Chapter 9 & 10: Subviral Agents Flashcards
Mutation
heritable change in genome
can lead to change in properties of an organism, can be beneficial, detrimental or have no effect
prokaryotes accumulate mutations ______ because of _________ growth
quickly, exponential
horizontal gene transfer
generates larger changes (than mutations)
2 factors thats fuel evolution
mutation and genetic exchange
bacteria can/cannot exchange genes
can
genomes of cells
contain double stranded DNA
viral genomes
contain double or single stranded DNA or RNA
wild-type strain
isolated from nature, refer to individual or one gene
mutant
cell/virus derived from wild type that carries genotype change
genome
nucleotide sequence
selectable mutations
contain an advantage, progeny cells outcompete parent, easy to detect and can be used as a genetic tool
example of selectable mutations
antibiotic resistance
non selectable mutations
do not contain an advantage, may still change phenotype, very hard to detect
example of non selectable mutation
color loss in pigmented organism
Ways to isolate mutants
selection and screening
spontaneous mutations
occur without external intervention, most result from errors by DNA polymerase during replication
induced muations
caused environmentally or deliberately, can result from exposure to radiation or chemicals that modify DNA
point mutations
change in only one base pair, occurs via single bp substitution, phenotypic change depends on location
types of base pair mutations
missense, nonsense, silent
silent mutations
do not affect sequence of amino acids due to degeneracy
no change in phenotype
almost always change in third base (in reading frame) of codon
missense mutation
changes sequence of amino acids
may alter activity
nonsense mutation
causes a stop codon where it doesn’t belong, results in truncated protein
lacks normal activity
frameshift mutation
single (or double) bp deletion or insertion that results in shift of reading frame
scrambles entire protein
can lead to a premature stop codon
insertion/deletion of three base pairs
adds or deletes an entire codon/amino acid, usually not as bad
insertions and deletions
can result in gain or loss off 100s to 1000s of bps, results in complete loss of gene function, can be lethal
may arise from errors during recombinations
large insertions may be from transposable elements
mutation rates
in microorganisms, 10^-6 to 10^-7 per kb
(single gene is ~1kb)
eukaryotes 10 fold lower
DNA viruses 100-1000x higher
RNA viruses even higher
Mutagens
agents that increase mutation rates
three types of mutagens
chemical, physical, biological
chemical mutagens
nucleotide base analogs: resemble nucleotide bases but have faulty base pairing (lack 3, -OH leading to chain termination)
replication errors occur at higher frequencies
results in mismatched base incorporation
cause chemical modifications
types of chemical modifications
alkylating agents and intercalating agents
intercalating agents
chemical mutation that cause frameshift mutations, push two bps apart
trigger insertions or deletions
alkylating agents
introduce changes in replicating or nonreplicating DNA
horizontal (lateral) gene transfer
gene movement between cells that are not descendants
what does horizontal gene transfer cause
quick acquisition of new characteristics
fuels metabolic diversity
three fates of DNA after horizontal gene transfer
degradation, replication by itself (viruses), recombination with host genome
genetic recombinations
physical exchange of DNA between genetic elements
homologous recombination
causes genetic exchange between homologous DNA from two sources
drives crossing over
genetic transformation
free DNA is incorporated into a recipient cell and brings about genetic change
typical size: 10 genes/10k nucleotides
competence
a cell that can take up DNA and be transformed
in bacteria, can be linked to pili
competence regulation
transformation
responds to cell density so not all cells become competent
electroporation
high voltage electrical pulses used to force cells to take up DNAm, key in gene cloning
Transduction
transfer of DNA from one cell to another by a bacteriophage
occurs in bacteria and archaea
modes of transduction
generalized and specialized
generalized transduction
DNA from any portion of host genome packaged inside virion
donor genes cannot replicate independently
will be lost without recombination
specialized transduction
DNA from specific region of host chromosome is integrated directly into virus genome, usually replaces viral genes
homologous recom. can occur
examples of transduction
multiple antibiotic resistance genes in salmonella, shiga like toxins in e. coli, virulence factors in vibrio cholerae, photosynthetic genes in cyanobacteria
process of general transduction
host DNA gets accidentally packaged into bacteriophage, forms transducing particle that is defective and cannot cause viral lytic infection
upon lysis, transducing particles and normal virons released
most cells receive virus, some get transducing particles that may recombine
low efficiency: one in 10^6 to 10^8 cells transduced
process of specialized transduction
phage genomes is integrated at specific site, viral replication under control of bacterial host chromosome
upon induction, viral DNA separates via process that reverses integration
sometimes excises incorrectly and takes adjacent host genes along with it, which can be transferred to another cell
limited amount of host DNA able to replace phage DNA, helper phages can assist
in mixed infection, fewer phage specific genes needed
extremely efficient transfer
benefits of transduction
allows cell to become immune to future infection by same phage
selective value for host because of resistance to further infection
gene transfer agents
defective bacteriophages that transfer DNA between prokaryotic cells
result from prokaryotes hijacking defective viruses specifically for DNA exchange
resemble tiny tailed bacteriophages
contain random small pieces of host DNA
do not produce viral plaques
found in many bacteria and some archaea
synthesis regulated by cell
may have evolved as mechanism for protected gene dispersion
conjugation
horizontal gene transfer that requires cell to cell contact
occurs between related cells
donor cell: contains conjugative plasmid
recipient cell: does not have plasmid
other genetic elements may be mobilized
conjugative plasmid
~99kbp circular DNA molecule, contains genes that regulate DNA replication, other transposable elements that allow plasmid to integrate into host cell, TRA genes that encode transfer function
Mobile DNA
discrete segments of DNA that move as a unit from one location to another within other DNA molecules
most are transposable elements, move by transposition, extremely abundant
transposable elements in bacteria
insertion sequences and transposons
encode transposase
contain short inverted repeats at ends required for transposition
genetic engineering
using in vitro techniques to alter genetic material in the laboratory
genetic engineering techniques
restriction enzymes, gel electrophoresis, nucleic acid hybridization, nucleic acid probes, molecular cloning, cloning vectors
nucleic acid hybridiztion
base pairing of single strands of DNA or RNA from two sources
nucleic acid probe
segment of single stranded DNA with predetermined identity used in hybridization
southern blot
hybridization procedure where DNA is in gel and probe is RNA or DNA
northern plot
gel contains RNA
applications of PCR
phylogenetic studies, surveying different groups of environmental organisms, amplifying small amounts of DNA, identifying specific bacteria, looking for a specific gene
molecular cloning
isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated
three steps of gene cloning
- isolation and fragmentation of source DNA
- insertion of DNA fragment into cloning vector
- introduction of cloned DNA into host organism
environmental gene mining
isolation of enzymes with industrial applications
examples of environmental gene mining
enzymes with resistance to industrial conditions (high temperatures, high or low ph, oxidizing conditions), enzymes with combinations of properties (heat stable lipases)
genome editing (CRISPR and CAS9)
add mutations to genomes that can be passed through generations
CRISPR
clustered regularly interspaced short palindromic repeats
type of prokaryotic immune system
region of bacterial chromosome containing DNA sequences similar to foreign DNA (spacers) alternating with identical repeated sequences
viroids
infectious RNA molecules that lack a protein coat
smallest known pathogens
cause plant diseases
small, circular ssRNA molecules
do not encode proteins, dependent on host encoded enyzmes
CAS proteins
obtain and store segments of foreign DNA as spacers
recognize and destroy foreign DNA
diseases caused by viroids
potato spindle tuber, chrysanthemum stunt, avocado sunblotch, coconut cadang-cadang
hepatitis D
examples of TSEs
scrapie, chronic wasting disease, bovine spongiform encephalopathy (mad cow disease), feline spongiform encephalopathy, creutzfeldt-jakob disease
possibly alzheimer’s and type II diabetes
prions
infectious proteins whose extracellular form contains no nucleic acid
cause transmissible spongiform encephalopathies
host cell contains gene that encodes native form of prion protein that is found in healthy animals
prion misfolding results in neurological symptoms of disease
