-
3 characteristics of Transformation
- Direct uptake of free DNA
- translocasome takes up DNA
- competence induced in different species
-
1 characteristics of Direct uptake of free DNA
it needs competent cells in order to occur. They can be natural or artificial
-
translocasome takes up DNA and is located in the nuclear envelope
-
3 characters in how competence is induced in different species
- gram + cells secrete signals
- has a competence factor
- stress incudes competence (starvation)
-
bacterial sex
conjugation
-
types of secretion system in conjugation
type IV system (pilus)
-
how conjugation works
- pilus proteins encoded on F factor
- transfers DNA on F factor (starts at oriT site)
- recipient cell's transferred DNA forms a new F plasmid
- recipient becomes donor
-
conjugations requires the presence of special transferable plasmids
ex F factor
-
F factor
fertility factor
-
2 sites of replication origins of F factor
-
When is the replication site used
oriV
oriT
- used in nonconjugating cells
- used during DNA transfer
-
conjugation begins with contact between the donor cell and recipient cell
-
plasmid transfer in conjugations requires specific genetic elements such as these 3
- transfer region (tra)
- origin of transfer (oriT)
- F plasmid (fertility)
-
when the F factor plasmid can integrate into the X, the cell is now a
Hfr (high frequency recombination)
-
this cell is capable of transferring X parts into a recipient cell
Hfr cell
-
3 characters of Hfr cell transfer
- genes transfer in order
- entire X takes 100 min to transfer
- the process can be used to map genes
-
3 characteristics of X mobilization
- transfer is rarely complete
- homologous recombo in the recipient cell following DNA transfer
- diff Hfr strains - X mapping
-
in Hfr, F factor integrates on bacterial X and tries to transfer entire X
-
2 characteristics of F' factor
- F plasmid contains extra genes
- Transfers extra genes to recipient
-
how transduction works
- virus injects DNA into cell (bacteriophage)
- package DNA into viral capsid (contains package bac DNA by mistake)
- transfer DNA to new host (can bring new bac genes to host)
-
process in which bacteriophages carry host DNA from one cell to another
transduction
-
2 types of transduction
- generalized transduction
- specialized transduction
-
this kind of transduction can transfer any genes from one donor to a recipient cell
generalized transduction
-
this kind of transduction can transfer only a few closely linked genes between cells
specialized transduction
-
4 characters of defense against transferred DNA
- bacteria cut entering DNA to pieces
- bacteria add methyl groups to DNA
- Entering DNA is destroyed
-
bacteria cut entering DNA to pieces at specific restriction sites
-
2 reasons bac add methyl groups to DNA
- prevents restriction at those sites
- adds as cell replicates X
-
in transferred DNA the entering DNA is destroyed unless these 2 occur
- it comes from entering species
- has methyl groups protecting DNA
-
entering DNA replaces X DNA
recombination
-
recombination occurs if
sequence is overall very similar
-
3 ways DNA can enter the cell
- transformation
- conjugation
- transduction
-
3 specific recombination proteins
-
2 reasons for recombo to occur besides DNA transfers
- replaces variable sized section of DNA
- repair damaged DNA
-
Two different DNA molecules in a cell can recombine by these 2
- generalized recombo
- site specific recombo
-
this kind of recombo requires that the 2 recombining molecules have a considerable stretch of homologous DNA sequences
generalized recombo
-
this kind of recombo requires very little sequence homology between the recombining DNA molecules
site specific recombo
-
how site specific recombo is diff from generalized
it requires a short sequence recognized by the recombo enzyme
-
heritable changes in DNA sequence
mutation
-
this mutation is due to a change in one base pair
point mutation
-
point mutation
transition ex
transversion ex
- purine>purine
- pyrimidine>pyrimidine
purine<>pyrimidine
-
3 examples of larger mutations
- insertion of DNA into X
- deletion of portion of the X
- inversion flipping a portion of X
-
4 ways in which there is no effect of a mutation
- mutations in regions between genes
- " " that change 3rd base of codon
- " " that change 1 amino acid into a similar one
- " " that change a protein that is not needed
-
change in genotype
mutation
-
effect of a mutation on an organism
phenotype
-
4 different causes of mutations
- spontaneous
- induced by mutagens
- electromagnetic radiation
- chemicals
-
single base change
point mutation
-
5 types of mutations
- silent
- missense
- nonsense
- transitions
- transversions
-
insertions and deletion mutations are known as
frameshifts
-
4 types of mutations on a molecular basis
- insertions/deletions
- translocation
- inversions
- reversions
-
suppressor mutations are known as
reversion
-
2 examples of electromagnetic radiation
-
3 characters to chemicals causing mutations
- similarities of bases
- base modifying
- intercalators insert between bases
-
chemical intercalators insert between bases causes what kind of mutations
frameshift
-
-
this kind of repair pathways prevents mutations
error proof
-
this kind of repair pathways risk introducing mutations
error prone
-
this repair operates only when damage is so severe that the cell undergoes apoptosis
error prone repair
-
examples of error proof repair
Methyl mismatch repair; photoreactivation; nucleotide excision repair; base excision repair; and recombinational repair
-
all microbial genomes have a mosaic nature
-
transfer free DNA from environment
transformation
-
DNA transfer after cell contact
conjugation
-
DNA transfer via bacteriophages
transduction
-
restriction modification systems are used to
protect bac from invasion by foreign DNA
-
transposable elements include these 2
-
genomes evolve primarily by horizontal gene transfer and by duplications follow by functional divergence through mutation
-
5 ways DNA repair occurs
- mismatch repair
- thymidine dimers
- damaged bases
- recombo repair
- sos repair
-
how does mismatch repair work
- mispaired base cut out of strand
- (strand w/o methyl groups is newer and assumed to be an error)
-
nucleotide excision repair that is induced by UV
thymidine dimers
-
what cuts out the nucleotide in the excision repair
UvrAB complex
-
this DNA repair is excised by specific enzymes in damaged bases
-
damaged bases are replaced by
DNA pol I
-
this kind of repair occurs after strand has replicated
recombo repair
-
when the strand is repaired in recombo it is copied and its catalyzed by RecA recombinase
-
3 characters of SOS repair
- extensive DNA damage inactivates LexA
- activation of many repair genes
- rapid polymerization of DNA
-
the rapid polymerization of DNA is error prone but better than no repair
-
this mobile genetic element moves from one DNA molecule to another
transposable elements
-
this element can move within and between X
transposable elements
-
these are simple transposable elements containing transposase gene which is flanked by short inverted repeat sequences
insertion sequence
-
these are targets for the transposase enzyme
insertion sequence
-
IS elements can transfer by 1 of 2 mechanisms
- replicative transposition
- nonreplicative transposition
-
are complex transposable elements that carry additional genes
transposons
-
small changes in gene sequence
- slow changes in a protein
- generally no new functions created
-
large changes in X occurs these 3
- insertions of new material
- duplications of genes
- genome reduction
-
large changes in X insertions of new material in genome evolution via
transposition
-
duplications if genes causes
the creation of new functions for the cell as long as copied gene maintains original function
-
movement of genes between cells
horizontal gene transfer
-
transposons carry genes to X
-
plasmid carries genes between cells
without having to become part of X
-
effects of gene transfer
spreads useful genes among bac
-
3 ways the effects of gene transfer spreads useful genes among bacteria
- antibiotic resistance genes
- pathogenicity islands
- genes to degrade special metabolites
-
archaea share many genes with bacteria
share other genes only with eukaryotes and midway between bac and eukary
-
genes in one cell may not have been inherited from parents
obtained instead from other bacteria
bacterial species are related through lateral transfer as well as through parentage
-
most cancer results from multiple mutations
-
ames test uses bacterial strain auxotrophic for histidine
has mutation in hisG gene and cannot grow unless histidine is supplied
-
mutagen causes reversion
changes mutation to normal form (rare)
-
more colonies on measurement of mutagens means
stronger mutagen
-
transposable elements can do these 3
- insert into X
- can jump from one site to another
- can copy itself to a new site
-
when transposable elements
jump from one side to another its known as
can copy itself to a new site is known as
- non replicative transposition
- replicative transpositions
-
4 characters of regulating gene expression
- microbes respond to changing environment
- sense environment
- must transmit info to X
- alter gene expression
-
2 ways microbes respond to the changing environment
- alter growth rate
- " " proteins produced
-
how microbes sense their environment
they have receptors on cell surface
-
the altering of gene expression in microbes leads to these 2
- change transcription rate
- change translation rate
-
5 ways microbes control gene expression at several levels
- alterations of DNA seq
- control of transcription
- control of mRNA stability
- translational control
- posttranslational controls
-
Cells use different mechanisms to sense and respond to conditions within or outside the cell.
-
help a cell sense internal changes and alter its gene expression to match.
Regulatory proteins
-
lacZ gene encodes
beta galactosidase
-
lacY gene encodes
lactose permase
-
the product of lacZ and Y genes both need proteins to digest lactose
-
2 characters of E.coli lac operon
- multiple genes transcribed from one promoter
- both genes are transcribed together
-
3 things that sigma factors are controlled by
- alternate transcription and translation
- proteolysis
- antisigma factors
-
Small regulatory RNAs can bind to mRNA and help stabilize it or make it susceptible to degradation.
-
bacteria can communicate with each other
quorum sensing
-
In quorum sensing, bacteria can communicate with each other at high cell densities via
autoinducers
-
DNA microarrays (RNA) and two-dimensional gels (protein) provide global snapshots of expression
-
The lacZYA operon is regulated as follows:
- - Operon is off when LacI binds to the operator.
- - Operon is on when allolactose binds to LacI; cAMP-CRP are bound to the promoter (and there is no glucose around).
-
The tryptophan operon is regulated by
repression and attenuation (premature transcript termination).
-
Induction of a quorum-sensing gene system requires the accumulation of a secreted small molecule called
autoinducer.
-
At a certain extracellular concentration, the secreted autoinducer reenters cells.
-
autoinducer reenters cells.
- It binds to a regulatory molecule. Once bound
The LuxR-autoinducer complex then activates transcription of the luciferase target genes that confer bioluminescence
-
2 characters if quorum sensing
- Cells work together at high cell density
- Send signal chemical to other cells
-
Cells work together at high cell density causes these 2
- V. fischeri becomes bioluminescence
- Many bacteria form biofilms
-
Autoinducer chemical for V. fishcheri
homoserine lactone
-
when microbes send signal chemicals to other cells these 2 occur
- chemical accumulation
- binds to sensor in cell
-
Chemical accumulation
high cell density
-
the binding to sensor in a cell in cell signaling does what
sensor activates transcription
-
this alloq bac to switch type of flagellum made
phase variation
-
In phase variation, the section of a X undergoes inversion in 2 orientations
- 1- element promotes transcription (H2 flagellin produced)
- 2- H2 is not transcribed (cell makes H1 flagellin instead)
-
trp operon in a cell must make
the amino acid tryptophan
-
trp operon requires many proteins made from one operon
when tryptophan is plentiful, the cell stops synthesis
-
trp repressor must bind tryptophan to bind DNA
its the opp of lac repressor
-
sensor kinase protein in PM function
binds to signal (food or chemical cue)
-
sensor kinases activates itself via phosphorylation
-
2 component signal transduction 2
- sensor kinases protein in PM
- cytoplasmic response regulator
-
3 functions of cytoplasmic response regulator
- takes phos from sensor
- binds X
- alters transcription rate of multiple genes
-
since lactose cannot pass through PM what2 ways does it undergo to enter the cell
-
lactose must be converted to glucose to be digested, what enzyme converts lactose
beta-galactosidase
-
this repressor protein blocks transcription
LacI
-
how LacI blocks transcription
- repressor binds to operator
- blocks sigma factor from binding promoter
-
how the repressor responds to presence of lactose
- binds inducer or DNA
- adds lactose which causes repressor to fall off operator
-
inducer of lac operon
allolactose
-
sigma factor guides RNA pol to initiate transcription at promoter
-
2 functions of sigma factors when it comes to proteins
- help guide sigma factor to promoter
- can block sigma factor from binding
-
3 characters when proteins can help guide sigma factor to promoter
- activates
- binds to DNA sites next to promoter
- increase freq of that gene's transcription
-
3 characters when protein block sigma factor from binding
- repressor
- binds to DNA sites next to promoter (operator)
- lowers freq of that gene's transcription
-
this acts as repressor to block transcription
araC
-
2 characters of ara operon
- araC acts as repressor to block transcription
- addition of arabinose changes conformation
-
2 occurances to when an addition of arabinose changes conformation on ara operon
- acts as activator
- stimulates binding of RNA pol
-
is the easiest sugar to digest
glucose
-
sugars converting to glucose require syn of proteins beta gala
-
if glucose is present, lac operon is not transcribed
-
how the presence of glucose affects signals inside cell 2
- entry of glucose directly affects adenylate cyclase
- lowers amount of cAMP
-
-
CRP protein is what kind of protein
activator
-
3 function of CRP protein
- binds next to promoter
- stimulates open complex
- increases transcription of lac and other operon
-
CRP responses to presence of cAMP
-
what happens when CRP responds to presence of cAMP
acts as activator only when bound to cAMP
-
high glucose>low cAMP levels>CRP inactive
-
When CRP is inactive, this happens
does not bind to operons and has low levels of lac transcription
-
trp operon weakening 3 steps
- leader peptide on mRNA encodes 2 tryptophan
- ribosomes binds to mRNA as transcription
- stalled ribosomes prevents mRNA from forming a transcription termintor
-
thiamine prevents translation of thiamine biosynthesis enzyme
feedback inhibition
-
translational control riboswitches function
binding of small molecules to the upstream regions can also induce stem loop structures that interfere with translation of the mRNA
-
small regulatory RNAs
- Small RNAs regulate transcription, translation
- RNA probably predates DNA and protein
- Made from ribose, prevalent sugar
- Some viruses have genome made only of RNA, no DNA
- Some RNAs have catalytic activity
- Major catalytic activity of ribosome is performed by RNA
-
Antisense RNA base-pairs to mRNA
Usually prevents translation
Until removed via endonuclease
Targets the mRNA for degradation
Universal method of gene control—found
in all creatures
-
sigma factors regulate transcription of all genes
control of ratio of sigma factor determines global control of protein synthesis
-
sigma factor 70 function
initiates transcription at most genes
-
Bs sigma 28 function
sporulation
-
sigma 38
stationary phase
-
sigma 32 function
heat shock and stress response
-
sigma 28
flagellar response
-
this structure allows translation of sigma 32 only at high temperatures
temperature sensitive mRNA
-
this rapidly removes sigma factors
the rapid turnover allows more exact control
sigma 70 degraded rapidly at 42 C
-
anti-sigma factors block sigma activity until needed and respond to the environment
-
what are the 3 mechanisms that set the levels of a particular protein in the cell
- transcription
- stability of mRNA transcripts
- translation of mRNA into protein
-
researchers won a nobel prize for discovering that genes can be induced
Jacob and monod
-
what happens during phase variation
a remodeling of an organism's DNA
-
the protein product of the lacI gene is a
regulatory protein
-
one advantage to using sRNAs to control protein expression is that
translation of the sRNA is not needed, thus saving amino acids
-
lac operon will be maximally transcribed when this occurs
lactose is present but not glucose
-
an inducer causes gene expression by
binding a repressor protein
-
cAMP receptor protein has binding sites for
the RNA pol alpha subunit
-
cell function is regulated at what level
both the biochemical and the genetic levels
-
AraC is one of a large family of AraC-like proteins. what do all of the proteins have in common
they all contain 2 helix turn helix DNA binding motifs
-
this occurs in response to low energy stores
stringent response
-
what is composed of DNA
operator (regulatory DNA sequence)
-
when weakening occurs at the trp operon, when will the ribosome pause at the leader sequence
when tryptophan levels are low
-
lac operon includes which 3 proteins
- lactose permease
- B-galactosidase
- transacetylase
-
what technique can analyze proteome patterns
2D gel electrophoresis
-
accumulation of the heat shock factor, sigma H, increases at high temps because
high temps sigma H mRNA adopts an unfolded secondary structure
-
to get maximal expression of the lac operon, low glucose is
necessary but not sufficient
-
enzyme activity can be most rapidly changed at what level
post translational modification of the enzyme
-
ppGpp serves as a signal to the cell that
ribosome synthesis should be curtailed
-
similar to trp operon, genes encoding proteins for histidine synthesis are in a his operon that is under weakened control. The his operon leader seq probably
contains adjacent codons for histidine
-
trp operon are regulated by these 2
- repressor protein
- attenuation
-
when glucose if present, enzyme IIA glc is not phosphor and LacY is inhibited
-
these factors bind anti sigma factors, releasing sigma factors to activate transcription
anti-anti sigma factors
-
what may indicate a DNA site that binds a regulatory protein
the DNA contain an inverted repeat
-
proteome refers to
complement of proteins present at a particular point of time
-
some sRNA are responsible for
regulating protein levels at the level of translation
-
what functional group do kinases transfer to other molecules
phosphate
-
a DNA microchip, scanned with cDNAs, can give info about
transcription of specific genes
-
Acyl homoserine lactone produced by LuxI of vibrio is an example of
an autoinducer
-
AraC-like regulator differ from the LacI repressor in that
only the AraC-like regulators can active transcription by direct interactions with RNA pol
-
bacteria may dontate DNA to
other bacteria of the same or of diff species, some eukary cells, and bacteriophages
-
during DNA synthesis, if DNA polymerase incorporates a wrong nucleotide
methyl mismatch repair will correct the unmethylated daughter strand to match the methylated template strand
-
pathogenicity islands (PAIs) are often found intergrated near
tRNA genes
-
DNA encoding for which capability is part of the flexible gene pool
antibiotic resistance
-
the enzyme photolyase repairs DNA damage caused by
UV radiation
-
if a bacterial cell is missing AP endonuclease genes, what could not occur
base excision repair
-
if a bacterial cell is competent, it means
the bacterium can import free DNA fragments and incorporate them into its genome
-
a mutation always results in
a change in genotype
-
a bacterial strain lacking the RecA protein will be
unable to perform generalized recombination
-
what type of DNA uptake is dependent on viruses
transduction
-
uptake of foreign DNA into a bacterium
may help or harm the bacterium
-
the relaxase enzyme, used during conjugation, has what enzymatic activity
endonuclease
-
in the basic ames test for mutagenesis, a mutagen is tested to see if it can
produce colonies on basic medium that lacks histidine, starting with a hisG mutant strain of bacteria
-
an insertion sequence contains a gene for which enzyme
transposase
-
one sign of horizontal gene transfer is
a CG base ratio different from flanking chromosomal DNA
-
which of the following is a laboratory technique for inducing transformation
electroporation
-
if a cytosine deaminates and becomes uracil, which enzyme will cleave the uracil from the DNA backbone in the first step of a repair process
glycosylase
-
transduction is an example of
horizontal gene transfer
-
which type of DNA uptake is dependent on transferable plasmids
conjugation
-
which of the following enzyme is used in methyl mismatch repair, nucleotide excision repair and base excision repair
DNA pol I
-
in generalized recombination, which protein complex is responsible for homology searching
RecA
-
overtime, the genome of a species
may change due to mutation or gene swapping
-
which of the following DNA repair mechanisms is error prone
sos repair
-
based on gel electrophoresis, a nonfunctional pro is found to have a smaller molecular weight than its wild type counterpart. A likely explanation for this observation is a
nonsense mutation in the DNA coding for the protein
-
what function do bacterial pheromones promote
conjugation
-
horizontal gene transfer can occur via these 3 ways
- conjugation
- transformation
- transduction
-
the process of importing free DNA from the environment into cells is called
transfomation
-
the mutation rate in a wild type E. coli cell is on the order of
10^-6 base pair replicated
-
this kind of recombination needs very little sequence homology between the donor and the recipient DNA
site specific recombination
-
transposable elements differ from plasmids in that
only plasmids may exist autonomously, not integrated into host DNA
-
how did so many different types of bacteria acquire such genomic blending? 3
- heavy horizontal gene transfer
- recombination events occurring w/in species
- variety of mutagenic and DNA repair strategies
-
reason for bac gene transfer
acquire genes that might be useful as the environmental changes
-
how to artificially manipulate the drive DNA into the cell like transformation?2
- -use CaCl2 to alter the membrane which makes the cells electrocompetent which allows DNA to pass
- -uses electroporation to shoot DNA across the membrane
-
3 reasons why species undergo natural transformation
- import DNA may use the transformed DNA as food
- use dead DNA seq that are compatible with their DNA to repair theirs
- help species adjust to new environment
-
vertical gene transfer is the same as cell division
-
natural transformation in gram+ typically involves the growth phase depend assembly of a translocasome complex across the cell membrane
-
this is composed of a binding protein that captures extracellular DNA floating in the environment
translocasome
-
once a translocasome is assembled, the cell can import free DNA fragments and incorporate them into its genome
-
gram- are capable of natural transformation and do not make competence factors, why?
they are either always competent or become when starved
-
gram- organisms import DNA through type IV pilus assembly
-
type IV pili assemble and disassemble at the cell surface expanding and contracting as a result. what does this do? 2
- happens constantly during the growth of a culture
- can help cells move across the solid surface
-
specificity is due to particular sequences in DNA that are recognized by part of the uptake apparatus, but not all gram- competence systems display such specifics
-
grams positives use nonpilis attachment proteins
-
bacterial conju requires the presence of special transferable plasmids that contain all the genes needed for pilus formation and DNA export
-
F factor contains 2 rep origins
oriV and oriT on the plasmid
-
plasmid
oriV function
oriT function
- replicate and maintain the plasmid in nonconj cells
- used to replicate DNA during DNA transfer
-
plasmid can exist in extra chromosomal and integrated forms, this is called
episome
-
refers to the fact that there are more cells capable of transferring chromosomal DNA in a Hfr pop
high frequency
-
why a F- never becomes an Hfr when an Hfr undergoes conjugation with it
the transfer never gets completed
-
Hfr was used to determine map genes based on how long it took the genes to transfer
-
the mapping process require mating an Hfr strain with a F- recipient that contained a mutation in the gene to be mapped. it is important in any genetic cross that the mutation create an observable phenotype
if the mutation is repaired by conjugation and recombination, it will grow and form a colony on an agar plate lacking the mutated amino acid
-
an aliquot of the mating mix is removed and the conjugation bridges are broken by blending
interrupted mating
-
The F factor derived from the plasmid that was inside of the chromosome (host DNA)
F' factor
-
F' plasmids do not have to recomb into the recipient X to be maintained, extra genes can be expressed a part of the F' plasmid
-
extra genes can be expressed as part of the F' plasmid, this established merodiploid situation
-
conjugal recipient of the F' factor contains 2 copies of those few genes, on the set of X and other on the F' factor
merodiploid
-
the conjugal recipient's 2 copies of those few genes could be used to
serve as raw material needed to evolve a new gene
-
1 group of plasmids that cannot transfer themselves can be mobilized if a transferable plasmid is also present in the same cell
-
mobilizable plasmids usually contain this
oriT like DNA rep origin recognized by the conjugation apparatus of the transferable plasmid. As a result when transferable plas begin conjugating so does the mobil plasmid
-
antibiotic resistance genes on plasmids
R factors
-
mobilizable plasmids along with transferable plasmids is one way in which antibiotic resistance genes can spread throughout a microbe pop
-
conjugation can be promoted by chem communication between cells
-
plasmid pAD1 produces a prot called aggregation substance in which it
catalyzes cell-cell contact and formation of conjug complex
-
when pheromone molecules are release by bac, they are these and how do they work?
- small pep
- they enter the donor cell through oligopeptide permeases present in the mem. Once in donor, pher stimulates transcript of pAD1 genes
-
Ti plasmid in the plant cell genome triggers the release of plant hormones that stimulate tumor growth of the plant. Plant cells within the tumor release this and do this
- AA derivatives, opines
- the microbe can then use as a source of C and N
-
bacteriophages can accidently move bac genes between cells as an offshoot of the phage life cycle
-
bac phages capable of gen transduc have trouble distinguishing their own DNA from that of the host when attempting to package DNA into their capsids. Host DNA gets packed in the phage capsid as well
-
this defines the ends of the phage genome, marking where packaging system cuts the P22 DNA and starts packing DNA into next empty phage head
pac site
-
specialized or restricted transduction is a phage mediated gene transfer mech that resembles the formation of the F' factors
-
how is specialized transduc diff from general
specialized can only move a limited number of host genes
-
specialized transduction will establish another merodiploid situation how
the new recipient contains 2 copies of a host gene, one originally present on its X and one brought in by the transduc DNA
-
safe sex approach to gene exchange
restriction and modification
-
the enzymatic cleavage of alien DNA and then protective methylation of self DNA
restriction and modification
-
Bac produce restriction enzymes that recognize short DNA seq and cleave DNA at or near those seq
-
how do bac avoid committing suicide
they protect themselves by produc matching modification enzymes that use S-adenosyl methionine to attach methyl groups to those same sex seq
-
only one strand of a seq needs to be methylated in order to be protected from cleavage from restriction enzyme
-
this type of restriction enzymes are used most for cloning and only possess endonuclease activity
type2
-
this type of restriction enzyme generally recognize palandromic
type 2
-
these 2 types of restriction enzymes have their restrict and mod activities in one multifunctional pro and cleave DNA from recog site
type 1& 3
-
if DNA is capable of autonomous replication
if DNA is not
- can coexist in cell sep from host X
- degraded by nucleases
-
kind of recomb requires two recomb molecules have a considerable stretch of homo DNA seq
general recomb
-
this recomb occurs via mech that requires very little sequence homology between the recomb DNA molecules
site specific recomb
-
this recomb requires a short seq recognized by recomb enzyme
site specific
-
a second CO that is some distance away from the first will lead to
an equal exchange of DNA in which neither molecule increases in length
-
3 ways recomb is advantageous
- DNA repair
- repair damaged genes
- enhance fitness
-
-
Before recA can find homology between 2 DNA molecules, the donor double stranded DNA molecule must
be converted to a single strand
-
the lacz+ gene product is normally used to
catabolize the carbo lactose
-
if lactose is the only carbo available the lacz mutant fails to grow
-
radiation resistant radiodurans is thought to use RcA protein to patch together homologous ends of fragmented DNA in a way that reconstructs the X after extreme radiation damage
-
phase variations are freq employed by pathogens to evade the host immune system by
changing expression of cell surface pro
-
a short 15bp seq known as the att site is present on these 2
-
this phage encoded protein engages the 2 att sites and through strand breakage and rejoining combines the molecule into 1
integras
-
a mutation that eliminates function
knockout mutation
-
spontaneous mutations are rare, and have a freq occurrence ranging from 10^-6 to 10^-8 per gen
-
this is a change in bonding properties of amino and keto groups
tautomeric shifts
-
a spon mutation can arise from tautomeric shifts in the chem structure of the bases
-
purine are particularly susceptible to spon loss from DNA by breakage of glycosidic bond connecting the base to sugar backbone. the result of this loss is formation of an apurinic site
-
apurinic
one missing purine
-
DNA damage can occur as result of metabolic activities of the cell that produce intermediates such as H2O2, etc
reactive oxygen species
-
naturally occurring intracellular methylation agents can spontaneously methylate DNA to produce a variety of altered bases
-
mutagens tend to increase the mutation rate by increasing the # of mistakes in a DNA molecule as well as by inducing repair pathways tat themselves introduce mutations
-
mutation rate for a given gene
# of mutations formed per cell doubling
-
tells you how many mutant cells are present in a pop
mutant freq
-
ratio of mutants per total cells in a pop
-
when a yeast cell rep to form a colony, a spon mutant yeast is unable to metabolize a compound which then accumulates and turns red
-
microbial survival depends on
its ability to repair
-
types of repair mech depends on 2 things
- type of mutation needing repair
- the extent of damage involved
-
some repair mech exise whole fragment og DNA or precisely excise the damaged bases or reverse them
-
if there has been extensive damage of DNA,
special emergency DNA polmerases are expressed that sacrifice rep accuracy to rescue the damaged genome
-
how can a cell repair a mutation after it has been introduced during rep
methyl mismatch repair which is base don recog of the methylation pattern in DNA bases
-
Dam methylates the palindromic seq GATC to produce GAmeTC
-
the pyrimidine dimers that form as a result of ultraviolet irradiation can be repaired by light activating mech
photoreactivation
-
photoreactivation function
- photolyase binds to the dimer and cleaves the cyclobutane ring linking the 2 adjacent damaged nucleotides.
- can be repaired without excising
-
this operates in the dark and is used to excise other kinds of damaged DNA as well as pyrimidine dimers
nucleotide excision repair
-
this 3 subunit exonuclease excises a patch of 12-13 nucleotides that includes the dimer
UvrABC
-
An RNA polymerase that encounters a UV dimer orother unrecognizable base on a template DNA strand willstall during transcription. The stalled RNA polymeraseis then recognized by a protein that mediates a processcalled transcription-coupled repair. The transcriptioncoupledrepair protein then snares a nearby UvrAB tobegin nucleotide excision repair.
-
examples of error proof pathways
- mismatch
- photoreactivation
- nucleotide excision
- base excision
|
|