1. Genetics: From Genes to Genomes
Powerpoint to accompany
Genetics: From Genes to Genomes
Third Edition
Hartwell ● Hood ● Goldberg ● Reynolds ● Silver ● Veres
Chapter 15
Prepared by Malcolm Schug
University of North Carolina Greensboro
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2. The Prokaryotic Chromosome: Genetic Analysis in Bacteria
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3. Outline of Chapter 15 General overview of bacteria
Range of sizes
Metabolic activity
How to grow them for study
The bacterial genome
Structure
Organization
Transcription
Replication
Evolution of large, circular chromosomes
Structure and function of small circular plasmids
Gene transfer in bacteria
Transformation
Conjugation
Transduction
A comprehensive example
Genetic tools to dissect bacterial chemotaxis
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4. General overview of bacteria
One of the three major lineages of life
Eukaryotes – organisms whose cells have encased nuclei
Prokaryotes – lack a nuclear membrane
Archea
1996 complete genome of Methanococcus jannaschii sequenced
More than 50% of genes completely different than bacteria and eukaryotes
Of those that are similar, genes for replication, transcription, and translation are same as eukaryotes.
Genes for survival in unusual habitats similar to some bacteria
Bacteria
Similar genome structure, morphology, and mechanisms of gene transfer to Archea
Evolutionary biologist believe earliest single celled organism, probably prokaryote, existed 3.5 billion years ago.
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5. A family tree of living organisms
Figure 15.1
Fig. 15.1
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6. Diversity of bacteria Outnumber all other organisms on Earth
10,000 species identified
Smallest – 200 nanometers in diameter
Largest – 500 micrometers in length (10 billion times larger than the smallest bacteria)
Habitats range from land, to aquatic, to parasitic.
Remarkable metabolic diversity allows them to live almost anywhere.
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7. Common features of bacteria
Lack defined nuclear membrane
Lack membrane bound organelles
Chromosomes fold to form a nucleoid body.
Membrane encloses cells with mesosome which serves as a source of new membranes during cell division.
Most have a cell wall.
Mucus-like coating called a capsule
Many move by flagella.
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8. Table 15.1
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9. Power of bacterial genetics is the potential to study rare events.
Bacteria multiply rapidly.
Liquid media – E. coli grow to concentration of 109 cells per milliliter within a day.
Agar media – single bacteria will multiply to 107 – 108 cells in less than a day.
Most studies focus on E. coli.
Inhabitant of intestines in warm blooded animals
Grows without oxygen
Strains in laboratory are not pathogenic.
Prototrophic – makes all the enzymes it needs for amino acid and nucleotide synthesis
Grows on minimal media containing glucose as the only carbon source
Divides about once every hour in minimal media and every 20 minutes in enriched media
Rapid multiplication makes it possible to observe very rare genetic events.
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10. The bacterial genome is composed of one circular chromosome.
4-5 Mb long
Condenses by supercoiling and looping into a densely packed nucleoid body
Chromosomes replicate inside cell and cell divides by binary fission.
Figure 15.4 b
Fig b
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11. E. coli lysed to release chromosome
Figure 15.4 a
Fig a
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12. How to find mutations in bacterial genes
Mutations affecting colony morphology
Mutations conferring resistance to antibiotics or bacteriophages
Mutations that create auxotrophs
Mutations affecting the ability of cells to break down and use complicated chemicals in the environment
Mutations in essential genes whose protein products are required under all conditions of growth
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13. How to identify mutations by a genetic screen
Genetic screens provide a way to observe mutations that occur very rarely such as spontaneous mutations (1 in 106 to 1 in 108 cells).
Replica plating – simultaneous transfer of thousands of colonies from one plate to another
Treatments with mutagens – increase frequency of mutations
Enrichment procedures – increase the proportion of mutant cells by killing wild-type cells
Testing for visible mutants on a petri plate
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14. Bacteria nomenclature
wild-type – ‘+’
mutant gene – ‘-’
three lower case, italicized letters – a gene (e.g., leu+ is wild-type leucine gene)
The phenotype for a bacteria at a specific gene is written with a capital letter and no italics (e.g., Leu+ is a bacteria that does not need leucine to grow, and Leu- is a bacteria that does need leucine to grow.)
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15. Structure and organization of E. coli chromosome
4.6 million base pairs
open reading frames (ORFs)
90% of genome encodes protein (compare that to humans!)
4288 genes, 40% of which we do not know what they do
almost no repeated DNA
427 genes have a transport function, other classes also identified
bacteriophage sequences found in 8 places (must have been invaded by viruses at least 8 times during history.
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16. Insertion sequences dot the E. coli chromosome.
Transposable elements place DNA sequences at various locations in the genome.
Geneticists use transposable elements to insert DNA at various locations in bacterial genomes.
If you were to insert a piece of DNA into a bacterial genome using a transposable element, can you think of a molecular method that you could use to find out which gene you inserted the DNA into?
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17. Transposable elements in bacteria
Figure 15.6 a, b,c
Fig. 15.6
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18. Transcription in bacteria
Transcription machinery moves clockwise.
Different strands code for different genes.
Several genes may be transcribed in one segment.
RNA polymerase may transcribe adjacent genes at the same time in a counterclockwise direction.
Highly transcribed genes generally oriented in direction of replication fork movement
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19. DNA replication in E. coli
Figure 15.7
Fig. 15.7
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20. Plasmids: smaller circles of DNA that do not carry essential genes
Plasmids vary in size ranging from 1kb – 3 Mb.
Plasmids can carry genes that confer resistance to antibiotics and toxic substances.
Plasmids are not needed for reproduction or normal growth, but they can be beneficial.
Plasmids can carry genes from one bacteria to another. Bacteria can thus become resistant to a drug, put the resistance gene in the plasmid, and transfer it to other bacteria. This transfer of plasmid DNA can even occur across species.
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21. Some plasmids contain multiple antibiotic resistance genes.
Figure 15.8 b
Figure 15.8 b
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22. Gene Transfer in Bacteria
Figure 15.9
Fig. 15.9
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23. Transformation
Fragments of donor DNA enter the recipient and alter its genotype.
Natural transformation – recipient cell has enzymatic machinery for DNA import
Artificial transformation – damage to recipient cell walls allows donor DNA to enter cells
Treat cells by suspending in calcium at cold temperatures
Electroporation – mix donor DNA with recipient bacteria and subject to very brief high-voltage shock
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24. Mechanism of natural transformation
Figure 15.10
Fig
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25. Conjugation – A type of gene transfer requiring cell-to-cell contact
Figure 15.11
Fig
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26. The F plasmid and conjugation
Figure a
Fig a
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27. The process of conjugation
Figure b
Figure b
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28. The F plasmid occasionally integrates into the E. coli chromosome.
Hfr cells have integrated part of chromosome.
Episomes – plasmids that can integrate into host chromosome
Exconjugate – recipient cell with integrated DNA
Integrated plasmid can initiate DNA transfer by conjugation, but may take some of bacterial chromosome as well.
Figure 15.13
Fig
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29. Gene transfer in a mating between Hfr donor and F- recipient
Figure 15.14
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Fig

30. Mapping genes in Hfr and F- crosses by interrupted mating experiments
Figure 15.15
Figure 15.15
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31. Interrupted mating studies confirm bacterial chromosome in a circle.
Cross between Hfr and F-
The F plasmid integrates into different locations in different orientations into the circular donor chromosome.
Figure a, b
Fig a, b
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32. Partial genetic map of the E. coli chromosome
Figure c
Fig c
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33. Recombination analysis improves accuracy of map
Interrupted mating experiments accurate to only 2 minutes
Frequency of recombination between genes is more accurate.
Start by considering only exconjugates that have all of the genes to be mapped (select for the last gene transferred).
Living cells must have even number of crossovers.
Consider as a three-point cross
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34. Mapping genes using a three-point cross
Figure a, b
Fig a-b
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35. F’ plasmids can be used for complementation studies.
F’ plasmids replicate as discrete circles of DNA inside host cells.
Transferred in same manner as F’ plasmids
A few chromosomal genes will always be transferred as part of the F’ plasmid
Can create partial diploids
Merizygotes – partial diploids in which two gene copies are identical
Heterogenotes – partial diploids carrying different alleles of the same gene
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36. Different classes of crossovers: quadruple crossover is least frequent
Figure c
Fig c
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37. F’ plasmid formation and transfer
Figure a, b
Fig a, b
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38. Complementation testing using F’ plasmids
Creation of a heterogenote
Phenotype of partial diploid establishes whether mutations complement each other or not.
Figure c
Fig c
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39. Table 15.2
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40. Transduction: Gene transfer via bacteriophages
Widely distributed in nature
Infect, multiply, and kill bacterial host cells
Transduction - may incorporate some of bacterial chromosome into its own chromosome and transfer it to other cells
Bacteriophage particles are produced by the lytic cycle.
Phage inject DNA into cell.
Phage DNA expresses its genes in host cell and replicate.
Reassemble into new phage particles
Cells lyse and phage infect other cells.
Lysate is population of phage after lytic cycle is complete.
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41. Generalized transduction
Figure 15.19
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Fig

42. Mapping genes by generalized transduction
Frequency of recombination between genes
P1 bacteriophage often used for mapping
90kb can be transduced corresponding to about 2% recombination or 2 minutes.
First find approximate location of gene by mating mutant strain to different Hfr strains.
P1 transduction then used to map to specific location
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43. Figure 15.20
Fig
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44. Temperate phage can integrate into bacterial genome through lysogenic cycle creating a prophage.
Figure 15.21
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Fig

45. Recombination between att sites on the phage and bacterial chromosomes allows integration of the prophage.
Figure b
Fig b
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46. Errors in prophage excision produce specialized transducing phage.
Adjacent genes are included in circular phage DNA that forms after excision.
Figure c
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Fig c

47. The isolation of mutants is the geneticist’s top priority.
Transposons create mutations.
Insertions into genes result in inactivation.
Used as mutagenic agents
Also contain antibiotic resistance selectable genes
Genetic screen using transposons
Introduce transposon into cell by transformation, transduction, conjugation.
Select for cells in which transposition has occurred.
Screen population of cells for mutant phenotype.
Locate position of transposon on chromosome.
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48. Mapping mutations Transposon insertion mapping
Identify mutant induced by transposition.
PCR amplify using primers in part of transposon and part of DNA.
Sequence PCR product and compare with E. coli genome database.
Hfr mapping of mutations not induced by transposons
Hfr strains with origins of transfer at different regions of chromosome, and a Tn 10 transposon for tetracycline resistance
Exconjugates screened for tetracycline resistance
Map location near the origin of transfer in a strain with loss of mutant phenotype
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49. Reverse genetics to determine function of unknown gene
Knockout mutation of mutant gene using recombinant DNA technology and homologous recombination in cell
Figure 15.23
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Fig

50. Genetic dissection helps explain how bacteria move.
How bacteria move to achieve chemotaxis
Straight run and tumble in a random walk
Addition of attractant or repellent causes biased random walk.
Figure 15.24
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Fig

51. Counterclockwise movement is achieved when flagella bundle.
Tumble is achieved when flagella are not bundled.
Figure 15.25
Fig
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52. Isolating bacterial mutants that cannot move towards food
Figure 15.26
Fig
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53. Bacterial mutants Flagellum mutants Motor mutants
More than 20 fla genes are required to generate a flagellum. Mutants prevent production of functional flagella.
Motor mutants
Mot genes are required to turn the flagellum. Mutants are paralyzed.
Signal transduction mutants
Che (chemotaxis) mutants have flagella that move only in one direction
Mutants prevent proper relay of messages from cell surface to motor where frequency and direction of rotation takes place.
Receptor mutants
Mutants in receptors that bind particular chemicals
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54. Bacteria have cell surface receptors that recognize particular attractants or repellents.
Figure 15.29
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Fig

55. The molecular basis of bacterial chemotaxis
Figure 15.30
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Fig

56. Genome analysis provides powerful new tools for understanding bacteria.
Comparative genome analysis can help dissect the genetic basis of bacterial behavior.
Examination and comparison of different species genomes
Evidence for the hypothesis that lateral gene transfer introduced disease causing genes into pathogenic species
Genomic analysis may help protect human health.
Identification of vaccine candidates
Aid the discovery of new drug targets
Provide new avenues for identification of bacterial strains; will provide a mechanism to trace the history of the infection
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57. Genome analysis provides powerful new tools for understanding bacteria.
Microbial ecology benefits from genomic methodologies.
Technology tools such as rapid sequencing, PCR, and DNA arrays provide a means to study composition of microbial communities previously difficult to study.
Bacteria in extreme environments that are difficult to culture in the lab
Studies of metabolic diversity in random DNA isolated from seawater reveal vast metabolic diversity among ocean microbes.
Complete genome sequencing of poorly understood bacteria that are major contributors to global nutrient cycles
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