Exam 3 Study guide

The Central Dogma (lect 1)

RNA polymerase is responsible for transcription of DNA into RNA
All genes encode RNA products

Why not just one? Chemistry generates differences

auto-catalytic cleavage facilitated by 2?-OH (RNA)
DNA has a wider/shallower major groove and is less rigid, easier to store
Why thymine? cytosine can easily degrade to uracil

Why not just one

Chemistry generates differences

Why thymine

cytosine can easily degrade to uracil

DNA ? information storage

Chemically stable, flexibility allows for easier storage (histones)
Double strand provides ? 1) protection from chemical attack 2) Redundancy of information (5? to 3? antiparallel)

RNA ? can be double or single stranded, but not redundant in total

Unstable RNA ? temporary messages ? mRNA
Stable RNA ? Enzyme components ? rRNA and Translator ? tRNA
Enzymatic Reactions ? Ribozymes

oriC ? origin of replication

Start at origin of replication (oriC)
Form Open Complex
Recognition of oriC by DnaA:ATP complex
Other proteins recruited; replication fork...
DnaB (helicase) ? unwinds the DNA
DnaG (primase) ? adds RNA priming strand...
DNA polymerase III ? copies the DNA
Replication Fork
Leading Strand ? copied in the direction of...
Lagging Strand ? copied in opposite...

Challenges ? Cellular functions do not stop during replication

RNA polymerases is copying genes
The replication complex must run into slower moving complexes of RNA polymerase
The replication complex somehow magically passes through such collisions

Leading Strand

? copied in the direction of the fork, adding on the 3? end (polymerase requires a 3? OH)

Managing the DNA ? 1.6mm of DNA (cell is at most 0.01mm long)

Tightly packed genome; DNA helix is supercoiled; histone-like proteins help to manage

Lagging Strand

? copied in opposite direction; repeated priming (every 1-2 kb) by primase and then elongation; DNA polymerase has 5? ? 3? exonuclease activity and degrades RNA replacing with DNA; finally all the breaks in the DNA are filled by DNA ligase

Segregation of DNA

Replication forks meet at the terminus and chromosomes separate
Membrane of the cell serves function of kinetochores

Involves

template DNA, RNA polymerase (bunch of subunits), Ribonucleoside Triphosphates – UTP, GTP, CTP and ATP

Core enzyme

alpha2
beta
beta prime

TRANSCRIPTION

? DNA to RNA

Holoenzyme

core and sigma subunit

STEPS

Recognition ? binding to promoter
Formation of the open complex
Elongation
Termination ? release from terminator

RNA polymerase

(bacterial)

Promoter Recognition

sigma subunit recognizes special sequence
promoter bound by RNA polymerase

Termination

Simple termination ? G-C rich stem loop; Series of Adenines; causes polymerase to fall off
Rho-dependent (complex) termination ? Stalled polymerase; involvement of protein Rho

Operons

Prokaryotic mRNA can encode multiple genes
Genes transcribed together are called operons
mRNA has one promoter, one terminator

DIFFERENCES between bacteria, archaea and eukaryotes

replication
promoters
mRNA to protein

Eukaryotic DNA Replication

Similarities with bacterial replication
Differences from bacterial replication

Similarities with bacterial replication

protein complex opens the DNA
bidirectional
RNA primase involved
Overall base insertion very similar (leading strand, lagging strand etc)

Segregation of DNA in Eukaryotes

Observable cellular program

Differences from bacterial replication

origin of replication every 50-100 kb
Cell cycle dependent (No competition with transcription)
one replication event at each origin

Observable cellular program

Prophase - chromosome condenses
Metaphase - nuclear membrane disappears and chromosomes align at center of cell
Anaphase - Sister chromosomes move apart to oppostie sides of the cell
Telophase - nuclear membrane reforms

Promoters

Archaea and Eukarya have different promoters than bacteria
RNA polymerase recognizes TATA
Proteins more important for guiding polymerase to site.

Transcription in eukaryotes

Generally similar
Have to deal with histones
Elongation affected by modifcation of histones
Transcripts typically only one gene
Gene splicing
Addition of 5?-cap and 3?poly A tail

The Genetic Code (lect 2)

Translation of mRNA to protein is performed by the ribosome
Functional structures of the ribosome are made of RNA

The Genetic Code

Nucleotides ? A, U, G, C
Constraints
must be co-linear
4 nucleotides
20 amino acids

Wobble Hypothesis ? 5? end of anti codon ( G , C, A, U ) - 3? end of codon ( U or C , G, U, A or G )

In the codon/anticodon interaction a G-U pair is tolerated in the third position

Players in translation

mRNA ? copy of relevant piece of DNA
Amino acids ? substrates of translation
Ribosomes ? machine where translation occurs

The real KEY of translation

formation of tRNA is true process of translation
Aminoacyl-tRNA synthetases
Recognize tRNA
Charge with correct amino acid

Overview of Translation

Elongation
assisted by release factors, ribosome falls off at stop codons

Elongation

Random insertion
GTP hydrolyzed per amino acid added

Wobble Hypothesis

? 5? end of anti codon ( G , C, A, U ) - 3? end of codon ( U or C , G, U, A or G )

Consider

Start (ribosome binding site & AUG)
Stop codon (UAA, UAG, UGA)
Frame

The moving polymerase problem

Transcription and translation are coupled in bacteria and archaea .

Differences in Eukaryotes

mRNA is processed
Translation initiation

EXCEPTIONS to the ?rules?

GUG, UUG can serve as start codons

Translation initiation

no Shine-Dalgarno sequence
go from first AUG

GUG, UUG can serve as start codons

Shine Dalgarno (RBS) not always necessary
Two different starts, Two proteins, one gene

21 st and 22 nd Amino Acids

Selenocysteine used in some proteins
Pyrrolysine
Translation Read-Through ? reads through a codon (stop for example and keeps going) cell hasn?t fixed this

Selenocysteine used in some proteins

Added at UGA codon

Polarity

blocking translation stops downstream transcription.

DNA sequence can be interpreted ? defined by computer

Promoter, start codon, protein, stop codon, terminator

Understanding the Central Dogma provides options

Computational approaches
Universal predictions
Manipulation of system
Commercialization of components

21

st and 22 nd Amino Acids

Discovery ? Allows prediction from sequence

Genes (ORFS)
Protein sequence

Manipulation ? molecular biology/biotechnology

Manipulation of organisms produces known result

Translation Read-Through

? reads through a codon (stop for example and keeps going) cell hasn?t fixed this

Mutations eliminate or alter function of protein

Enzymes in the central process key to biotechnology

Discovery

? Allows prediction from sequence

Manipulation

? molecular biology/biotechnology

Data for ORF

licheniformis complete genome.

Promoters -

These are not always the same.

Gi

56182545

From

657000 to 667000

Mutations: Heritable information can be changed (Lect 3)

mutations occur randomly, mostly at replication errors
cells have mechanisms to repair mutations,

What is a mutation

A change (from defined control) in the DNA sequence
Gene (genetic unit of function)
Regulatory region
Non-coding/extragenic sequence

cells have mechanisms to repair mutations,

DNA polymerase proofreading
Recombination
Error-prone DNA polymerase
Mismatch repair

Genotypes and Phenotypes

Genotype : DNA sequence of an organism.
Phenotype Behavior or appearance of an organism.

Genotype

DNA sequence of an organism.

Phenotype

Behavior or appearance of an organism.

Base substitutions

(transitions or transversions)

Frameshifts

-unique insertion or deletion gain or loss of non(x3) base pairs

Deletion

abc dk lmn

Duplication

abcde bcde fghi

Insertion

abcde xyz fghi

Inversion

abcde ihgf jkl

Base Substitutions

Replacing one base for another
Transition ? purine to purine -- pyrimidine to pyrimidine (A to G or G to A) (C to T or T to C)
Transversion ? purine to pyrimidine (A to C or T; T to G or A)

CAUSES of mutations

Errors in process (Replication/recombination)
Environmental inputs (mutagens/carcinagens)
Human manipulation (molecular biology)
Replication (substitutions)
Spontaneous mutation every 1/1010 bases synthesized

Mismatch repair system)

Frameshifts occur in redundant runs of bases
Electromagnetic Radiation
Deletions ? caused by abberant recombination
Duplications - another consequence of aberrant recombination (further amplification)

Frameshifts occur in redundant runs of bases

Strand slippage in replication

Electromagnetic Radiation

Ultraviolet light

Causes reactions between pyrimidines
Form T-T or C-C dimers
Cause double stranded breaks in DNA

Form T-T or C-C dimers

rays, gamma rays

Result in ?non-standard? base pair

Transposons
Oxidative Damage
Chemical Mutagens

Transposons

Mobile genetic elements
Insert into DNA and disrupt genes
Often have antibiotic resistance encoded.
Needs transposase, and end sequences

Oxidative Damage

Reactive oxygen compounds
If not quenched by cell, react with DNA

Reactive oxygen compounds

Singlet oxygen (O-)
Hydrogen peroxide (H2O2)
Super oxide (O2-)
Hydroxy radical, and more (OH-)

If not quenched by cell, react with DNA

Deamination, depurination
Random methylation of bases

Base analogs

(cause transitions)

Alkylating agents

(variety)

MUTATION FIXATION

When an error occurs the base looks ?odd?
For the mutation to become permanent, a second round of replication must occur
It is a race between repair mechanisms and DNA polymerase
Almost always repair mechanisms win

Repair Mechanisms

Mismatch repair
Photo repair
Methyl transferase
Base excision repair, nucleotide excision repair
Recombination repair (SOS repair)
Mismatch repair
Problem, which base is wrong

Mismatch repair

Repairs base substitutions
RECOGNITION (MutS)
EXCISION (MutL, MutH)
REPAIR (Pol I)

- Photo repair

Photolyase enzyme binds dimer
Light photon activates reaction
Base repaired
Methyltransferase
Base excision repair
DNA glycolase, DNA glycolase, AP...
Recombination Repair
DNA lesion ? replication fork demise ? RecA...
SOS Repair
DNA damage ? DNA pol III stalls and falls off...

Methyltransferase

Repair of methylated DNA
Several different enzymes for each type of methylation
Protein binds to damaged base
Transfer methyl to itself
Protein inactivated by reaction

Base excision repair

Removal of damaged bases
Multi-enzyme process

Multi-enzyme process

Damaged base removed
Sugar-phosphate backbone removed
Gap repaired

Recombination Repair

Growing cells will often have more than one active replication round
requires 17 proteins
Damaged region borrowed from good chromosome and lined up with bad
Damaged areas resynthesized
Found in eukaryotes, but has a different purpose -- Genetic re-assortment during meiosis

Damaged region borrowed from good chromosome and lined up with bad

Rec A very important in this process

SOS Repair

DNA damage
DNA Polymerase stops
RecA binds
SSB and PolV (umuC D complex)
Error prone repair
Polymerase begins again

Horizontal Transfer: Heritable Info can be changed (Lect 4)

DNA can be transferred between bacteria, and that this DNA is often not chromosomal.
modes of DNA transfer.
DNA elements, such as transposons and insertion sequences, can jump into the chromosome and cause mutations
if the DNA that transfers into a cell is homologous to DNA in the chromosome, the transferred DNA can recombine into the chromosome.

DNA can be transferred between bacteria, and that this DNA is often not chromosomal.

horizontal transfer of DNA is one mechanism of evolution.

modes of DNA transfer.

transduction
transformation
conjugation

Eliminate function

missense, nonsense, deletion, frameshift etc.

Change (gain) function

missense

Genome The complete genetic content of a cell or organism including chromosomes, plasmids and prophages.

Vertical transfer ? by replication and cell division
daughter cells inherit copy of parental chromosome
cell in population obtains a plasmid

Horizontal Transfer

Heritable Info can be changed (Lect 4)

Change levels

mutations in regulatory region

daughter cells inherit copy of parental chromosome

Horizontal transfer ? other than by reproduction

Bacterial Genomes

Chromosome

Large stretch of DNA
Haploid - 1 copy per cell
Codes for essential genes
Often circular
Some microbes have more than one

Often circular

Can be linear

Summary of Genomes

- Most bacteria and archaea have one circular chromosome

Genome

The complete genetic content of a cell or organism including chromosomes, plasmids and prophages.

Vertical transfer

? by replication and cell division

- (Some have linear chromosomes )

Many microbial species have plasmids

Horizontal transfer

? other than by reproduction

Plasmids

Close circular double-stranded DNA
Can direct their own replication
Some code for useful functions

DNA pol III, ssb protein, helicase

The addiction system ensures plasmid distribution

Plasmids in the natural world

Range in size from 2 kb to 1,000 kb
Maintained at one to hundreds of copies per cell
Code for non-essential functions
Some can conjugate -- move between cells

Plasmids are vehicle of horizontal transfer

acquired and/or lost by the cell
kept around by selective pressure

Code for non-essential functions

Drug resistance
Infection
Catabolism of unusual compounds

kept around by selective pressure

Example If you take a strain carrying a plasmid that codes for ampicillin resistance and grow it in a rich medium with no ampicillin, Most of the microbes will lose the plasmid

Darwinian definition: natural selection acts on pre-existing variables to favor advantageous traits

pre-existing variation? Large population size

Variation is pre-existing in microbial populations

induced mutations
spontaneous mutations

Microbial communities evolve together

Photosynthetic cannot fix nitrogen
Nitrogen fixer is not an autotroph
Separately do not do well, together

Darwinian definition

natural selection acts on pre-existing variables to favor advantageous traits

pre-existing variation

Large population size

Evolution of populations can be rapid and dramatic

The DNA make-up of a bacteria can change dramatically
Two ways to become resistant
Change
Addition

To be relevant in a population DNA must be

transferred
transformation ? uptake of naked DNA
conjugation ? movement of DNA involving cell to cell contact
transduction ? movement of DNA by virus
Independent replication
Recombination into the chromosome

transduction ? movement of DNA by virus

captured

Homologous recombination

Main function is in repair of severely damaged DNA
Also has role incorporating foreign DNA
System induced because DNA from transduction, transformation and conjugation (linear DNA here) looks like damaged DNA
Induces RecA and recombination system

General model of homologous recombination ?

DNA lined up with target stretch in chromosome
Double strand break*
5? to 3? exonuclease
Strand invasion (RecA)
Holliday junctions, branch migration, DNA synthesis
Ligation
Branch migration
Resolution

Natural Transformation

Competence to accept DNA occurs only at certain stages
DNA binds and one strand is incorporated
Preserving the DNA requires recombination into cell chromosome

Natural transformation

Streptococcus pneumoniae, Neisseria, Haemophilus . (pathogens )

coli

CaCl2 treatment

First discovered system F+

Joshua Lederberg 1950?s

Special Case ? Hfr ? high frequency of transfer

Hfr - High Frequency of Transfer
Integration of F plasmid into chromosome
Transfers chromosomal DNA
usually aborts before complete transfer

The transfer of genetic information between cells through the mediation of a virus (phage) particle.

DNA packaged in protein coat
Injected into host cell, with or without accompanying viral (phage) genes
Form (linear, plasmid) and source of the DNA determines what happens to it

HOMOLOGOUS RECOMBINATION AFTER TRANSDUCTION

phage capsid with host DNA attaches
injects DNA
enters host by homologous recombination
can change genes this way

Mobile genetic elements

Sequences of DNA found in all organisms that are capable of moving
Types
Insertion sequences ? only code for function for moving DNA
Transposons ? have useful genes for host ? antibiotic resistance (Tn5 ? a bacterial transposon)

general mechanism of transposition

Transposoase binds to recognition site
Cleavage
Target bound
TRANSPOSONS can cause mutations and/or bring in new DNA
MGEs are 700 to 10,000 bp
Have transcription and translation stop sites near ends
Selectable markers (drug resistance)

Target bound

Insertion

MGEs are 700 to 10,000 bp

Disrupt genes and could kill host

Variation

Spontaneous or induced mutation
Horizontally acquired DNA (that becomes fixed)

Features of a virus

infectious, obligate, intracellular parasite
small; 25 to 900 nm in diameter
package their genomes inside a shell
genomes of DNA or RNA
The genomes contain information for initiating and completing an infectious cycle
The virus replicates inside the host, parasitizes necessary host functions and directs the synthesis of new virion components .

CLAssification

Host range
Size/shape of virion
Genome properties

Viral Stuctrues

Capsid - Protective shell around the virion
Nucleic Acid- genomic information
Envelope (eukaryotic viruses)- lipid coat about the virion

Capsid

Protection from temperature, chemical and physical damage
Important in delivering the viral nucleic acid into the host.

Capsids can be non-enveloped or encased in an envelope

Primarily present on animal viruses
Made of lipids,carbohydrates, and proteins.
Lipids and carbohydrates taken from the host on extrusion.

Viron Content

genome
virally encoded proteins

Rational for Regulation

static system ? deal with what you can
flexible system ? be prepared and able to change
name of the game - Sense a need (or an opportunity) and change behavior to respond to it.

Allosteric proteins ? ?other state?

Changes conformation in response to binding something (usually small molecule)
Change effects activity - (i.e., enzymatic activity, DNA binding activity etc.)

Helical (filament)

Capsomers arranged as a hollow spiral

Regulations of Transcription ? regulatory proteins

Initiation is the most common point of regulation

MICROBES RESPOND TO STIMULI

BY REGULATION (lect 6)

- NEGATIVE regulation

Repressor binds near promoter
Binding of repressor blocks promoter
RNA polymerase cannot transcribe operon

Inactivation of Repressor

Inactive repressor falls off of binding site
Promoter open
RNA polymerase can now proceed

Positive regulation with activator

activator on RNA polymerase is binded to and transcription occurs
if the activator on is not on RNA polymerase and is not binded to then transcription will not occur

Allosteric proteins

? ?other state?

Regulatory proteins have two stages

Competent to bind/incompetent to bind

DNA binding proteins

- Sequence specific contacts with DNA, can have consequences for other interactions

Regulations of Transcription

? regulatory proteins

Regulatory possibilities

binds and represses
binds and activates
fails to bind

Proteins

Repressor, activator

Positive

Activator
Binds operator (usually upstream of promoter)
Recruits polymerase for transcription

Negative

Repressor
Binds operator (usually downstream of promoter)
Blocks polymerase from transcribing

Genes required to synthesize the amino acid methionine?

transcribed when methionine is LOW
these genes are regulated by a REPRESSOR

OPERONS

Often more than one protein is needed for a functional group (I.e. pathway)
Prokaryotes will often organize into cotranscribed groups called operons
Easy regulation

Regulatory proteins impact RNAP ability to act.

sigma factors are regulators

Archaea/ Eukarya Promoters

Promoter recognition proteins not associated with RNAP
TATA binding protein [TBP] and Transcription Factor B [TFB] and E
Bind first, then recruit RNA polymerase

RIBOSWITCHES

mRNA
untranslated region binds to metabolite
can hide ribosome binding site
can generate terminator

Regulation by small RNAs

6S RNA
binds to 70 subunit and reduces transcription at some promoters (primarily in stationary phase)

Recap

Sense stimulus and convert it to appropriate action (behavior change)
So, far simple binding and conformational change which as consequences? but it gets more sophisticated

ATTENUATION ? the status of a process (translation) is monitored.

Remember Polarity

- 6S RNA

- binds to ?70 subunit and reduces transcription at some promoters (primarily in stationary phase)

Consider Tryptophan biosynthesis

Short sequence of amino acids (leader peptide)
Contains multiple trp codons
If concentration of trp is low, ribosome pauses
If concentration of trp high, ribosome reads through and pauses at end
HIGH Trp = low expression (transcription termination)
LOW Trp = high expression (transcription of trp genes)

Antisense RNA regulatory mechanisms

- ryhB down regulates iron storage genes in absence of iron

Catabolic Pathways ? lactose utilization genes

ON when substrate is present
OFF when substrate is absent

Anabolic Pathways ? methionine biosynthesis

ON when product is absent
OFF when product is present

HIGH Trp

low expression (transcription termination)

LOW Trp

high expression (transcription of trp genes)

Regulatory proteins have two states: ON (can bind) and OFF (can?t bind)

most can?t bind when there is a higher concentration of effectors
most can bind when there is a low concentration of effectors

Catabolic Pathways

? lactose utilization genes

Other Post-Transcriptional

mRNA stability
Translation
rate of protein synthesis (codon usage)
strength of ribosome binding site.

Anabolic Pathways

? methionine biosynthesis

Other

Post-Transcriptional

Example

often found in amino acid biosynthetic pathways – Stops the first “committed” step

Post-Translational Modification

inactivates central metabolic enzyme

Variations in regulating enzyme ACTIVITY

Feedback inhibition (end product inhibition)
Protein modification (tag protein to change function)
Regulated proteolysis

null

STANDARD

Repressor (eg.

Sensing the Environment

Moving the signal inside can be done directly (metabolites) or indirectly (pH, temperature)

TWO COMPONENT SYSTEM

Histidine protein kinase
Response regulator
Phosphatase removes phosphate from response regulator

Histidine protein kinase

Often in membrane
Senses environmental stimulous
Phosphorylates itself
Transfers phosphate to response regulator

Response regulator

Carboxy-terminal domain carries out the regulator role
Amino-terminal domain inhibits the carboxy-terminal domain.
Phosphorylation of amino-terminal domain stops blocking of activity

Sporulation

Several genera capable of forming endospores
Best understood system in gram-positive microbe Bacillus subtilis
Involves a complex regulatory cascade controlling over 200 genes
Think of temporal and spacial constraints of the whole process
Requires sequential expression
Quorum sensing

Regulatory interplay at one site

Tryptophan biosynthetic pathway (consider in isolation)

Regulatory systems define

disrupt the system, see how it responds, make conclusions about function

Constitutive (always on) expression

A mutation that disrupted attenuation would be a deletion of the attenuator (gives constitutive expression)

Global regulation strategies, methods or rationale

? or adaptive responses Starvation is the initial signal

Adaption

Ability to change behavior in response to demands placed on the individual by the environment.

Marine invertebrates will move away from polluted waters
If an animal is straving, its metabolism will change, use fat, slow catabolism/anabolism, become infertile

In Microbes?

photosynthetic apparatus of Rhodobacter changes with light conditions

GLOBAL REGULATION

strategies, methods and rationale or adaptive responses (lect 8)

MICROBES ADAPT

Pre-exposure to stress often allows survival of lethal dose

Things to consider

Readiness is expensive - enzyme expression
Probability of encountering the stress
Speed of adaptation
Normal tolerance to stress

Molecular Considerations

Single pathway strategies (binded ? ON, not binded ? OFF)

Regulatory proteins

catabolite repression, SOS, iron stress, etc?.

Alternative sigma factors;

heat shock, nitrogen regulation, envelope stress, etc?.

Small RNAs;

iron stress, oxidative stress (more to come??)

GENERAL principles of global regulation

Parallels what happens at single locus
Solves a cellular (global) problem,
Often integrated with multiple systems, both global and linear

Catabolite Repression

Regulation provides functional information
stress occurs
regulation occurs (change in gene expression)
induced functions address the stress
changed ?behavior?

To determine the genes regulation in response to a heat shock you would like to monitor

DNA sequence
Protein synthesis
mRNA synthesis
Level of tRNAs

? The Theory?

cells growing under relevant condition
isolate mRNA
Determine what genes the mRNA represents
sequenced genome (ability to identify hybridization)

Time Genomics

analysis of all genes (genome) at the same time

Miccroarrays (DNA chip) ? identifies the genes that are being transcribed

extract mRNA
add fluorescent dye
mix with DNA array
detect

Virulence Genes

Incubate with and without host
Those genes expressed in host are good candidates for virulence factors
Technology should facilitate, not drive the biology
Global techniques are powerful hypothesis generators
In general they do not test hypotheses

Miccroarrays (DNA chip)

? identifies the genes that are being transcribed

Genetics as a thought process

Start with a system (cell)
Remove a component of the system (mutation)
Observe the resulting perturbation of the system (growth phenotype)
Deduce role of the relevant component in the functional system.

Mutations

help us understand phenotypes, and phenotypes help us understand mutations

SELECTION

Define a condition where ONLY the cells with the phenotype of interest grow

SCREEN ?

by replica plating ? easy
by enzyme assay ? hard
indicator plates ? easy

SCREEN

“Look” at all cells and find the ones with the phenotype you are interested in

Molecular Biology, biotechnology

Understanding of central dogma provided by classical microbial genetic approaches
Technical advances
Specific phenotype > identify mutations
Specific mutations > identify phenotype

MICROBES ARE KEY

Essential for the emergence of the field
Molecular biology is the ?perversion? of normal processes identified in microbes.
The ?tools? were first defined and understood in microbial systems.

(NOTE: outcome of basic microbiological research.)

Microbes remain a reagent in the field -- Microbes provide a ?bag of enzymes?.
Genetic and biochemical manipulation facilitate many tasks when the natural host organism is difficult.

(NOTE

outcome of basic microbiological research.).

Manipulation of DNA to create a desired product.

Different sequence
Increased production (of DNA or protein product)
Heterologous systems (move into different organism)

Needed in general

DNA
restriction enzymes
a replicon
polymerase
ligase

Restriciton enzymes in reality

Part of restriction/modification pairs
Recognizes self (modified) and ?restricts? others

DNA ? can isolate it from organism of interest

can ?make? it using PCR technology (polymerase)
can ?move? it , which allows manipulation (restriction enzymes, ligase, plasmid replicons)

PCR- make it

Amplification of DNA using replication-like conditions
4 nucleoside triphosphates ? GTP, TTP, ATP, CTP
DNA polymerase

Move it

restriction enzymes ? cut
ligase ? pastes
replicon ? allows propagation allowing subsequent manipulation

Restriction Enzymes ? recognize and ?cut? DNA at a specific sequence

either ?blunt? ends or ?sticky? ends

Exam 3 Study guide

Microbiology 303 with Downs/forest/rondon at University of Wisconsin - Madison

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