the transfer of genetic material from one cell to another via naked DNA

Transfer is unidirectionl from donor to recipient cell - no fusion of cells as occurs in eukary

defining criteria:

• it is sensitive to DNase
• it does not require cell to cell contact
• it does not incolce bacteriophage particles

primary mechanism of spread of antibiotic resistance within a bacterial species and

exchange of virulence factors

But one of several mechanisms of antigenic variationto overcome host responses

Gene transfer is rare (10^-2 to 10^-6 per cell)

but in the lab we use genetic markers (mutations) that provide us with the ability to select for transferred genes (i.e. antibiotic resistance); the cell wont grow in the presence of the antibiotic unless they have acquired the resistance GENE

The transformation efficiency is small (10^-3 transformants per recipient cell for a given marker)

the cells develop competence --> the ability to bind and uptake DNA on their own, often at limited periods during growth in a culture

DNA is FRAGMENTED during uptake into recipient cell

Gram negative bacteria and Gram postive (there is a list on the slide) 

competence is developed only under unusual lab conditions for ex, high salt, heat shock for E. coli

DNA is taken up in an INTACT form (not fragmented)

Allow Ecoli to transfer naked DNA

exposure of cells to very short burst of high electirc feild that transiently permeablizes the cells and causes them to take up molecules from the surrounding medium

used in labs to transform cells that will not take up DNA

Competence is NOT involved

DNA is taken up in an INTACT form

* Works for both prok and euks

• Very similar to Gram + except for two big exceptions:

(best studied in Haemophilus and Neisseria)

• DNA binding requires specific sequences in the double stranded DNA in order to bind to the receptor
• DNA is taken up in a double stranded form in to a special membrane vesicle called a transformasome --> one strand is integrated into chromosome
• in  Haemophilus the sequence is 11bp long and is present frequently in Haemophilus DNA but rarely in the DNA of other bacteria 

membrane vessicle that takes up double straned DNA during Gram (-) transformation

This dsDNA is later intgrated as one strand into the host chormosome

• DNA is released from the cell by lysis or extraction - usually as large DNA fragments due to shearing
• DNA binds to cell surface receptor (does not have to be specific) 
• The ssDNA invades the homologous region and displaces one original strand which is then degraded
• the heteroduplex is replicated, producing one transformant and one unaltered recipient genome
• Genome markers close together in the genome can be transformed together on a single strand
• ( ex: if orginial strand is A-B- but the new sstrand is A+B+, both genetic markers can be transfered to genome--> which now becomes A+B+ )

direct transfer genetic material from one cell to another via CELL to CELL CONTACT

requires:

• specific surface protiens (including pili in the donor cell)
• DNase resistant (while transformation is sensitive)
• its does not involve a phage 

The ability to conjugate is usually encoded by a plasmid on the donor cell, these plasmids may be autonomous,in a freely replicating state or they can be integrated into the host chromosome

covalently closed, circular supercoiled DNA that replicate autonomously

they are stably inherited in an extrachromosomal state

(can also be intergrated into chromosome)

some can be linear

vary in size (2kb to 1 Mb)

usually NOT essesntial

Many carry genes that confere detectable phenotypes:

• Anibiotic resistance
• toxin production
• abiltiy to degrade organic compounds
• bacteriocin production
• metal ion resistance
• virulence
• ability to conjugate (tra gene)

ability to promote their own transfer from one cell to another (tra, oriT)

they can cause a high frequency of transfer of chromosomal markers if integrated into chromosome

unable to promote their own transfer

2 Types: Mobilizable  and Nonmobilizable

cannot transfere themselves but they can mobilize to transfer by another conjugative plasmid in the same cell

they are oriT + (nic+ or bom +)

may produce one or more auxillary plasmid-specific mobilization (Mob) protiens that let them use the Tra protiens of the conjugate plasmid

the number of copies of the plasmid per cell, determined by the plasmid replication system, not the plasmid size

BUT: often small plasmids have large copy numbers while large plasmids have small copy numbers

Tra genes encode pilus protiens, Dna transfer protiens, and cell surface protiens( includes exclusion protiens)

IS sequences confer the aility of F to integrate into the host chromosome

no f factor, they are good recipients for DNA transfer by conjugation, the other three (F +, Hfr, and F' ) are poor

due to their synthesis of surface exlusion protiens

contains the F factor and it is integrated into its chromosome

F' the extrachromosomal F factor also carries some chorosomal genes incorporated during exicsion of an integrated plasmid

*the F+, Hfr and F' states are interconveratble by homologous or nonhomologous recombination

1. Hfr cell contact the F- cells with the F pillus --> retracting it to make close contact and the conjugation bridge
2. Nicking occurs at the oriT, rolling circle replication ensuse and the 5' end of the transfered strand enters into the recipent
3. In this case sequences needed for circularization are not transfered bc the conjugation bridge usually breaks before the whole chromosome can be transfered
4. transfered strand finds its homologue in the recipient chromosome and recombines into it to form the recombinant  --> multiple contiguous genes are transfered 

1. F+ cell contact the F- cells with the F pillus --> retracting it to make close contact and the conjugation bridge
2. Nicking occurs at the oriT, rolling circle replication ensuse and the 5' end of the transfered strand enters into the recipent --> where the complementary strand is made 
3. When the entire plasmid DNA has been transfered it circularizes and becomes stably replicates in the recipient cell 
4. Conjugation bridge is broken and both the donor and the recipient now carry identical plasmids

*pili also serve as receptors for male -specific phages such as F1 and M13

Ex: Enterococcus (strep) faecalis

both plasmid and chromosomal genes are involves

there are NO pili involved

recipient cells make small peptides pheromones and release them into medium, Donor cells respond making adhesins that interact with receptors on both donros and recipient cell walls --> clumping of cells

Conjugation bridges for btw clumps and DNA is trasferred from donor to recipient

multiple pheromones, BUT each is plasmid specific --> cells containing a plasmid do not make the pheromone specific for that plasmid

No! multiple drug resistance are encoded by a single plasmid

the IS elements can lead to transposition of the intervening DNA segment into other DNA

any IS element can serve as a region of homology for recombination into the host chomosome -->homologous recombination btw two IS elements in the plasmid can lead to excision or inversion of the intervening DNA depending on the realtive IS orientations

• discrete DNA elements that carry PROMOTERLESS antibiotic resistance gene
• in contrast to transposons, integrons integrate into specific sites and do not encode trasposase
• instead the target plasmid contains the 8 bp integration site and encodes the integrase protien (int)
• the plasmid must provide the promoter for the transcription of the anitbiotic resistant genes
• plasmids with integrons become frequent targets for integration b more integrons which use a conserved 60bp sequence (box) for integration
• *** more mulit drug resistant plasmids are created by integrons than by transposition of individual transposons

large continguous blocks of DNA encoding groups of genes involved pathogenesis

often encodes virulence factors such as toxins, secretion systems, pili ects

Often have a G+C content different from the rest of the chormosome

beleived to originiate by horizontal transfer --> not stepwise adaptive evolution of host genes by mutation

dsDNA

most of the time is a single sircular molecule -- 50% (1-9 Mb)

Can be more than circular molecule (40%) (0.5-5Mb)

single linear molecule (ex: Borrelia) ~1Mb <5%

more than one linear molecule (ex: Streptomcyces) 0.5 - 5 Mb <5%

No!

dsDNA

most of the time is a single sircular molecule -- 50% (1-9 Mb)

Can be more than circular molecule (40%) (0.5-5Mb)

single linear molecule (ex: Borrelia) ~1Mb <5%

more than one linear molecule (ex: Streptomcyces) 0.5 - 5 Mb <5%

*usually dsDNA but not always, NOT essential 

plasmids --> 2-500 kb usually circular

bacteriophages--> 5-250kb ~50% circular

Can be..

integrated into plasmids

integrated into bacteriophages

remnants of plasmids and bacteriophages

transposable elements

pathogenicity islands

requires extensive regions of DNA sequences of homology

*homology= identical od very similar for 50-500 bp

AND  host or phage encoded homologouse recombination protiens

(these are used for generalized transduction, transformation, and conjugation)

insertion of transposable elements into diiferent non-homologous sites in the host DNA; mediated by transposase protiens encoded by the ransposable element

- used by insertion sequences, transposons and phage Mu

requires very little homology (25-50 bp) and uses enzymes that recognize specifice sites and recombine them

- lambda intergration

Contain an upstream regualtory region - which usually contains a promoter, and a repressor binding site

after the start site there is a coding region of variable length

the coding region ends with the termination/stop signal

After the coding region there is a termination region (50-100bp)

the DNA segment is transcribed to produce a single mRNA which codes for more than one polypeptide

Activator/activation site - binding site of the activator protien

operator- binding site of the repressor protien

promoter - binding site of the RNA polymerase

rbs- ribosome binding site

Nope! but it is in prokaryotes

as the DNA transcribes the mRNA, ribosomes bind in sucession and begin translation -- mRNA degredation follows close behind

When does translation initiate in prokaryotes?

most mRNAs have 10-50bp leaders prior to protiens coding sequences

begins translation when ribosomes recognizes a rbs ~6bp upstream of the initiation codon (usually AUG, can be GUG, CUG)

prok: no introns, the gene and ploypeptide products are colinear,

genes are often group together into multicistronic operon--co-ordinate regulation

transcription and translation are coupled

NO mRNA GTP cap or poly-A tail

prok protiens initiate with N-formylmet which act as chemoattractants for neutrophils and monocytes

1. purine--> purine (G to A)

ect.

2. purine --> pyrimidine (G or A to C or T)

these mutatations may or may not change the aa and therefore the protien product (third position is often variable)

insertion or deletion of one or more bases --> change the reading frame of the ribosome. This leads to the incorportation of the wrong aa --> usually results in stop codon

*severely defective

hundreds to thousands of contiguous bases are lost

** severly defective or undetectable protiens

insertions of transposable elements

* like deletions they cause severe defects in protien fxn

• Mistake made during DNA replication and repair--> called spontanerous mutations (10^{-6 -9})
• Mutations caused by natural exposure to mutagens--> also called spontaneous mutations (10^{-6 -9}) (ex: UV, cosmic rays, heat, transposable elements) 
• Caused by purposful exposure to mutagens: Xrays, UV, chemicals ( occur at a higher frequency 10^-3-5)
• caused by error prone repair of DNA damage -->SOS system is highly error prone

Conformational change in a protien can result from binding of another protien or small molecule,

conformational changes alter the aility of the protien to interact with its substrate or other interaction partners--> such as DNA, RNA or other protiens

Yes!

regulation is accomplised by the concentration dependence of binding of small molecule inducers and co-repressors to the regulatory protiens

small molecule signals are transproted into the cell or are made by the cell

• Catabolite repressor -- CAP  catabolite activator protein  (also called CRP, cAMP receptor protein
• CAP is an activator protein that bnds to the promoter region of the lac operon interacting with the RNA polymerase and turns on transcription
• CAP alon cannot bind to the promoter DNA ***olny CAP in a cAMP-CAP complex can bind to DNA -->CAP is a monitor of cAMP levels
• specificity is exerted at the level of each sugar catabolic operon by the binding of a receptor  --> so if the glucose levels are high =low cAMP
• This results in both positive and negative regulation /control

yes! this is the form of negative control on the lac operon (catabolite repression) to ensure that the BEST (glu) is used as a source for carbon

when given a mix of glu and lac they will use up all the glu before turning on the lac operon

** catabolite repression is mediated by the concentration of cAMP

When glu is high --> cAMP is Low

it is transcribed from the promoter as a polycistronic mRNA

which is then TRANSLATED into three proteins (beta-galactosidase, permease, acetylase)

inducer binds to the repressor and induces a conformational change that makes the repressor unable to bind to the operator DNA

*because the lac promoter is poor only a small amount of transcript is made -- they are translated making three proteins

high levels of cAMP which binds to CAP  forming the CAP-cAMP complex and binds to the promoter

BUT since there is no lactose = no inducer therefore the repressor protien is still bound to the operator and prevents the transcription of the lac operon mRNA ect.

the lac repressor binds to the inducer and can no longer bind to the operator DNA

Due to the absence of glu --> there are high levels of cAMP which can comples with CAP and bind to the promoter and interact with RNA polymerase causing high levels of transcript to be made= large amount of lac proteins

encodes five enzymes --> biosynthesis of aa tryptophan

between the operator and the first gene is the short region encoding a leader peptide which contains two tryptophans (W) near its C-terminus

Trp operon is regulated by a combination of mechanisms using both repression and attenuation

it is a biosynthetic operon --> it is default ON (anabolic)

the repressor protein by itself is inactive --> does not bind to DNA

When trp (co-repressor) is in excess its binds to the repressor protein changing its conformation allowing it to bind to DNA

the ribosomes stall at the 2 trp codons allowing for the formation of 2-3 hairpin loop --> an anti-terminator structure

this allows transcription to proceed in to the downstream structural genes

*** if there is no protien synthesis hairpins 1-2 and 3-4 form resulting in termination

the leader region of the trp operon contains complementary sequences 1,2,3,4

when W levels are high, ribosomes translate the leader mRNA quickly, preventing the formation of the more stable 2-3 hairpin, and leading to the formation of the 3-4 hairpin that serves as a terminator ( this prevents the transcription of the downsteam structral genes)

Bacterial pathogens possess these, allow them to survive in multple host. They have evolved a regulatory system that specifically allows them to turn them on and off only when they are in the host

EX: phosphorlating type two component regulatory systems enavle bacteria to monitor their external enviroment for signals that differ significantly btw the host and the external enviroment and adjus their virulence gene accordingly

Bacteria communicate with eachother by releasing specific signaling molecuels and by measuring the local concentrations of signaling moelcules in their enviroments

As the cell density of the pop increases the concentration of the signaling molecules also increases, when a concentration threshold of the signalng molecules is reached, the cells respond by turning on a new set of genes, This ability of the cells to sense the cell density is called quorum sensing

How does quorum sensing affect the bacterial pathogens and their abiltiy to evade the human host?

They use it to co-ordinate their expression of virulence genes in order to evade the immune response and establish a successful infection

**chemical agents that interfere with quorum sensing are potential alternatives to anitbiotics  --> inhibit the ability of the pathogen to be harmful not kill it

it is esp important in biolfilm!

biofilms are organized microbial systems of layers of microbial cells embedded in to polysaccharide matrix of microbial orgin associated with surfaces

Biofilm Formation:

1. Attachment (adhesion of few cells to a solid surface)
2. Colonization (intercellular communication, growth, and polysaccaride formation
3. Development ( more growth and polysaccharide)

Yes!

biofilms form on catheter, teeth, contact lenses, toilet bowls, GI, GU tract and in the lung

They are usually much more resisitant to antibiotics bc they dont reach the bacteria in the center --> they also ompede access of the host immune response factors and cell to those bacteria

Ex: psuedomonas aerginosa uses quorom sensing to for biofilms and produce alginate!

with out the ability to quorom sense they would not be pathogenic (would not cause pnuemonia)

obligate intracellular parasites that replicate by self-assembly of individual components rather than by binary fission

they cannot make energy or protiens independ of the cell

contain a genome of the limited size that is eiher RNA or DNA but NOT BOTH

the physical and biochemical characteristics of the virus partical

size, morphology (shape and presence of an envelope), type of genome (RNA, DNA) and mechanism of replication

Circular, or linear ss RNA:

+ RNA genomes are the same sense as messenger RNA

-RNA genomes are the oposite sense as mRNA

Linear dsRNA

linear ssDNA

circular or linear dsDNA... so you cant have ss circular DNA

*some contain segmented RNA which are effectively like several chromosomes within a single virion

• Into protien shell known as capsids: rigid structures that withstand enciromental conditions
• capsids are the result of self assembly virally encoded capsomeres
• they can come in three forms:isosahedral, helical or complex * the shape is dictated by the capsomere
• Genome+capside = nucleocapsid =viron for "naked" viruses

Some viral nucleocapsids can be surronded by a lipid envelope that comes from the cellular membrane

nucleocapsid + membrane = viron for enveloped virus

virally encoded glycoprotiens are inserted in the membrane and serve as virus attachment protiens and membrane fusion protiens

**enveloped viruses are less stable than naked viruses --> more susceptible to drying, sensitive to detergents and alcohols and cannot survive in the GI

Enveloped viruses spread in large droplets, secretions, organ transplants and blood transfusions

Which are more stable, enveloped or naked viruses?

naked!

Enveloped viruses are less stable than naked viruses --> Due to the lipid composition : more susceptible to drying, sensitive to detergents and alcohols and cannot survive in the GI

1. Attachment
2. penetration: endocytosis, membrane fusion
3. uncoating
4. early transcription and synthesis of nonstructural proteins: RNA viruses--> virally encoded RNA dependent RNA polymerases/ DNA viruses --> uses host RNA polymerase *except pox
5. Genome replication: RNA viruses (cytoplasmic) v DNA viruses (nuclear *pox)
6. late transcription and synthesis of structural proteins
7. assembly of virus particles: RNA viruses (cytoplasmic) v DNA viruses (nuclear *exc pox and hepadnaviruses)
8. release of viruse particles: cell lysis and budding (enveloped viruses)

1. Inhibition of cellural protien synthesis
2. Inhibition and degradation of cellular DNA
3. alteration of cell membrane structure
4. disruption of cytoskeleton
5. formation of inclusion bodies
6. toxicity of viron replication

• Functions as mRNA and is immediately translated by cellular ribosomes
• Tranlated as a polyprotein which must be cleaved into individual protiens
• one of these proteins is a RNA-dependent RNA polymerase that transcribes -RNA strands from the +RNA strand
• (-) RNA strands are used as a template to make many copies of the +RNA genome
• +RNA copies are used as mRNA to make structural protiens and are encapsilated to produce nucleocapids

cannot be used a mRNA, must be used as a template to transcribe +RNA (mRNA) strand

to transcribe the (-)RNA genome the incoming viruse particle must CARRY a RNA-dependent RNA polymerase

resulting +RNA strand can now be translated into protiens and more  (-) RNA to be used as templates for more transcription

the newly produced (-) RNA genomes can now be packaged with the newly translated viral protiens and encapsilated = nucleocapsids

They must carry reverse transcriptase

+RNA genome is reverse transcribed into dsDNA and integrated into the host genome

retrovirus protien and +RNA genomes are produced by host enzyes

• ***Except poxviruses
• transcribed by host DNA-dependent RNA polymerases
• many viruses have a host shutt-off mechanism that degrades mRNA
• many viruses use specific transcript factors that redirect host polymerases to viral genes and away from cellular genes
• viral genomes replication is dependent on virally encoded DNA dependent DNA polymerases for larger viruses but for smaller viruses - they can use host DNA polymerase
• newly produced DNA genomes are encapsilated

 hole in confluent monolayer of cells --> plaque and can be used to measure viral growth in labs

Plaque assays: measure the # of infectious virions in a given volume of lysate

1. Generally measured as plaque-forming units (pfu) per ml of lysate =titer

This is a biological assay of infectivity

lysate is the suspension of virions in culture medium that results from unrestricted growth of the viruses on the cell monolayer

not all viruses produce in a lysate are infectious

is the ratio of number of infectious particles to the number of target cells to be infected

an MOI =1 will only infect about 60%

MOI =5 -10 is needed to infect 100% of the monolayer

Single-cycle growth curve

can be divided into 2 periods:

eclipse: post penetration phase until virus can be detected intracellularly -->cooresponds to uncoating, early transcription and genome replication steps, ends at virus assembly

latent period: post penetration phase until virus can be detected extracellularly, incudes the eclipse period, corresponds to uncoating, early transcription, genome replication, virus assemble, release

Viral Mutations

exchange of protiens

• if genome wtih a lethal mutation in gene "X" arises along with the wild type genome in the same cell --> then the wild type copy of gene X can provide the funtion for the mutatnt genome
• Result: the mutant genome will be packagesd into a capsid and will be able to infect a second cell upon release
• BUT: the mutant will not be able to replicate in the second cell bc the genome still contains the lethal mutation in gene X

an exchange of genetic material on the same segment of genome

• if the genome with a lethal mutation in gene X arises along with a wild type genome in the same cell then the mutation can be removed by recombination with wild type for gene X
• the virus will now be able to replicate in a second cell bc it now contains the wild type in its genome
• recomb occurs often in DNA viruses
• True recombination does not occur in RNA viruses

an exchange of genetic material on a different segment of genome

• if two segmented viruses infect the same cell they can exchange some of their segments at the time of virus assembly
• a novel strain of virus is created that is composed of segments of each virus "parent"
• this is an important mechanism for the flu!!

• Fecal-oral
• respiratory - inhalation is the most common route of infection
• blood borne
• sexual
• maternal neonatal
• animal reservoir or arthropod vector

They enter through breaks in the skin or thru the mucosa and initial replication occurs in cells that express viral receptors and contain appropriate cellular factors for replication

Localized spread of virus can be acheived by:

Release of virus from an infected cell and subsequent infection of surrounding cells

some enveloped viruses can fuse an infected cell with uninfected cells to directly spread to surrounding cells (syncytia formation)

• Virus may spread from the original site of infection by gaining access to the bloodstream or lymph system

• the prescence of virions in blood is termed viremia

• viruses can gain access to the central nervous system by circumventing the BBB, thru CSF, or by direct uptake in the peripheral nerves

• The period postinfection prior to onset of the symptoms
• 1-2 days is a short incubation period and is characteristic of viruses that do not require secondary spread for symptomology 
• 12-14 days is usually the minimum for viruses that require secondary spread
• some viruses have extended incubation periods of months or even years (HIV)
• most patients are infectious during incubation period

acute phase: in symptomatic phase of infection  - can be very mild or asymptomatic; most viral infections completely resolve following acute

persistant: immunocomp patients, 3 forms

Chronic(productive) virus is produced at low levels but may not continue to cause disease sympt (Hep B)

Latent: viral genome remains in cells indef, but viruses particles are not produced, can be reactivated!(herpes)

Transforming: intact or partial virus genome integrates in cellular DNA or is otherwise maintained in the cell adn immortilizes the cell - altering growth properties (ex oncogenic viruses --> RNA =retroviruses, Hep C or DNA = Hep B, papilloma, poloma, adeno type 2,

First defense, three types (alpha, beta, gamma) but all are secreted cytokines that regulate immune and inflammatory responses

the secreted IFN bind to surronding, uninfected cells and induce pathways that prevent replication

Non-specific: NK cells and interferon

viral PAMPs  are recognized by TLRs and cytomplasmic PRR to induce alpha and beta IFN

main PAMP is ds RNA, along with unmethly DNA and 5'modified RNA ss

the secreted IFN bind to surronding, uninfected cells and induce pathways that prevent replication

arise several days post infection

• CD8 cytotoxic cells are the most important cellular response to primary viral infections - lysis results after Ag presentation
• Ab can neutralize --> but this is not that important (bc children that are defective in Ab production are not more susceptible to viruses)
• Viruses have evolved to evade host responses: Ag variations allow them to escape humoral responses, inhibtion of Ag presentation, cytokine homologs that down regulate or block CD8, latent infections in neurons - no class I MHC

Flu - like symptoms are caused by IFN and lymphokines

inflamation is caused by Tcells and macrophages (+ PMNS)

Immunocomplex disease is caused by Ab - complement

Hemorrhagic disease caused by Tcells , Ab and complement

immunosupression

1. vaccines
2. immune globulins
3. drugs

active immunization

• not practical if a large number of virus strains cause a disease ( many serotypes - 100 strain of common cold)
• not always practical if virus undergoes much anitgenic variation due to high mutation rates in a dominant anitgenic structure 
• there are 3 basic types -->attenuated , killed, subunit

strains cause subclinical infections

advantages: cheap to produce, strong response (IgG, IgA and Tcell) that is long lasting

Disadvantages: can be labile in transport, cannot be given to immunocomp, can revert to virulence

cannot cause illiness

advantages: very stable, rare side effects , cannot revert to virulence

Disadvantages: more expensive to prepare, shorter term immunity that is mostly limmited to IgG

exist for hep B and human papilloma virus

composed of sinlge viral protein that are expressed in yeast using recombinant DNA tech

advantages: cannot cause disease, are not derived from blood

disadvantages: requires multiple injections

passive immunization is used in both pre and post exposure prophylaxis

postexposure: Hep A or B, measles, rabies, and chicken pox

challenge: find a unique target that is not toxic to us (since viruses use so much of our materials)

in theory any step in the viral infection can be inhibited

1. attachment
2. uncoating: picornavirus -- disoxaril - -which fits in to cleft in the recepetor - binding canyon of the capsid
3. uncoating - flu A - amantadine and rimantadine - inhibit viral ion channel
4. transcription and translation- Hep B, C, and papilloma viruses - IFN alpha 
5. translation - CMV mRNA - anitsenseRNA
6. *majority of antivirals target DNA replication and are composed of nucleotide analogs that inhibit DNA polymerase -- herp, Hep B and HIV
7. polyprotien cleavage and virus assemably -- protease inhibitors (HIV)
8. Nucleoside biosynthesis - ribavirin - cap formation and some RNA polymerases (Hep C, flu)
9. viral release - Relenza and Tamiflu -- flu A and B neurominidase
10. *** new is protease inhibitor cocktails and combination therapies

there are two types of bacteriophages that differ by their lifestyles :

Virulent and temperate

infection can result in either of two outcomes:

lytic development and progeny phage production

formation of lysogen containing a repressed prophage

Bacteriophages:

1. Adsorption
2. injection of DNA
3. Transcription of phage DNA --> mRNA
4. phage protien are made, replication of phage DNA conversion of bacterium to phage factory
5. Factory --> bacterium produces phage structures
6. Dna is packages into phage --> assembly of phage particle
7. Lysis
8. Culture of fluid resulting from phage-induced lysis is called a lysate. it can be titered to determine concentration of phage particles 

the phage DNA molecule is injected into the bacterium and circularizes

after a breif period of early transcription for synthesis of repressor protiens and integrase enzyme, phage mRNA synthesis for lytic fxn is turned OFF by the repressor

A phage DNA molecule - typlically a replica of the injected molecule is inserted into the chromosome of the bacterium by the phage integrase protien

bacterium continues to grow and divide and the integrated phage genes are replicated as part of the bacterial chromosome

phage lambda is the prototype

- after injection and circularization --> repression of transcription (stays as DNA --> no mRNA)

there is no integrase or integration, the phage DNA replicate at a low level as a plasmid

the plasmid prophage replicates and sergregates into both daughter cells upon division

Ecoli prophage  P1 is the prototype

1. Destruction of the phage repressor protien often as a result of DNA damage and induction of the SOS repair system which leads to cleavage of the repressor --> the phage can now enter into the lytic cycle 
2. chromosome with integrated prophage --> induction, excision  of integrated prophage --> replication: phage is produced* this is the same for both the plasmid prophage and the integrated prophage

once the integrated segement is excised the process is the same for both plasmid prophage and integrated prophage

 encoded by ctxA and ctx B genes on a temperate filamentous phage CTX O which uses Vibrio cholera TCP pillus as its receptor for infection

this phage can be found intergrated into the chromosome or as a plasmid

*it is integrates at different chromosal locations in different strains

• All are immune to superinfection 
• (superinfection is the process by which a cell, that has previously been infected by one virus, gets coinfected with a different strain of the virus, or another virus at a later point in time)
• some have altered cell wall porperties and new phage resistances due to lysigenic conversion. Salmonella phage E 15 alters the O-Ag of the LPS
• a small number of phage particles are released spontaneously by a few cells in each culture
• many are sensitive to DNA damage resulting in induction of phage development and cell lysis
• for some the prophage may confer or enchance pathogenicity

the transfere of genetic material by a phage particle

there are two types: Generalized and Specialized

occurs by mistaken packaging--> piece of host DNA into a phage particle instead of phage DNA

transducing particels are found in all lysates --> this does not mean is will be the same gene! 

all host makers can be transuced, at frequencies of

10^{-5 - -8} per pfu

ex: the host is leu+ and pahge is leu- but some how the phage heads package fragments of the host chromosome including the leu+ gene

aberrant excision of prophage leading to incorporation of small piece of host DNA into the phage genome. This host DNA is replicates as part of the phage DNA 

prototype = lambda

when specialized particle infects a cell the phage integrates into the chromosome by 1) phage integration OR 2) homologous recomb within the host gene on the phage and the host gene in the chromosome

sometimes the transduced DNA doesnt recombine into the chromosome

if it circularizes instead - it can be passed down to one of the two daughter cells at each division

but since it cant replicate it cant be stably inherited in all the daugher cells

therefore this results in the formation of microcolonies instead of normal sized colonies

lambda integration is site-specific recombination between attachment sites (att) on the lambda DNA and host DNA and is catalyed by the lambda integrase protien

Excision is the reverse of integration and requires both integrase and XIS protien

when lambda lysogen is induced the normal excision mechanims occasionally fails

instead an illegit crossover occurs btw non-homo regions of the phage and bacterial chromo

this produces a phage missing - for example- the HIJ gene region having instead a segment adjacent bacterial DNA which includes the gal gene (for example)

the resulting phage is often lambda-d-gal --> the d indicates is it defective for growth and plaque formation

Methods of Transfere of DNA based on Size:

Transduction: small pieces of DNA, about 1-2% of chromosomes

Transformation: transfers medium sized DNA pieces, 5-10% of chromosomes

Conjugation: transfers large pieces of DNA, 25-50% of chromosome

Since transducing particles arise by the aberrant prophage excision --> they are found only in the lysate made by induction of the lysogen (unlike generalized - which have transducing particles in all lysates)

Only genes next to the prophage attachement site are transduced (in general all host markers can be transduced)

often the phage genes are lost during excision, so the transducing phages may be defective for phage growth--> in this case "we" provide a helper phage to make the missing functions to help it grow

*Can result in a high frequency transducing lysate (HFT) if both the special and the helper infect a new host --> the resulting lysate will have more transducing particles

discrete segments of DNA that move from one site to another in genome without requirement for DNA sequence homology

Transposition is catalyzed by transposase enzyme - usually encoded by this element

all have short (15-30 bp) inverted repeats at their ends which are recognized by transposes for transposition

Mulitple types: insertion sequences, transposons, Mu-like bacteriophages

Transposons

Type I composite v. Tn3-like

Type I: Larger 2-10 kp with long 800-1500bp repeats - direct or inverted --at the ends and unique DNA in the middle often encoding anitibiotic resistance and occasionally toxin production, heavy metal ion resist ect

Tn3:  shorter (15-50bp) inverted repeats at the ends these ends cannot transpose independently

replicate by transposition - Mu the mutator phage - causes mutations by insertion into host genes (phage is 30-50kb long)

* the invertible G segment repeats do not function in transpostion they are site specific recombination sites for G segment inversion)

• All have short inverted repeast at their outside ends (15-30 bp) these ends are required for transposition - they bind transposase which recombines the donar Tn into target DNA
• Some also have long inverted or direct repeat (1 kb) in these the short inverted repeat ends are stil present --at the outisde end of these long repeats
• Transpostion to a new site involves duplication of a short segment of DNA at the target site so that one copy flanks each end of the inserted transposon in directly repeated orientation
• insertion into a gene disrupts the gene fxn (causes a mutation ) and often eliminates fxn of downstream gene by polarity
• insertion can occur at many sites

insrtion in a gene disrupts the gene function -- causes a mutation - and often eliminates function downstream of genes by POLARITY

*MOST transposons contain transcription termination signals that cause RNAP to stop transcribing

ok so I am guessing this means that the protien cut short

1. donor and recipient make contact and form a conjugation bridge
2. Triggers excision of the transposon, which forms a covalent bond closed supercoiled molecule
3. this molecule is transferred to recipient and may integrate at many possible sites in the genome of the recipient cell ( with out target site duplication)

• Sometimes more than one transposon is found in the transconjugant
• In Gram + bacteria, conjugative transposons are as important as plasmids to the spread of antibiotic resistance
• Many conjugative transposons can transfer btw Gram + and gram - bacterial species

Hybridization with labeled specific DNA probe

PCR with specific primers

Naked eye- some parasites

light microscope - bacterial, viral aggergates

electron micoscope - individual viruses

identification by light microscopy is often facilitated by use of 1) differential staing with dye or 2)immunofluo staining with specific Ab tagged with dye

1. Growth of bacteria on selective media or indicator media often followed by battery of test for biochemical characteristics-- computerized and automated
2. for viruses and strict intracellular bacteria must culture in living euk cells

select from site fo actua lactive infection

avoid / minimize contamination with indigenous flora

collect at optimum time for pathogen recovery

collect before antimicrobial therapy

collect sufficient amount for test required

limit submissions to acceptable, required amounts

limits swabs to skin mucous membrane surfaces

aspirates/tissue preferable from other sites

use acceptable transport/devices/conditions

minimize time from collection to assay

1. Take sample for virus frown in culture and infectivity titer determined
2. add virus sample to test tube containing Ab to KNOWN virus and to control (A, B, control)
3. incubate for 1 hr
4. inoculate into cell culture each test tube (Virus Ab A, Ab B and control)
5. If there is no cytopathic effect --> that is the virus in culture (Ab A + virus = cytopathic effect, as did the control --> virus is B  * I guess haveing the Ab present would prevent the infection and lysis of the host cells?
6. CPE = structural changes in a host cell resulting from viral infection. CPE occurs when the infecting virus causes lysis (dissolution) of the host cell or when the cell dies without lysis because of its inability to reproduce

that pathogen-specific Ag may be fixed to the surface of RBC or latex particles. patient serum containing Ab directed against Ag will linkt he particles together causing a visible agglutination

Advanatge: fast, takes only 5-10 mins

Disadvantages: false negs could be obtained if bacterial count it low

1. Detection of viral Ag!
2. fix cells on the slide
3. add fluoresein-conjugated antiserum and incubate
4. unattached Ab removed by washing
5. examine for flourescence

1. Detection of viral Ag
2. fix cells on slide
3. add unconjugated Ab from rabbit source--> is Ag specific but NOT labeled
4. wash
5. specific Ab is attached to Ag
6. Add anti-rabbit Ab with Flourescein and allow to bind
7. wash
8. Now observe the binding via flourescein

pathogen specific Ag fixed on solid surface

detection of patient Ab response

Detection based on attached enzyme involves addtion of the enzyme's substrate whic his then converted to insoluble colored precipitate

Enzyme used: Horseradish peroxidase

Alkaline phosphatase

Beta-galactosidase

Seroconversion:in acute infection Ab begin to be made within several days but levels rise dramatically with in 2-3 weeks, Compare patients Ab titer in ealy illnes and again later

If titer against pathogen-specific Ag increases dramatically it demonstrates seroconverson and identifies pathogen

in acute infection Ab begin to be made within several days but levels rise dramatically with in 2-3 weeks, Compare patients Ab titer in ealy illnes and again later

If titer against pathogen-specific Ag increases dramatically it demonstrates seroconverson and identifies pathogen

assay is preformed on serial dilutions of patiens serum, titer reported is the highest serum dilution that still gives a detectable Ag-Ab reaction based on color observed

1. known Ag attached to tube surface
2. Ab in patien serum  -- specific Ab in serum should bind to Ag
3. wash excess Ab
4. Enzyme labeled Anti-Ab  (ie Rabbit Anti-human Ab)--> this will bind to the primary Ab and contains the color changing Enzyme
5. wash and Add enzyme substrate, detect color change

Pretreatment: Denature to separate DNA strands

Hybridization: Add DNA probe, wash off unbound probes (* probes are pathogen specific commercially avaible)

Complementary base pairing provides the specifity

Direct color development: Probe itselt contains the lable to be detected--> radioactive or color change

Secondary reaction: Probe was labelled with biotin which bind avidin --> reacts with sample with avidin -fluoro dye and color!

or an enzyme with color change

SDS gel electrophoresis to seperate HIV protiens by size

Electrophoretically transfer to nitrocellulos paper

cut paper into thin strips

incubate with patient serum

wash thoroughly to remove unbound Ab

Incubate with secondary enzyme - coupled Anti-human Ab

wash

Add substrate enzyme  and observe color change

CDC interpretation guidlines:

Positive for HIV: p24, gp41, and or gp120, 160

flourescence in situe hybridization with PNA probes:

• in probes the phosphate-sugar polynucleotide backbone of nucleic acid is replaced by flexible psuedo-peptide ploymer to which the nucleobases are linked with spacing similar to that of the bases in DNA and RNA.
• PNA hybridize with high affinity and specifity to complementary sequences of DNA and RNA
• PNA probes are also relavtively resistant to nucleases and proteases
• In current diagnostic application flourescently-labelled PNAs are designed to hybridize to species-specific sequences in ribosomal RNA. Since cells have many ribosomes the target RNA is already naturally amplified.

1. Fix cells from blood culture to a slide,
2. do Gram stain to identify cell shape,
3. hybridize with PNA probe, wash,
4. detect by examination with flourescence  microscope

Strand extension by DNA polymerases begins at the end of the primer and continues until the end of the template. After 30-40 cycles the reaction tube will contain large amounts of DNA fragments whose length and ends are determined by the pathogen specific primers

1. First seperate strands and anneal primer
2. exten primers with polymerase
3. and repeat

Amplicon Detection - not in real time?

Sorry this slide was a little weird

Samples were removed from the PCR reaction tubes after 30 to 35 cycles and run on a gel --> now you can see how much is there?

you can use:

Agarose gels and Ethidium Bromide or Polyacrylamide gels and 32P probes

PCR

Advantages v. Disadvantages

advantages:

several orders of magnitude more sensitive than direct hybridization (can detect 1-10 copies of gene in 10⁶copies of other DNA)

need very small amounts of DNA in specimen

Very fast - hrs not days

Disadvantages:

expensive

False positive from contamination, false negatives from improper conditions * must use controls in every experiment

Applications:

For pathogens with no existing test

for pahtogens with poor test - long culture times, high cross-reactions; for diseases with low antigen and antibody production

Chronic Hep B detection

chlamydia tachomatis, Neisseria gonorrhoeae

HIV - esp in neonates (maternal Ab prevent use of Elisa)

Lyme disease ( Ag production is low)

Quantitative and Fast

You can now have amplicon detection in real time --> PCR products as they accumulate

Uses DNA intercalating dyes (SYBR green) or fluorogenic DNA probes

allows for accurate quantitation based on the time it takes until defined threshold level of PCR products are detected

the more copies of nucleic acid present the sooner and increase in fluoro is detected

Amplification and detection are done simultaneously in a closed system --> reducing contamination and time; the amplicon tube is never open

Real time PCR takes about 1/3 the time and half the cost of the standard PCR assay which is -10$ for reagents, 9 hr start to result, pay tech - 30$ ..seriously?)

St judes and some other childrens hospitals are beginning to use these real time PCR and they are becoming more available

1. Transformation
2. Transduction
3. Bacterial conjugation
4. gene transfere elements

transfer unidirectional !

• Transformation, the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign genetic material (DNA or RNA). This process is relatively common in bacteria, but less so in eukaryotes. Transformation is often used in laboratories to insert novel genes into bacteria for experiments or for industrial or medical applications.
• Transduction, the process in which bacterial DNA is moved from one bacterium to another by a virus (a bacteriophage, or phage).
• Bacterial conjugation, a process in which a bacterial cell transfers genetic material to another cell by cell-to-cell contact.
• Gene transfer agents, viruslike elements encoded by the host that are found in the alphaproteobacteria order Rhodobacterales.^[10]