lmw movie scripts

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MOVIE SCRIPTS

LIVIng In microbial world

Bruce V. Hofki

This document contains the transcripts o the voice-over narration or the

movies that accompanyLiving in a Microbial World.

The movies can be ound at http://www.garlandscience.com/lmw


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2.1 Noncovalent Bonds

Molecules in solution undergo random thermal movements and mayencounter each other requently i the concentration is sufciently high. Itwo molecules with poorly matched suraces collide, only a ew weak bonds

will orm between them. Thermal motion o the molecules rapidly breaksthese bonds apart, and the molecules separate.

I the suraces o two molecules are well matched, many weak bonds willorm between the two. The bonds hold the molecules together or a longtime beore thermal jolting tears them apart.

Tightly bound molecules will spend most o their time associated although

they will go through cycles o association and dissociation. The afnity o thetwo molecules or one another is a measure o the relative time they spendbound together.

Video: David Roger, Vanderbilt University.

Diital capture: Tom Stossel, Brigham and

Womens Hospital, Harvard Medical School.

1.1 Neutrophil Chase

Neutrophils are white blood cells that hunt and kill bacteria. In thisspread, a neutrophil is seen in the midst o red blood cells. Staphylococcusaureus bacteria have been added. The small clump o bacteria release achemoattractant that is sensed by the neutrophil. The neutrophil becomespolarized, and starts chasing the bacteria. The bacteria, bounced around bythermal energy, move in a random path, seeming to avoid their predator.Eventually, the neutrophil catches up with the bacteria and enguls them byphagocytosis.

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2.2 Glucose

A glucose molecule is a six-carbon sugar, consisting o a total o 24 atoms.

It is very polar due to its hydroxyl groups to which water molecules can

hydrogen bond.

By convention, carbon atoms are shown in gray, oxygen in red and hydrogenin white.


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Video: Howard C. Berg, Harvard University.

3.1 Bacterial Flagellum

Many species o bacteria propel themselves through their environment byspinning helical motorized agella. Rhodobacter cells have one agellumeach, whereas E. colicells have multiple agella that rotate in bundles. Eachagellum consists o a helical flament that is 20 nanometers wide and up to15 microns long and spins on the order o 100 times per second.

2.3 Lipids

Phospholipids contain a head group, choline in this case, that is attached viaa phosphate group to a 3-carbon glycerol backbone. Two atty acid tails areattached to the remaining two carbons o the glycerol.

The head groups and the phosphate are polar, that is, they preer to be in an

aqueous environment.

In contrast the atty acid tails are hydrophobic, that is, they are repelled romwater. The atty acid tails on phospholipids can be saturated, with no doublebonds, or unsaturated, with one or more double bonds. The double bondsare usually in the cis-confguration, which introduces sharp kinks. Whenorming a bilayer, unsaturated atty acid tails pack loosely, which allows thebilayer to remain uid. I there were no double bonds, bilayers would solidiyto a consistency resembling bacon grease.

3.2 Lipid Bilayer

In a lipid bilayer, lipids arrange themselves so that their polar head groupsare exposed to water and their hydrophobic tails are sandwiched in themiddle.


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4.1 Biosynthesis 1: Double-stranded DNA(dsDNA) Viruses

The biosynthesis o double-stranded DNA virus takes place in the nucleus othe host cell and uses the host cells replication machinery.

Ater entering the host cell, the virion uncoats in the cytoplasm, releasingthe double-stranded DNA. The double-stranded DNA then moves into thenucleus where it will begin biosynthesis.

RNA polymerase transcribes one o the strands o the viral DNA to createa messenger RNA molecule. Ater transcription is complete, the messengerRNA leaves the nucleus, and is then translated into capsomere proteins usedto assemble new viral capsids.

Moving back to the nucleus, the host cells DNA polymerase replicatesthe double-stranded viral DNA, producing multiple copies o the originalgenome.

Ater replication, the newly synthesized double-stranded DNA viral genomesmigrate to the cytoplasm, where they are moved into the new capsids,orming complete, intact virions.

Video reproduced from: M. Maniak,

R. Rauchenberger, R. Albrecht, J. Murphy, and

G. Gerisch. Coronin involved in phagocytosis.

Cell83:91924. 1995, with permission from

Elsevier Science.

3.3 Phagocytosis

Phagocytosis allows cells to take up large particles, such as these yeast cellsthat are being enguled by the slime mold Dictyostelium.


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4.3 Biosynthesis 3: (+) Single-stranded RNA(+ssRNA) Viruses

Single-stranded RNA viruses contain either a negative RNA strand or apositive RNA strand. In this animation, we will look at the biosynthesis o apositive RNA strand virus.

Ater entering the host cell, the virion uncoats in the cytoplasm, releasingthe positive, single-stranded RNA.

Since the positive RNA strand is already in the orm o messenger RNA, it canbe translated immediately into capsomere proteins used to assemble newviral capsids.

Replication o the positive RNA strand is accomplished in two steps. First,through complementary base pairing, RNA polymerase creates a templatestrand.

Second, the template strand is used to make many complementary positiveRNA strands.

The newly made positive strands combine with the capsids to complete theconstruction o new positive, single-stranded RNA virions.

4.2 Biosynthesis 2: (-) Single-stranded RNA(-ssRNA) Viruses

Single-stranded RNA viruses contain either a positive RNA strand or anegative RNA strand. In this animation, we will look at the biosynthesis o anegative RNA strand virus.

Ater entering the host cell, the virion uncoats in the cytoplasm, releasingthe negative, single-stranded RNA.

Unlike positive, single-stranded RNA, the negative stranded RNA is not inthe orm o messenger RNA. So RNA polymerase must frst make templatestrands through complementary base pairing.

These template strands, which are in the orm o messenger RNA, can nowbe translated into capsomere proteins used to assemble new viral capsids.

The template strands are also used to produce new negative RNA strands.

The newly made negative RNA strands combine with the capsids to complete

the construction o new, negative stranded RNA virions.


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6.1 DNA Structure

Two DNA strands intertwine to orm a double helix. Each strand has abackbone composed o phosphates and sugars to which the bases areattached. The bases orm the core o the double helix, while the sugarphosphate backbones are on the outside.

The DNA backbone is assembled rom repeating deoxyribose sugar unitsthat are linked through phosphate groups. Each phosphate carries a negativecharge, making the entire DNA backbone highly charged and polar.

A cyclic base is attached to each sugar. The bases are planar and extend outperpendicular to the path o the backbone. Pyrimidine bases are composedo one ring and purine bases o two rings. Adjacent bases are aligned so thattheir planar rings stack on top o one another. Base stacking contributessignifcantly to the stability o the double helix.

In a double helix, each base on one strand is paired to a base on the otherstrand that lies in the same plane. In these base pairing interactions, guaninealways pairs with cytosine, and thymine with adenine.

A GC pair is stabilized by three hydrogen bonds ormed between amino andcarbonyl groups that project rom the bases.

In contrast, an AT pair is stabilized by two hydrogen bonds.

The specifcity o base pairingthat is, C always pairing with G, and A alwayspairing with Tensures that the two strands are complementary. This isimportant or DNA replication and transcription.

4.4 HIV Infection

HIV is an important example o a retrovirus. The inection cycle begins whenHIV binds receptors on the host cell.

Interactions o the virus with these membrane receptors allow viral uncoatingand the entry o the nucleocapsid, containing the viral genome, into the cell.

The viral reverse transcriptase, which is an integral part o the viral particle,copies the RNA genome o HIV into double-stranded DNA. The viral genomethen integrates into the DNA o the host cell.

In this state the virus is latent; that is, it can persist in the cell in an inactivestate.

Reactivation o the virus occurs when the host cell becomes activated andviral transcription is initiated. This results in the accumulation o viralproteins as well as genome-length RNA transcripts o the virus.

Viral proteins assemble at the cell membrane with copies o the RNA genome,and bud o to create a new viral particle


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6.2 Replication

Replication begins when enzymes uncoil the double-stranded DNAmolecule and separate the strands. These strands serve as templates orsynthesizing new DNA molecules. The area where the strands separate iscalled the replication ork.

The two template strands are anti-parallel; that is, they are oriented inopposite directions. One strand is oriented in the 3 to 5 direction, andcalled the leading strand template; the other strand is oriented in the 5 to 3direction, and called the lagging strand template.

On the leading strand template, DNA polymerase attaches at an area wherea small piece o RNA, called a primer, has been attached to the DNA. As theDNA polymerase moves down the leading strand in the 3 to 5 direction, itsynthesizes a complementary strand. This synthesis o a new DNA strand,called replication, proceeds continuously toward the opening replicationork.

Replication o the lagging strand is more complicated because DNA

polymerase only works in the 5

to 3

direction. Thus, the lagging strandmust be completed in segments using a backstitching mechanism. The DNApolymerase begins replicating at an RNA primer attached to the DNA andcontinues until it reaches the end o the ragment. Each segment o DNAreplicated on the lagging strand is called an Okazaki ragment. As the laggingstrand is being made, the enzyme RNAase H degrades the RNA primer, andthen a DNA polymerase molecule flls in the gap with nucleotides. Once thegap is flled, the ends o the separate DNA pieces are linked together by theenzyme DNA ligase. Though the leading strand is made continuously andthe lagging strand is made in Okazaki ragments, the process is simultaneousand continuous and replication o each strand keeps up with the uncoilingo the parental DNA at the replication ork.

Animation produced for DNA Interactive (www.

dnai.org) 2003 Howard Hughes Medical

Institute (www.hhmi.org) All rights reserved.

6.3 Translation I

In a dazzling display o choreography, all the components o a molecularmachine lock together around the RNA to orm a miniature actory calleda ribosome. It translates the genetic inormation in the RNA into a string oamino acids that will become a protein. tRNA molecules the green trianglesbring each amino acid to the ribosome. The amino acids are the small redtips attached to the tRNAs. There are dierent tRNAs or each o the twentyamino acids, each o them carrying a three-letter nucleotide code that ismatched to the mRNA in the machine. Now we come to the heart o theprocess. Inside the ribosome, the mRNA is pulled through like a tape. Thecode or each amino acid is read o, three letters at a time, and matched tothree corresponding letters on the tRNAs. When the right tRNA plugs in, theamino acid it carries is added to the growing protein chain. You are watchingthe process in real time. Ater a ew seconds the assembled protein starts toemerge rom the ribosome. Ribosomes can make any kind o protein. It just


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Electro microraph: John Heuser, Washinton

University in St Louis.

6.5 Polyribosome

Ribosomes translate RNA into amino acids. Typically, many ribosomestranslate the mRNA simultaneously. Each ribosome begins at the 5 endo the mRNA and progresses steadily toward the 3 end. New ribosomesattach to the 5 end at the same rate as the previous ones move out o the

way. These multiple initiations allow the cell to make much more proteinrom a single message than i one ribosome had to complete the task beoreanother could begin. When a ribosome reaches a stop codon, the ribosomeand the new protein dissociate rom each other and rom the mRNA. Thiselectron micrograph depicts a membrane-bound polyribosome rom a

eukaryotic cell.

6.4 Translation II

To extend a growing polypeptide chain, the ribosome must select the correctamino acids that are specifed by the messenger RNA.

A tRNA amino acid complex enters the ree site on the ribosome. I theanticodon o the charged tRNA does not match the codon in the messengerRNA, the tRNA is rejected.

The process o trial and error repeats until the correct tRNA is identifed.

I the tRNA is correctly matched and remains bound or a long enough time,it is committed to be used in protein synthesis.

The ribosome catalyzes the ormation o the new peptide bond andundergoes a dramatic conormational change. This switches the ribosomeback to the state in which it can accept the next incoming tRNA.

depends on what genetic message you eed in on the mRNA. In this case,the end product is hemoglobin. The cells in our bone marrow churn out ahundred trillion molecules o it per second! And as a result, our muscles,brain and all the vital organs in our body receive the oxygen they need.


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7.1 ATP

ATP molecules store and supply energy or cellular processes. An ATPmolecule contains three building blocks: the at purine ring systemcontaining multiple nitrogen atoms shown in blue, the ribose sugar in themiddle, and the three phosphate groups with the phosphorus atoms shownin yellow.

Electro microraph: Charles C. Brinton, Jr

and Judith Carnahan.

6.6 Conjugation

In this electron micrograph, one bacterium has contacted another by a longprotein tube known as a sex pilus. Such contact initiates a type o bacterialmating in which one bacteriumthe donor with the sex pilitransers DNAto a recipient, which lacks sex pili.

The donor cell contains a circular piece o DNA, called an F plasmid, thatis transerred to the recipient cell. Only one strand o the double-strandedF plasmid is tranerred. The complementary strand is synthesized by therecipient cell.

Because the F plasmid contains all the genes required or making sex pili andor transerring the DNA, both bacteria are now potential DNA donors.

The F plasmid can also bring along other genes rom the donors chromosome,thereby allowing even more extensive genetic transer between the two cells.

7.2 Glycolysis

Cells break down ood molecules, such as glucose, through multi-steppathways. In the process o glycolysis, the breakdown o one glucosemolecule into two three-carbon molecules produces a net gain o energythat is captured by the molecules ATP and NADH. The breakdown product,pyruvate, next enters the Krebs Cycle, where it can be used to generate more

energy.

Glycolysis involves a sequence o 10 steps. In the frst three steps, energyin the orm o ATP is invested to be recouped later. In the ourth and fthsteps, this energy allows glucose to be split into two smaller molecules rom

which energy can be harnessed efciently. And in the last fve steps, energyis released step-wise as ATP and NADH. The elegant chemistry that evolvedto catalyze these reactions ensures that energy is released in small portionsthat can be efciently captured. Less controlled combustion reactions wouldrelease most o the energy as heat.

The chemistry o glycolysis is conserved all the way rom bacteria to animalcells.


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Video: Henry Bourne and John Sedat, University

of California at San Francisco; Orion Weiner,

Harvard Medical School.

11.1 Chemotaxis of Neutrophils

These human neutrophils, taken rom the blood o a graduate student, aremobile cells that will quickly migrate to sites o injury to help fght inection.They are attracted there by chemical signals that are released by other cellso the immune system or by invading microbes.

In this experiment tiny amounts o chemoattractant are released rom amicropipette. When neutrophils sense these compounds they polarize andmove towards the source. When the source o the chemoattractant is moved,the neutrophil immediately sends out a new protrusion, and its cell bodyreorients towards the new location.

TEM: Eye of Science/Photo Researchers Inc.

10.1 Cholera Exotoxin

Vibrio cholerae is a Gram-negative bacteria that produces a powerulexotoxin. Once in the hosts intestine, the bacteria excrete the exotoxin intothe surrounding environment.

Each exotoxin molecule consists o two parts: an A subunit and a B subunit.The B subunit allows the exotoxin to bind to membrane proteins on intestinalepithelial cells. Binding o the B subunit stimulates the host cell to engul theexotoxin by endocytosis.

Following endocytosis, the A and B subunits separate. The A subunit isreleased to the cytoplasm while the B subunit returns to the cell membrane

where it is released by exocytosis.

The A subunit interacts with a host protein which ultimately causes waterto leave the cell, and enter the intestine. This water loss results in severediarrhea, which is the primary symptom o a cholera inection.

7.3 ATP Synthase

ATP synthase is a molecular machine that works like a turbine to convert theenergy stored in a proton gradient into chemical energy stored in the bondenergy o ATP.

The ow o protons down their electrochemical gradient drives a rotor that

lies in the membrane. It is thought that protons ow through an entry opento one side o the membrane and bind to rotor subunits. Only protonatedsubunits can then rotate into the membrane, away rom the static channelassembly. Once the protonated subunits have completed an almost ullcircle, and have returned to the static subunits, an exit channel allows themto leave to the other side o the membrane. In this way, the energy stored inthe proton gradient is converted into mechanical, rotational energy.

The rotational energy is transmitted via a shat attached to the rotor thatpenetrates deep into the center o the characteristic lollipop head, the F1

ATPase, which catalyzes the ormation o ATP.


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11.2 The Immune Response

An immune response involves events that unold both locally, at the site oan inection, and at more distant sites, such as nearby lymph nodes. We cansee the integration o the dierent parts o the immune response i we ollowthe course o a typical inection.

Most pathogens are kept outside o the body by epithelial barriers, such asthe epidermis, and are crossed only when there is an injury or tissue damage.

Ater an injury, bacteria cross the epidermis and establish an inection in theunderlying tissue. Phagocytic cells in the tissues, such as macrophages andneutrophils, engul the pathogen.

Dendritic cells are also phagocytic, and are activated by binding pathogensto leave the site o inection and migrate to a lymph node. The migratingdendritic cells enter the lymphatic vessels and are collected in a draininglymph node. In the lymph node, T cells are activated by antigen presented bythe dendritic cells, and in turn activate B cells to secrete antibody.

Eector T cells and antibody molecules return to the circulation. They leave

the circulation again at the site o inection, where inammatory mediatorshave induced changes in the blood vessel endothelium.

T helper cells activate macrophages to become more cytotoxic, whileantibody enhances the uptake o pathogens by phagocytes. In the case o aviral inection, activated cytotoxic T cells would kill any inected cells present.

11.3 Leukocyte Rolling

Leukocytes are white blood cells that help fght inection. At sites o injury,inection, or inammation, cytokines are released and stimulate endothelial

cells that line adjacent blood vessels.

The endothelial cells then express surace proteins, called selectins. Selectinsbind to carbohydrates displayed on the membrane o the leukocytes, causingthem to stick to the walls o the blood vessels. This binding interaction is osufciently low afnity that the leukocytes can literally roll along the vessel

walls in search or points to exit the vessel. There, they adhere tightly, andsqueeze between endothelial cellswithout disrupting the vessel wallsthen crawl out o the blood vessel into the adjacent connective tissue.

Here, leukocyte rolling is observed directly in an anaesthetized mouse. The upand down movement o the rame is due to the mouses breathing. Two bloodvessels are shown: the upper one is an arterywith blood owing rom right to

let. The lower one is a veinwith blood owing rom let to right. Leukocytesonly adhere to the surace o veins; they do not crawl out o arteries.

Some leukocytes are frmly attached and are in the process o crawlingthrough the vessel walls, whereas others have already let the vessel and areseen in the surrounding connective tissue.

When the blood ow is stopped temporarily by gently clamping the vessels,we can appreciate how densely both vessels are flled with red blood cells.Red blood cells do not interact with the vessel walls and move so ast undernormal ow that we cannot see them. When the blood ow is restored,some o the leukocytes continue rolling, whereas all noninteracting cells areimmediately washed away by the shear.

Video: Marko Salmi and Sami Tohka, MediCity

Research Laboratory, University of Turku,

Finland.


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11.5 Phagocytosis/Opsonization

The uptake o bacteria by phagocytes is an active process, which requires thetriggering o specifc receptors on the phagocyte. Special receptors, whichbind antibody-coated bacteria, trigger phagocytosis.

Binding o the aggregated antibody molecules to the receptors on thephagocyte causes the cell to engul the bacterium.

The phagocyte frst produces pseudopods or rues that surround thebacterium and then use, trapping the bacterium within what is now anintracellular vesicle, the phagosome.

Within the phagocyte, lysosomes use with the vesicle, delivering theirenzymatic contents to degrade the enguled bacterium

11.4 Dendritic Cell Migration

Dendritic cells, the key antigen-presenting cells o the immune system, aregenerated rom progenitors in the bone marrow that migrate into peripheraltissues through the blood stream.

There the immature dendritic cells lie in wait or pathogens entering the

body, through sites o injury, or example. Dendritic cells express variouspattern recognition receptors that can recognize common eatures o manybacterial and ungal pathogens. Through these receptors they are able tobind to and phagocytose pathogens.

When these receptors bind pathogens, they activate the dendritic cells,which then start to mature. In this process they migrate rom the tissues andchange their behavior to stop phagocytosis and to start expressing immunestimulatory molecules.

The activated dendritic cells migrate rom the tissues into lymphatic vessels,where lymphatic uid drains through lymph nodes, carrying the dendriticcells with it.

T cells, migrating through the lymph nodes, inspect the dendritic cells orthe presence o antigen. T cells that ail to recognize antigen on one dendriticcell carry on to inspect others and eventually return again to the circulation.T cells that do recognize their specifc antigen become activated, and bothprolierate and dierentiate into eector cells.


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Video: Matthais Gunzer and Peter Friedl,University of Muenster, Germany.

11.7 Activated T Cell

In this video we can see a T cell that becomes activated when it interactswith a dendritic cell. The T cell is labeled with a dye that uoresces when itbinds calcium ions. At the moment the T cell is not activated. Its intracellularcalcium concentrations are low, and so little green uorescence is visible.

As the T cell contacts the surace o the dendritic cell, we can see it suddenlyuoresce bright green as it becomes activated. However, it still continues tomove, crawling over the surace o the dendritic cell, perhaps to sample thecells display o peptide-MHC complexes.

Eventually the T cell loses interest. While it is still contacting the dendriticcell you can see the uoresence start to ade. The T cell then migrates awayrom the dendritic cell.

Video: Michael Redd and Paul Martin,

University College London.

11.6 Lymphocyte Homing

To visualize lymphocyte homing to a site o injury, a zebrafsh larva wasanaesthetized and its fn pierced with a needle to introduce a small wound.

A vein is seen at the bottom o the rame.

Because the fn is very thin and transparent, we can watch directly as

lymphocytes crawl out o the blood vessel and migrate towards the wound.They are attracted there by chemicals released rom damaged cells, invadingbacteria, and other lymphocytes.

In a zoomed out view we can appreciate that lymphocyte invasion isrestricted to the wounded area. The static cells that are dispersed in theconnective tissue are fbroblasts. In these movies, 60 minutes o real time arecompressed into 15 seconds.

11.8 MHC

MHC proteins display their bound antigen on the cell surace or immunesurveillance. Immune cells, called cytotoxic T cells express T-cell receptorsthat bind to the MHC head domain and the bound antigen. I the cellexpressing the MHC protein displays antigen oreign to the immune system,the T cell is activated by this receptor-MHC interaction. The activated T cellthen proceeds to destroy the inected cell. Cut-away views o this antigen-bound MHC protein complexed with a T-cell receptor reveal the exquisiteprecision with which the interacting suraces ft together.


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11.10 Antibodies

Antibodies o the immunoglobulin G class are Y-shaped glycoproteins thatcirculate in the blood stream. They bind to and inactivate oreign moleculesthe antigensand mark them or destruction. Each IgG molecule consists otwo light chains and two heavy chains. The heavy chains have carbohydratesattached. The regions o the antibody that bind to antigens are located at thevery tips o the two arms.

Antigens bind to the tip o each antibody arm, generally two molecules perantibody. In the example shown here, the antigen binds to the antibody via alarge contact surace, providing a tight and highly specifc association.

11.9 T Cell Killing

Viruses are intracellular pathogens that inect cells o the body, in thisexample epithelial cells, usurping their biosynthetic machinery to producenew viral proteins.

Degradation o virus proteins within the cell allows viral peptides to be

displayed at the cell surace bound to MHC molecules. Cytotoxic T cells thatrecognize these MHC-antigen complexes are activated and kill the inectedcell. Having killed one cell, the T cell can move to a new target, kill that cell,and move on again.

The killing process is initiated when the T cell receptor binds the MHCmolecule bearing a viral antigen, producing signals that activate the T cell.

Cytotoxic T cells contain membrane vesicles called cytotoxic granules,which package the proteins that kill target cells. The most important o theseare a protein called perorin, and a set o proteases called granzymes. Theseproteins are complexed with a scaolding protein called serglycin. Activationo the T cell causes the release o the content o these vesicles, delivering the

proteins directly onto the surace o the target cell.

Although the exact mechanism is not known, the perorin acilitates thedelivery o the granzymes into the cytosol.

At this point the target cell is destined or death, and the T cell can migrateonward to fnd a new target cell.


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Part I:

Julie A. Theriot, Stanford University School

of Medicine; Daniel A. Portnoy, University of

California at Berkeley.

Part II:

Julie A. Theriot, Stanford University School of

Medicine; Frederick S. Soo, Stanford University.

12.1 Intracellular Listeria Infection

In this video, we see a mammalian cell inected with Listeria monocytogenes.The bacteria are the small, darkly stained organisms seen moving rapidly

within the larger animal cell. This Gram-positive bacterial species is ableto grow at low temperatures and humans oten become inected aterconsuming contaminated moist, rerigerated ood such as sot cheeses.Most inections are asymptomatic, but occasionallyListeria can invade thecentral nervous system where it can cause meningitis.

Listeria can inect new cells when an uninected cell phagocytoses abacterium rom an inected cell. Once inside a new cell, the bacterium isable to replicate. Because Listeria transer directly rom cell-to-cell, they alsoavoid immune detection and attack.

13.1 Antigenic Drift

Pathogens, such as the inuenza virus, have receptors that enable them tobind to host cell suraces.

Antibodies to these viral receptors prevent the virus rom binding to andinecting cells. These are neutralizing antibodies, since they neutralize theability o the virus to inect the cell.

However, some viruses will have mutations that alter the receptor in ways

that prevent the binding o neutralizing antibodies while leaving the virusable to bind to, and inect, host cells.

In this way the pattern o antigens expressed by a virus can change over time.This process o accumulation o small changes is called antigenic drit, andcontributes to our susceptibility to inuenza inections year ater year.

13.2 Antigenic Shift

Pathogens, such as the inuenza virus, have receptors that enable them tobind to host cell suraces.

Antibodies to these viral receptors prevent the virus rom binding to andinecting cells. These are neutralizing antibodies, since they neutralize theability o the virus to inect the cell.

In some cases, viruses arise that are able to escape the eects o neutralizingantibodies. This can happen when two dierent strains o inuenza virus areable to inect the same host cell.

The progeny viruses produced rom such doubly-inected cells can containsegments o genome rom either o the two original viruses.


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14.1 PCR

The polymerase chain reaction, or PCR, amplifes a specifc DNA ragmentrom a complex mixture.

First, the mixture is heated to separate the DNA strands. Two dierentspecifc oligonucleotide primers are added that are complementary to shortsequence stretches on either side o the desired ragment. Ater lowering thetemperature, the primers hybridize to the DNA where they bind specifcallyto the ends o the desired target sequence. A heat stable DNA polymerase andnucleotide triphosphates are added. The polymerase extends the primersand synthesizes new complementary DNA strands. At the end o this frstcycle, two double-stranded DNA molecules are produced that contain thetarget sequence.

This cycle o events is repeated. The mixture is again heated to melt thedouble-stranded DNA. The primers are hybridized and the DNA polymerasesynthesizes new complementary strands.

At the end o the second cycle, our doubled-stranded DNA molecules areproduced that contain the target sequence. In the third cycle, the mixtureis heated, the primers are hybridized and DNA polymerase synthesizes newcomplementary strands. At the end o the third cycle, eight double-strandedDNA molecules are produced that contain the target sequence. Two o thesemolecules are precisely the length o the target sequence. In uture cyclesthis population increases exponentially.

Cycle 4heating, hybridization, DNA synthesis.

At the end o the fth cycle there are 22 double-stranded DNA ragments othe correct length and 10 longer ones.

Cycle 6, 10, 15, 20 . . .

Ater 30 cycles there are over 1 billion ragments o the correct length butonly 60 longer ones. The product thereore consists o essentially pure targetsequence.

Some viruses will acquire a segment o genome rom the other strainencoding the receptor or host cell suraces.

Neutralizing antibodies that block the binding o the original virus will beunable to recognize the receptor rom the second strain and will be unableto prevent the virus binding to and inecting host cells. This process, in whichlarge changes in the antigenicity o the virus occur, is known as antigenic

shit. These large changes can mean that much o the immunity against theoriginal virus is ineective, and such antigenic shit mutations are otenassociated with large-scale virus epidemics.

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