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1. Genetics of Virusesand Bacteria
2. Microbial Model Systems Viruses called bacteriophages can infect and set in motion a genetic takeover of bacteria, such as Escherichia coli
E. coli and phage model systems frequently use by researchers in studies that reveal broad biological principles
Viruses and bacteria have unique genetic mechanisms
3. Characteristics of Viruses Recall that bacteria are prokaryotes with cells much smaller and more simply organized than those of eukaryotes
Viruses are smaller and simpler still
Smallest viruses are only 20 nm in diameter
The virus particle, or virion, is just nucleic acid enclosed by a protein coat
4. Characteristics of Viruses A virus has a genome but can reproduce only within a host cell
Scientists detected viruses indirectly long before they could see them
The story of how viruses were discovered begins in the late 1800s
Tobacco mosaic disease stunts growth of tobacco plants and gives their leaves a mosaic coloration
In the late 1800s, researchers hypothesized that a particle smaller than bacteria caused the disease
In 1935, Wendell Stanley confirmed this hypothesis by crystallizing the infectious particle, now known as tobacco mosaic virus (TMV)
5. Characteristics of Viruses Viruses are very small infectious particles consisting of
Nucleic acid - genome
Protein coat which encloses the genome
And in some cases, a membranous envelope
Viral genomes may consist of
Double- or single-stranded DNA
Double- or single-stranded RNA
6. Capsids A capsid is the protein shell that encloses the viral genome, it can have various structures
May be rod-shaped, polyhedral or complex
Composed of capsomeres protein subunits; from one or a few types of proteins
Spikes or glycoproteins like the herpes shown
7. Membranous Envelope Some viruses have envelopes which are membranous coverings derived from the membrane of the host cell
Maybe a single layer or double layer envelope
Bilipid bilayer with glycoproteins spikes protruding from the outer layer
8. Membranous Envelope Many animal viruses have a membranous envelope
The membrane cloaks the viral capsid, helps viruses infect their host
Derived from host cell membrane which is usually virus-modified
Viral glycoproteins on the envelope bind to specific receptor molecules on the surface of a host cell
9. Bacteriophages Also called phages (T2, T4, T6) have the most complex capsids found among viruses
Icosohedral head encloses the genetic material; the protein tailpiece w/tail fibers attaches the phage to its bacterial host and injects its DNA into the bacterium
10. Viral Reproductive Cycles Although a virus has a genome it can only reproduce within a host cell
Viruses are obligate intracellular parasites
Each virus has a host range - a limited number of host cells that it can infect
Recognize host cells by a complementary fit between external viral proteins and specific cell surface receptor sites
Viruses use enzymes, ribosomes, and small molecules of host cells to synthesize progeny viruses
11. Viral Reproduction
12. Reproductive Cycles of Phages Phages are the best understood of all viruses
They through two alternative reproductive mechanisms: the lytic cycle and the lysogenic cycle
Lytic cycle - culminates in the death of the host
Lysogenic cycle - replicates the phage genome without destroying the host
13. The Lytic Cycle A phage reproductive cycle that culminates in the death of the host cell
Produces new phages and digests the hosts cell wall, releasing the progeny viruses
A phage that reproduces only by the lytic cycle is called a virulent phage
Bacteria have defenses against phages, including restriction enzymes that recognize and cut up certain phage DNA
14. The Lysogenic Cycle The lysogenic cycle replicates the phage genome without destroying the host
The viral DNA molecule is incorporated by genetic recombination into the host cells chromosome
This integrated viral DNA is known as a prophage
Every time the host divides, it copies the phage DNA and passes it to the daughter cells
Phages that use both the lytic and lysogenic cycles are called temperate phages
15. Viral Classification The nature of the genome is the basis for the common classification of animal viruses
16. 3 patterns of viral replication DNA ? DNA: If viral DNA is double-stranded, DNA replication resembles that of cellular DNA, and the virus uses DNA polymerase produced by the host.
RNA ? RNA: Since host cells lack the enzyme to copy RNA, most RNA viruses contain a gene that codes for RNA replicase, an enzyme that uses viral RNA as a template to produce complementary RNA.
RNA ? DNA ? RNA: Some RNA viruses encode reverse transcriptase, an enzyme that transcribes DNA from a RNA template.
17. RNA As Genetic Material - Retroviruses / Proviruses The broadest variety of RNA genomes is found among the viruses that infect animals
Retroviruses, such as HIV, use the enzyme reverse transcriptase to copy their RNA genome into DNA
The viral DNA that is integrated into the host genome is called a provirus
Unlike a prophage, a provirus remains a permanent resident of the host cell
18. The Reproductive Cycle Of An Enveloped RNA Virus The hosts RNA polymerase transcribes the proviral DNA into RNA molecules
The RNA molecules function both as mRNA for synthesis of viral proteins and as genomes for new virus particles released from the cell
19. The Reproductive Cycle Of HIV, A Retrovirus
20. Viral Diseases in Animals Viruses, viroids, and prions are formidable pathogens in animals and plants
Viruses may damage or kill cells by causing the release of hydrolytic enzymes from lysosomes
Some viruses cause infected cells to produce toxins that lead to disease symptoms
Emerging viruses are those that appear suddenly or suddenly come to the attention of medical scientists
Outbreaks of new viral diseases in humans are usually caused by existing viruses that expand their host territory
Severe acute respiratory syndrome (SARS) recently appeared in China
21. Bacterial Genetics Rapid reproduction, mutation, and genetic recombination contribute to the genetic diversity of bacteria
Bacteria allow researchers to investigate molecular genetics in the simplest true organisms
The bacterial chromosome is usually a circular DNA molecule with few associated proteins
In addition to the chromosome, many bacteria have plasmids, smaller circular DNA molecules that can replicate independently of the bacterial chromosome
22. Mutation and Genetic Recombination Since bacteria can reproduce rapidly new mutations can quickly increase a populations genetic diversity
Further genetic diversity can arise by recombination of the DNA from two different bacterial cells
23. Mechanisms of Gene Transfer and Genetic Recombination in Bacteria Three processes bring bacterial DNA from different individuals together
Transformation - Is the alteration of a bacterial cells genotype and phenotype by the uptake of naked, foreign DNA from the surrounding environment
Transduction - Phages carry bacterial genes from one host cell to another
Conjugation - Is the direct transfer of genetic material between bacterial cells that are temporarily joined
24. Transformation Transformation is the alteration of a bacterial cells genotype and phenotype by the uptake of naked, foreign DNA from the surrounding environment
For example, harmless Streptococcus pneumoniae bacteria can be transformed to pneumonia-causing cells
26. Conjugation and Plasmids Conjugation is the direct transfer of genetic material between bacterial cells that are temporarily joined
The transfer is one-way: One cell (male) donates DNA, and its mate (female) receives the genes
Maleness, the ability to form a sex pilus and donate DNA, results from an F (for fertility) factor as part of the chromosome or as a plasmid
Plasmids, including the F plasmid, are small, circular, self-replicating DNA molecules
27. The F Plasmid and Conjugation Cells containing the F plasmid, designated F+ cells, function as DNA donors during conjugation
F+ cells transfer DNA to an F? recipient cell
Chromosomal genes can be transferred during conjugation when the donor cells F factor is integrated into the chromosome
28. The F Plasmid and Conjugation A cell with a built-in F factor is called an Hfr cell
The F factor of an Hfr cell brings some chromosomal DNA along when transferred to an F cell
Thr transfer of part of the bacterial chromosome from an Hfr donor to an F recipient results in recombination
29. R plasmids and Antibiotic Resistance R plasmids confer resistance to various antibiotics
When a bacterial population is exposed to an antibiotic, individuals with the R plasmid will survive and increase in the overall population
30. Transposition of Genetic Elements The DNA of a cell can also undergo recombination due to movement of transposable elements within the cells genome
Can move around within a cells genome
Are often called jumping genes
Contribute to genetic shuffling in bacteria
31. Insertion Sequences The simplest transposable elements, called insertion sequences, exist only in bacteria
An insertion sequence contains a single gene for transposase, an enzyme that catalyzes movement of the insertion sequence from one site to another within the genome
32. Transposons Transposable elements called transposons are longer and more complex than insertion sequences
In addition to DNA required for transposition, transposons have extra genes that go along for the ride, such as genes for antibiotic resistance
33. Control of Gene Expression Every cell contains thousands of genes which code for proteins.
However, every gene is not actively producing proteins at all times.
To be expressed, a gene must be transcribed into m-RNA, the m-RNA must be translated into a protein, and the protein must become active.
Gene regulation can theoretically occur at any step in this process
34. Control of Gene Expression Two categories of gene regulation:
Transcriptional controls - factors that regulate transcription
Post-transcriptional controls factors that regulate any step in gene expression after transcription is complete
It is most efficient to regulate genes during transcription.
Both prokaryotes and eukaryotes rely primarily on transcriptional controls.
35. Regulating Prokaryotic Gene Expression Prokaryotes can quickly turn genes on and off in response to environmental conditions.
This metabolic control occurs on two levels
Adjusting the activity of metabolic enzymes already present
Regulating the genes encoding the metabolic enzymes
36. Response is facilitated by:
Simultaneous transcription and translation
Functionally related genes are often located next to each other and are transcribed as a unit.
For example E. coli,
5 different enzymes are needed to synthesize the amino acid tryptophan
The genes that code for these enzymes are located together
37. Operons: The Basic Concept In bacteria, genes are often clustered into operons, composed of
An operator, an on-off switch
Genes for metabolic enzymes
Is usually turned on
Can be switched off by a protein called a repressor
38. A single promoter serves all 5 genes. (region where RNA polymerase binds to DNA and begins transcription)
The genes are transcribed as a unit, - one long mRNA molecule which contains the code to make all 5 enzymes
39. There is also a single regulatory switch, called the operator.
The operator is positioned within the promoter, or between the promoter and the protein coding genes. It controls access of RNA polymerase to the genes.
40. Transcription of the 5 coding genes in the tryptophan operon is blocked when a transcriptional repressor binds to the operator.
The repressor binds to the operator only when there is a high level of tryptophan present: Prokaryotic Gene Regulation
41. Two Types of Negative Gene Regulation In a repressible operon, binding of a specific repressor protein to the operator shuts off transcription
Repressible enzymes usually function in anabolic pathways
In an inducible operon, binding of an inducer to a repressor inactivates the repressor and turns on transcription
Inducible enzymes usually function in catabolic pathways
42. Prokaryotic Gene Regulation: Inducible Operon The lactose operon in E. coli is an inducible operon
It controls the production of 3 enzymes needed to digest lactose (catabolism of a disaccharide made of glucose and galactose)
When lactose is absent, the repressor is active and the operon is off.
The lac repressor is innately active, and in the absence of lactose it switches off the operon by binding to the operator.
43. lac Operon If lactose is present, the repressor is inactivated and the operon is on
Allolactose, an isomer of lactose, turns on the operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced.
44. Positive Gene Regulation Regulation of both the trp and lac operons involves the negative control of genes, because the operons are switched off by the active form of the repressor protein
Some operons are also subject to positive control via a stimulatory activator protein, such as catabolite activator protein (CAP)
CAP (catabolite activator protein) stimulates transcription of genes that allow E. coli to use other food sources when glucose is not present such as lactose
45. Positive Transcriptional Control In E. coli, when glucose is scarce, the lac operon is activated by the binding of a regulatory protein, catabolite activator protein (CAP)
Low levels of glucose lead to high levels of cAMP
cAMP binds to CAP, CAP binds to CAP binding site, and transcription of lac mRNA is stimulated for catabolism of lactose
46. Positive Transcriptional Control When the glucose level is high, cAMP is low. CAP is not activated and transcription is not stimulated:
When glucose levels in an E. coli cell increase, CAP detaches from the lac operon, turning it off
47. Lab 9A/B - How Are Plasmids Used In Recombinant DNA Technology
48. Recombinant DNA Formed by joining DNA from 2 different individuals into a single molecule.
Various natural mechanisms can combine DNA from 2 individuals of the same species
Scientists have also developed techniques to combine DNA from any 2 individuals.
49. Two key enzymes are used to make artificially recombined DNA.
Restriction enzymes (also called restriction endonucleases) cut DNA into fragments so called molecular scissors
Each one recognizes and cuts DNA only where a specific sequence of base pairs occurs
A restriction enzyme will usually make many cuts in a DNA molecule yielding a set of restriction fragments
The most useful restriction enzymes cut DNA in a staggered way leaving unpaired bases at both ends.
These fragments are called sticky ends and can bond with complementary sticky ends of other fragments
DNA ligase is used to join DNA fragments together. This is the molecular glue
50. Procedure for Recombining DNA Isolate DNA from 2 different sources
Cut the DNA from both sources into fragments using the same restriction enzyme.
Mix the DNA fragments together. Since they were cut with the same restriction enzyme, fragments from different sources will have the same sticky ends and can pair up.
Use the enzyme DNA ligase to join the paired fragments together
51. Recombinant Plasmids Recombinant DNA technology can be used to create recombinant plasmids (or other agents such as viruses) used to insert foreign genes into recipient cells.
Plasmids (or other recombinant agents) used to insert foreign DNA into recipient cells are called vectors
Recombinant plasmids can then be used to produce multiple copies of the DNA fragment
52. Lab 9-A Transformation bacteria absorb fragments of DNA from surrounding media
Transform E. Coli with 3 unknown media samples
One solution contains no DNA at all
One solutuion contains normal pUC18 plasmid
Gene for ampicillin resistance
Lac Z gene which codes for ?-galactosidase, lactose digestion enzyme
One solution contains recombinant pUC18
Contains a fragment of foreign DNA from ? phage
Inserted in to middle of Lac Z gene, inactivating it
53. Transformation Procedure Add E. Coli to all three unknown solutions
Chill then heat shock samples to facilitate uptake of plasmid
Incubate then inoculate agar plates
Agar plates contain nutrients, ampicillin, Xgal (analog of lactose that release blue color when digested)
54. Using Restriction Enzyme EcoRI Procedure will cut the plasmids in the three unknown samples with the restriction enzyme EcoRI
Add EcoRI to the three unknown plasmid stock solutions and incubate
Separate the DNA fragments using gel electrophresis
Small fragments move faster farther
Similar to proteins except instead of MW we use base pairs (bp) to reference size
55. Plasmid pUC18 2686 base pairs in size
56. Plasmid pUC18 2686 base pairs in size