bacterial genetics
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Bacterial Genetics. Overview. Two general mechanisms of genetic change in bacteria: Mutation - alteration in existing DNA sequence DNA transfer - acquisition of DNA from another source. Spontaneous Induced (caused by mutagens). Overview. - PowerPoint PPT PresentationTRANSCRIPT
OverviewTwo general mechanisms of genetic change in bacteria:
Mutation - alteration in existing DNA sequence
DNA transfer - acquisition of DNA from another source
SpontaneousInduced (caused by mutagens)
OverviewTwo general mechanisms of genetic change in bacteria:
Mutation - alteration in existing DNA sequence
DNA transfer - acquisition of DNA from another source
SpontaneousInduced (caused by mutagens)
Why study bacterial genetics?Model system
•Spontaneous mutations occur in all cells at a very low frequency (≈one per billion nucleotides)
•Bacteria quickly grow to high concentrations (109/ml) in culture, making it possible to study rare occurrences
•Test chemicals for potential carcinogens
Understand bacterial adaptation•Resistance to antimicrobial drugs
Agency Urges Change in Antibiotics for Gonorrhea
By LAWRENCE K. ALTMANPublished: NY Times April 13, 2007
The rates of drug-resistant gonorrhea in the United States have increased so greatly in the last five years that doctors should now treat the infection with a different class of antibiotics, the last line of defense for the sexually transmitted disease, officials said yesterday…..
No new antibiotics for gonorrhea are in the pipeline, officials of the centers told reporters by telephone.
“Now we are down to one class of drugs,” said Dr. Gail Bolan, an expert in sexually transmitted diseases at the California Department of Health Services. “That’s a very perilous situation to be in.”
OverviewTwo general mechanisms of genetic change in bacteria:
Mutation - alteration in existing DNA sequence
DNA transfer - acquisition of DNA from another source
SpontaneousInduced (caused by mutagens)
Why study bacterial genetics?Model system
•Spontaneous mutations occur in all cells at a very low frequency (≈one per billion nucleotides)
•Bacteria quickly grow to high concentrations (109/ml) in culture, making it possible to study rare occurrences
•Some mutagens are carcinogens
Understand bacterial adaptation•Resistance to antimicrobial drugs•Acquisition of disease-causing traits
Terms
Phenotype - the observable characteristics of an organism
Genotype - the sequence of nucleotides in the DNA of an organism
Prototroph His- auxotrophdisrupt gene required for histidine synthesis
Wild type - characteristics similar to the organism as it occurs in nature. Prototroph - requires the same nutrients as the wild type.
Auxotroph - a strain that has lost the ability to synthesize a specific compound; as a consequence, that compound must be supplied as a nutrient in the growth medium.
When studying mutations, you only see what you look for
Part I Mutation
•How we can select (and therefore, study) mutants
•How cells can repair errors/damage
•How mutations occur, and their consequences
Spontaneous Mutation
Mistakes during replicationBase substitution
TGGtryptophan
TGCcysteine
TGAStop codon
TGTcysteine
No consequence Consequence varies Truncated protein; generally non-functional
Silent mutation Missense mutation Nonsense mutation
Spontaneous Mutation
Mistakes during replicationBase substitutionRemoval or addition of nucleotides
TGTTTGACCTAGGT
Spontaneous Mutation
Mistakes during replicationBase substitutionRemoval or addition of nucleotides
TGTTTGACCTAGGT
TGTTGACCTAGGT
TGT TTG ACC TAG GT
TGT TGA CCT AGG T
Frameshift mutation•Generates an entirely different set of triplets•Often, a stop codon is generated
Spontaneous Mutation
Transposons
• Insertional inactivation of the gene in which the transposon lands• A transposon can insert elsewhere in the same DNA molecule, or
into an entirely different DNA molecule • Some transposons simply “hop”; others replicate then hop
“jumping genes”
Mutationsspontaneous
mistakes during replicationbase substitutionaddition/removal of nucleotides
transposable elements
Summary
induced
Induced Mutation
Alter the base-pairing properties
Chemical mutagens (potential carcinogens)Chemicals that modify purines and pyrimidines
Induced Mutation
Example: nitrous acid strips the amino group from nucleotides
:G :A
Alter the base-pairing properties
Chemical mutagensChemicals that modify purines and pyrimidines
Induced Mutation
Resemble nucleotide bases; erroneously incorporated into DNA
Example: nitrous acid strips the amino group from nucleotidesBase analogs
Alter the base-pairing properties
Chemical mutagensChemicals that modify purines and pyrimidines
Analog base-pairs with a different nucleotide
T C
Induced Mutation
Intercalating agents Insert between base-pairs, pushing nucleotides apart; extra nucleotide may then be erroneously added during replication
Analog base-pairs with a different nucleotideResemble nucleotide bases; erroneously incorporated into DNA
Example: nitrous acid strips the amino group from nucleotidesBase analogs
Alter the base-pairing properties
Chemical mutagensChemicals that modify purines and pyrimidines
Induced MutationTransposons
Intentional use of an agent that naturally creates spontaneous mutations
Ultraviolet irradiationCauses formation of covalent bonds (thymine dimers) between adjacent thymine bases
Distorted DNA can be repaired, but the process (SOS repair) may introduce errors
High doses are used to sterilize surfaces, lower doses to introduce mutations
X raysCauses double- and single-stranded breaks in DNA
Induced Mutation
Radiation
Mutationsspontaneous
mistakes during replicationbase substitutionremoval or addition of nucleotides
transposable elementsinduced
chemical mutagensradiationtransposons
Summary
Repair of errors in base incorporationDNA polymerase
proofreadingMismatch repair
excision/replacement
DNA Repair
Repair of thymine dimmers
DNA Repair
Repair of thymine dimmers
Repair of errors in base incorporationDNA polymerase
proofreadingMismatch repair
excision/replacement
Light reactivation (photorepair)
DNA RepairRepair of errors in base incorporation
DNA polymeraseproofreading
Mismatch repairexcision/replacement
Repair of thymine dimmersLight reactivation (photorepair)Excision repair (dark repair; light-independent repair)
DNA Repair
Repair of Modified BasesGlycosylase removes oxidized guanine
SOS repair Induction of SOS system
New polymerase (tolerates “slop”)
Repair of errors in base incorporationDNA polymerase
proofreadingMismatch repair
excision/replacement
Repair of thymine dimersLight reactivation (photorepair)Excision repair (dark repair; light-independent repair)
Mutant Selection
Direct selectionObtain resistant mutants (ex. antibiotic resistant)
Obtain prototrophs that have reverted from auxotrophs Auxotrophs
Prototroph (revertant)
Enriched complexmedium
Minimal medium (glucose-salts)
Application of direct selectionAmes Test - screens for mutagens(used to narrow down list of possible carcinogens)
Mutationsspontaneous
mistakes during replicationtransposons
inducedchemical mutagensradiationtransposons
Repairrepair of errors in base incorporationrepair of thymine dimmersSOS repair
Selecting mutantsdirect - obtain antibiotic resistant mutants, Ames testindirect - obtain prototrophs
Summary
DNA Transfer
1920s; Frederick Griffith- strains of Streptococcus pneumoniae that produce capsules kill mice
“transforming principle”(DNA)
DNA Transfer
To be stably maintained, transferred DNA must either be a plasmid (has an origin of replication), or integrate into the host cell’s genome
Recipient Donor
DNA Transfer
To be stably maintained, transferred DNA must either be a plasmid (has an origin of replication), or integrate into the host cell’s genome
Recipient Donor
DNA Transfer
Donor Recipient
Integrate into host genome byHomologous recombination (site-specific recombination)
DNA Transfer
Donor Recipient
Integrate into host chromosome byHomologous recombination (site-specific recombination)
DNA Transfer
Donor Recipient
Integrate into host chromosome byHomologous recombination (site-specific recombination)
DNA Transfer
Donor Recipient
Integrate into host chromosome byHomologous recombination (site-specific recombination)
replication
heteroduplex
DNA Transfer
Donor Recipient
Integrate into host chromosome byHomologous recombination (site-specific recombination)
DNA Transfer
Donor Recipient
A+, B- A-, B+
A+, B+
A-, B-
A+, B-
B- A-
Horizontal (lateral) gene transfer
DNA-Mediated Transformation
Recipient cell must be competent
Uptake of naked DNA
Observed in only certain speciesExample - Streptococcus pneumoniae (GPC)
Example - Haemophilus influenzae (GNR)
Natural competence
Artificial competenceIn the laboratory, treat cells with specific chemicals
Process is sensitive to the addition of DNAse
(plasmids taken up)
•Becomes competent in late log phase•Competent cell binds ds DNA•Enzymes cut DNA into smaller fragments (5 - 15 kb)•Single strand is taken up by cell
•Cell binds DNA only from related species•Takes up ds DNA
Conjugation
Requires cell-to-cell contactInvolves a conjugative plasmid
F plasmid (fertility plasmid) serves as a modelThree types of donors:
F+
HfrF’
In donor cell, replication replaces strand that’s being transferred
In recipient cell, complement to transferred strand is synthesized
Conjugation: F+ donor
F+ + F- F+ + F+
In donor cell, replication replaces strand that’s being transferred
In recipient cell, complement to transferred strand is synthesized
Note: some R plasmids (encode resistance to one or more antibiotics) are conjugative
Conjugation: F+ donor
Conjugation: Hfr donor
•Some F plasmid DNA is transferred first, followed by chromosomal DNA
• In donor cell, replication replaces strand that’s being transferred
• In recipient cell, complement to transferred strand is synthesized
Cells inevitably separate before entire
chromosome is transferred
Conjugation: Hfr donor
•Some F plasmid DNA is transferred first, followed by chromosomal DNA
• In donor cell, replication replaces strand that’s being transferred
• In recipient cell, complement to transferred strand is synthesized
Cells inevitably separate before entire
chromosome is transferred
Conjugation: Hfr donor
Hfr + F- Hfr + F-
Significance of Hfr strains:•Chromosomal transfer•Allowed mapping of E. coli chromosome
Fundamental Tools Used in Biotechnology
•Restriction Enzymes - used to cut DNA at specific sequences•Gel Electrophoresis - used to separate nucleotide (or protein) fragments•DNA Probes - used to “find” specific nucleotide sequences•Primers - used to initiate DNA synthesis at a specific location
vectorinsert
Self-replicating DNA (ex. plasmid)
recombinant molecule
Techniques Used in Genetic Engineering
Cloning Overview
• Cut out the gene of interest from donor
• Put the gene into a vector
• Transfer the vector into a recipient
• Select for the recipient from a mixed population
Cloning Vectors
1) Plasmids
2) Bacteriophage lambda
3) P1 Phage
4) Cosmids
5) Yeast artificial chromosomes (YAC)
Characteristics of cloning vectors
1) Should have it’s own replicon i.e., be capable of autonomous replication in the host cell
2) Should carry one or more selectable markers that permit identification of parent and recombinant vectors
3) Restriction sites in non-essential regions of DNA into which foreign DNA can be inserted
Applications of molecular cloning
• Medical application– gene therapy– production of drugs (insulin, antibiotics, hormones)
• Agricultural application– Nutrients enriched food– Nitrogen fixing plants
• Scientific research
(PCR) Polymerase chain reaction
ds DNA containing the target
Taq polymerase (Thermus aquaticus)
nucleotides
primers
thermocycler
Amplifies target sequence
PCR
• Medical application: – Genetic diseases– Infectious disease
• Forensic science:– Identify criminals– parental identification
• Research application
DDC: DNA Test Sets Inmate Free After 18 Years
Forensic Resources DNA Diagnostics Center’s Forensics Division provided the DNA testing that eventually resulted in the release of inmate Robert McClendon. McClendon had spent 18 years in prison, convicted of a child rape that he has always maintained he did not commit.McClendon’s is 1 of 30 cases in Ohio that were identified to have “legitimate claims of innocence” in an investigation conducted by The Columbus Dispatch together with the Ohio Innocence Project (OIP). DDC has volunteered to provide DNA testing for the OIP's post-conviction cases free of charge.