o Nitrogenous base (varies between different nucleotides)(Fig 10.7).+ adenine and guanine are purines (double ring).+ thymine, cytocine and uracyl are pyrimidines (single ring).# thymine present in DNA, and uracyl present in RNA.* The nucleotides found in DNA are shown below:*** To form RNA and DNA polynucleotides, nucleotides are connectedby a covalent bond (called phosphodiester bond ) between the 5phosphate of one nucleotide and the 3-OH of another.
The DNA double helix* In 1953, James Watson and Francis Crick showed that DNA is madeup of two single strands wrapped around one another as a doublehelix. The data they used came from two sources:o Base composition studies:o Erwin Chargaff analyzed DNA from many sources by hydrolysing itand quantifying the nucleotides. He found that in all cases there was a1:1 ratio of purines to pyrimidines. Also, the A/ T ratio and the C/Gratio was 1. These equivilencies are known as Chargaffs rules (Table10.2).o X-ray diffraction studies:+ Rosalind Franklin and Maurice Wilkins used X-ray diffraction toobtain information about DNAs atomic structure (Fig 10.10). Theydiscovered that DNA was helical and had two distinctive regularitiesof 0.34 nm and 3.4 nm along the axis of the molecule. In addition, itwas shown that DNA had a uniform thickness of 2 nm.* Watson and Crick used this information to build models which wereconsistent with all the data. Their double-helical model of DNA hasthe following features (Fig 10.11 and 10.12):1. DNA consists of two polynucleotides chains wound around eachother in a right-handed helix.2. Chains are in antiparallel conformation (one strand goes 5 to 3, theother goes 3 to 5).
3. Sugar-phosphate backbone is on outside of helix, and bases areoriented toward central axis. Bases are flat, and stack up on top of oneanother. Double helix caused naturally when bases stack up duringcomplementary base-pairing.o stacking adds stability to DNA by excluding water away fromspaces between base pairs.4. Strands held together by complementary base pairing through H-bonds. Adenine forms 2 H-bonds with thymine and cytosine forms 3H-bonds with guanine. Because of complementary base pairing, thesequence of one strand can be inferred from the other.o important that strands be held by weak bonds, so that DNA strandscan be separated during replication and transcription.5. Base pairs are 0.34 nm apart. A complete turn of the helix takes 3.4nm, and the diameter of the helix is a uniform 2 nm.6. Double helix contains a major and a minor groove. The bases areexposed in these grooves, allowing sequence-specific interaction withproteins.* Watson and Cricks double -helical model is also known as B-DNAwhich closely approximates how DNA is found in the cell. DNA canalso exist in slightly different conformations called A-DNA and Z-DNA (Fig 10.23), but we will not concern ourselves with these in thiscourse.Organization of DNA in chromosomes* In cells, DNA is organized into physical structures calledchromosomes. The full DNA sequence of an organisms haploid set ofchromosomes is called the genome. The organization of DNA in thegenomes of prokaryotes, eukaryotes and viruses vary considerably.Before studying the nature of genomes more closely, it is helpful tohave a clear idea of what is a gene.What is a gene?* A gene is a region of chromosomal DNA that can be transcribed intoa functional RNA at the correct time and place during development.* Genes vary in size and function. For most genes, certaintopographical features can be delineated:
o Regulatory region : unique sequences upstream of coding regionthat bind proteins and initiate transcription according to signals fromother parts of genome and environment.o Coding region art of gene which actually encodes functionalproduct (RNA or protein)o Transcription termination region* Coding regions of eukaryotic genes may be interrupted bynoncoding regions called introns (must be spliced out to make maturetranscript).o frequency of introns varies greatly within eukaryotic lineages.Fewest in fungi, most in mammals.Genome size* Generally, genome size increases with complexity of group;considerable variation exists (up to 1000-fold within a group)(Table10.4 )* Generally, the larger the genome, the more genes present (manyexceptions).* Viral genomes smallest. Prokaryotic genomes are 100-1000xsmaller than eukaryotes.Plasmids
* Small circular DNA molecules common in the cytoplasm ofbacteria.o not essential for basic operation and survival of the cell.o some linear plasmids are known.* Plasmids often contain useful genes for bacterial host: eg, sex pilus,antibiotic resistance, production of toxins.* Plasmids are occasionally found in fungal and plant cells:o most are inside mitochindria or chloroplast.o unlike in prokaryotes, provide no benefit to host.* Plasmids rely on cellular machinery of host for replication andmaintenance.* Plasmids are very useful in recombinant DNA technology.Organellar DNA* Both mitochondria and chroloplasts are derived from once free-living bacteria which became endosymbionts. They retain theircircular genome containing many genes (although far fewer thantypical prokaryote).* Many organellar genomes have introns.* Contain little intergenic space.* Individual mitochondria and chloroplasts contain identical multiplecopies of chromosomes (i.e high copy number).* Genes in organellar DNA are inherited from one parent only, usuallythe mother. Therefore, do not show mendelian pattern of inheritance.Viral genomes* Viruses are nonliving particles that must infect and subvert cellularmachinery of host to reproduce.* Viruses are composed of protein coat and a core that contains thegenome.* Genome of viruses are the most diverse due to diverse evolutionaryhistories as well as diversity in strategies for packing genome in coat.Genomes can be can be made of ssDNA, dsDNA, ssRNA, or evendsRNA. May be linear or circular. In most viruses, the genome ismade up of a single chromosome, but in a few, it is segmented intomore than one chromosome.* Compared to organismal genomes, viral genomes are very small,
have little intergenic space, and few genes (herpes virus contains ~200genes, and many others have far fewer genes).* Examples of viral genomes:o T-even bacteriophage (T2, T4, T6) (Fig 9.11a)+ consists of a single linear chromosome consisting of double strandedDNA In T4, genome size is 168 kb. DNA is packaged within proteincoat (viral head).o Phage FX174 (Fig 10.14)+ has single 5.3 kb chromosome packaged into phage head.+ DNA is single stranded. The ratio of A:T or G:C is not 1:1.+ DNA is circular. Therefore, resistant to exonucleases ( enzymes thatremove nucleotides from the ends of DNA molecules).o Lambda (l) phage+ similar to T-even phage in structure, but the double-stranded DNAgenome alternates between linear and circular forms (Fig 10.15).# within phage particle, chromosome is linear and the two ends have a12 nt single stranded regions that are complementary.# when lambda infects E. coli, the sticky ends base pair andchromosome becomes circular. Chromosome reverts back to linearform during phage replication and packaging.Eubacterial Genomes* Genome size is relatively small (average is 4 Mb) , but theresconsiderable variation.o Bacillus megasterium = 30 Mbo Yeast = 12.1 Mb* In most cases, made up of a single circular chromosome; but thereare exceptions:o Vibrio Cholera has 2 different circular Xsomes.o Borrelia burgdoferi (Lyme disease) has one linear chromosome, and17 plasmids (combined size is 0.53 Mb).o Agrobacterium tumefaciens (causes crown gall disease in plants) has3 Mb circular genome and a 2.1 Mb linear chromosome.* High gene density ; i.e. genes close togethero 85-90% of DNA is codingo less than 1% if DNA is noncoding, made up of transposableelements.o In E coli, 3 cases of overlapping genes (this phenomena more
common in viruses)* Introns extremely rare* Functionally related genes located together as a group (operons).o in E. coli, 27% transcriptional units are polycistronic; genes inoperons functionaly relatedo in Aquifex aeolicus, operons are present, but genes not functionalyrelated (redefines our ideas on operons).* Bacterial genomes have associated proteins used to pack it into anucleoid (not like histones, though).* The DNA in the nucleoid is supercoiled (ie twisted about its ownaxis)(Fig 10.17 and 18).o supercoiled DNA is under some tension. If one strand is cut, DNAbecomes relaxed.o Supercoiling makes for more efficient packing in the nucleoidregion.o Supercoiling in all organisms is controlled by topoisomerases.* Chromosomes in bacteria are further compacted 10-fold because it isorganized into looped domains (Fig 10.19).* Plasmids are common in bacteria.o some plasmids carry essential geneso in some cases, the same plasmid often found in different species.Archaean Genomes* Share similarities to both eubacterial and eukaryotic genomes.However, no linear chromosomes have been found in archaea.* Example: Methanococcus jannaschii (extreme thermophile)o genome sequenced in 1996o has 3 circular Xsomes+ one 166 MB Xsome, plus two smaller Xsomes, of 58.4 and 16.5 kb.o 58% of genes do not match any known geneso majority of genes involved in energy production, cell division, andgeneral metabolism more closely resemble eubacterial genes.o similarities to eukaryotes:+ genes involved in RNA synthesis, protein synthesis, and DNAsynthesis.+ contain histone genes and DNA organized into chromatin+ have introns in tRNA genes
Eukaryotic Nuclear genomes* Most eukaryotes carry either 1 set (haploid ) or 2 sets (diploid ) ofnuclear chromosomes.* In diploids, members of a chromosome pair are called homologues.Members of homologous pair are very similar in size and genecontent, carrying the same genes in the same relative positions.However, homologs can carry different alleles of a gene.* Different sets of chromosomes carry different sets of genes. Alsomay differ size, location of centromere, and staining pattern.* The number of chromosomes varies considerably in eukaryotes.Each species has a characteristic number. Human have 46chromosomes (23 pairs).* Total amount of DNA in haploid genome is called the C value(Table 10.24)o C value does not vary much within nor between mammals, birds andreptiles.o In plants, amphibians and insects show much greater variationwithin and between groups.o In eukaryotes, there is not much correlation between amount ofDNA and organismal complexity. This is known as the C valueparadox.+ e.g. human genome has 3.2 billion bp. Amoeba has 290 billion bp.* DNA in each eukaryotic chromosome is linear and extensivelyassociated with histone proteins, which help condense DNA intochromatin (Fig 2.22).* Chromatin structureo chromatin is made up of 50% DNA, 50% protein. Most of theseproteins are histones. Others are scaffold proteins, which furthercondense chromosomes into the structures visible during cell division.Certain DNA sequences along the chromosomes serve as attachmentssite to scafold (Fig 10.20 and 21 ).o Histones are the most abundant proteins in chromatin. Five maintypes of histones: H1, H2A, H2B, H3 and H4. All are basic proteins(have positive charges that interact with negative charge of phosphatebackbone of DNA). All histones are very conserved in all eukaryotes,suggesting that they perform the same function.o DNA condensation varies depending on stage of cell cycle. Least
compact form of chromatin is 10 nm thick, and has "beads-on-a-string" appearance (Fig 10.20). Beads are nucleosomes. Eachnucleosome is made up of 147 bp of DNA wound 1.65 times around ahistone octamer (made of two of each H2, H2A, H3 and H4) (Fig10.21).o Nucleosomes connected by linker DNA (in humans its about 38-53bp).o Next level of packing occurrs when H1 binds linker DNA, causingadjacent nucleosomes interact to form a 30 nm chromatin fiber.o Chromatin packing beyond 30 nm is less understood. However, it isknown that 30 nm chromatin form loops that are bound to scafoldproteins (Fig 10.22, 23 and 24).o Overall packing results in chromosomes that are 10,000 timesshorter and 400 thicker than naked DNA. This makes it easier fordividing cell to partition DNA into daughter cells.* Euchromatin and Heterochromatino Degree of DNA packing changes throughpout cell cycle. Its mostcompact during metaphase (stains more intensely), and least compactduring the S phase (stains least intensely).o Euchromatin refers to chromosomes or parts of chromosomes thatshow normal condensation and decondensation during cell cycle.+ In most cells, most of the genome is euchromatin.+ euchromatin is actively transcribed.+ euchromatin is devoid of repetitive regions.o Heterochromatin is made up of chromosomes or chromosomalregions that usually remain condensed throughout cell cycle. Stainmore darkly than euchromatin. Genes in heterochromatin are usuallytanscriptionaly inactive. Two types:+ Constitutive heterochromatin: mostly repetitive DNA; present atidentical positions on both homologues (e.g. centromertes)+ Facultative heterochromatin: varies between cells and at differentdevelopemntal stages or from one homologue to another. Representsinactivated segments of euchromatin (e.g. Barr bodies).* Centromereso DNA regions arounf attachment point of spindle fibers; responsiblefor proper segregation of chromsomes during cell division.o made up of highly repetitive DNA.* Telomeres
o telomeres are repetitive sequences at ends of chromosomes requiredfor replication and chromosome stabilityo characteristically heterochromatic
Chapter 1: Introduction* Outlineo Introductiono Classical and Molecular Geneticso Important Concepts of Genetics+ DNA, Genes , and Chromosomes+ Transmission of Genetic Information+ Expression of Genetic information+ Sources of Genetic Variationo Geneticists and Genetic Research+ Basic and Applied research+ Genetic databases and maps+ Model organisms in genetics+ BiotechnologyIntroductionGenetics is the scientific study of heredity. It aims to understand thebiological properties that are passed from one generation to the next.Genetics is diverse in its scope, dealing with the transmission ofgenetic information, as well as the nature of the information that isbeing passed and how that information determines the characteristicsof organisms. Genetics is central to biology because gene activityunderlies all life processes.This course focuses on molecular genetics. Molecular genetics is thestudy of genes and their function at the molecular level. Moleculargenetics has provided some of the most important unifying themes forthe whole of biology. Like no other biological discipline, it hasbecome central to numerous aspects of human affairs, be they social,political or moral. Our understanding of DNA structure and function,and how the information it stores ultimately manifests itself into anorganism has brought us a long way. We live in an age in which DNAcan be manipulated directly to synthesize entirely novel strains oforganisms, tailored exclusively to human needs. This technology iscalled molecular genetic engineering and will have a profound impacton human life.Classical and Molecular Genetics
* Genetics is often divided into classical and molecular genetics.* Classical genetics refers to the use of crosses to breed new strains oforganisms and to understand how traits are transmitted. An importanttool of classical genetics is the isolation of mutants affecting particulartraits which can then be compared to normal strains. This approachhas been very fruitful and has led to the discovery of genes involvedin many biological processes. The first genetic maps of genes onchromosomes were generated through this approach.* Molecular genetics refers to the study of genes at the molecularlevel. It has been made possible because of advances in cloning andsequencing of genes. Modern genetics has provided an enormousunderstanding about the physical nature of genes, their expressionpatterns, their role in development and disease, and how their productsinteract. In the last decade the complete genomic sequence for avariety of prokaryotes and eukaryotes has been determined. This newexciting field of genomics is already reaping enormous benefits, andwill lead to a better understanding of how genes control manybiological processes, from development and evolution to the treatmentof human disease.* The distinction between classical and molecular genetics is oftenblurred. For example, many genes are first identified through theireffect on phenotype, and then later cloned and sequenced. In addition,molecular geneticists often have to perform crosses to determine ifengineered strains have received one or multiple gene inserts.Important concepts in geneticsDNA, Genes , and Chromosomes* With the exception of some RNA viruses, the genetic material of allviruses, prokaryotes and eukaryotes is composed of DNA(deoxyribonucleic acid).* Genome = full DNA (or RNA) sequence of an organism.* DNA structure (Fig 1.1)o DNA is composed of two strands (chains) wrapped around oneanother to form a double helix. Each strand is made up of monomerscalled nucleotides. Each nucleotide consists of a deoxyribose sugar, aphosphate and a base. There are four bases in DNA: Adenine (A),guanine (G), cytosine (C), and thymine (T). Genetic information is
conveyed in the sequence of nucleotides, much like information canbe conveyed by a sequence of letters in this sentence.o A gene is a segment of DNA bearing a specific sequence ofnucleotides. Genes encode the traits that are passed from parent tooffsrping.o The genetic material is organized into chromosomes.+ Prokaryotes usually contain a single circular chromosome.+ Eukaryotes contain many linear chromosomes within a nucleus.Each chromosome is a single molecule of DNA complexed withhistone proteins. Histones allow efficient packing of the genome andare also involved in gene regulation. Eukaryotes also containextranulear DNA in their mitochondria and chloroplasts (plants andalgae).Transmission of Genetic Information* The laws of inheritance were first elucidated by Gregor Mendel. Hemade careful crosses between pea plants that differed in particularcharacteristics (traits), counted the number of times the traits appearedin subsequent generations, and interpreted the results. He made thefollowing conclusions:o Inherited characteristics are determined by factors (now calledgenes).o Each organism contains two copies of each gene: one inherited fromthe father, the other from the mother.o Alternative versions of genes (alleles) account for variations ininherited characters.* Homozygous: organisms having a pair of identical alleles for a trait(e.g. CC or cc for petal colour)* Heterozygous: organisms having two different alleles for a gene(e.g. Cc)* Genotype: complete genetic makeup of an organism.* Phenotype: all the observable characteristics of an organism.* The genotype interacts with environment (internal and external) toproduce the phenotype. This means that in many cases geneticallyidentical organisms may differ in phenotype because theirenvironment may differ.* Mendel had no idea what the material basis of his "factors" were.However, he did correctly infer their behaviour in crosses, and came
up with his two laws:1. Mendels First Law (aka the principle of segregation): members of agene pair segregate randomly into gametes.2. Mendels Second Law (aka principle of independent assortment):different genes assort indepnedently during gamete formation (wenow know this only applies to genes on different chromosomes orgenes that are located very far apart on the same chromosome).* Mendels discoveries remained unknown to the scientific communityuntil the early 1900s when others independently came up with thesame results. In 1902, Sutton and Boveri proposed the chromosometheory of segregation, which provided a material basis for genesegregation.Expression of Genetic information* Gene expression is the process by which a gene produces its productand that product carries out its function. Although Mendel inferred thepresence of "genes" and how they behaved from one generation to thenext, many questions remained. What are genes made of? What dothey encode? How do genes encode specific traits?* In 1941, Beadle and Tatum working with nutritional mutants ofNeurospora crassa, showed that each step in a biochemical pathwaywas under genetic control. They proposed the one-gene-one enzymehypothesis. Their hypothesis was subsequently modified into the one-gene-one-polypeptide hypothesis, because not all proteins areenzymes. So genes provide instructions for making proteins.* The process by which proteins are made from genes involves twomain steps: transcription and translation.* Transcription: process by which a segment of DNA (a gene) iscopied into RNA (Fig1.3).o DNA is locally unwound and RNA polymerase uses one strand as atemplate to synthesize an RNA copy.o Three major kinds of RNA are transcribed:+ ribosomal RNA (rRNA): used to make ribosomes, the translationmachines of the cell.+ transfer RNA (tRNA): decode DNA language into protein languageby bringing amino acids to ribosome.+ messenger RNA (mRNA): specify the amino acid sequences ofproteins. Most genes encode for mRNA.
* Translation: process by which the base sequence of mRNA isconverted into an amino acid sequence.o Occurs on ribosomes (large complexes of RNA and protein).o Genetic code: base sequence information that specifies the aminoacid sequence of a protein.o Codon: three -nucleotide sequence of mRNA that specifies an aminoacid. There are 64 codons: 61 that specify 20 amino acids and threethat are used as translation stops.* Not all genes in a cell are expressed at the same time. In amulticellular organism, each cell type (tissue type) only expresses asubset of all genes. The unique set of proteins made is also whatmakes the cell type unique. What determines which genes are "turnedON" or "turned off"?* Our first glimpse into gene regulation began in 1961 with Jacob andMonods operon model invoving molecular switches in E. coli. Aswitch is made up of a protein (a transcription factor) whoseconformation can be altered by ligand binding (lactose, in the lacoperon) and a DNA sequence to which it binds (promoter). Thepromoter is just upstream of the gene it helps regulate. Whentranscription factors are bound to promoters, they affect thefrequencey with which RNA polymerase binds and initiatestranscription. The operon model describes many gene systems inbacteria and viruses.* In eukaryotes, gene regulation is much more compex, manymechanisms are eukaryotic specific, the genetic switches are muchmore complicated, but the same general principles apply. Much aboutgene regulation remains unswered.Sources of Genetic Variation* Many of the differences between organisms are the results ofdifferences in the genes they carry. Genetic differences arise throughthe process of:o mutation: change in genetic material (subsitutions, deletions,insertions, translocations, etc...)o recombination: exchange of chromosomal material betweenhomologous chromosomes at meiosis.o selection: different genotypes contribute alleles to the nextgeneration in proportion to the selective advange they confer on those
organismns who possess them. Over long periods of time, this processleads to a change in frequencies of genes affecting certain traits.* All genetic variation ultimately is derived from mutation. As such,mutation provides the raw material for natural selection and hasimportant implications for the evolutionary process.Geneticists and Genetic Research* Virtually all areas of biology have been impacted by geneticresearch. Over the last 100 years geneticists have learned much abouthow life is organized at the molecular level. Along the way, tools weredeveloped that enabled direct manipulation and analysis of that whichgives form, the genetic material. These genetic tools are employed bygeneticis in such fields as evolution, ecology, behavior, cell biology,biochemistry, embryology, medicine, and increasingly biotechnology.* The success of genetic research is due to the rigorous use of thescientific approach (hypothetico-deductive method of investigation).o Involves making observations about the natural world, andgenerating hypotheses to explain observations. Hypothesis mustgenerate experimental predictions which can be tested undercontrolled conditions. The results can be used as further observationsleading to a refinement of hypothesis.o In most reseach, the exact path of a research project can not bepredicted. Its a highy interactive and dynamic process. Thats whatmakes it so interesting and motivating.Basic and Applied research* Research in general may either be basic or applied, although oftenthere is no sharp dividing line between the two. Both use similartechniques and depend on accumulated knowledge.* Basic research: experiments done with the goal of understandingfundamental phenomena. Most of the facts in the text come from basicresearch. This knowledge is largely used to fuel more basic research,but often leads to unanticipated avenues of knowledge which can leadto applied reseach. For example, it was basic research into howbacteria defend themselves against phage that lead to the discovery ofrestriction enzymes. These enzymes form the cornerstone ofrecombinant DNA technology, which has many practical benefits.
o Much basic research is often done on model organisms.* Applied research: experiments done with the goal of overcomingspecific problems in society or exploiting discoveries derived frombasic research. Searching for cures to diseases, producing improvedstrains of animals and plants for consumption are good examples.Virtually all applied research depend on the accumulated knowledgederived from basic research.Genetic databases* The amount of genetic information has grown exponentially in thelast couple of decades. Much of this information is stored in geneticdatabases and computers are indispensable in accessing and analyzingthis information. An important set of databases resides at the NationalCentre for Biotechnology Information (NCBI) athttp://www.ncbi.nlm.nih.gov, whose mandate is to "create publicdatabases, conduct research in computational biology , developsoftware tools for analyzing genome data, and disseminate biomedicalinformation-all for the better understanding of molecular processesaffecting human health and disease". A powerful feature of the NCBIdatabases is that they are interlinked. Some of the search toolsavailable at NCBI include:o Pubmed: access literature citation and abstract; has links toelectronic research articles. Very useful site for students going on inbiology.o OMIM (Online Mendelian Inheritance in Man): database of humangenes and genetic disorders.o BLAST: serach engine used to compare DNA or protein sequenceinfo with all sequences in database to find possible matches.o GenBank: a genetic sequence database containing all publiclyavailable protein and DNA sequences.o Entrez: system for searching several linked databases.Genetic Maps* A genetic map shows the relative locations of genes along achromosome as well as the distance between genes (Fig 1.6). Thereare different kinds of maps. The first genetic maps were calculatedfrom crosses by counting recombination frequencies.
* Genetic maps give us an understanding of the organization of genesalong chromosomes. They have also proven useful in cloning genesand as part of genome projects to obtain the complete sequences ofgenomes.Model organisms in genetics* DNA works virtually the same way in all organisms. What we learnfrom one organism can often be applied in principle to others. Manyadvances in human genetics made possible because of advances madein model organisms. Each model organism has its own strengths (ex.frogs make large eggs that are easy to observe and manipulate in thestudy of early embryonic development). They are selected becausethey lend themselves to the study of a particular question and arerepresentative of a larger group.* Many organisms are used in genetic research. Some of the qualitiesthat make an organism a good model for genetic experimentationinclude:o well known genetic history, allowing extrapolation to other relatedorganisms.o short life cycle.o matings produce large amount of offspring.o easy to handleo genetic variation must exist between individuals in a population.Without genetic variation, genetic crosses are uninformative.* Model organisms include representatives from all the main lineagesof the tree of life (Fig 1.7-10).o Eukaryote model organisms: Saccharomyces cereviceae, Drosophilamelanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Musmusculus, Zebra fish, Neurospora crassa, Tetrahymena, paramecium,Chlamydomonas reinhardtii, Pisum sativum, Zea maize, and Gallus.o Prokaryotic model organisms: Most common is E. coli, but manyothers as well, including members of Archaea.Biotechnology* Biotechnology is the application of genetic engineering tocommerce and it is poised to become one of the most profitableindustries of the coming decades. Biotechnology will be important in
areas such as:o Food Production+ modify nutritional content+ introduction of resistance genes+ herbicide resistance+ growth on marginal habitats+ better storage and processingo Industry+ production of strains producing mammalian substances (e.g.,insulin, human growth hormone, blood-clotting factors, etc...)+ production of enzymes, vitamins, solvents, antibiotics, etc...+ land reclamation, bioremediation, mining, oil spills, fuelso Medicine+ pharmacological drugs+ Large portion of human ill health has a genetic basis. Byunderstanding the disease at the molecular level, better treatments canbe designed based on a more targeted and rational approach to drugdiscovery.+ Many inherited genetic diseases are caused by abnormal forms ofsingle genes inherited through the gametes. Many such genes havebeen mapped on the human chromosomes (Fig 1-6) and theirnucleotide sequence is known. Sequence information can be used to# deduce structure and function of the encoded protein and henceunderstand the physiological effect caused by the mutation.# Develop DNA tests for clinical diagnosis# Genetic screening+ Gene therapy (normal gene inserted transgenically into cellscarrying the defective version)+ Human Genome Projecto Understanding the molecular genetic basis of other diseases, such ascancer, AIDS, and others, will undoubtedly also lead to more effectivetreatments.Ethical implications of genetic technologies* Like all technological advances, genetic technologies also bringswith it its own share of ethical dilemmas:o human cloningo animal cloning
o genetically modified foodso growth of spare body partso discrimination by insurance companies based on geneticinformation.o genetic pollution