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75 Cards in this Set
- Front
- Back
Mutation
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an inherited change in a nucleotide base or base sequence in the DS DNA of the genome
can be beneficial or harmful |
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Genetic recombination
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genes from two separate genomes that are brought together
a larger degree of genetic change occurs through recombinationt han through mutation |
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Genetic Change in Prokaryotes
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do not reproduce sexually
prokaryotes can undergo genetic change due to gene transfer |
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Later Gene Flow
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mechanisms that allow for gene transfer and recombination are based on this
genes are transferred from donor to recipient horizontally rather than vertically from mother to daughter cell |
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Mutant
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a strain carrying a mutation
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Wild type strain
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A strain isolated from nature
mutant derviatives can be derived from this |
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Genotype of organism designated by this
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three lowercase letters followed by a capital letter all in italics
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Phenotype of organism is designated by this
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capital letter follwed by two lower case letters with either a plus or minus subscript indicating the presence of absence of a property
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Selectable mutations
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confer somee type of an advantage to the organisms possessing them
example of this is srug resistance |
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Unselectable mutations
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no selective advantage is incurred
example of this is the loss of color in a pigmented organism |
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Spontaneous Mutations can occur because of this
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1) exposure to nautral forms of radiation
2) oxygen radicals 3) base pairing errors during DNA replication |
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Point Mutation
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if a mutaiton affects just a single base pair
if point mutations are carried through transcription and translation they can end up altering the protein sequence can have severe implications or can be unharmful |
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Reversion
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point mutations may be reversed by this process
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Same site revertants
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site that is mutated that reverts back to the original base
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Second Site revertants
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second mutation occurs at a second site in the sequence
compensates for the effect produced by the first mutation |
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Transition Changes
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occur when a base mutation involves the substitution of one purine base for another purine base or one pyrimidine base for another
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Transversion Changes
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purine for pyrimidine or vice versa
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Base insertions
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mutations involving the addition of a base or bases
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Base deletions
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mutations involving the deletion of a base or bases from the nucleotide sequence
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Frameshift Mutations
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alter the reading frame of the mRNA by mvoing the reading frame up or down a single base or several bases
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Mutations in RNA genomes
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occur at a level that is 1000 fold higher than in DNA genomes
there are no repair mechanisms but there are proof reading functions leads to a heightened mutation rate in the RNA genomes of RNA viruses makes RNA viruses able to eveolve more rapidly |
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Mutagenesis
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induced by agents called mutagens
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Nucleotide base analogs
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molecules that resemble DNA purine and pyrimidine bases in structre yet display faulty base pairing properites
result in mismatched base pairs |
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Alkylating Agents
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react with amino, carboxyl, and hyroxyl groups in proteins and nucleic acids and substitute alkyl groups in their place
can induce changes even in nonreplicating DNA |
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Radiation
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1) ionizing
2) nonionizing both are used to create mutations |
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Nonionzing radiation
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more widely used
used in the form of UV light maximum absorption is at 260 nm |
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Pyrimidine dimers
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one of the effects of UV light on DNA is the formation of this
when two adjacent pyrimidine bases become covalently bonded to one another such that the probability of the DNA polymerase misreading the sequence is greatly increased |
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Ionizing
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1) more powerful form of radiation than UV light
cause the ionization of compounds which brings about the formation of potent chemical species such as free radicals has greater penetrating power than UV light and is potentially more destructive |
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Free radicals
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react with DNA causing "hits" to occur that result in mutations
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Examples of short wavelength
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1) X-rays
2) Cosmic Rays 3) Gamma Rays |
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DNA Repair
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if an error in DNA synthesis is correct before the cell divides, no mutation occurs
if DNA replication stalls, teh cell will die |
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SOS regulatory system
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an example of DNA repair process that has some aspects that occur in the absence of template instruction
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Hyperaccurate Phenotype
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where an organism has an extremely efficient system of DNA fidelity repair and replication
can be advantageous or disadventageous |
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Advantageous
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when making repairs caused by exposure to radiation
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Disadvantggeous
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when it prevents an organism from evolving at a apce that is dictated by a changing environment
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Homologous Recombination
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genetic exchange between homologous DNA dequences from two different sources
invovles crossing over uses RecA protein occurs only after the transfer when DNA from the host is in the recipient cell |
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Transformation
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occurs in BActeria and some archae
process by which free DNA is incorporated into a recipient cell and bring about genetic change occurs in B. subtilis fragmented DNA only 1% of certain strains and species of prokaryotes are naturally competent (can be transformed) |
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incorporation of donor DNA into recipient cell
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requires the activity of a single-stranded binding protein, RecA protein, and several other enzymes
SS binding proteins prevent transforming DNA from degradation by nucleases |
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RecA
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allows homologous recombination to occur which allows transforming DNA to be integrated into the genome of the recipient
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Transduction
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bacterial DNA is transferred from cell to cell by a bacterial virus
occurs by generalized transduction or specialized transduction |
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Generalized transduction
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virus incorporates random fragments of the host bacteria cell's chromosomal DNA into the viral genome
efficiency is low |
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Specialized transduction
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DNA from a specific region of the host bactera chromosome is integrated idrectly into the virus genome and ususally replaces some of the virus genes
efficiency may be very high only occurs in some temperate viruses |
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Plasmids
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small circular or linea DNA molcules that carry any of a variety of unessential genes
vary in size from 1kb to >1 mb smaller than bacterial chromosome double stranded (DS) majority are circular and in a supercoiled conformation present in cells in various copy numbers (few as 1 or as many as 100 copies present in a cell) |
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Multiple Plasmid Types
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cells can contain more than one type of plasmid
each is genetically distinct |
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Incompatible Plasmids
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if a plasmid is transferred to a cell that already carries another type of plasmid the second plasmid may not be maintained and consequently lost during subsequent cell replication
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F Plasmid
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found in E. coli
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Conjugation
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involves cell to cell contact
a replicative process whereby both cells end up with copies of the plasmid process of genetic transfer |
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Conjugative Cells
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Plasmids that govern their own transfer by cel to cell contact
not all plasmids are conjugative |
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Resistance Plasmids
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Known as R plasmids
among the most widespread and well-studied groups of plasmids confer risistance to antibiotics and other growth inhibitors resistance is spread between bacteria through conjugation |
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Colonziation factor antigen
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allows certain bacgterial pathogens to colonzie the small intestine prior to secreting endotoxins
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Bacteriocins
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plasmids may also allow code for genes that produce proteins that allow bacteria to kill or inhibit related or unrelated strains of bacteria
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Features of Conjugation
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sometimes other genetic elements such as all or parts of the host chromosome can be transferred during conjugation
DNA replication must take place during conjugation such that both the donor and recipient cells have fully formed plasmids at the conclusion of conjugation |
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Pilus
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produced by the donor cell
allows for direct contact between the cells |
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Rolling Circle Method
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Method of DNA replication during conjugation
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Episome
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it can integrate into the host chromosome
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Mobilization of F Plasmid
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When integrated into the chromosome, the chromosome becomes mobilized
this can lead to a transfer of chromosomal genes to a recipient cell |
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F+ Plasmids
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Cells that have an unintegrated F plasmid
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Hfr Plasmids
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High frequency recombination
F plasmid integrated into the chromosome |
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F' Plasmids
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previously integrated F plasmids that have deintegrated and excised some chromosomal genes
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Insertion Sequence (IS)
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integration of the F plasmid into the host chromosome occurs at this site
these sites are regions of DNA sequence homology between chromosomal and F plasmid DNA |
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Complementation
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recombination takes place between the donor chromosome and the recipient chromosome
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How do we clone?
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1) isolating a gene of interest onto a fragment of DNA
2) experimentally control the replication 3) amplify copies |
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Gene Cloning
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Involves molecular manipulation in vitro of vectors using PCR, RE, synthesizing DNA probes, DNA sequencing, gel electrophoresis etc.
purpose is to isolate multiple copies of specific genes or regions of a chromosome in pure form |
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How are plasmids used as cloning vectors?
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1) they are easy to isolate and purify
2) can multiply to high copy numbers in bacterial cells 3) presence of selectable markers like antibiotic resistant genes |
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Plasmid pBR322
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replicates in e. coli
small genome size can be maintained stably in its host can by amplifies to very high copy numbers reasonable amount of foreign DNA can be inserted into it |
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Insertional Activation
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foreign DNA is inserted into one of these restriction sites contained within an antibiotic resistant gene, antibiotic resistance will be lost
can be used as a means of selecting transformed bacterial clones that have picked up the insert |
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Second Generation Plasmid Vectors
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Higher utility and even easier to use than pBR322
Example is pUC19 |
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pUC19
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contains a multiple cloning site
polylinker is contained within the coding region of the gene that encodes B galactosidase |
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Blue colonies
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bacterial clones that contain pUC19 without the DNA insert (B galactosidase is not disrupted and remains active)
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White colonies
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contain pUC19 with the DNA insert (B galactosidase is disrupted and inactivated)
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Bacteriophage Lambda as a cloning vector
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modified bacterial phage
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Transfection
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introduces bacteriophage lambda into host cell
allows DNA fragments up to 20 kb to be clones into lambda |
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Why are bacteriophages like lambda advantageous?
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1) transfection rate is greater than the transformation rate of plasmid vectors
2) can hold larger amounts of DNA than most plasmid vectors |
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Five steps of cloning with lambda
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1) isolate vector DNA from phage particles and digest with the appropriate RE
2) connect the two lambda fragments with the fragments of foreign DNA using DNA ligase 3) packaging of DNA insert by adding cell extracts containing the phage head and tail proteins and allowing viable phage particles to form 4) transfecting E. coli and isolating phage clones by selecting plaques on a host strain 5) checking the recombinant phage for the presence of the foreing DNA insert sequence |
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use of site directed mutagenesis
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comparison of wild type protein versus a mutated protein
examine the efficiency of porteins and/or enzymes with a particualr amino acid substitution examine how mutation of a gene promoter affects transcription |