Genetics: From genes to genomes - Chapter 14: How genes travel on chromosomes

PowerPoint to accompanyGenetics: From Genes to GenomesFourth EditionLeland H. Hartwell, Leroy Hood, Michael L. Goldberg, Ann E. Reynolds, and Lee M. SilverPrepared by Mary A. BedellUniversity of Georgia*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th editionHow Genes Travel on Chromosomes*PART IVCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14Prokaryotic and Organelle Genetics14.1 A General Overview of Bacteria14.2 Bacterial Genomes14.3 Gene Transfer in Bacteria14.4 Bacterial Genetic Analysis14.5 The Genetics of Chloroplasts and Mitochondria14.6 Non-Mendelian Inheritance of Chloroplasts and Mitochondria14.7 mtDNA Mutations and Human HealthCHAPTER OUTLINECHAPTERStudies of bacteria were critical to the development of the field of geneticsClassical bacterial genetics – 1940s to 1970sVirtually all knowledge of gene structure, expression, and regulation came from studies of bacteria and bacteriophagesAdvent of recombinant DNA technology – 1970s and 1980sDepended on understanding of bacterial genes, chromosomes and restriction enzymesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Bacteria have adapted to a range of habitatsDifferent habitatsOn land, in aquatic environments, as parasites or symbionts inside other life-formsSome bacteria cause hundreds of animal and plant diseaseMost are crucial to maintenance of earth's environmentRelease oxygen to atmosphereRecycle carbon, nitrogen, and other elementsDigest human and other animal wasteNeutralize pesticides and other pollutantsProduce vitamins and other materials essential to humans and other organismsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Bacteria sizes and characteristicsVast range of sizesSmallest has 200 nm diameterLargest is 500 μm in lengthAll bacteria are prokaryotes, which lack a defined nuclear membraneAll bacteria lack membrane-bounded organellesBacterial chromosomes fold to form a nucleoid body that excludes ribosomesMost bacteria have a cell wall made of carbohydrate and peptide polymers that surrounds the cell membraneCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Metabolic diversity of bacteriaPlay essential roles in many natural processesBalance of microorganisms is key to success of ecological processes that maintain the environmentNitrogen cyclingDecomposing bacteria break down plant and animal matter and produce ammonia (NH3)Nitrifying bacteria use NH3 as source of energy and release nitrate (NO3), which is used by some plantsDenitrifying bacteria convert nitrate into atmospheric nitrogen (N2)Nitrogen-fixing bacteria live in roots of some plants and convert N2 to ammonium (NH4+) for their host plant to useCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Bacteria must be grown and studied in culturesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.1Fig. 14.2Bacteria grown as a cell suspension in liquid mediaBacteria grown as colonies on solid nutrient-agar in a petri dishEscherichia coli (E. coli) is a versatile model organismE. coli is the most studied and best understood bacterial speciesInhabits intestines of warm-blooded animalsCan grow in complete absence of oxygen or in airLab strains are not pathogenic, but other strains can cause variety of intestinal diseasesPrototrophic, can grow in minimal mediaSingle carbon and energy source (e.g. glucose)Inorganic saltsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Examples of phenotypic variation in bacteria due to mutationsAltered colony morphology Large or small; shiny or dull; round or irregularResistance to bactericides Antibiotics, bacteriophagesAuxotrophs – unable to reproduce in minimal mediaDefective in enzymes required to synthesize complex compounds (e.g. amino acids, nucleotides)Defective in using complex chemicals from the environment Example - breaking down lactose into glucose and galactoseDefective in proteins essential for growthConditional lethal mutations, e.g. temperature-sensitive (ts)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Finding mutations in bacterial genesRapid bacterial multiplication allows detection of very rare genetic eventsIn minimal media, bacteria divide every 60 minIn rich media, bacteria divide every 20 minutesEffectively haploid – straightforward relationship between mutation and phenotypic variationSelection – establish conditions in which only the desired mutant will growe.g. Select for streptomycin resistance (Strr) by plating on media containing streptomycin, select for prototrophic revertants by plating auxotrophs on minimal mediaScreen – examine each colony for a particular phenotypeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Techniques used to identify rare mutantsSpontaneous mutations in specific bacterial genes occur very rarely (1 in 106 to 1 in 108)Replica plating – simultaneous transfer of thousands of colonies from one plate to another (see Fig 7.5a)Mutagens – used to increase the frequency of mutations (see Fig 7.10)Enrichment – increases the proportion of mutant cellse.g. Penicillin kills only cells that are dividing but not cells that are unable to divide (Fig. 14.4)Testing for visible phenotypese.g. β-galactosidase from wild-type lacZ gene breaks down X-Gal substrate into a blue pigmentCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Penicillin enrichment for auxotrophic mutantsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.4The typical bacterial genome is composed of one circular chromosome4 to 5 Mb of DNA in most commonly studied bacterial speciesDNA molecule condenses by supercoiling and looping Each bacterium replicates and then divides by binary fission into two daughter cells Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.5The E. coli genome was sequenced in 1997Genome of K12 strain was sequenced4.6 Mb~ 90% of genome encodes protein4288 genes, but function known for only 60%On average, 1 gene per kbNo intronsVery little repetitive DNASmall intergenic regionsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.6Insertion sequence (IS) elements dot the genomes of many types of bacteriaBacterial strains have different numbers and distributions of IS elements e.g. 15-25 in E. coli, none in B. subtilisSmall transposable elements (700-5000 bp length)Inverted repeats (IRs) at endsCarry transposase geneCan move to other locations in genomeCan disrupt genes by insertion into coding regions (Fig 14.7b)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.7aTn elements in bacteria are composite transposable elements Contain transposase gene and genes conferring resistance to antibiotics or toxic metals (e.g. mercury) e.g. Tn10 – two different IS elements flank 7 kb of DNA that includes a gene for tetracycline resistanceEasily scored marker for genetic analysis (gene disruptions and mapping experiments) and for transferring a disrupted gene to another strainCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.8Identifying a gene that was disrupted by insertion of a Tn elementCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.9Genomic analyses in bacteria have created an information explosionComplete genome sequence known for hundreds of prokaryotic species and partial genome sequence known for thousands of speciesNew avenues of research are possible with genome studiesMetagenomics – analysis of genomic DNA from a community or habitatMicrobial ecology and communities - DNA sequencing of bacteria in extreme and unusual environment (Fig. 14.10)Comparative genome analysis – identify similarities and differences between genomes of different speciesGenome studies and public health – aid in development of vaccines, identify new drug targets, identify specific bacterial strains in epidemiological studiesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*New analyses for assessing microbial diversityBacteria in extreme environments are difficult to culture in labRapid DNA sequencing, large-scale PCR amplification, and DNA arrays can be used to survey composition of microbial communities and metabolic status Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.10Plasmids are smaller circles of DNA that carry genes beneficial to the host cellPlasmids vary in size from 1 kb to several Mb in lengthPlasmids don't carry genes that are essential to the hostExamples of plasmid genes that are beneficial to the host Genes that protect host against toxic chemicals (e.g. mercury) and metabolize environmental pollutants (e.g. toluene, napthalene, petroleum products)Pathogenic genes (e.g. toxins produced by S. dysenteriae)Genes encoding resistance to antibioticsMultiple antibiotic resistance often due to composite IS/Tn elements on a plasmid (see Fig. 14.12)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Some plasmids contain multiple antibiotic resistance genes and transposonsMovement of antibiotic resistance genes to the plasmid was facilitated by transposons Multiple antibiotic resistance genes can be transposed from the plasmid as a unitCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.12Gene transfer in bacteriaLateral (or horizontal) gene transfer – traits are introduced from unrelated individuals or from different species Vertical gene transfer occurs in sexually reproducing organisms – traits are transferred from parent to offspringThree mechanisms for gene transfer in bacteria (Fig. 14.13)In all three mechanisms:Donor bacterium provides the DNA that is transferredRecipient bacterium receives the DNA, which can result in altered phenotypeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Gene transfer in bacteria: An overviewCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.13In transformation, the recipient takes up DNA that alters its genotypeTransformation – competent cells can take up DNA fragments from surrounding environmentNatural transformation occurs in some bacterial speciese.g. B. subtilis, S. pneumoniae (Griffith's experiments, see Chapter 6), H. influenzae, N. gonorrhoeaeIn B. subtilis, competence occurs only in nearly starved cells at specific times in growth culture1% - 5% of cells become competentArtificial transformation can be accomplished in the lab by making the cells competent Treat cells with calcium to make the cell walls and membranes permeable to DNA or use electroporation Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Natural transformation in B. subtilisCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.14Selection for His+ and/or Trp+ is used to identify transformantsThen, screen for His+ Trp+ co-transformantsGenes close together have a higher frequency of co-transformation than genes that are further apartSelecting and screening for transformationTo select for Trp+ transformants, plate on minimal media with histidine and no tryptophanTo select for His+ transformants, plate on minimal media with tryptophan and no histidineTo screen for His+ Trp+ co-transformants, test Trp+ transformants and His+ transformants for growth on minimal media with neither tryptophan nor histidine Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.14aSelection and screening for gene transfer from His+ Trp+ donor to His− Trp− recipient:Demonstration of gene transfer by Joshua Lederberg and Edward Tatum (late 1940s)This type of gene transfer requires direct cell-to-cell contact and was later shown to be conjugationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.15The F plasmid contains genes for synthesizing connections between donor and recipient cellsDonors for conjugation are F+ (carry an F plasmid)Recipients for conjugation are F− (don't carry an F plasmid)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.16aThe process of conjugationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.16bFormation of an Hfr chromosomeF plasmid has three IS elements, which are identical to IS elements found at various positions on the bacterial chromosomeHigh frequency recombinant (Hfr) cells are formed when an F plasmid integrates into the bacterial chromosome through recombination between IS elementsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.17Different Hfr chromosomes20-30 different Hfr strains can be generated that differ in the location and orientation of the integrated F plasmidsHfr strains retain all F plasmid functions and can be a donor for conjugation with an F− strainCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.18Gene transfer between Hfr donors and F− recipientsTransfer of DNA starts in the F plasmid at the origin of transferChromosomal genes located next to F plasmid sequences are transferred to the recipientTransferred chromosomal DNA recombines into homologous DNA in recipientCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.19Interrupted-mating experiments with Hfr and F− strainsGenes closest to origin of transfer in F plasmid are transferred firstOrder of transfer reflects the gene order on the chromosomeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.20Mapping genes by interrupted-mating with Hfr and F− strainsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*(a) Time of gene transfer(b) Map based on mating resultsFig. 14.21In transduction, a phage transfers DNA from a donor bacterium to a recipient bacteriumBacteriophages (aka phages) are viruses that infect, multiply in, and kill various species of bacteriaWidely distributed in natureMost bacteria are susceptible to one or more phagesTransduction – process by which a phage transfers DNA from one host cell to another host cellVirulent phages – always enter lytic cycle after infecting cell, multiply rapidly, and kill cellTemperate phages – can enter either lytic cycle or enter an alternative lysogenic cycleCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*The lytic cycle of phage multiplicationLytic cycle - results in cell lysis and release of progeny phagePhage injects its DNA into bacterial cellPhage proteins are expressed and take over protein synthesis and DNA replication machinery of infected cellPhage DNA replication occursPhage particles are assembled with phage DNA and phage proteinInfected cell bursts (lyses) and releases 100-200 new viral particles able to infect other cellsLysate – the population of phage particles released from host bacteria at the end of the lytic cycleCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Generalized transductionIncorporation of random fragments of bacterial DNA from donor into bacteriophage particlesDNA from donor cell injected into infected recipient cellTransduced chromosomal DNA recombines into homologous DNA in recipientCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.22Mapping genes by cotransduction frequencies~90 kb of DNA (~ 2% of genome) can be transducedFrequency of cotransduction is higher for genes that are close together compared to genes that are further apartCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.23Temperate phagesChoice of lytic or lysogenic cycle (Fig 14.24) depends on many factors, including environmental conditionsLysogen – bacteria that harbor an integrated temperate phageProphage – temperate phage that has integrated into host chromosomeBacteriophage lambda (λ) is the most commonly used temperate phageCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Lytic and lysogenic modes of reproductionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.24Prophages:Do not produce the viral proteins needed for more virus particlesLysogens can be induced to enter lytic cycleProphage excises from chromosome, undergo replication, form new virus particlesIntegration of the phage DNA initiates the lysogenic cycleRecombination between att sites on phage λ and the bacterial chromosome allows integration of the prophageCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.26aExcision of a prophage from a lysogenAbnormal excision produces a specialized transducing phageBacterial DNA adjacent to integration site can be packaged with viral DNA and then transferred to a recipient cell Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.26bComparison of generalized and specialized transductionWhen donor DNA is packaged by phage:Generalized transduction – during lytic cycleSpecialized transduction – during transition from lysogenic to lytic cyclesWhich donor DNA can be packaged with phage:Generalized transduction – any bacterial gene or set of genes on the correct size of DNA fragmentSpecialized transduction – only those bacterial genes near the integration site of the phageCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Evolutionary implications of lateral gene transferGenomic analysis has revealed widespread occurrence of gene transfer mechanisms in many bacterial speciesGene transfer is an important mechanism for rapid adaptation to environmental changes and to development of pathogenic strains of bacteriae.g. presence of diptheria toxin of Corynebacterium diphtheriae on a lysogenic phageCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Genomic islands originated from transfer of foreign DNALarge segments of DNA (10-200 kb in size) G+C content is different from the rest of the genomePresence of direct repeats at each endFound at sites where tRNAs genes are locatedContain integrase genes and sites for integrationPathogenicity islands are a subtype of genomic islandsLateral transfer of a "package" of genes from a pathogenic species to a nonpathogenic species Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.27Pathogenicity islands in Vibrio choleraVariation in genes present in pathogenicity islands of different strains of V. choleraSeverity of cholera epidemic depends on genes present in the strainEnterotoxin interferes with host-cell functionInvasion proteins for travel of bacterium through mucus of the intestinal tractPilus formation to allow phage attachmentPhage-related integrases Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Pathogenicity islands in E. coliIntegrative and conjugative elements (ICE) allow transfer of DNA between many different speciesHave features of conjugative plasmids (like F factor), encodes an integrase (like lambda phage) and machinery for conjugationOne pathogenic E. coli strain has an ICE that is similar to Yersinia pestis and Y. pseuodotuberculosisContains genes for mating pair formation, presumed origin of transfer, integrase for excision of the elementE. coli strain O157:H7 – causes diarrhea or meningitisEncodes proteins for attachment to epithelial cells, changes to cytoskeleton, loss of fluid, toxin from Shigella that damages kidneys and intestinesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Bacterial genetic analysisTransposons allow manipulation of bacterial genomesUseful mutagenic agents because they can disrupt genes and carry genes for antibiotic resistance Reverse genetics provides a way to insert synthetic genes to test functionRecombineering (Fig. 14.28) - replacement of a wild-type gene with a knockout gene through in vivo recombinationGenomic and genetic approaches can be combinedCreate large scale mutant library by random transposon mutagenesis, identify sites of insertion by PCR amplification and comparing sequence to genome sequenceCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Recombineering(b) In vivo: Introduce DNA fragment into cells, induce recombination, and select for antibiotic resistanceCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.28(a) In vitro: Use recombinant DNA techniques to create a defective allele of a gene by insert of a selectable marker (e.g. antibiotic resistance) Mitochondria and chloroplasts in eukaryotes have characteristics of prokaryotic cellsEndosymbiotic theory – mitochondria and chloroplasts are descended from bacteria that fused with nucleated cellsMitochondria – organelles that produce energy for metabolic processes, found in all eukaryotic cellsEach cell has many mitochondria, highest number in cells with high energy requirementsSimilar in size and shape to modern aerobic bacteriaProduces energy in the form of ATPChloroplasts – organelles that capture energy from light and store it as carbohydrates, found in plant and algal cellsStructural similarities to certain cyanobacteria40-50 per cellsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*The genomes of mitochondria (mtDNA)Located within highly condensed structures (nucleoids)Number of nucleoids per mitochondria varies depending on growth conditions and energy needs of cellmtDNA replication occurs independent of cell cycleRandom occurrence of replication – some mtDNAs replicate many times, and other mtDNAs don't replicate at allIn most species, mtDNA is circularSome species (Tetrahymena, Paramecium, Chlamydomonas, Hansenula) have linear mtDNAProtozoan parasites have single mitochondrion (kinetoplast) with large network of minicircles and maxicircles (Fig. 14.30) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*The size and gene content of mitochondrial genomes varies from organism to organism Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Table 14.1Table 14.2Mitochondrial genome variation across speciesMitochondrial genome in humans is very compactAdjacent genes either abut each other or overlap slightlyVirtually no intergenic regionsNo intronsMitochondrial genome of S. cerevisiae is 4X larger than in humans and other animalsLong intergenic regionsHas intronsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Editing of RNA transcriptsRNA editing first discovered in mtDNA transcripts of trypanosomes (a protozoan parasite)Transcription of mtDNA produces pre-mRNA that is converted to mature mRNAs by RNA-editingRNA editing produces start and stop codons for translation as well as internal codonsRNA editing also identified in some plants and fungiCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.31Mitochondrial translation differs from translation of mRNAs from nuclear genesSimilar aspects of translation in prokaryotesInitiation of translation by N-formyl methionine and tRNAfMetTranslation in prokaryotes and mitochondria is inhibited by chemicals (e.g. chloramphenicol and erythromycin) that don't affect translation of nuclear mRNAsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Table 14.3The genetic code for nuclear and mitochondrial genes is differentThe genomes of chloroplasts (cpDNA)Includes genes for some photosynthetic enzymes and for gene expressionRanges in size from 120-217 kb, but most are 120-160 kb (see Table 14.4)Closely packed genes with little intergenic sequence (like human mtDNA) but has introns (like yeast mtDNA)Similarities to bacteriaRNA polymerases of choloroplasts and bacteria are similarTranslation in prokaryotes and chloroplasts is inhibited by chemicals (e.g. chloramphenicol and streptomycin) that don't affect translation of nuclear mRNAsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Potential uses of transformed chloroplastsTechniques for introducing genes into organellesGene gun – coat 1 μm metal particles with DNA and then "shoot" the DNA into cellsBiolistic transformation – DNA released from particle, enters nucleus or organelle, recombines into the genomeCan alter properties of commercially important crop plantsHerbicide resistanceMaternal inheritance (not through male pollen) limits risk of escape Turn chloroplasts into protein-production factories (i.e. for vaccines)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Nuclear and organellar genomes cooperate with each otherAssembly and maintenance of functional organelles depend on both organelle and nuclear gene productse.g. the 7 subunits of cytochrome c oxidase in most organisms are encoded by 3 mitochondrial genes and 4 nuclear genesOrganelles don't carry all the genes needed for translation (semiautonomous)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.32Number and genomic location of oxidative phosphorylation genesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.32Gene transfer between organelles and the nucleusEvidence for transfer via an RNA intermediateCOXIII gene in plants – encodes component of mitochondrial electron transport chainPresent in the nuclear genomes of some plants but in the mitochondrial genomes of other plantsSome plant species have a non-functional mitochondrial gene that contains an intron and the functional nuclear gene doesn't have the intronEvidence for transfer at the DNA levelSome plant mtDNAs contain large fragments of cpDNANonfunctional, partial copies of organellar genes are present in the nuclear genomeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*High rate of mutation in mtDNAmtDNA evolves ~10X more rapidly than does nuclear DNAMore errors in replication and less efficient repairProvides valuable tool for studying evolutionary relationships of closely-related speciesBut, has little value for studying evolutionary relationships of distantly-related speciesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Non-Mendelian inheritance of organelles1909 – green vs. variegated leaves in four-o'-clocksVariegated offspring produced when ovules of variegated plants were fertilized with pollen from green plantsNo variegated offspring produced when ovules of green plants were fertilized with pollen from variegated plants1949 – when S. cerevisiae grown on glucose, 95% of colonies were large (grande) and 5% were small (petite)Mating grande x grande produced grande diploid, sporulation produced 4 grande sporesMating petite x petite produced petite diploids, but they were defective for respiration and couldn't sporulateMating grande x petite produced grande diploids, sporulation produced 4 grande spores and 0 petite sporesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Maternal inheritance of Xenopus mtDNATwo closely-related species of frogsDNA probes from mtDNA were used to identify mtDNA present in F1 offspringCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.34A maternally inherited neurodegenerative disorder in humansLeber's hereditary optic neuropathy (LHON)G-to-A substitution in gene for an NADH subunit causes Arg-to-His missense substitutionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.35Distribution of organelles during mitosisHeteroplasmic cells contain a mixture of organelle genomesHomoplasmic cells contain only one type of organelle genomeMitotic progeny of homoplasmic cells are also homoplasmicMitotic progeny of heteroplasmic cells can be either heteroplasmic, homoplasmic wild-type, or homoplasmic mutantUneven distribution of organellar genomes has distinct phenotypic consequencesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Some organisms exhibit biparental inheritance of organellar genomes1909 – reciprocal crosses between green and variegated geraniumsBoth types of crosses produces green, white, and variegated offspring in varying proportionsChloroplast traits inherited from both parentsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.37mtDNA mutations and human healthMaternal pattern of inheritanceSymptoms vary enormously among family membersMyoclonic epilepsy and ragged red fiber disease (MERFFF) Range of symptoms: uncontrolled jerking, muscle weakness, deafness, heart problems, kidney problems, progressive dementiaMutations in mitochondrial tRNAs (e.g. tRNALys)Disruption of mitochondrial transport chainIndividuals affected by MERFF are heteroplasmicSeverity of phenotype depends on percentage of mutant mtDNA (see Fig. 14.39 and 14.40)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*The proportion of mutant mitochondria determines the severity of the MERFF phenotype and the tissues affectedTissues with higher energy requirements are less tolerant of mutant mitochondriaTissues with low energy requirements are affected only when the proportion of wild-type mitochondria is greatly reducedCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*Fig. 14.40Mitochondrial mutations may have an impact on agingOxidative phosphorylation system in the mitochondria generates free radicals, which can damage DNAAccumulation of mtDNA mutations over time may result in age-related decline in oxidative phosphorylationEvidence in support of role of mtDNA and aging:Percentage of heart tissue with a mitochondrial deletion increases with ageBrain cells of people with Alzheimer’s disease (AD) have abnormally low energy metabolism20% to 35% of mitochondria in brain cells of most AD patients have mutations in cytochrome c oxidase genes, which may explain the low energy metabolism Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14*