Chapter 18 - Genetics of Viruses and Bacteria Questions 1. The proteins that encapsulate the genetic material of a virus is known as the _____________. 2. Draw a general structure of a eukaryotic virus and label 3. An parts. individual protein of the structure mentioned in question number 1 is known as a _______________. 4. A bacteriophage can reproduce via two different life cycles known as the ________________ and _________________. 5. The genetic material of viruses can be ______, ______, _____ or ______. 6. This general structure is found to be part of some viruses like Influenza and not part of other viruses like Chapter 18 - Genetics of Viruses and Bacteria NEW AIM: Viruses: Packaged Genes… Viruses : Packaged Genes Chapter 18 - Genetics of Viruses and Bacteria NEW AIM: Viruses: Packaged Genes… What is a virus? 1. Obligate intracellular parasite - A small [20 to 250nm in diameter] infectious agent that requires a host cell to replicate (make more of itself). **1/1000th the diameter of a eukaryotic cell. If the classroom was a cell, a virus would be about the size of a paperclip. 2. General Structure Nucleic acid enclosed in a protein coat and, in some cases, a membranous envelope 3. Host Range - Each virus can only infect a specific range of cell types Ex. HIV can only infect CD4+ Helper T- SEM of adenovirus Chapter 18 - Genetics of Viruses and Bacteria NEW AIM: Viruses: Packaged Genes… Size Comparison Virus: 20 to 250nm (.02 to .25um ) Prokaryote: 1 to 10um Eukaryote: 10 to 100um Chapter 18 - Genetics of Viruses and Bacteria NEW AIM: Viruses: Packaged Genes… CAPSID 1. All viruses contain genetic material (DNA or RNA) encapsulated by a protein coat called a capsi 2. An individual protein in the capsid is called a capsomere. 3. Bacteriophage (phage) have the most complex capsids Capsomere of capsid Membranous envelope RNA Capsomere DNA Head RNA 18 Glycoprotein 70–90 nm (diameter) 250 mm 20 nm 50 nm (a) Tobacco mosaic virus (b) Adenoviruses DNA Tail Capsid sheath Glycoprotein 80–200 nm (diameter) 50 nm (c) Influenza viruses Tail fiber 80 225 nm 50 nm (d) Bacteriophage T4 Chapter 18 - Genetics of Viruses and Bacteria NEW AIM: Viruses: Packaged Genes… Influenza looks different…it has an envelope. What’s up with that? Capsomere of capsid Membranous envelope RNA Capsomere DNA Head RNA 18 Glycoprotein 70–90 nm (diameter) 250 mm 20 nm 50 nm (a) Tobacco mosaic virus (b) Adenoviruses DNA Tail Capsid sheath Glycoprotein 80–200 nm (diameter) 50 nm (c) Influenza viruses Tail fiber 80 225 nm 50 nm (d) Bacteriophage T4 Chapter 18 - Genetics of Viruses and Bacteria NEW AIM: Viruses: Packaged Genes… Envelopes 1. Only some viruses have cell membrane-like envelopes Membranous envelope Capsid RNA Ex. Influenza (shown right) 2. The envelope is derived (comes from) the cell membrane of the host cell Glycoprotein 80–200 nm (diameter) 50 nm (c) Influenza viruses Chapter 18 - Genetics of Viruses and Bacteria NEW AIM: Viruses: Packaged Genes… How do viruses replicate (reproduce)? Viruses Hijack Cells They gain access and use the enzymes, ribosomes, and small molecules (ATP, nucleotides, amino acids, phospholipids, etc…) of host cells. Simplified viral reproductive cycle Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… Let’s begin with the best understood virus: T4 Phage infecting E. coli 1. Bacterial virus (bacteriophage or just phage) How do they reproduce? Chapter 18 - Genetics of Viruses and Bacteria NEW AIM: Viruses: Packaged Genes… Fig. 10.17 Bacteriophage reproductive cycle (two methods of reproduction) Bacteriophage binds to the surface of the bacterium using the tail fibers and injects its DNA into the cell… Chapter 18 - Genetics of Viruses and Bacteria NEW AIM: Viruses: Packaged Genes… Fig. 10.17 Bacteriophage reproductive cycle (two methods of reproduction) Lysogenic cycle Chapter 18 - Genetics of Viruses and Bacteria NEW AIM: Viruses: Packaged Genes… Bacteriophage reproductive cycle (two Fig. 10.17 methods of reproduction) Lytic cycle Lysogenic cycle Chapter 18 - Genetics of Viruses and Bacteria NEW AIM: Viruses: Packaged Genes… Lysogenic cycle - After the bacteriophage injects its DNA, it might get incorporated into the bacterial chromosome and is now called a prophage. Now when the bacterial cells replicates, the phage DNA replicates with it. Lytic cycle - After the bacteriophage injects its DNA or when the prophage jumps out of the DNA, it can hijack the cell and use it (its ribosomes and other enzymes) to make more viral DNA and proteins to in turn make more viral particles. The cell will lyse and the viruses will be released. Temperate Phages - Phages that can do both lytic and lysogenic methods of reproduction Ex. Lambda (λ) phage Chapter 18 - Genetics of Viruses and Bacteria NEW AIM: Viruses: Packaged Genes… What causes a temperate phage like lambda to switch from lysogenic to lytic? We observed the switch to be caused by environmental factors like radiation or certain chemicals causing DNA damage, which would promote the lytic phase as the bacterial cell will likely die soon and the phage needs to get out quick. In addition, lytic is favored when nutrients are plentiful allowing the phage to makes lots more of itself, while the lysogenic is favored when nutrients are in low concentration within the bacterium. This makes sense as the virus can lay low until better times. Can’t make more of yourself if the materials are simply Chapter 18 - Genetics of Viruses and Bacteria NEW AIM: Viruses: Packaged Genes… Can prokaryotes defend themselves against this attack? Of course. They contain enzymes that attempt to hydrolyze the viral DNA known as restriction enzymes like little molecular scissors. Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses A. Anatomy Genetic Material – Can be ssDNA/dsDNA or ssRNA/dsRNA depending on the virus. Codes for polypeptides/proteins needed by the virus to enter and hijack the cell as well as the proteins of the capsid and envelope. Capsid – made of proteins and surrounds the genetic material in the core. Envelope – Phospholipid bilayer similar to a cell membrane with embedded proteins (protein spikes) surrounding the capsid. Not all virus types have envelopes Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses DNA Capsid Protein spikes Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses They are classified by their genetic material. Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses DNA viruses Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses B. DNA viruses DNA capsid envelope Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses B. DNA viruses Ex. Adenovirus - Causes upper respiratory - Symptoms range from those infections similar to the common cold to bronchitis or pneumonia. (Common cold is caused by rhinovirus, an RNA virus) Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses B. DNA viruses Ex2. Herpesviruses (family of related viruses) These can cause: 1. Oral herpes (cold sores) or genital herpes (an STD) Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses B. DNA viruses Ex2. Herpesviruses (family of related viruses) These can cause: 2. Chicken pox (varicella zoster virus) Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses B. DNA viruses Ex3. Poxvirus (family of related viruses) Can cause: 1. Small pox This is the only human infectious disease to ever be eradicated (removed from the face of the planet) – we did this through extensive Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses B. DNA viruses Ex4. HPV – Human Papillomavirus A. Over 200 different types…many are STDs (sexually transmitted) 1. Some of these STD viruses can lead to cancers of the cervix, vagina, and anus in women or cancers of the anus and penis in men. a. Nearly all cases of cervical cancer are caused by HP 2. Others cause genital warts Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses B. DNA viruses HPV Vaccine Recommended by CDC for all females and males age 11 to 26. http://www.nytimes.com/2011/10/26/health/policy/26vaccine.html Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… What do viruses need to accomplish to continue to exist? 1. Gain access to a cell 2. Use the cell’s workers (ribosomes, RNA polymerase, etc…) to make more of itself. a. Synthesize viral proteins b. Replicate its genome c. Assemble these into new viral particles Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Life cycle of a DNA virus What is the first thing a virus must be able to do? 1. Viral Attachment and Entry a. If the virus does not have an envelope, protein spikes on the capside will act as ligands and bind cell receptors, triggering receptor mediated endocytosis. Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Life cycle of a DNA virus 1. Viral Attachment and Entry b. If it does have an envelope, the protein spikes in the envelope will act as ligands and bind to cell receptors resulting in fusion of the viral membrane and cell membrane, injecting the capsid into the cell… Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Life cycle of a DNA virus 1. Viral Attachment and Entry Analogy: Cell receptors = door lock Protein spikes = the key In either case, the protein spikes on the surface need to bind receptors to gain access to the cell, which is why specific viruses can only infect specific cells with matching receptors. Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Life cycle of a DNA virus A. Viral attachment and entry B. Uncoating The capsid fall apart and the viral DNA enters the C.nucleus Transcription and translation of the viral DNA The viral DNA is transcribed and translated by our workers (our RNA polymerases, ribosomes/tRNAs/etc…) using our ATP made by our mitochondria!! Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Life cycle of a DNA virus D. Replication of the viral DNA E. Viral protein sorting Capsid proteins are brought into the nucleus while envelope proteins get into nuclear membrane via endomembrane system. Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Life cycle of a DNA virus F. Viral assembly Capsid forms around DNA and then buds out of nucleus picking up its H.envelope Release How the virus, now in the cytoplasm, gets out of the cell is not understood yet. Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Life cycle of a DNA virus This process typically happens over and over and over again until the cell dies…The cell is a virus producing factory. DNA integration In certain viruses, like Herpes virus, the viral DNA can integrate (become part of) the cell’s DNA (your DNA), and sit quietly similar to the lysogenic cycle of bacteriophages. Almost all adults carry Herpes Simplex 1 virus (oral herpes). Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses RNA viruses Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses C. RNA viruses Ex1. Mumps virus - Member of the paramyxovirus family - Causes the mumps Extreme swelling of salivary glands Before infection Contagious via respiratory secretions (coughing/sneezing/sharing After infection glass/kissing/etc…) Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses C. RNA viruses Ex2. Rubella virus - Member of the togavirus family - Causes rubella (German measles) Rash on body Flu-like symptoms Highly Contagious Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses C. RNA viruses Ex3. Measles - Caused by a member of the paramyxovirus family like mumps - Highly contagious through respiratory secretion just like mumps Symptoms: Rash on body, cough, runny nose, red eyes, four day fevers Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses C. RNA viruses If these viruses are so easily contagious, why haven’t you gotten them? You have all been vaccinated against them (MMR shot) MMR = measles, mumps, rubella Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses C. RNA viruses Ex4. Poliomyelitis (polio) - Highly contagious through fecal-oral route (feces to the mouth) It is easier than you think…the chef prepares your food and didn’t wash - Inhis 1%hands of infections, virus enter neurons and destroys motor function – lose control of your muscles You are vaccinated against this one too… Animal RNA virus life cycle 1. Viral attachment and entry Similar to DNA virus – protein spikes act as ligands for cell receptors. 2. Uncoating Capsid falls apart releasing the 3. RNA RNA synthesis A viral enzyme will make the complementary RNA strand (purple) using the genomic RNA (red) as a template 4. Protein synthesis Complementary RNA can act as mRNA and your ribosomes will translate it, making new viral proteins. Fig. 10.18a Animal RNA virus life cycle 5. Synthesizing more genomic RNA The complementary strand (purple) can also act as a template to back synthesize the more genomic RNA (red) 6. Assembly The viral proteins and genomic RNA come together to make new viral particles. Some of the viral proteins made were sent through the endomembrane system to the cell membrane. Fig. 10.18a Animal RNA virus life cycle 7. Exit The capsid/RNA pinch off from the cell, which is how it acquires the envelope with embedded viral proteins. -Notice that the nucleus is not involved. -This process happens again and again until the cell is dead. -There can be no integration of standard RNA viruses into our genome as RNA cannot be integrated into DNA Fig. 10.18a The reproductive cycle of an enveloped RNA virus 1 Glycoproteins on the viral envelope bind to specific receptor molecules (not shown) on the host cell, promoting viral entry into the cell. Capsid RNA Envelope (with glycoproteins) 2 Capsid and viral genome enter cell HOST CELL Viral genome (RNA) Template 5 Complementary RNA strands also function as mRNA, which is translated into both capsid proteins (in the cytosol) and glycoproteins for the viral envelope (in the ER). 3 The viral genome (red) functions as a template for synthesis of complementary RNA strands (pink) by a viral enzyme. mRNA Capsid proteins ER Glycoproteins Copy of genome (RNA) 4 New copies of viral genome RNA are made using complementary RNA strands as templates. 6 Vesicles transport envelope glycoproteins to the plasma membrane. 8 New virus 7 A capsid assembles Figure 18.8 around each viral genome molecule. Chapter 18 - Genetics of Viruses and Bacteria AIM: Viruses: Packaged Genes… 2. Animal viruses C. RNA viruses Ex5. Retrovirus Ex. HIV (human immunodeficiency virus)– you will need to know the details on this one Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Retroviruses - A special family of RNA viruses - Retro implies Reverse - These viruses have an RNA genome, but use a special enzyme called Reverse Transcriptase to make a DNA copy of the RNA (the reverse of transcription; hence the name) Ex. HIV (human immunodeficiency virus) Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Retroviruses Fig 10.21A Attachment protein is called GP120 HIV - Enveloped RNA virus HIV - Capsid houses two identical RNA molecules and the enzyme Reverse Transcriptase as well as others needed for the virus to function. Why do you think the virus needs to carry its own Reverse Transcriptase? Because our cells do not have the gene for reverse transcriptase Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Retroviruses HIV How is HIV transmitted? The virus is transmitted through contact of a bodily fluid containing HIV like blood, semen, vaginal fluid, and breast milk with a mucous membrane or the bloodstream. A. ~33 million people are HIV positive in the world. B. Estimated 1.1 million people are HIV positive in the US. C. ~2.2 million people, 330,000 of which were children, died as a result of the virus last year – 75% of deaths occurred in Sub-Saharan Africa. Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Retroviruses Fig 10.21A HIV What disease does HIV cause? - AIDS – Acquired Immune Deficiency Syndrome Immune system gradually declines leaving the individual susceptible to opportunistic infections like tuberculosis (5 – 10% of Americans test positive for the bacterium that causes tuberculosis, but the immune system keeps it in check and the person is fine)and tumors (many cells that would have caused cancer are destroyed by the immune system). Therefore, HIV/AIDS does not kill anyone directly, it is the opportunistic infection or cancer that kills the person. Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Retroviruses HIV How does HIV cause AIDS? HIV (blue dots) infects, hijacks and in the end destroys Helper T-cells (red) (special type of cell of the human immune system required for proper function). Let’s look at how HIV infects Helper-T cells… Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… HIV Life Cycle GP120 Attachment and Entry: HIV envelope glycoprotein GP120 (ligand) binds to the CD4 receptor on the surface of the Helper T-cell resulting in fusion of the viral envelope with the cell membrane thereby allowing the capsid to enter the cell and fall apart releasing the viral RNA and Reverse transcriptase enzymes. Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… HIV Life Cycle This figure skips the “attachment and entry” and “uncoating” of the viral particle. Fig 10.21B Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… HIV Life Cycle 1. Reverse Transcriptase makes a DNA copy (blue) of the viral RNA genome (red). 2. Reverse Transcriptase then removes the RNA and synthesizes the complementary DNA strand. 3. Integration: the dsDNA enters the nucleus and gets integrated (inserted) into the DNA. Fig 10.21B Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… HIV Life Cycle 4/5. Transcription/Translation: viral RNA and proteins are synthesized from the provirus (analogous to prophage) DNA. 6. Assembly: viral particles are assembled and bud off the cell This process happens over and over again as long as the Helper T-cell lasts… Fig 10.21B The reproductive cycle of HIV, a retrovirus HIV Membrane of white blood cell 1 The virus fuses with the cell’s plasma membrane. The capsid proteins are removed, releasing the viral proteins and RNA. 2 Reverse transcriptase catalyzes the synthesis of a DNA strand complementary to the viral RNA. HOST CELL 3 Reverse transcriptase catalyzes the synthesis of a second DNA strand complementary to the first. Reverse transcriptase Viral RNA RNA-DNA hybrid 4 The double-stranded DNA is incorporated as a provirus into the cell’s DNA. 0.25 µm HIV entering a cell DNA NUCLEUS Chromosomal DNA RNA genome for the next viral generation Provirus mRNA 5 Proviral genes are transcribed into RNA molecules, which serve as genomes for the next viral generation and as mRNAs for translation into viral proteins. 6 The viral proteins include capsid proteins and reverse transcriptase (made in the cytosol) and envelope glycoproteins (made in the ER). Figure 18.10 New HIV leaving a cell 9 New viruses bud off from the host cell. 8 Capsids are assembled around viral genomes and reverse transcriptase molecules. 7 Vesicles transport the glycoproteins from the ER to the cell’s plasma membrane. Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… What determines the damage a virus does? One item is the type of cell it infects… Examples: HIV – immune system cells Influenza – respiratory cells Polio – neurons (can’t divide) Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Vaccinations 1. Edward Jenner A. Credited with discovering the first vaccine in 1798 B. The disease was small pox C. He observed that milk maids (people that milked cows) did not get small pox. D. Took the pus from these people infected with cow pox (a similar virus to small pox that you catch from cows) and injected it into other people. E. The cow pox pus somehow protected these people against small pox Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Vaccinations 2. How do vaccines work? - By injecting the cowpox pus, the immune system mounts an attack against the virus in the pus. - The immune system remembers the foreign substances it attacks and is prepared if it attacks again… - Since the small pox virus is so similar to the cow pox virus, the immune system is prepared for the small pox virus as well... Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Vaccinations 2. How do vaccines work? - Most modern day vaccines are typically an injection of dead or weakened (attenuated) viruses or viral proteins…more about this when we look into the immune system in detail. Chapter 24: The Immune System NEW AIM: How does the body defend itself against MO’s? I. Nonspecific vs. Specific Immunity B. Specific immunity (The Immune System) OVERVIEW Memory T-cells are also made from Tcells activated by Helper T-cells. For a future encounter with the same antigen carrying pathogen. Chapter 24: The Immune System NEW AIM: How does the body defend itself against MO’s? I. Nonspecific vs. Specific Immunity B. Specific immunity (The Immune System) vii. Memory cells a. Memory B and T-cells are reservists for next time that specific antigen shows up: Primary immune response The first time the lymphocytes see the antigen. Antibodies are made, but relatively slowly due to the small number of B-cells activated and only a relatively small number of antibodies are made compared to the second time the lymphocytes see the antigen for the same reason. Secondary immune response The secondary response results upon reexposure to the antigen. You have millions of memory B-cells. Most of them will be activated and antibodies are made quickly and in large number thanks to the large number of cells. You do not get sick. It must be the same antigen. Any mutation that changes the structure of the antigen will not elicit the secondary response. Fig. 24.8 Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Vaccinations Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Vaccinations Chapter 18 - Genetics of Viruses and Bacteria NEW: Viruses: Packaged Genes… Fig 10.19 Tobacco Mosaic Virus – Plants get viruses t Chapter 18 - Genetics of Viruses and Bacteria Transmission Bacterial and Viral Transmission 1. Droplet Contact - coughing or sneezing on another person Ex. Chicken pox, common cold (rhinovirus), influenze (flu), Tuberculosis, Measles, Mumps, Rubella, Pertussis, Strep throat Chapter 18 - Genetics of Viruses and Bacteria Transmission Bacterial and Viral Transmission 2. Direct Physical Contact - touching an infected person, including sexual contact Ex. Sexually transmitted diseases, Athlete’s foot (fungal), Warts Chapter 18 - Genetics of Viruses and Bacteria Transmission Bacterial and Viral Transmission 3. indirect contact - usually by touching a contaminated surface like a door knob or your desk. (ex. Rhinovirus…common cold) 4. airborne transmission - if the microorganism can remain in the air for long periods (essentially droplet transmission) 5. fecal-oral transmission - usually from contaminated food or water sources (cholera, hepatitis A, polio, rotavirus, salmonella) 6. vector borne transmission - carried by insects or other animals (malaria – a protist) Chapter 18 - Genetics of Viruses and Bacteria Transmission Bacterial and Viral Transmission This is why surgeons look like this… Chapter 18 - Genetics of Viruses and Bacteria Transmission Bacterial and Viral Transmission …and people working in a biosafety level 4 laboratory look like th Chapter 18 - Genetics of Viruses and Bacteria Transmission Bacterial and Viral Transmission Biosafety Levels Examples Non-pathogenic E. coli (Escherichia coli Hepatitis A, B, C, influenz Tuberculosis, West Nile Virus, Anthr Ebola virus, small pox , Argentine hemorrhagic fevers, Marburg virus, Lassa fever, Crimean-Congo hemorrhagic fever Chapter 18 - Genetics of Viruses and Bacteria Transmission Viroids 1. Circular RNA molecules that infect plants (only several hundred nucleotides long) proteins 2. DO NOT encode 3. The RNA molecules replicate inside plant cells using their machinary THEY ARE JUST SINGLE MOLECULE!! TEM of circular viroid RNA (black Plants infected with varying degrees of viroid particles (control Chapter 18 - Genetics of Viruses and Bacteria Transmission Prions 1. Infectious Protein!! 2. Cause a number of degenerative brain diseases in various animals Ex. scrapie in sheep, mad cow disease in cows, Creutzfeldt-Jakob disease in humans 3. Transmitted through ingestion of food with these prions in them like eating beef from cattle that had mad cow disease. ALARMING CHARACTERISTICS 1. They are slow-acting - Takes about 10 years until you see symptoms 2. Virtually Indestructable - They are not destroyed (denatured) by heating to normal cooking temperatures Chapter 18 - Genetics of Viruses and Bacteria Transmission Prions How can a protein, which cannot replicate itself, be a transmissible pathogen? Hypothesis: - A prion is a misfolded form of a protein normally present in brain cells - When the prion gets into a normal cell, with the normal form of the protein, it converts the normal protein to the prion form. You eat prion infected beef Prion gets into neurons in your brain and turn normal protein into prion form…chain reaction. Chapter 18 - Genetics of Viruses and Bacteria Transmission BACTERIAL GENETICS Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? How do bacteria (prokaryotes) they take up DNA… (it is more than just mutation that gives certain species of bacteria their genetic diversity) Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Reproduce by binary fission Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Reproduce by binary fission How do bacteria maintain genetic One way is through mutation since they can diversity? Replication of single, circular bacterial reproduce so quickly leading to millions upon billions chromosome preceding binary fission of slightly different individuals in only a days time. Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Reproduce by binary fission Is this the only way they maintain diversity? Replication of single, circular bacterial Absolutely not…let’s look at other ways to do this… chromosome preceding binary fission Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Look at this experiment and explain what is being observed: How were these bacteria able to exchange genes (DNA)? Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Three major methods have evolved by which bacteria take up foreign DNA to enhance diversity: 1. Transformation 1. Bacteria can take up a free piece of bacterial DNA 2. Crossing-over will occur between exogenous DNA and the bacterial chromosome. Fig. 12.1A-C Recall Griffith’s experiment Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? There are three methods by which bacteria take up DNA in n 2. Transduction Bacteriophage is mistakenly packaged with bacterial DNA. Injects this DNA into another bacteria. Recall Hershey and Chase Fig. 12.1A-C Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? 2. Transduction Bacteriophage is mistakenly packaged with bacterial DNA. Injects this DNA into another bacteria. Fig. 18.6 Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? There are three methods by which bacteria take up DNA in n 3. Conjugation “Male” (F+) bacteria extend sex pili called a mating bridge (long tube) to “female” (F-) bacteria. Part of chromosome is replicated and transferred. F+ F- Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? There are three methods by which bacteria take up DNA in n 3. Conjugation F+ means the cell has the so-called F (fertility) factor What is an F factor? It is a special segment of DNA that can be part of: F+ F- 1. The bacterial chromosome OR 2. A plasmid Now what’s a plasmid? Bacteria can have small, circular extrachromasomal (not the chromosome) pieces of DNA. Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Lysed bacterium The majority of the DNA above that has spilled out of the bacterium is chromosomal, but you can see smaller circular pieces not part of the chromosome…plasmids. Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Plasmid - Small, circular piece of DNA distinct from bacterial chromosome - has own origin of replication (ori) - carries genes in nature or humans can modify them and insert genes into the so-called polylinker region - called vectors when used by humans as tools of genetic engineering… Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? There are three methods by which bacteria take up DNA in n 3. Conjugation F+ means the cell has the so-called F (fertility) factor The F plasmid A special plasmid containing the F factor plus some 25 other genes needed for the production of sex pili F+ F- ***This plasmid has the ability to integrate into the chromosome of the bacterium or remain separate (see next slide). F+ cells have the F plasmid and can form sex pili and exchange DNA with an F- cell. Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? There are three methods by which bacteria take up DNA in n 3. Conjugation The F- cell is now and F+ cell because it now has the F plasmid and can form sex pili with other F- cells and pass along DNA. Fig. 18.18 Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? There are three methods by which bacteria take up DNA in n 3. Conjugation As mentioned earlier, the F plasmid has the potential to integrate into the chromosome of the bacterium as shown above resulting in what we call an Hfr (High frequency of recombination) cell. Fig. 18.18 Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? There are three methods by which bacteria take up DNA in n 3. Conjugation Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? There are three methods by which bacteria take up DNA in n 3. Conjugation Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? There are three methods by which bacteria take up DNA in n 3. Conjugation Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? There are three methods by which bacteria take up DNA in n 3. Conjugation Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? There are three methods by which bacteria take up DNA in n 3. Conjugation Now when the plasmid begins to replicate, it will also replicate part of the bacterial chromosome giving new genes to the recipient cell. Crossing over and therefore recombination will occur within the Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? There are three methods by which bacteria take up DNA in n 3. Conjugation Complete picture of the two possibilities Fig. 18.18 Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? 1. Transformation 2. Transduction 3. Conjugation Where have we observed transformation before in this class? The Griffith experiment when he mixed the R strain with the heat-killed S strain… Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? β-lactam ring R plasmids (aside) 1. R stands for resistance 2. These are bacterial plasmids that carry genes that confer resistance to antibiotics like ampicillin ampicillin 3. The gene that confers resistance is called AmpR (ampicillin resistance). What protein does is code for? It encodes the protein β-lactamase Guess what is does: β-lactamase with ampicillin bound in the active site Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Transposable Elements (Transposons) 1. Also known as “jumping genes” Insertion sequence Nobel Prize, Cold Spring Harb 5 A T C C G G T… A C C G G A T… 3 3 TAG G C CA… TG G C CTA… 5 Transposase gene Inverted Inverted repeat repeat (a) Insertion sequences, the simplest transposable elements in bacteria, contain a single gene that encodes transposase, which catalyzes movement within the genome. The inverted repeats are backward, upside-down versions of each other; only a portion is shown. The inverted repeat sequence varies from one type of insertion sequence to another. Figure 18.19a Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Transposable Elements (Transposons) 1. Also known as “jumping genes” Nobel Prize, Cold Spring Harb Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Transposable Elements (Transposons) 1. Also known as “jumping genes” Transposon Insertion sequence Antibiotic resistance gene Insertion sequence 5 3 3 5 Inverted repeats Transposase gene (b) Transposons contain one or more genes in addition to the transposase gene. In the transposon shown here, a gene for resistance to an antibiotic is located between twin insertion sequences. The gene for antibiotic resistance is carried along as part of the transposon when the transposon is inserted at a new site in the genome. Figure 18.19b Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Transposable Elements (Transposons) DNA-transposons vs Retrotransposons Almost 50% of the human genome is composed of retrotransposons Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Transposable Elements (Transposons) DNA transposon: Chapter 12 - DNA Technology and the Human Genome How can we use bacteria to manipulate DNA and protein? Transposable Elements (Transposons) DNA-transposons Important in gene duplication during S phase of meiosis Chapter 11 - The Control of Gene Expression NEW AIM: How are genes regulated (controlled) in prokaryotes? Bacteria, like all other organisms, respond to their environment by regulating gene expression and protein/enzyme activity… (a) Regulation of enzyme activity Precursor Feedback inhibition (b) Regulation of enzyme production Enzyme 1 Gene 1 Enzyme 2 Gene 2 Enzyme 3 Gene 3 Enzyme 4 Gene 4 (a) Negative Feedback: You have already seen how the product of a biosynthesis pathway like the amino acid tryptophan (trp) can allosterically inhibit an enzyme in its production pathway thereby shutting down its own production (negative feedback). (b) Regulating gene expression: Regulation of gene expression – Genes can also be turned on/off. – Let’s look at how bacteria regulate gene expression first in relation to lactose and then trptophan… Figure 18.20a, b Enzyme 5 Tryptophan Gene 5 Chapter 18 - Genetics of Viruses and Bacteria Questions 1. “Jumping genes” are known as __________________ and always code for the enzyme known as _________________. 2. An F+ cell is said to be “fertile” because it carries with it the ________________. 3. SRP RNA is found where in the cell? 4. How many different aa-tRNA synthetases are there? 5. Amino acids are added to what end of the tRNA by aatRNA synthestase. 6. If the anticodon for a given tRNA is 3’-GCG-5’, what letters would you look for on the genetic code chart to determine the amino acid attached to this tRNA? Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? In order to begin to understand this process, we will look at a set of three genes involved in Glucose and galactose lactose metabolism (the hydrolysis of lactose to _______________) called the… Lactose (Lac) Operon Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Fig. 11.1B LacA Anatomy of an operon (only prokaryotes have operons) The terminator An operon typically contains a: sequence 1. Promoter 2. Operator 3. A set of genes (3 in this specific case) A. LacZ B. LacY C. LacA 4. What critical gene part is missing from this figure? The terminator sequence The regulatory gene (LacI) is found OUTSIDE of the operon. Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Fig. 11.1B LacA The three gene products (can you guess what they might be?): 1. LacZ codes for β-galactosidase - The enzyme that hydrolyzes lactose to glucose and galactose 2. LacY codes for permease - A passive lactose transporter protein that sits in the membrane and allow lactose to diffuse into the cell. 3. LacA codes for transacetylase - Exact function not yet known… Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Fig. 11.1B QUESTION If lactose is present around the cell (perhaps it is one of the bacterium in your mouth and you just drank a glass of milk), should these genes be turned on or off? They should be ON since lactose is present and will need to be hydrolyzed so the glucose and galactose can be used to make ATP of for biosynthesis. Let’s look at how this operon works to control expression of these three genes… Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? 1. The regulatory gene codes for the repressor protein. A. What does repress mean? - To prevent B. What will this protein do then? - It will prevent the expression of the genes (turn them Fig. 11.1B - Any guess how it might do this? Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? 1. The regulatory gene codes for the repressor protein. C. It represses by binding to the Operator sequence and in doing so blocks the promoter sequence. Fig. 11.1B Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Fig. 11.1B 1. The regulatory gene codes for the repressor protein. C. It represses by binding to the Operator sequence. -When it binds the operator, it will interfere with RNA polymerase binding to the promoter. The genes are off. Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Fig. 11.1B ALL FOR ONE AND ONE FOR ALL Notice that all three genes are turned on/off together. Eukaryotes do not typically do this. They turn genes on/off individually. Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Fig. 11.1B Q1. How do you suppose these genes will be turned ON when lactose is present? A1. Somehow the repressor needs to fall off. Q2. How can we get it to fall off? (HINT: you are changing its function) A2. You need to change its structure. Q3. How can we change the structure? A3. Bind something to it…a ligand. Q4. What should the ligand be? Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? The ligand should be lactose itself since in the presence of lactose these genes should be turned ON. Fig. 11.1B Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Activating the operon: 1. Lactose binds the repressor. 2. A conformational (shape) change occurs and the repressor falls off the operator. 3. RNA polymerase now binds to the promoter and begin transcription of all three genes in one long mRNA. 4. Ribosomes translate the mRNA into proteins. Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Q1. What will happen when β-galactosidase breaks down most of the lactose? A1. Lactose will fall off the repressor and the repressor will once again bind to the operator and turn the genes off. Q2. Why not just leave these genes on all the time? A2. This would be a huge waste of resources…ATP, amino acids, ribosomes, nucleotides, RNA polymerases and space. Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? To be more detailed about it… A small amount of lactose is converted to allolactose by an enzyme in the cell. It is actually allolactose that is what we call the inducer, which simply means it inactivates the repressor (aka induces transcription). Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Lac repressor protein Repressor bound to the operator Lac operon – The Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? In reality, the presence of lactose alone is not enough to induce the transcription of the lac gene…why would this be logical? Because there could be other sugars in excess like glucose. Why waste ATP going after lactose if you are already overloaded. How does the bacterium sense the levels of glucose and translate this information to the genome you ask… Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? When glucose is absent and lactose present, cAMP levels are1.high… cAMP is an allosteric activator of CAP (catabolite activator protein) 2. CAP will bind to the CAP-binding site on the promoter and recruit RNA polymerase resulting in the production of much mRNA: Promoter DNA lacl lacZ CAP-binding site cAMP Inactive CAP RNA Operator polymerase can bind Active and transcribe CAP Inactive lac repressor (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized. If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces Figure 18.23a large amounts of mRNA for the lactose pathway. Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? When glucose is present with lactose, cAMP levels are low… 1. CAP is inactive and RNA polymerase will not bind well to the promoter even if the repressor is not present. 2. Little mRNA made Promoter DNA lacl lacZ CAP-binding site Operator RNA polymerase can’t bind Inactive CAP Inactive lac repressor (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized. When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription. Figure 18.23b Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? How exactly does glucose lower the levels of cAMP? 1. Obviously the activity of adenylyl cyclase needs to be lowered, but glucose does not interact directly with this enzyme… Not something you should memorize, just understand… Figure X. Control of adenylate cyclase via the phosphotransferase system. A. IIA, IIB, IIC, and HPr comprise the phosphotransferase system. When glucose is present, the phosphorylated forms of IIAGlc are low because glucose siphons off the phosphate. IIAGlc then interacts with and inhibits adenylate cyclase activity. B. In the absence of glucose, the phosphorylated forms of glucose-specific IIAGlc and IIBCGlc accumulate because they cannot pass the phosphate to substrate (there is no glucose). Adenylate cyclase functions in this situation to produce cAMP. The inset on the right shows the conversion of ATP to cyclic AMP by adenylate cyclase. Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Tryptophan (Trp) operon - This operon contains fours genes whose protein products are responsible for synthesizing (making) the amino acid tryptophan. Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Tryptophan (Trp) operon When would you want to turn these genes on? When tryptophan is NOT present, because that is when you need to make it… when trp is present, it will bind to and activate the repressor: Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Tryptophan (Trp) operon How does this compare to the lac operon? Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Tryptophan (Trp) operon Inducible operon You can turn ON (induce) the operon by adding something (lactose in this case) Repressible operon You can turn OFF (repress)the operon by adding something (Tryptophan in this case) Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Tryptophan (Trp) operon I do not recommend memorizing the difference. Think about is logically: 1. The repressor bind to the operator 2. When it is bound the genes are off 3. You need the lactose break down genes when lactose is present. 4. Therefore, when lactose binds to repressor, it should fall off operator 5. Likewise, when trp is present, the trp synthesis genes are unnecessary because you have it already 6. Therefore, Trp when Trp binds to the repressor, the repressor should bind the operator and shut the genes off. Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Trp operon in detail… Tryptophan (Trp) is a corepressor since it represses along with the repressor. Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? The trp repressor (with trp bound) binding to the operator sequence. Chapter 11 - The Control of Gene Expression AIM: How are genes regulated (controlled) in prokaryotes? Both cases are examples of repression… …but there can also be activation by activator proteins as we shall see in the next slide. Chapter 18 - Genetics of Viruses and Bacteria Questions 1. A functioning unit of genomic DNA containing a cluster of genes under the control of a single promoter. 2. The lac genes in E. coli are turned on when what two conditions are present in the cell? 3. The major difference between how the trp genes are regulated compared to how the lac genes are regulated. 4. When glucose concentrations are low within an E. coli cell the concentration of _________ is _________ causing the activation of _____________, which is required RNA to pol. 5. What isfor Χ2recruiting analysis used determine?