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PowerPoint to accompany Genetics: From Genes to Genomes Fourth Edition Leland H. Hartwell, Leroy Hood, Michael L. Goldberg, Ann E. Reynolds, and Lee M. Silver Prepared by Mary A. Bedell University of Georgia Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition 1 PART IV How Genes Travel on Chromosomes CHAPTER Prokaryotic and Organelle Genetics CHAPTER OUTLINE 14.1 A General Overview of Bacteria 14.2 Bacterial Genomes 14.3 Gene Transfer in Bacteria 14.4 Bacterial Genetic Analysis 14.5 The Genetics of Chloroplasts and Mitochondria 14.6 Non-Mendelian Inheritance of Chloroplasts and Mitochondria 14.7 mtDNA Mutations and Human Health Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 2 Studies of bacteria were critical to the development of the field of genetics Classical bacterial genetics – 1940s to 1970s • Virtually all knowledge of gene structure, expression, and regulation came from studies of bacteria and bacteriophages Advent of recombinant DNA technology – 1970s and 1980s • Depended on understanding of bacterial genes, chromosomes and restriction enzymes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 3 Bacteria have adapted to a range of habitats Different habitats • On land, in aquatic environments, as parasites or symbionts inside other life-forms • Some bacteria cause hundreds of animal and plant disease Most are crucial to maintenance of earth's environment • Release oxygen to atmosphere • Recycle carbon, nitrogen, and other elements • Digest human and other animal waste • Neutralize pesticides and other pollutants • Produce vitamins and other materials essential to humans and other organisms Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 4 Bacteria sizes and characteristics Vast range of sizes • Smallest has 200 nm diameter • Largest is 500 μm in length All bacteria are prokaryotes, which lack a defined nuclear membrane All bacteria lack membrane-bounded organelles Bacterial chromosomes fold to form a nucleoid body that excludes ribosomes Most bacteria have a cell wall made of carbohydrate and peptide polymers that surrounds the cell membrane Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 5 Metabolic diversity of bacteria Play essential roles in many natural processes • Balance of microorganisms is key to success of ecological processes that maintain the environment Nitrogen cycling • Decomposing 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 plants • Denitrifying 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 use Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 6 Bacteria must be grown and studied in cultures Bacteria grown as a cell suspension in liquid media Bacteria grown as colonies on solid nutrient-agar in a petri dish Fig. 14.2 Fig. 14.1 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 7 Escherichia coli (E. coli) is a versatile model organism E. coli is the most studied and best understood bacterial species Inhabits intestines of warm-blooded animals Can grow in complete absence of oxygen or in air Lab strains are not pathogenic, but other strains can cause variety of intestinal diseases Prototrophic, can grow in minimal media • Single carbon and energy source (e.g. glucose) • Inorganic salts Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 8 Examples of phenotypic variation in bacteria due to mutations Altered colony morphology • Large or small; shiny or dull; round or irregular Resistance to bactericides • Antibiotics, bacteriophages Auxotrophs – unable to reproduce in minimal media • Defective 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 galactose Defective in proteins essential for growth • Conditional 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 9 Finding mutations in bacterial genes Rapid bacterial multiplication allows detection of very rare genetic events • In minimal media, bacteria divide every 60 min • In rich media, bacteria divide every 20 minutes Effectively haploid – straightforward relationship between mutation and phenotypic variation Selection – establish conditions in which only the desired mutant will grow • e.g. Select for streptomycin resistance (Strr) by plating on media containing streptomycin, select for prototrophic revertants by plating auxotrophs on minimal media Screen – examine each colony for a particular phenotype Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 10 Techniques used to identify rare mutants Spontaneous 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 cells • e.g. Penicillin kills only cells that are dividing but not cells that are unable to divide (Fig. 14.4) Testing for visible phenotypes • e.g. β-galactosidase from wild-type lacZ gene breaks down X-Gal substrate into a blue pigment Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 11 Penicillin enrichment for auxotrophic mutants Fig. 14.4 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 12 The typical bacterial genome is composed of one circular chromosome 4 to 5 Mb of DNA in most commonly studied bacterial species DNA molecule condenses by supercoiling and looping Each bacterium replicates and then divides by binary fission into two daughter cells Fig. 14.5 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 13 The E. coli genome was sequenced in 1997 Genome of K12 strain was sequenced 4.6 Mb ~ 90% of genome encodes protein 4288 genes, but function known for only 60% On average, 1 gene per kb No introns Very little repetitive DNA Small intergenic regions Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 Fig. 14.6 14 Insertion sequence (IS) elements dot the genomes of many types of bacteria Bacterial strains have different numbers and distributions of IS elements • e.g. 15-25 in E. coli, none in B. subtilis Small transposable elements (700-5000 bp length) • Inverted repeats (IRs) at ends • Carry transposase gene • Can move to other locations in genome • Can disrupt genes by insertion into coding regions (Fig 14.7b) Fig. 14.7a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 15 Tn 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 resistance Easily scored marker for genetic analysis (gene disruptions and mapping experiments) and for transferring a disrupted gene to another strain Fig. 14.8 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 16 Identifying a gene that was disrupted by insertion of a Tn element Fig. 14.9 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 17 Genomic analyses in bacteria have created an information explosion Complete genome sequence known for hundreds of prokaryotic species and partial genome sequence known for thousands of species New avenues of research are possible with genome studies • Metagenomics – analysis of genomic DNA from a community or habitat Microbial 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 species • Genome studies and public health – aid in development of vaccines, identify new drug targets, identify specific bacterial strains in epidemiological studies Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 18 New analyses for assessing microbial diversity Fig. 14.10 Bacteria in extreme environments are difficult to culture in lab Rapid 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 19 Plasmids are smaller circles of DNA that carry genes beneficial to the host cell Plasmids vary in size from 1 kb to several Mb in length Plasmids don't carry genes that are essential to the host Examples 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 antibiotics • Multiple 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 20 Some plasmids contain multiple antibiotic resistance genes and transposons Movement of antibiotic resistance genes to the plasmid was facilitated by transposons Multiple antibiotic resistance genes can be transposed from the plasmid as a unit Fig. 14.12 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 21 Gene transfer in bacteria Lateral (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 offspring Three mechanisms for gene transfer in bacteria (Fig. 14.13) • In all three mechanisms: Donor bacterium provides the DNA that is transferred Recipient bacterium receives the DNA, which can result in altered phenotype Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 22 Gene transfer in bacteria: An overview Fig. 14.13 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 23 In transformation, the recipient takes up DNA that alters its genotype Transformation – competent cells can take up DNA fragments from surrounding environment Natural transformation occurs in some bacterial species • e.g. B. subtilis, S. pneumoniae (Griffith's experiments, see Chapter 6), H. influenzae, N. gonorrhoeae • In B. subtilis, competence occurs only in nearly starved cells at specific times in growth culture 1% - 5% of cells become competent Artificial 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 24 Natural transformation in B. subtilis Selection for His+ and/or Trp+ is used to identify transformants Then, screen for His+ Trp+ cotransformants Genes close together have a higher frequency of co-transformation than genes that are further apart Fig. 14.14 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 25 Selecting and screening for transformation Selection and screening for gene transfer from His+ Trp+ donor to His− Trp− recipient: Fig. 14.14a To select for Trp+ transformants, plate on minimal media with histidine and no tryptophan To select for His+ transformants, plate on minimal media with tryptophan and no histidine To 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 26 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 conjugation Fig. 14.15 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 27 The F plasmid contains genes for synthesizing connections between donor and recipient cells Donors for conjugation are F+ (carry an F plasmid) Recipients for conjugation are F− (don't carry an F plasmid) Fig. 14.16a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 28 The process of conjugation Fig. 14.16b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 29 Formation of an Hfr chromosome F plasmid has three IS elements, which are identical to IS elements found at various positions on the bacterial chromosome High frequency recombinant (Hfr) cells are formed when an F plasmid integrates into the bacterial chromosome through recombination between IS elements Fig. 14.17 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 30 Different Hfr chromosomes 20-30 different Hfr strains can be generated that differ in the location and orientation of the integrated F plasmids Hfr strains retain all F plasmid functions and can be a donor for conjugation with an F− strain Fig. 14.18 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 31 Gene transfer between Hfr donors and F− recipients Transfer of DNA starts in the F plasmid at the origin of transfer Chromosomal genes located next to F plasmid sequences are transferred to the recipient Transferred chromosomal DNA recombines into homologous DNA in recipient Fig. 14.19 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 32 Interrupted-mating experiments with Hfr and F− strains Genes closest to origin of transfer in F plasmid are transferred first Order of transfer reflects the gene order on the chromosome Fig. 14.20 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 33 Mapping genes by interrupted-mating with Hfr and F− strains (a) Time of gene transfer (b) Map based on mating results Fig. 14.21 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 34 In transduction, a phage transfers DNA from a donor bacterium to a recipient bacterium Bacteriophages (aka phages) are viruses that infect, multiply in, and kill various species of bacteria • Widely distributed in nature • Most bacteria are susceptible to one or more phages Transduction – process by which a phage transfers DNA from one host cell to another host cell Virulent phages – always enter lytic cycle after infecting cell, multiply rapidly, and kill cell Temperate phages – can enter either lytic cycle or enter an alternative lysogenic cycle Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 35 The lytic cycle of phage multiplication Lytic cycle - results in cell lysis and release of progeny phage • Phage injects its DNA into bacterial cell • Phage proteins are expressed and take over protein synthesis and DNA replication machinery of infected cell • Phage DNA replication occurs • Phage particles are assembled with phage DNA and phage protein • Infected cell bursts (lyses) and releases 100-200 new viral particles able to infect other cells Lysate – the population of phage particles released from host bacteria at the end of the lytic cycle Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 36 Generalized transduction Incorporation of random fragments of bacterial DNA from donor into bacteriophage particles DNA from donor cell injected into infected recipient cell Transduced chromosomal DNA recombines into homologous DNA in recipient Fig. 14.22 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 37 Mapping genes by cotransduction frequencies ~90 kb of DNA (~ 2% of genome) can be transduced Frequency of cotransduction is higher for genes that are close together compared to genes that are further apart Fig. 14.23 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 38 Temperate phages Choice of lytic or lysogenic cycle (Fig 14.24) depends on many factors, including environmental conditions Lysogen – bacteria that harbor an integrated temperate phage Prophage – temperate phage that has integrated into host chromosome Bacteriophage lambda (λ) is the most commonly used temperate phage Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 39 Lytic and lysogenic modes of reproduction Prophages: Do not produce the viral proteins needed for more virus particles Lysogens can be induced to enter lytic cycle Fig. 14.24 • Prophage excises from chromosome, undergo replication, form new virus particles Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 40 Integration of the phage DNA initiates the lysogenic cycle Recombination between att sites on phage λ and the bacterial chromosome allows integration of the prophage Fig. 14.26a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 41 Excision of a prophage from a lysogen Abnormal excision produces a specialized transducing phage • Bacterial DNA adjacent to integration site can be packaged with viral DNA and then transferred to a recipient cell Fig. 14.26b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 42 Comparison of generalized and specialized transduction When donor DNA is packaged by phage: • Generalized transduction – during lytic cycle • Specialized transduction – during transition from lysogenic to lytic cycles Which donor DNA can be packaged with phage: • Generalized transduction – any bacterial gene or set of genes on the correct size of DNA fragment • Specialized transduction – only those bacterial genes near the integration site of the phage Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 43 Evolutionary implications of lateral gene transfer Genomic analysis has revealed widespread occurrence of gene transfer mechanisms in many bacterial species Gene transfer is an important mechanism for rapid adaptation to environmental changes and to development of pathogenic strains of bacteria • e.g. presence of diptheria toxin of Corynebacterium diphtheriae on a lysogenic phage Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 44 Genomic islands originated from transfer of foreign DNA Large segments of DNA (10-200 kb in size) • G+C content is different from the rest of the genome • Presence of direct repeats at each end • Found at sites where tRNAs genes are located • Contain integrase genes and sites for integration Pathogenicity islands are a subtype of genomic islands • Lateral transfer of a "package" of genes from a pathogenic species to a nonpathogenic species Fig. 14.27 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 45 Pathogenicity islands in Vibrio cholera Variation in genes present in pathogenicity islands of different strains of V. cholera Severity of cholera epidemic depends on genes present in the strain • Enterotoxin interferes with host-cell function • Invasion proteins for travel of bacterium through mucus of the intestinal tract • Pilus formation to allow phage attachment • Phage-related integrases Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 46 Pathogenicity islands in E. coli Integrative and conjugative elements (ICE) allow transfer of DNA between many different species • Have features of conjugative plasmids (like F factor), encodes an integrase (like lambda phage) and machinery for conjugation One pathogenic E. coli strain has an ICE that is similar to Yersinia pestis and Y. pseuodotuberculosis • Contains genes for mating pair formation, presumed origin of transfer, integrase for excision of the element E. coli strain O157:H7 – causes diarrhea or meningitis • Encodes proteins for attachment to epithelial cells, changes to cytoskeleton, loss of fluid, toxin from Shigella that damages kidneys and intestines Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 47 Bacterial genetic analysis Transposons allow manipulation of bacterial genomes • Useful mutagenic agents because they can disrupt genes and carry genes for antibiotic resistance Reverse genetics provides a way to insert synthetic genes to test function • Recombineering (Fig. 14.28) - replacement of a wild-type gene with a knockout gene through in vivo recombination Genomic and genetic approaches can be combined • Create large scale mutant library by random transposon mutagenesis, identify sites of insertion by PCR amplification and comparing sequence to genome sequence Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 48 Recombineering (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) (b) In vivo: Introduce DNA fragment into cells, induce recombination, and select for antibiotic resistance Fig. 14.28 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 49 Mitochondria and chloroplasts in eukaryotes have characteristics of prokaryotic cells Endosymbiotic theory – mitochondria and chloroplasts are descended from bacteria that fused with nucleated cells Mitochondria – organelles that produce energy for metabolic processes, found in all eukaryotic cells •Each cell has many mitochondria, highest number in cells with high energy requirements •Similar in size and shape to modern aerobic bacteria •Produces energy in the form of ATP Chloroplasts – organelles that capture energy from light and store it as carbohydrates, found in plant and algal cells •Structural similarities to certain cyanobacteria •40-50 per cells Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 50 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 cell mtDNA replication occurs independent of cell cycle • Random occurrence of replication – some mtDNAs replicate many times, and other mtDNAs don't replicate at all In most species, mtDNA is circular • Some species (Tetrahymena, Paramecium, Chlamydomonas, Hansenula) have linear mtDNA • Protozoan 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 51 The size and gene content of mitochondrial genomes varies from organism to organism Table 14.1 Table 14.2 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 52 Mitochondrial genome variation across species Mitochondrial genome in humans is very compact • Adjacent genes either abut each other or overlap slightly • Virtually no intergenic regions • No introns Mitochondrial genome of S. cerevisiae is 4X larger than in humans and other animals • Long intergenic regions • Has introns Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 53 Editing of RNA transcripts RNA 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-editing • RNA editing produces start and stop codons for translation as well as internal codons RNA editing also identified in some plants and fungi Fig. 14.31 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 54 Mitochondrial translation differs from translation of mRNAs from nuclear genes Similar aspects of translation in prokaryotes • Initiation of translation by N-formyl methionine and tRNAfMet • Translation in prokaryotes and mitochondria is inhibited by chemicals (e.g. chloramphenicol and erythromycin) that don't affect translation of nuclear mRNAs The genetic code for nuclear and mitochondrial genes is different Table 14.3 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 55 The genomes of chloroplasts (cpDNA) Includes genes for some photosynthetic enzymes and for gene expression Ranges 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 bacteria • RNA polymerases of choloroplasts and bacteria are similar • Translation in prokaryotes and chloroplasts is inhibited by chemicals (e.g. chloramphenicol and streptomycin) that don't affect translation of nuclear mRNAs Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 56 Potential uses of transformed chloroplasts Techniques for introducing genes into organelles • Gene gun – coat 1 μm metal particles with DNA and then "shoot" the DNA into cells • Biolistic transformation – DNA released from particle, enters nucleus or organelle, recombines into the genome Can alter properties of commercially important crop plants • Herbicide resistance Maternal 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 57 Nuclear and organellar genomes cooperate with each other Assembly and maintenance of functional organelles depend on both organelle and nuclear gene products • e.g. the 7 subunits of cytochrome c oxidase in most organisms are encoded by 3 mitochondrial genes and 4 nuclear genes Organelles don't carry all the genes needed for translation (semiautonomous) Fig. 14.32 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 58 Number and genomic location of oxidative phosphorylation genes Fig. 14.32 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 59 Gene transfer between organelles and the nucleus Evidence for transfer via an RNA intermediate • COXIII gene in plants – encodes component of mitochondrial electron transport chain • Present in the nuclear genomes of some plants but in the mitochondrial genomes of other plants • Some plant species have a non-functional mitochondrial gene that contains an intron and the functional nuclear gene doesn't have the intron Evidence for transfer at the DNA level • Some plant mtDNAs contain large fragments of cpDNA • Nonfunctional, partial copies of organellar genes are present in the nuclear genome Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 60 High rate of mutation in mtDNA mtDNA evolves ~10X more rapidly than does nuclear DNA • More errors in replication and less efficient repair Provides valuable tool for studying evolutionary relationships of closely-related species But, has little value for studying evolutionary relationships of distantly-related species Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 61 Non-Mendelian inheritance of organelles 1909 – green vs. variegated leaves in four-o'-clocks • Variegated offspring produced when ovules of variegated plants were fertilized with pollen from green plants • No variegated offspring produced when ovules of green plants were fertilized with pollen from variegated plants 1949 – 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 spores • Mating petite x petite produced petite diploids, but they were defective for respiration and couldn't sporulate • Mating grande x petite produced grande diploids, sporulation produced 4 grande spores and 0 petite spores Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 62 Maternal inheritance of Xenopus mtDNA Two closely-related species of frogs DNA probes from mtDNA were used to identify mtDNA present in F1 offspring Fig. 14.34 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 63 A maternally inherited neurodegenerative disorder in humans Leber's hereditary optic neuropathy (LHON) G-to-A substitution in gene for an NADH subunit causes Arg-to-His missense substitution Fig. 14.35 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 64 Distribution of organelles during mitosis Heteroplasmic cells contain a mixture of organelle genomes Homoplasmic cells contain only one type of organelle genome Mitotic progeny of homoplasmic cells are also homoplasmic Mitotic progeny of heteroplasmic cells can be either heteroplasmic, homoplasmic wild-type, or homoplasmic mutant Uneven distribution of organellar genomes has distinct phenotypic consequences Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 65 Some organisms exhibit biparental inheritance of organellar genomes 1909 – reciprocal crosses between green and variegated geraniums • Both types of crosses produces green, white, and variegated offspring in varying proportions • Chloroplast traits inherited from both parents Fig. 14.37 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 66 mtDNA mutations and human health Maternal pattern of inheritance Symptoms vary enormously among family members Myoclonic epilepsy and ragged red fiber disease (MERFFF) • Range of symptoms: uncontrolled jerking, muscle weakness, deafness, heart problems, kidney problems, progressive dementia • Mutations in mitochondrial tRNAs (e.g. tRNALys) • Disruption of mitochondrial transport chain • Individuals affected by MERFF are heteroplasmic • Severity 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 67 The proportion of mutant mitochondria determines the severity of the MERFF phenotype and the tissues affected Tissues with higher energy requirements are less tolerant of mutant mitochondria Tissues with low energy requirements are affected only when the proportion of wild-type mitochondria is greatly reduced Fig. 14.40 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 14 68 Mitochondrial mutations may have an impact on aging Oxidative phosphorylation system in the mitochondria generates free radicals, which can damage DNA Accumulation of mtDNA mutations over time may result in age-related decline in oxidative phosphorylation Evidence in support of role of mtDNA and aging: • Percentage of heart tissue with a mitochondrial deletion increases with age • Brain cells of people with Alzheimer’s disease (AD) have abnormally low energy metabolism • 20% 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 69