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75 Cards in this Set

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Mutation
an inherited change in a nucleotide base or base sequence in the DS DNA of the genome

can be beneficial or harmful
Genetic recombination
genes from two separate genomes that are brought together

a larger degree of genetic change occurs through recombinationt han through mutation
Genetic Change in Prokaryotes
do not reproduce sexually

prokaryotes can undergo genetic change due to gene transfer
Later Gene Flow
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
Mutant
a strain carrying a mutation
Wild type strain
A strain isolated from nature

mutant derviatives can be derived from this
Genotype of organism designated by this
three lowercase letters followed by a capital letter all in italics
Phenotype of organism is designated by this
capital letter follwed by two lower case letters with either a plus or minus subscript indicating the presence of absence of a property
Selectable mutations
confer somee type of an advantage to the organisms possessing them

example of this is srug resistance
Unselectable mutations
no selective advantage is incurred

example of this is the loss of color in a pigmented organism
Spontaneous Mutations can occur because of this
1) exposure to nautral forms of radiation

2) oxygen radicals

3) base pairing errors during DNA replication
Point Mutation
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
Reversion
point mutations may be reversed by this process
Same site revertants
site that is mutated that reverts back to the original base
Second Site revertants
second mutation occurs at a second site in the sequence

compensates for the effect produced by the first mutation
Transition Changes
occur when a base mutation involves the substitution of one purine base for another purine base or one pyrimidine base for another
Transversion Changes
purine for pyrimidine or vice versa
Base insertions
mutations involving the addition of a base or bases
Base deletions
mutations involving the deletion of a base or bases from the nucleotide sequence
Frameshift Mutations
alter the reading frame of the mRNA by mvoing the reading frame up or down a single base or several bases
Mutations in RNA genomes
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
Mutagenesis
induced by agents called mutagens
Nucleotide base analogs
molecules that resemble DNA purine and pyrimidine bases in structre yet display faulty base pairing properites

result in mismatched base pairs
Alkylating Agents
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
Radiation
1) ionizing

2) nonionizing

both are used to create mutations
Nonionzing radiation
more widely used

used in the form of UV light

maximum absorption is at 260 nm
Pyrimidine dimers
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
Ionizing
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
Free radicals
react with DNA causing "hits" to occur that result in mutations
Examples of short wavelength
1) X-rays

2) Cosmic Rays

3) Gamma Rays
DNA Repair
if an error in DNA synthesis is correct before the cell divides, no mutation occurs

if DNA replication stalls, teh cell will die
SOS regulatory system
an example of DNA repair process that has some aspects that occur in the absence of template instruction
Hyperaccurate Phenotype
where an organism has an extremely efficient system of DNA fidelity repair and replication

can be advantageous or disadventageous
Advantageous
when making repairs caused by exposure to radiation
Disadvantggeous
when it prevents an organism from evolving at a apce that is dictated by a changing environment
Homologous Recombination
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
Transformation
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)
incorporation of donor DNA into recipient cell
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
RecA
allows homologous recombination to occur which allows transforming DNA to be integrated into the genome of the recipient
Transduction
bacterial DNA is transferred from cell to cell by a bacterial virus

occurs by generalized transduction or specialized transduction
Generalized transduction
virus incorporates random fragments of the host bacteria cell's chromosomal DNA into the viral genome

efficiency is low
Specialized transduction
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
Plasmids
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)
Multiple Plasmid Types
cells can contain more than one type of plasmid

each is genetically distinct
Incompatible Plasmids
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
F Plasmid
found in E. coli
Conjugation
involves cell to cell contact

a replicative process whereby both cells end up with copies of the plasmid

process of genetic transfer
Conjugative Cells
Plasmids that govern their own transfer by cel to cell contact

not all plasmids are conjugative
Resistance Plasmids
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
Colonziation factor antigen
allows certain bacgterial pathogens to colonzie the small intestine prior to secreting endotoxins
Bacteriocins
plasmids may also allow code for genes that produce proteins that allow bacteria to kill or inhibit related or unrelated strains of bacteria
Features of Conjugation
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
Pilus
produced by the donor cell

allows for direct contact between the cells
Rolling Circle Method
Method of DNA replication during conjugation
Episome
it can integrate into the host chromosome
Mobilization of F Plasmid
When integrated into the chromosome, the chromosome becomes mobilized

this can lead to a transfer of chromosomal genes to a recipient cell
F+ Plasmids
Cells that have an unintegrated F plasmid
Hfr Plasmids
High frequency recombination

F plasmid integrated into the chromosome
F' Plasmids
previously integrated F plasmids that have deintegrated and excised some chromosomal genes
Insertion Sequence (IS)
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
Complementation
recombination takes place between the donor chromosome and the recipient chromosome
How do we clone?
1) isolating a gene of interest onto a fragment of DNA

2) experimentally control the replication

3) amplify copies
Gene Cloning
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
How are plasmids used as cloning vectors?
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
Plasmid pBR322
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
Insertional Activation
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
Second Generation Plasmid Vectors
Higher utility and even easier to use than pBR322

Example is pUC19
pUC19
contains a multiple cloning site

polylinker is contained within the coding region of the gene that encodes B galactosidase
Blue colonies
bacterial clones that contain pUC19 without the DNA insert (B galactosidase is not disrupted and remains active)
White colonies
contain pUC19 with the DNA insert (B galactosidase is disrupted and inactivated)
Bacteriophage Lambda as a cloning vector
modified bacterial phage
Transfection
introduces bacteriophage lambda into host cell


allows DNA fragments up to 20 kb to be clones into lambda
Why are bacteriophages like lambda advantageous?
1) transfection rate is greater than the transformation rate of plasmid vectors

2) can hold larger amounts of DNA than most plasmid vectors
Five steps of cloning with lambda
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
use of site directed mutagenesis
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