BIOL200Brazill Notes

BIOL200 Notes Professor Derrick Brazill
Fall 2011
August 30 Description: Dr. Brazill started the class by going over some housekeeping stuff, such as his office hours and email. He also went over his grading policy. There is no final exam, just three midterms each worth 200 points, plus 400 points from lab, out of 1000 total points. Exams are based solely on lectures, not on labs, and not on anything he hasnt covered in class. And he says that if we have any questions, we may just ask; he likes an informal class. Should be fun . He had no idea that the textbook had a third edition out, either. I think Ill like this professor. And at the end of class, someone told us about some event hes putting together. Notes: This is Cell Biology, so the focus will be on microorganisms. The goal of the course is to take a look at the cell processes involved in microorganisms. Normally, when we think of microorganisms, we think of bacteria that cause disease. Microorganisms, however, are also used in such diverse places as manufacturing (creating insulin and other hormones), to monitor ecosystems, and for bioremediation (cleaning oil spills and collecting heavy metals from the soils and environment). There are lots of microorganisms, so we will just look at a few examples of them, and look at similarities and differences between those different examples. Whenever he talks about a microorganism, we should think about the similarities and differences between that and other microorganisms. In review of previous information: we will see ionic chargebased chemical bonds, seen in salts. Covalent bonds are bonds where two atoms in a compound share electron orbitals. We will see hydrogen bonds, polar bonds formed between hydrogen and generally either oxygen or nitrogen. These may not be strong individually, but all together they will have a large impact when in large numbers. They are important in protein folding, for example making nonpolar sections of the protein fold inward together. Biological molecules: great diversity and complexity can be achieved in a biological context while using a limited number of building blocks. For example, there are only 4 nucleotides in DNA, the genetic material for all life. There are only 20 different amino acids, and many different proteins can be formed from them. A large variety of biological products can be made from a small set of tools. Because creating amino acids takes energy, it is helpful for organisms to require only a small set of molecules. An organism creates polymers of multiple building block subunit molecules through condensation. Condensation involves the hydrogen on one end of a monomer binding with the hydroxide group on the end of another monomer, producing a water molecule and bonding the two monomers. This dimer is now ready for another round of condensation; another monomer can now bind to the end. Thus, polymers are created from these subunits. Hydrolysis is the reverse of this, depolymerization, where a water molecule enters and breaks the bond between two monomers, putting a hydroxide group on one monomer, and a hydrogen atom on the other. These monomers may then be reused; this saves energy in creation and destruction of polymers because monomers do not need to be broken down and recreated. This energy can then be used in other ways by the organism. Carbohydrates are organic molecules, made of multiples of carbon and water in sugar form. Sugars are normally 5- or 6-carbon sugars, with hydrogens and hydroxide groups attached. Sugar molecules can be combined through condensation to create dimers and polymers. The linkage between two sugar molecules is a glycosidic bond, made during the condensation reaction. It comes in two different forms: alpha and beta, depending on whether the bond is on one side or the other of the carbon ring. Glucose monomers linking in a 1-4 alpha bond gives us
By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com
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glycogen and starch. Glucose monomers linking in a 1-4 beta bond gives us cellulose, which our bodies cannot digest. Lipids come in two types: 1. Regular lipids they are made from condensation reactions between glycerol and fatty acid tails. Glycerol is a 3-carbon alcohol with three hydroxyl groups. A fatty acid is a long chain of carbon atoms with hydrogen atoms attached, with a carboxylic acid group at one end (the other end just has hydrogens, a methyl group). A condensation reaction links the carboxyl group with one of the hydroxide groups on the glycerol. Lipids are generally poor hydrogen bonders, and are not water-soluble. They can be saturated or unsaturated in a saturated fat, the fatty acids have no double bonds. In an unsaturated fat, the fatty acid has at least one cis double bond. 2. Phospholipids instead of using glycerol, the phospholipid uses glycerol phosphoric acid. In place of the third hydroxyl group, a phosphate group is attached, and only two hydroxide groups condense with fatty acids. So half the molecule is hydrophobic, while the head (phosphate group and polar head) are hydrophilic. Therefore, phospholipids are amphipathic, with both hydrophobic and hydrophilic halves. When phospholipids are placed in water, they will naturally form membranes, or lipid bilayers. These are the membranes for all biological organisms that have membranes. Proteins are the work horses of the cell. They are made from amino acids, each of which consists of a carbon attached to a hydrogen, an amine group on one end and a carboxyl group on the other end, and a side chain that varies depending on the amino acid. Of the amino acids, there are hydrophobic (non-polar), hydrophilic (polar), charged as acidic or basic, or special amino acids. And we must memorize which ones fall into which category. Polypeptides are chains of amino acids, held together by peptide bonds. The primary level of polypeptide bonding involves direct condensation reactions between amino acids, reactions between the amine group of one amino acid and the carboxyl group of another amino acid; this is called a peptide bond in the case of two amino acids. Proteins are very long polypeptides that have folded together through secondary, tertiary, and quaternary structure. Secondary structure involves alpha helices and beta pleated sheets with only hydrogen bonds within a single polypeptide chain. Tertiary structure involves other bonding within a polypeptide, such as disulfide bridges, ionic bonds, other hydrogen bonds, and nonpolar interactions between side groups. Tertiary structure causes the polypeptide to form a three-dimensional shape. Quaternary structure is bonding between different polypeptide chains in a protein whole, driven by the same sort of bonding as in tertiary structure, but between different polypeptide chains. Nucleic acids are mainly used in the cell for information storage and retrieval. The two nucleic acids that we normally work with are DNA and RNA. They have two parts, the bases and the sugars. The bases can be two-ring purines (adenine and guanine) or single-ring pyramidines (cytosine and thymine). The sugar can be a ribose or a deoxyribose 5-carbon sugar. A nucleoside (base and sugar) attached to a phosphate group is a nucleotide. Nucleic acid wholes are polynucleotides, formed through condensation reactions between the phosphate groups and 3 sugars of consecutive nucleotide monomers. DNA has deoxyribose as its sugar. DNA
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makes a double-stranded helix, held together by hydrogen-bonds between bases A and T and between bases C and G. RNA is generally single-stranded (some double-stranded RNA viruses), with ribose as its sugar. There are several types of RNA, mRNA (messenger RNA), tRNA (transfer RNA) and rRNA (ribosomal RNA), all used in the creation of proteins. The rRNA gives the ribosome its structure, while the tRNA translates the mRNA (that was transcribed from DNA) into polypeptide. In summary: There are several basic building blocks of the cell. Carbohydrates give structure and energy. Lipids are used for membranes. Proteins give structure and act as the work horses of the cell. And nucleic acids store and translate cell information. All of them are basic units that make polymers through condensation reactions. This allows for numerous combinations of monomers with minimal number of monomers and energy-requiring reactions. September 1 Description: Brazill started todays class by teaching us some of the history of microbiology, with names and dates of inventions of microscopes and other such historical crap. He then talked about resolution and contrast, and drew boxes on the projector as examples of poor and good resolution and contrast. After we covered microscopy, we moved on to discuss microbial diversity. At one point, he talked about the difference between transcription and translation: Nothing is more infuriating to a professor than a student using the wrong term. Because the terms we use are very important. Now Ill get off my soapbox, thank you for indulging me! In an unrelated amusing occurrence, at the beginning of the class I overheard a couple girls talking about Brazill. It seems hes quite popular among themthey said that they wanted to ask him about his workout routineand that when hes writing, Im watching his arm moving. I swear, those are direct quotes! Notes the first half of todays notes is on microscopy; if your course doesnt require learning it then feel free to skip until just above the table. The second half is about the domains of life and the theory of the origin of eukaryotes, feel free to skip that too. But, todays notes: The normal eye has an eye resolution of approximately 0.2 millimeters; the eye can resolve images of this size and larger. E coli bacteria, however, are approximately .002 millimeters. The microscope allows for the identification and characterization of microbes. Robert Hooke made the first microscope in 1665, with a maximum 30x magnification, allowing to resolve images of .007 millimeters. Antonie van Leeuwenhock, in 1676, developed better lenses, and could see single-celled organisms. Robert Koch, in 1877, used the microscope to determine that microbes cause disease, in his Germ Theory of Disease. He identified that bacillus anthracis caused anthrax, and that mycobacterium tuberculosis caused tuberculosis. Koch created postulates in determining whether or not microbes do indeed cause disease; criteria for linking a microorganism to a disease: 1. The microbe is seen in all cases of the disease, and is absent from healthy people. 2. The microbe can be isolated from a diseased host and cultured. 3. It is possible to introduce the microbe into a healthy organism, and this introduction will often cause the healthy organism to become sick with this disease. 4. This same microbe can be isolated from the newly infected host and can infect others.
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Thus, it was possible for him to classify diseases by the organisms that caused them, and it was possible for him to link specific microbes to certain diseases. Contrast and resolution: Contrast is the ability to easily visually distinguish two different things, the cell and its background. The resolution is the minimum distance by which two objects can be separated and still be distinguished. Resolution is equal to .61lamda/(nsin(theta)), where n is the index of refraction that depends on the medium through which it is viewed, theta is the half-angle of the light captured by the objective lens, and lamda is the wavelength of the light being used to illuminate the object. A good resolution is a small number. A microscope consists of several things: 1. Light source the light source illuminates the object. 2. Condenser lens it focuses the light onto the specimen. 3. Objective lens it collects the light after it has passed through the specimen, and makes the image look larger. 4. Ocular lens this focuses the light onto the eye. The only difference between the resolutions of a 100x lens and a 10x lens is that the angle of the light is much larger. But this doesnt improve contrast, only resolution. In order to improve contrast, in order to tell the difference between the watery cell and its watery background, we: 1. Fix kill the cell, polymerize its cytoplasm, and attach the cell to its slide. 2. Stain two types of stains: a) Direct stain that stains the bacteria, not its background. These stains (methylene blue, crystal violet) are positively charged dyes which bind to the negatively charged surface of the cells. b) Indirect stain the bacteria are left unstained while the background is stained, allowing for contrast. One such stain is nigrosine. c) Spore stain some bacteria form spores, special structures (which we will talk about later) that can be stained with a spore stain such as malachite green. d) Gram stain some bacteria can be stained by a stain called gram stain, those are called gram-positive. Gram-negative bacteria cannot be stained by this gram stain which is based on the structure of the cell wall. e) Fluorescence microscope: Fluorescence is the ability of a compound to absorb light at one wave length and emit light at a second wavelength. With a fluorescence microscope, we are observing the light emitted by a dye emitting this light. We stain bacteria with a fluorescent dye, so that the bacteria are bright, but there is no other light source so the background is dark. This provides far better contrast than does a light microscope. Two fluorescent stains are the gram stain and viability stain. Microorganisms are an incredibly diverse group of organisms. Microbial diversity: There are three domains of life, archaea, bacteria, and eucarya. Archaea and bacteria are prokaryotes. All three domains contain microorganisms, and the three domains were originally related. Bacteria Archaea Eucarya Information All three domains use the same method of information processing. Processing DNA is transcribed to RNA, which is translated to protein.
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Chromosome material Chromosome structure Nucleus Operons Membrane-bound organelles Ribosome Histones/Nucleosomes Introns RNA Polymerases Peptidoglycans DNA Circular No Yes No 70S No No 1 Yes DNA Circular No Yes No 70S Yes Yes >1 No DNA Linear Yes No Yes 80S Yes Yes >1 No
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Eucarya and archaea are more closely related to each other than they are to bacteria. The Endosymbiotic Theory explains why and how eukaryotes got organelles. It states that anaerobic prokaryotes enveloped aerobic oxygen-using prokaryotes in an internal (endo) symbiosis that allowed both to survive. In this theory, pre-eukaryotic anaerobic prokaryotic species were fed by aerobic prokaryotes. At one point, a pre-eukaryote ingested a prokaryote but didnt digest it. They formed a symbiosis, where the prokaryote gets shelter and aerobic nutrition, and the preeukaryote gets ATP production and oxygen scavenging so that it can now survive in an oxygencontaining environment. Thus, according to this theory, mitochondria were formed. According to this theory, chloroplasts were formed similarly, when a mitochondria-containing preeukaryote ingested photosynthetic bacteria. The photosynthetic bacteria are sheltered, while the pre-eukaryote gets food production. Evidence for this theory includes: both mitochondria and chloroplasts have double membranes, both have their own genomes, and both have their own ribosomes. September 6/8 Descriptions: Not many jokes, fairly boring two classes with quite a lot of writing. We continued on the 8th by completing the list begun on the 6th, so I kept the two days notes together. Notes: Although there are differences between the types of microorganisms, they all have similarities. They all have: 1. Cell membranes all cells have a plasma membrane, made of two parts that slide past each other in a fluid mosaic model, phospholipids and plasma proteins: a) Phospholipids amphipathic molecules with hydrophilic heads and hydrophobic tails that come together to form a bilayer with the hydrophobic tails on the inside of the membrane. Phospholipids in the bilayer can be in the inner leaf, on the side of the bilayer that is inside the cell, or on the outer leaf, on the external side of the bilayer. Phospholipids can have different head groups, and can even be attached to each other, but all phospholipids are amphipathic with a hydrophilic polar head and a hydrophobic tail.
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b) Proteins there are several types of plasma membrane proteins. There are peripheral proteins that are only attached to the polar heads of one side or the other of the bilayer and dont cross the bilayer. There are also integral membrane proteins that span the lipid bilayer. The peripheral proteins are hydrophilic, while the integral membrane proteins have a hydrophobic core with hydrophilic ends that allow it to associate with the water on the outside. Transmembrane proteins actually completely cross the membrane, while some integral proteins are only stuck in the bilayer. Integral proteins can be on the outer leaf or on the inner leaf. Proteins on the outer leaf are involved in substrate binding and transport. Proteins on the inner leaf are generally more involved in metabolism and energy production. The membrane is fluid, but the membrane should not be too fluid lest it fall apart at high temperatures. Fluidity can be controlled by adding molecules to increase rigidity. One example of such a rigid molecule is a hopanoid, which is a planar molecule that cannot move as easily, making the membrane more rigid. They are normally seen in prokaryotes; eukaryotes use sterols to do the same. Archaea use different lipids from eukaryotic bacteria. Bacteria and eukaryotes use ester linkages to link their fatty acids to the glycerol molecule, while archaea use ether linkages. Archaea can use and create phospholipids with tails that are bound to the ends of the tails of other phospholipids, allowing for a formation of a monolayer with polar heads on both ends and a hydrophobic core. This linkage of tails can be done through the two ends forming carbon-carbon bonds or through cyclopentane rings. One purpose of the plasma membrane is to act as a permeability barrier allows only certain molecules in and out. It has a hydrophobic core and hydrophilic heads, and makes a very good barrier. Gases pass through easily, and small hydrophobic molecules can pass. Water passes without too much difficulty, but polar glucose cannot pass easily, and ions can barely pass at all. Thus, the membrane allows for the controlled transport of molecules. It uses transport proteins, transmembrane proteins that have regions on their inside that allow molecules to pass. Transport can be active or passive, depending on whether the molecule is going up or down its concentration gradient. If it is going down its gradient, the energy comes from the concentration gradient; this is passive transport. In active transport, the energy comes from ATP, or from the transport of a second molecule. There are three different types of transporters: a) Uniporters move 1 molecule across the membrane in one direction. b) Antiporters move 1 molecule across a membrane while moving a second molecule in the opposite direction. c) Symporters move two molecules in the same direction 2. Deal with osmotic stress, whether with a cell wall in prokaryotes or the many other methods in eukaryotes. In most cells, the concentration of solutes inside the cell is far higher than is the concentration of solutes outside the cell. Osmotic pressure seeks to balance out the concentrations, water attempts to flow into the cell. This drive is called osmotic pressure. The cell would burst were it to be unable to deal with it, as water would flow into the cell until the membrane ruptures. Most microbes use a cell wall to deal with osmotic pressure. In bacteria, there are two basic types of cell walls: those made by gram-positive bacteria and those made by gram-negative bacteria. The stain
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results are from two different cell wall components. In bacteria, cell walls contain peptidoglycan, with a peptide part and a sugar part. The sugar part is made of two sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). When put together, sugars and amino acids form a repeating unit called glycantetrapeptide. There are cross-linkages between amino acid chains that give this cell wall its strength. Transpeptidization is a transpeptide bond between two different glycantripeptides. As the cell grows, it makes more crosslinked cell wall molecules. (Penicillin, for example, blocks this transpeptidization). In gram-positive bacteria, 90% of the cell wall is comprised of peptidoglycan, while gram-negative bacteria cell walls are only 10% peptidoglycan. In gram-positive bacteria, the peptidoglycan layer is very thick, with a large amount of teichoic acids that have a large negative charge inside, making it difficult for charged molecules to cross. Gram-negative bacteria have very thin peptidoglycan layers, but have a lipopolysaccharide layer. There is an outer membrane, attached to the peptidoglycan layer by lipoproteins. And porins can exist that allow ions to cross both membranes. The periplasm, between the two membranes, can be accessed through the lipopolysaccharide layer. (And Brazill notes that the lipopolysaccharides cause intestinal problems in pathogenic gram-negative bacteria, as an endotoxin). The gram stain is based on insoluble violet-iodine crystals. These crystals can enter the cell through the cell walls, so that both the gram-positive and gramnegative bacteria can take up the stain. These crystals can then be extracted using ethanol. Gram-negative bacteria allow their crystals to be washed away with the ethanol. However, with gram-positive, the ethanol dehydrates the thick peptidoglycan layer. When this occurs, the pores through which the iodine crystals would flow, shrink, causing the iodine to be trapped, leaving the stain still visible. The gram stain can give structural information about bacterial cell walls. This only works for bacteria, not for archaea or eukaryotes. For archaea and eukaryotes, the gram stain tells nothing of the cell wall architecture. Archaea dont have peptidoglycans; they only have pseudopeptidoglycans in a different organization from that seen in bacteria. In addition, some archaea have polysaccharide cell walls, and others have glycoprotein- or proteinbased cell walls. Some eukaryotes have cell walls made of cellulose, such as algae and other plants. Some have cell walls made of chitin, others have cell walls made of silicates, and still others only have pellicles, which while providing the same function as a same wall, has far more fluidity. Either way, whether or not an archaea or eukaryote stains with gram stain implies little about its cell wall structure. 3. Some method of DNA organization nucleoid in prokaryotes and a nucleus in eukaryotes. A nucleoid is a complex of DNA and proteins, with DNA compacted into surpercoils. 4. Method of motility flagella in prokaryotes, combined with other methods in eukaryotes. Flagella are used in most prokaryotes, allowing rapid movement (up to 60 cell lengths per second, faster than any land animal). Flagella orientation: several different types: a) Polar/monotrichous one flagellum, located at one end of a bacterium. b) Lophotrichous multiple flagella located at one end. c) Peritrichous flagella surrounding the bacterium, not just at one end.
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d) Amphitrichous two flagella on opposite ends of the bacterium.
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The structure of a flagellum: it has both a plasma membrane and a cell wall. It has different rings, along with a rotor and a hook. Rings sit in the membrane or cell wall; the C ring sits in the cytoplasm while the P ring sits in the peptidoglycan layer and the L ring sits in the LPS (lipopolysaccharide) layer. The rotor provides rotational movement. The rod is the axle that transmits rotor movement to the flagella. And the hook attaches the rod to the flagella. The flagellum filament provides the motive force, and is made from a protein called flagellin. (Note, this is the structure of flagella in bacteria; archaean and eukaryotic flagella look quite different.) 5. Dealing with stressful environments microorganisms have several ways of dealing with stressful environments, where there might be a lack of food or other dangers: a) Spore formation the spore is the storage form of an organism. It is resistant to heat, ultraviolet light, dessication, and radioactivity. It has very low metabolism, so it can be very long lived. Endospores are formed by some bacteria. They have a core, made of what was formerly the cell wall, membrane, cytoplasm, and nucleoid. Surrounding that core is another layer, the cortex, made of peptidoglycan. Outside the cortex is the spore coat, made of proteins. Outside the spore coat is the exosporangium, also made of proteins. Inside the core, water is replaced with dipicolinic acid, which is 10% of the spore mass and provides dessication and heat resistance. Also in the core are small acid soluble proteins (SASPs) that protect the DNA, binding tightly to it. They prevent the DNA from denaturing, protect it from radiation, and are used as an energy source when the spore eventually germinates. In sporulation, instead of undergoing normal replication with two split cells, after replication the smaller forespore is engulfed as an internal compartment within the mother cell, although still with a full complement of DNA. During this process, the DNA in the mother cell begins to be degraded. After the forespore is fully engulfed, it becomes the cortex and begins to make dipicolinic acid, as the exosporangium is formed. The spore coat is then made, and the mother cell releases the spore, which is now prepared to remain such until germination, where it can become a naturally dividing vegetative cell. The mother cell sacrifices itself in order to form a spore, which can then last protected for a long period of time. Germination has several steps: activation is triggered by its environment, with a cue. Germination causes a loss of spore resistances, with an increase in metabolism and hydration. The spore coat is absorbed and the SASPs (small acid-soluble proteins) are degraded. In outgrowth, the new vegetative cell emerges. What defines prokaryotic species? A species is normally defined as being able to mate with each other and produce fertile offspring. But single-celled organisms do not normally reproduce by mating (asexually). They are classified by several things: one way is by differentiating according to common characteristics, including shape, structures, and biochemistry. The members of the same species will have the same characteristics, while members of related species will have similar characteristics. But characteristics might be simply due to adaptation to an environment, might have little to do with relatedness. DNA sequencing is used more
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often in order to determine relatedness between species in addition to determining that the members of the same given species will have very similar DNA sequences. The closer two species are, the more similar their DNA sequences will be. Divergence occurs through mutation, when mistakes happen during DNA replication. Most of these mutations are harmful, but some are beneficial, and those that give an evolutionary advantage to organisms with this mutation will gain a larger share of the gene pool. Such a mutation could make an enzyme more effective, or causes the loss of an unnecessary gene, removing the need of an organism to waste energy on an unnecessary gene. This process occurs again and again, and if isolation occurs, a group of organisms are very different from the species from which they evolved, as this group has been accumulating mutations. This is called speciation. Most mutations are neither positive nor negative; they are neutral, and have no impact on the viability of an organism. These mutations can accumulate in an isolated population, in areas of the genome not under selective pressure. Mutations accumulate at a relatively constant rate, based upon the DNA replication error rate and the lengths of the generations (frequency of reproduction). By comparing two genomes in two different species, those more closely related will show fewer differences. Organisms in species farther apart will show more differences. Mutations occur at a relatively constant rate. We can use DNA similarities to measure how related two organisms are. And by measuring the number of differences and multiplying by the DNA mutation rate, we can measure how long ago they shared a common ancestor. Choosing a DNA sequence for a molecular clock: 1. We want the gene to have the same function in all organisms examined if it has different functions, it could be under selective pressure, and wouldnt make a good molecular clock without a constant mutation rate. 2. The generation time of the two organisms must be relatively similar a more rapid generation time would allow two organisms to accumulate mutations at different rates. 3. Constant mutation rate across generations. We normally use a small subunit of rRNA (named 16S or 18S) that is conserved across species, highly conserved in some portions (been selected for) but not conserved and not selected for in other portions. These unconserved, unselected portions are used for a molecular clock. September 13 Description: My notes are a bit shorter than usual today. Why? Because on my way over to school, I was videotaping, trying to beat the 4-minute mark in biking from home to school on camera. I was trying to incorporate some of the road maneuvers that I learned in last weekends bike safety class into my everyday riding. Riding downhill on my Fast Sidewalk Path in Central Park, there was a biker ahead of me on the crossing West Drive. I put on my brakes, but my back brake couldnt stop me from plowing into him. My left hands middle fingertip is rather swollen, so typing the letter e hurts a little. Anyway, we all got a good laugh out of Brazill saying that nodes occur when ancestors way way back in the future existed. September 15 Description: For todays notes, I just continued the table started last time. At the beginning of the class, Brazill didnt make everyone be quiet until class actually started. But then we went straight into the material. We finished discussing the characteristics of microorganisms, then started discussing our first specific microorganism, E. coli. But of course,
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Brazill went into a long tangent about cell respiration, which takes up the majority of a singlespaced page. September 13/15 Notes: My apologies for my slight lateness to class. Explanation is in the description. Phylogenetic trees show the tree of evolutionary descent with modification, showing what organisms are most closely related to what other organisms. In order to construct such a tree, we use a sequence of DNA that is of adequate length, serves the same function in all studied organisms, and is not genetically selected for. We look for the number of differences in the sequences, and the degrees to which various pairs of sequences are related to each other. This gives us a likely tree. The distances along the branches represent how related two organisms are. Trees can be rooted or unrooted; to get a rooted tree, a very distantly related outgroup must be included in the analysis, to give a perspective from which we can view the relationships within the tree. An unrooted tree shows relative relationships among the organisms, but tells you nothing about the evolutionary history of the organisms. A rooted tree shows relative relationships on an evolutionary path. A phylogram contains nodes, branching points in evolutionary paths. Internal nodes symbolize extinct ancestors, a point of divergence. External nodes represent species which exist today. Branches define the order of descent and ancestry of the nodes. And clades are groups of species which share a common ancestor (An organism may be in several different clades). How to define bacterial species: there is no universally accepted answer. The consensus is that if there is less than 3% divergence in the 16S rRNA, and more than 70% similarity in the overall genome, then the two organisms are in the same species. Vertical gene transfer is the transfer of genetic material from the parent to offspring. This is the more common type of genetic transfer. Horizontal gene transfer is mostly seen in prokaryotes. They can pick up huge chunks of the genome from other species and incorporate it into their own genome. This is not a slow, progressive series of mutations. This is a rapid, sudden change in the genome. It can occur through plasmids, transposable elements, bacteriophages, or transformation. Large stretches of DNA can get taken in and integrated into the bacterial genome. This can explain sudden antibiotic resistance; a bacteria can pick up antibiotic resistance from another bacteria. A framework to compare and contrast organisms discussed: characteristics of living organisms: 1. Reproduction the generation of offspring. Through reproduction, genetic material is transferred. In most microorganisms, reproduction occurs through binary fission. In binary fision, one parent splits into two identical daughter cells, each of which may then split again into two more cells, in exponential growth 2n where n is the number of generations. Total number of organisms after n number of generations: NT = N02n, where NT is the total (final) number of organisms, N0 is the initial number of organisms, and n is the number of generations. Reproduction in culture has several phases: a) Lag phase in this phase, the bacteria are adapting to their environment. Reproduction is slow as the bacteria learn to use their surroundings effectively. They are detecting the environment and changing gene expression accordingly. b) Log/Exponential phase in this phase, the cells have fully adapted. They reproduce at the maximum rate during this period, filling their surroundings until the surroundings are saturated with microorganisms.
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c) Stationary phase in this phase, the cell population is no longer growing. Due o a lack of nutrients and increase in waste products, the rate of reproduction equals the rate of death. d) Death phase in this phase, the population size decreases, reproduction occurs at a slower rate than death. The cells have saturated the environment to the extent that they cannot exist there any more. 2. Metabolism the way that an organism takes in and uses energy, using it to fight entropy. (Here Brazill went off on a tangent about energy: Gibbs free energy, dG0 = dH TdS. dH is enthalpy change, positive when heat is absorbed, negative when heat is released. T is the temperature in Kelvins. S is the entropy (so dS is the change in entropy) or disorder. For the reaction to occur spontaneously, dG0 must be negative. The reaction is favored when dH is negative and dS is positive. Diffusion occurs because of a difference in concentrations, which is an ordered state, diffusing into a more randomized state. Organism growth increases order, so dH must be sufficiently negative to overcome dS.) Energy, dH, is used to fight disorder, dS. Metabolism uses energy to fight chaos. Two parts of metabolism: a) Catabolism breaks down molecules into energy. b) Anabolism using energy to make molecules. Adenosine triphosphate, ATP, is a molecule that stores of energy, used as sort of an energy currency. It consists of a nucleoside, adenosine, attached to three phosphate groups. Because of the negative charge of the phosphate groups, the outer two phosphate bonds hold a lot of energy. In catabolism, ATP is made, with phosphate groups added to AMP (adenosine monophosphate) or ADP (adenosine diphosphate). This requires energy. In anabolism, ATP or ADP is used, the phosphate bond breaking releasing energy. This reaction can be coupled with an anabolic reaction in order to cause the overall coupled reaction to spontaneously occur. 3. Cellular structure every living organism has some sort of structure of organization. They can all use basic molecules to make intricate forms. They make different substances, with basic molecules arranged in special, spatial relationships. The existence of the organism depends on maintaining structure. 4. Respond to external stimuli most living things must be able to respond to external stimuli, whether this be in obtaining food or fending off predators or moving toward a food or light source. 5. Grow, develop, and differentiate energy from metabolism can be used to increase the organisms mass and complexity. Existing structures must be replicated and modified. For example, production is modified during endospore formation. 6. Adapted to environment all living things evolve to exploit their environment. Factors in their environment include access to water and oxygen, the presence of heat and light, predators, and competitors. Species that still exist today have specific characteristics used to take advantage of their physical surroundings. The survival of the species depends on their having these beneficial characteristics. These characteristics develop due to the selective pressures of evolution.
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7. Respond to environment individual microorganisms must be able to respond to respond to stimuli in their environment. They must be able to detect changes in their environment and respond accordingly. The lag phase is an example of this, as the microorganisms get used to their environment and adjust gene expression accordingly. September 20 Description: I figured here was a nice place to make the break between days of work. I dont like to interrupt a list, but the break was somewhere toward the end of #4 in the E. coli list below. Tonight, there was a full section that he declared to be difficult enough to warrant saying twice once for us to hear, a second for us to understand. He made a mistake at the end of it, I corrected him, and to much class laughter, said good boy as he corrected his mistake. September 15/20 Notes: Now, to discuss our first organism, E. coli. Every species has a first name (a genus) and a last name (a species name). List of stuff for E. coli: 1. Full Name Escherichia coli. 2. Grouping belongs to a class called gamma proteobacteria. It belongs to the group of intestinal enterobacteria. Family members include salmonella (causes typhoid and gastroenteritis), shigella (dysentery), klebsiella (pneumonia), and yersinia (plague). 3. Organization it is a rod-shaped bacteria. It has no nucleus, no organelles, but does have a cell wall. It is gram-negative, with a thin peptidoglycan layer and a lipopolysaccharide layer. It has flagella, and is peritrichous, with flagella surrounding it at several points. 4. Metabolism it uses glycolysis like most other organisms. (And here Brazill went off on a tangent about glycolysis: glycolysis is catabolic. It turns the 6-carbon glucose sugar into two 3-carbon pyruvate molecules, creating two ATP molecules and two NADH molecules along the way. First, two ATP are added to the glucose molecule, releasing the ADPs to create fructose 1,6 bisphosphate, which is turned into two molecules of glyceraldehyde-3 phosphate, which are each turned into molecules of 1,3 bisphosphoglycerate creating two NADH molecules from NAD+ molecules, and then each 1,3 bisphosphoglycerate picks up a phosphate and moves the phosphate groups onto ATP, leaving 4 ATPs for a net gain of two. These 1,3 bisphosphoglycerate molecules are converted into pyruvate 3carbon sugar molecules. If no NAD+ is left for use (NADH is normally converted back to NAD+ during the electron transport chain but if this doesnt occur), fermentation occurs to regenerate NAD+ from NADH. Fermentation creates either lactic acid or formic acid from its pyruvate molecule, in mixed acid fermentation. This fermentation occurs in the absence of the oxygen that would normally allow cell respiration to occur. Normally, the Citric Acid/Krebs/TCA cycle would create 3 carbon dioxide, 4 NADH, an FADH2, and a GTP. And normally, in oxidative phosphorylation in the electron transport chain, electrons are moved through proteins to oxygen (electrons join the oxygen, which forms water with hydrogen), releasing energy in each protein that powers proton pumps, creating a proton gradient that is used to power ATP synthase enzymes to create ATP. (F1/F0 ATPase, called such because it has two components, F0 is a proton channel, the flow through which can cause the F1 part, the rotor that produces ATP, to spin. This can
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be reversed, burning ATP to rotate F1, as a flagella does) NADH molecules yield three ATP molecules each by being turned into NAD+, and FADH2 yields approximately two ATP per molecules by being turned back into FAD+, for a total of approximately 30 ATP per glucose molecule. But in the absence of oxygen, the electrons transferred to NADH have nowhere to go. Therefore, in the absence of oxygen, the ETC (Electron Transport Chain) cannot occur.) E. coli can grow in both aerobic and anaerobic environments. It is facultative anaerobe that can grow both in the presence and in the absence of oxygen. It prefers the presence of oxygen, but can create energy through fermentation in its absence. Strict aerobes require oxygen to make ATP, and cannot perform fermentation (or fermentation cannot provide the amount of energy required for life of the organism). There are strict anaerobes, for whom oxygen is poisonous. There are aerotolerant anaerobes, that dont use oxygen although oxygen is not poisonous for them. Microaerophiles need low levels of oxygen. Some organisms can use oxygen in the creation of ATP. For others, diatomic oxygen creates oxygen radicals that cause DNA damage and cell structure damage. Organisms that can tolerate oxygen can scavenge, or can repair the DNA and cell structure damage. In an oxygen utilization test, we put bacteria into a vial of liquid. Aerobes will congregate at the top. Anaerobes will congregate at the bottom. Facultative anaerobes will congregate mostly at the top, with some scattering around the rest of the vial. Microaerophiles will form a band midway through the vial. And aerotolerant bacteria spread throughout the vial, evenly. 5. Reproduction and gene transfer E. coli performs binary fission, where a bacterium replicates its haploid genome, then increases its cytoplasm and creates a septum of plasma membrane and cell wall between the two cells that then split. E. coli has a circular genome. In its DNA replication, vertical gene transfer, rolling circle replication occurs, with the replication fork starting in the circular genome, becoming larger and larger (through the Theta structure, named after the way it looks like the Greek letter) until two circles are formed. In horizontal gene transfer, conjugation occurs. In conjugation, there is a donor cell and a recipient cell. The donor cell sends out a protein pilus that reels in the recipient cell and forms a special pore between them. The transferred plasmid is replicated and transferred into the recipient cell through this pore. Now, each cell can donate a copy of this plasmid to other cells. 6. Adapts to environment this is what occurs in the gut of a mammal, with a constant temperature and pH, where food is plentiful. Thus why E. coli survives so well in mammalian intestines. 7. Responds to environment chemotaxis is the ability to move towards or away from different chemicals. Attractants signal the bacteria to move toward such an attractant, while repellents signal the bacteria to move away from the repellent. Bacteria are very sensitive to these signals, up to extraordinarily small concentration, using chemoreceptors on the cell surface that span the plasma membrane. Signals received are magnified, and secondary signals and proteins interact with cellular proteins that control the actual motility. There are 5 known chemoreceptors, each of which can bind multiple ligands. Tar is one of them binds to aspartate, maltose, cobalt, and nickel. Mechanism of chemotaxis: in the absence of attractants, the cell moves randomly. It can run and tumble in basic motility. Run is motion in any direction, with the flagella
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working together, moving counterclockwise in a direction. Tumble is a turn-around flip, with the flagella moving clockwise. The cell modifies its runs and tumbles to move towards an attractant. It must be able to sense when the attractant is present, and it must be able to tell whether it is moving up the concentration gradient of the attractant in order to know that it is moving toward that attractant. Chemotaxis occurs: in the presence of an attractant, its runs will be longer in the direction of the chemoattractant. Important and supposedly complicated enough for him to repeat it in order for us to understand it: Signaling for chemotaxis: chemoreceptors are MCPs, methyl-accepting chemotaxis proteins. MCP binds two proteins (with the prefix Che for chemotaxis) CheW and CheA. CheA is a protein kinase that phosphorylates other proteins. In the absence of a ligand, CheA is active. It phosphorylates two proteins: CheY and CheB. When phosphorylated, CheY binds the flagellar motor, causing clockwise rotation of the flagella to cause a tumble. But CheY is regularly dephosphorylated by CheZ, existing in both states, the phosphorylated and unphosphorylated forms. CheY is phosphorylated by CheA, resulting in clockwise rotation and tumbles. It is dephosphorylated by CheZ, returning it to a dephosphorylated state that allows for counterclockwise motion of the flagella, causing run. The relative activities of CheA and CheZ determine runs and tumbles. The activity of CheZ is constant. But CheA is active when there is no ligand, and inactive when a ligand is present. So in the presence of a ligand, CheA is inactive, and CheZ is allowed to dephosphorylate CheY. CheY cannot interact with the flagellar motor, allowing for counterclockwise rotation of the flagella, resulting in a run. Thus, the presence of a ligand results in a run. To restate: when CheY is phosphorylated, it binds the flagellar motor, causing the clockwise rotation. CheY gets phosphorylated by CheA, and dephosphorylated by CheZ. So CheA and CheZ operate reverse reactions upon CheY. The decision whether to run or to tumble depends on which protein is more active. When CheA is inactive, having bound a ligand, CheZ will have more of an effect, dephosphorylating CheY and causing counterclockwise rotation and a run. When CheA is active, it is more active than CheZ, phosphorylating CheY and causing clockwise rotation, causing a tumble. Short-term memory is controlled by CheR. CheR is a methylase, which adds a methyl group to MCP. When MCP is not methylated, it has increased affinity for the ligand. When MCP is methylated, it has decreased affinity for the ligand. CheR adds a methyl group to MCP. When CheB is phosphorylated by CheA, it demethylates MCP, increasing affinity. The affinity state therefore depends upon the relative activities of CheR and CheB. When CheR is at a higher activity level than CheB, MCP will be methylated more often and will need a higher concentration of attractants in order to become more active. CheA phosphorylates CheB when CheA is activated in the absence of ligands, demethylating MCP and making it more receptive to the attractant. There is this feedback regulation, wherein CheA controls its own activity through a feedback loop. It is a 2-component system with a sensor kinase (CheA) and response regulators (CheY and CheB). And that ends this lecture. Cmjohns712@gmail.com
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September 22 Description: Today there werent many funny things to mention, besides the fact that there was a handout from the Biochem class on the desk along with our handout. I thought it was for us, and was rather confused. But besides thatwe talked about the material, and covered quite a lot. September 27 Description: Today we continued our discussion of Chromatium vinosum and anyoxygenic photosynthesis, after starting late due to Brazill attempting something with a projector. Notes: Bacillus anthracis: 1. Organization it is a rod-shaped bacteria, without any organelles. It has a cell wall, and is gram-positive, with a large peptidoglycan (PG) layer but no lipopolysaccharide (LPS) layer. Google states that it is peritrichous, but Im not sure Brazill actually said so. 2. Metabolism it is a chemoheterotroph, getting energy from oxidation. Phototrophs get their energy from light, chemotrophs get their energy from oxidation, chemolithotrophs get their energy from inorganic molecules. Bacillus anthracis is a chemoheterotroph, getting its energy through the oxidation of carbon compounds. Bacillus anthracis is an infectious organism. It normally infects cattle, goats, and sheep. Occasionally it infects humans (only if they come into contact with infected meat). But according to Brazill, it can be weaponized. This bacteria secretes a substance called zinc metalloprotease to break peptide bonds and reduce proteins into small peptides. Then, it can ingest these peptides for energy and carbon. This is far more energy efficient than making its own amino acids. Zinc metalloprotease breaks down the collagen in the connective tissue, causing capillaries to weaken and burst, causing liquid to leak into the surrounding tissue, providing more food for the bacteria. Zinc metalloprotease also destroys red blood cells and macrophages in order to make more food and weaken the bodys immune response. Bacillus anthracis also secretes exotoxins: a) Lethal Factor (LF), directly kills cells. b) Edema Factor (EF) causes tissue swelling. c) Protective Antigen (PA) allows LF and EF to enter the cells. 3. Growth and Development Bacillus anthracis grows by elongation in preparation for cell division. They can form endospores, which as previously discussed are heat, dessicant, and UV protected, and metabolically inactive. Endospores are only formed outside the host. Endospores germinate when they are ingested, signaled by an increase in carbon dioxide levels. Carbon dioxide activates the transcription of genes required for emergence and infection. 4. Reproduction binary fission 5. Adapted to environment it is an infectious bacteria that requires a host in order to multiply. There are 3 modes of infection: a) Cutaneous anthrax infected through a cut or abrasion on the skin. Ulceration will be visible, ulcer caused by metalloproteases. b) Gastrointestinal anthrax infected through ingestion of tainted meat
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Germination occurs in a host. Before the bacteria exits the spore, it forms a protective capsule that helps to protect it from phagocytosis by macrophages. (As a result, it is normally treated by antibiotics, such as penicillin and its derivatives.) 6. Respond to environment Bacillus anthracis germinates in response to increased levels of carbon dioxide; therefore it must be able to sense the levels of carbon dioxide. There are two transcription factors that get activated by carbon dioxide: AcpA and AtxA control the transcription of toxins and the capsule. To summarize: Bacillus anthracis is a bacteria, gram-positive, rod-shaped, peritrichous, and with a capsule. It is a strict aerobe, a chemoheterotroph that feeds on mammals. It can develop into endospores, and reproduces through binary fission. It is adapted to be infectious, and can sense carbon dioxide levels. Next bacteria: Chromatium vinosum: 1. Organization it is an oval-shaped (ovoid) bacteria, with no nucleus, no organelles, and a cell wall. This cell wall is gram-negative, with only a thin peptidoglycan (PG) layer and a lipopolysaccharide (LPS) layer. It only has one flagellum, and is polar monotrichous. It has intracellular sulfur granules that store sulfur and are located in the periplasm. They have chromatophores, membrane-bound vesicles that contain photosynthetic proteins. 2. Metabolism it is a strict anaerobe. Oxygen is poisonous to it, and it cannot perform oxidative phosphorylation. It is a photoautotroph, receiving its energy from light, and its carbon from carbon dioxide. Chromatium vinosum performs anoxygenic photosynthesis, not producing oxygen. It captures light to convert carbon dioxide into sugars. This requires reducing power (usually in the form of NADH or NADPH) and energy (usually in the form of ATP). Chromatium vinosum uses sulfur. It uses light to oxidize H2S (instead of oxygens H2O, water) to sulfate SO42-, as reducing power to regenerate NAD(P)H from NAD(P)+. More on this process: to do this, Chromatium vinosum must be able to capture light. Bacteriochlorophylls and carotenoids absorb light energy, and are held in chromatophores. These molecules are ringlike, and the side groups on the rings determine the wavelength of the light that can be absorbed. Whereas for a normal plants chlorophyll, the best absorptions are for blue and red light, for bacteriochlorophyll the absorption peaks are in the infrared and ultraviolet spectra. Chlorophyll is organized into antenna systems. Around the reaction center of the photosynthesis complex, there is a circle of light harvest 1 complexes, and then a circle of light harvest 2 complexes. All of these molecules have chlorophyll in them, but photosynthesis only occurs in the reaction center. Light harvest complexes 1 and 2 funnel light into the reaction center. The antenna has larger area than the reaction center, thereby having a better chance of capturing a photon, and the larger the amount of food that it can thereby create. To convert carbon dioxide into sugar, ATP is required. ATP is made by the electron transport chain. When an electron is excited by light, it is transferred to the protein
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3. 4. 5.
6. 7.
quinol. Electrons are then transferred to cytochrome bc, then to cytochrome c, then back to the light reaction center, as protons are pumped against their gradient, a gradient which can then drive ATP synthesis. Since electrons do a complete cycle, this process is called cyclic photophosphorylation. To create reducing power, some electrons leave from quinols to NAD(P)+, creating NAD(P)H, requiring the energy that would otherwise be given to the proton gradient and oxidative phosphorylation. Those electrons are replenished from H2S, which donates its electrons to chlorophyll. Anoxygenic photosynthesis uses light to both create energy and create reducing power; energy in the form of ATP, and reducing power in the form of NAD(P)H from the conversion of H2S to sulfate. To make sugars from this place, the Calvin Cycle is used, with 3 parts: a) Carboxylation in carboxylation, a carbon dioxide molecule is attached to an organic molecule. This is done by adding the carbon dioxide molecule to an existing 5-carbon sugar, done by the enzyme rubulose bisphosphate carboxylase, or Rubisco. b) Reduction in the reduction step, the highly oxidized carbon dioxide in the new 6carbon sugar must be reduced to a usable form. This requires ATP and NADPH. c) Regeneration in this final step, the 5-carbon sugar is made again when the reduced carbon dioxide (in the form of a sugar called G3P) breaks off with two other such reduced carbon dioxides, regenerating the original 5-carbon sugar with the help of ATP. Growth and development there is no development in this bacteria. It grows through elongation, and splits. Reproduction binary fission. Adapted to the environment Chromatium vinosum dwells primarily in deep lakes and lake sediment, where there isnt much oxygen. But there is competition with other photosynthesizers for light. In order to get some amount of light, it absorbs only specific wavelengths of light. It absorbs the light that other photosynthesizers dont use. It absorbs UV light, which has more energy than other normal wavelengths of light, along with absorbing the infrared wavelengths that arent used by others. Responds to the environment instead of chemotaxis, Chromatium vinosum performs phototaxis (method unknown) to move toward light. Summary Chromatium vinosum is a gram-negative ovoid bacteria, with one polar flagella. It has sulfur granules to hold the sulfur that it uses in photosynthesis. It has chromatophores with photosynthetic proteins in it. It is a strict anaerobe, for which diatomic oxygen is poisonous; it does not perform oxygenic phosphorylation. It is a photoautotroph that performs anoxygenic sulfur-using photosynthesis. It uses the bacterial chlorophyll and carotenoids to harvest light (at less commonly used wavelengths) and convert it into ATP, using H2S to convert NADP+ into NADPH. Carbon dioxide, NADPH, and ATP are converted by the Calvin cycle into sugars.
Next bacteria: Myxococcus xanthus: 1. Organization this organism is a rod-shaped bacteria, with no nucleus, nor organelles, but having a cell wall. It is gram-negative, with a thin peptidoglycan layer but containing
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2.
3.
4. 5.
a lipopolysaccharide layer. It has no flagella, moving by gliding motility, the smooth but slow movement of a cell on a surface without the need for flagella. Myxococcus xanthus secretes a slime trail to ease its movement over a surface. It has two different types of motility: a) Adventurous single-cell movement, working best on firm surfaces. This method of motility depends on a protein called CglB, an outermembrane lipoprotein (in the LPS layer), and of course we dont know how it works. (He says that it is believed that cells release a surfactant that breaks the water tension behind it, allowing water to move it without any use of energy on the part of the cell.) b) Social cells moving in groups. In order to do this, the cells use a type 4 pilus, similar to the pilus used during conjugation. This pilus attaches to the surface, dragging the cell forward by pulling upon the surface. Metabolism Myxococcus xanthus is a strict aerobe, requiring oxygen. It performs glycolysis and oxidative phosphorylation, but cannot perform fermentation. It is a chemoheterotroph, taking carbon from organic molecules, and energy from the oxidation of these organic molecules. It is a predator that feeds on other bacteria or fungi, secreting digestive enzymes that lyse nearby organisms. It secretes antibiotics, using peptides and amino acids from its prey as a source of carbon, nitrogen, and energy. But as these enzymes cannot be in high concentration, the bacteria must hunt in packs, with large groups of cells all producing these enzymes. This is called swarming, and trail blazers lay down a slime trail, letting remaining cells follow this trail. Growth and development these cells grow by elongation in preparation for cell division. And they also develop into spores, but not endospores. They form a fruiting body, with spores in a fruit atop a stalk. This fruiting body formation can hold around 100,000 cells. This formation is triggered by starvation. When starvation is sensed, these cells form an aggregate and differentiate into spores. These spores can then germinated when food is more plentiful. These cells need to: a) Sense starvation it senses when there is no more food, likely sensed by a decrease in metabolic activity, such as monitoring the ATP/ADP ratio. b) Form aggregate the cells must move towards each other, using chemotaxis towards the phospholipid phosphatidylethanolamine, which is only secreted by starved cells. c) Differentiate into spores this differentiation starts when the mound is formed. Not all of these cells differentiate, with many cells sacrificing themselves in order to feed the rest of the remaining cells. New genes are transcribed in this differentiation into spores. These spores can survive for as much as 10 years, with low metabolism, resisting dessication, heat, and UV (due to the presence of carotenoids that absorb the UV damage instead of the cells). Thus, the spores can germinate at the same place and time, with a ready-made pack ready to hunt. d) Germinate when food returns. Reproduction binary fission. Adapted to environment Myxococcus xanthus are soil bacteria. The soil is rich with other bacteria and fungi upon which it can prey. The soil doesnt have much water for flagella to be of much use, so they developed other methods of motion.
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6. Respond to environment they go through development into spores when starved. Cells chemotax to P.E. (phosphatidylethanolamine) with the MCP receptor FrzCD, the sensor kinase FrzE, and the response regulators FrzZ and FrzS. 7. Summarize: Myxococcus xanthus is a soil bacteria. It is gram-negative, rod-shaped, with no flagella. It is a strict aerobe, a chemoheterotroph that performs glycolysis and oxidative phosphorylation, but not fermentation. It eats bacteria and fungi by swarming. October 6 Description: Brazill started the class by informing us about the first exam, which will be a week from Tuesday. It will cover up to the middle of this lecture, up to Hyphomicrobium facilis, a bacteria we will cover today. He has posted a previous exam on Blackboard, and he says that he will use questions verbatim from that exam on our exam. It was not an easy class for Brazill, he had to answer lots of questions, first about the exam then about other things. At one point, I was asking him a question that he didnt seem to know, kept stammering, then said any questions I do have the answer to? to general laughter. With ten minutes left, he commented that you guys are really working me today to more laughter. It was a decent class, at least we know that we wont be tested on what he doesnt know. Notes: New bacteria: Hyphomicrobium facilis: 1. Organization - Hyphomicrobium facilis is an ovoid bacteria, with no nucleus or organelles. It has a gram-negative cell wall, with a thin peptidoglycan (PG) layer and a lipopolysaccharide (LPS) layer. It only has flagella during part of its life cycle, and is polar monotrichous when it does have flagella. 2. Metabolism - Hyphomicrobium facilis is a strict aerobe that requires oxygen in order to live. It performs oxidative phosphorylation, and does not perform fermentation. Unlike most of the bacteria we have discussed thus far, Hyphomicrobium facilis does not perform glycolysis. It is a chemoheterotroph that gets its energy from oxidizing organic molecules, and gets its carbon from organic substances. It is a facultative methylotroph, meaning that it gets its carbon and energy from singlecarbon compounds, from things such as methanol, formate, and formaldehyde. In normal glycolysis, glucose is converted to two molecules of pyruvate, which is eventually converted to three molecules of carbon dioxide, during which energy in the form of ATP is produced (whether through substrate-level phosphorylation or through oxidative phosphorylation). In Hyphomicrobium facilis, methylotrophy occurs. Methanol is converted to formaldehyde, which is converted to formic acid, which is converted to carbon dioxide. In each step of this process, NAD+ is converted to NADH. 3. Growth and development Hyphomicrobium facilis grows in preparation for cell division. There is no development, and it grows through hyphal extension. 4. Reproduction - Hyphomicrobium facilis sends out a hypha. As the hypha extends, it replicates its DNA. Once the hypha reaches an appropriate size, it becomes a bud. The DNA moves to the new bud, a septum forms, and the bud buds off of the end of the hypha. The bud will have a flagella, that will eventually fall off. Thus, offspring can move away and colonize new areas.
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5. Adapted to environment - Hyphomicrobium facilis is found in fresh and salt water, especially in areas with poor nutrition. It uses single-carbon compounds that other organisms cannot use. Thus, they dont much need to compete with other organisms. 6. Responds to environment buds can perform chemotaxis with their flagella, moving toward such molecules as methylamine, dimethylamine, and trimethylamine. 7. Summary: Hyphomicrobium facilis is a gram-negative, ovoid bacteria with polar monotrichus flagellum on the young buds. It is a strict aerobe, a chemoheterotroph. It is a facultative methylotroph that performs oxidative phosphorylation, and no fermentation. It can use glucose (and break it down) if necessary, but it prefers singlecarbon compounds. It grows through hyphal extension, reproducing through hyphal budding, and is adapted to nutrient-poor conditions by taking single-carbon molecules that are not used by other organisms. With this, we have finished the material that we will cover on the first exam. From here on, we are covering material for the second exam: Archaea is the next domain of organisms. They are prokaryotic, and are extremophiles. They are found in extreme environments. For example, they are found in areas with high levels of salts and dissolved minerals, high temperature, very acidic or basic conditions, and high pressures. The first archaea we will study is Thermoplasm volcanium: 1. Organization - Thermoplasm volcanium is a prokaryotic, spherical archaea. As such, it has no nucleus or organelles. It has flagella and is peritrichous. But it has no cell wall, a cell wall that would normally prevent a cell from lysing. It lives in temperatures of as much as 58 degrees Celsius, and lives in a low-pH acidic condition. It requires acidic condition in order to live. It is an obligate acidophile it lyses if the pH is above 4. (And here we go on a tangent about temperature categories: a) Psychrophiles live and grow in temperatures below 15 degrees Celsius. b) Mesophiles live and grow in temperatures between 15 and 45 degrees Celsius. c) Thermophiles live and grow in temperatures between 45 and 80 degrees Celsius. d) Hyperthermophiles live and grow in temperatures above 80 degrees Celsius. The optimal temperature is one where enzymatic reactions are maximized. At the minimum temperature, the membrane forms a gel, and there is no membrane transport. Past the maximum temperature, the proteins denature and the membrane ruptures.) 2. Metabolism - Thermoplasm volcanium is a facultative anaerobe. It can grow with or without oxygen. But it does not perform fermentation, using sulfur respiration to produce electrons for the electron transfer chain and produce a proton gradient, entirely bypassing the need for fermentation. It turns H2S to sulfuric acid in sulfur respiration, but Brazill cant answer and cant remember how exactly this occurs. Thermoplasm volcanium is a chemoheterotroph, a chemoorganotroph, receiving its energy from oxidizing organic compounds, and taking its carbon from organic molecules.
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3. Growth and development grows by increasing diameter in preparation for cell division, and doesnt develop. 4. Reproduction binary fission. 5. Adapted to its environment it grows in solfatara fields, extinct volcanoes. These areas have lots of sulfur, high temperatures, and high acidity. It uses sulfur respiration on the sulfur in the area. In order to cope with the high temperatures, it must prevent several problems: a) Membrane denaturing the membrane could denature and rupture. Instead of having a lipid bilayer, Thermoplasm volcanium has a lipid monolayer. In a normal lipid bilayer, the phospholipids can easily slide past each other; this would allow the bilayer to easily be disrupted by temperature. Thermoplasm volcanium has a monolayer, which is difficult to disrupt by temperature. Instead of having two layers that slide past each other, in a monolayer the phospholipids are attached to the ones on the other side of the layer. They are tetraethers, not normal phospholipids essentially phospholipids with tails bound to each other, but instead of ester linkages, they only have simple ether linkages. b) DNA unwinding and denaturing in order to prevent this, the DNA is tightly wrapped around histone proteins. The DNA is negatively charged due to the phosphate backbone, using positively charged proteins to wind the DNA. Thermoplasm volcanium must also cope with high acidity. In order to counteract this, the membranes have negatively-charged glycoproteins on its surface. These glycoproteins attract and bind the protons, leaving an area of relative neutrality just outside of the membrane. But in a non-acidic environment, these negatively-charged glycoproteins wont have protons to attract, and will instead repel each other. Thus, Thermoplasm volcanium is an obligate acidophile. One problem of high-acidity environments that Thermoplasm volcanium must deal with is the problem of protons potentially leaking into the cell, because the cytoplasm has an approximately neutral pH. To remove these protons, it uses a proton-potassium antiporter, and it pumps out potassium ions to create the gradient through a different method. 6. Responds to its environment it has flagella, so presumably it can chemotax with the flagella. Chemotaxis can be tested by placing a capillary with a chemical into a culture of the bacteria; if the chemical is a chemoattractant, lots of cells will move into the capillary. If the chemical is not a chemoattractant, few cells will move into the capillary (no more than in a control), and if the chemical is a repellant, no cells will move into the capillary. But we dont know what acts as attractants and repellants for Thermoplasm volcanium. 7. Summary - Thermoplasm volcanium is a prokaryotic archaea. It is spherical, with peritrichous flagella, but no cell wall. It is a facultative anaerobe, a chemoheterotroph that performs sulfur respiration. It grows with increasing diameter, no development, reproduction through binary fission. It is adapted to high temperatures, large amounts of sulfur, and is an obligate acidophile. It probably chemotaxes.
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October 11 Description: The notes for today actually start above, with the proton-potassium pump in the acidophile archaea, and they continue below. And Brazill spent the first 10 minutes doing what we had already done yesterday, so it didnt matter that I was slightly late. Notes: Another archaea, Halobacterium salinarum: a salt-loving, halophilic extremophile organism. 1. Organization - Halobacterium salinarum is a prokaryotic, rod-shaped archaea. It doesnt have a nucleus, nor does it have organelles. It has a cell wall that is gram-negative, but its gram-negativity does not tell us anything about its structure. But we know that its cell wall is made of glycoprotein. It has flagella and is lophotrichous, with multiple flagella on one side only. 2. Metabolism - Halobacterium salinarum is a chemoheterotroph that gets its carbon from organic molecules and its energy from the oxidation of organic molecules. A strict aerobe, Halobacterium salinarum requires oxygen, and performs glycolysis and oxidative phosphorylation. It doesnt perform fermentation, but unlike most aerobes, it can live for short periods of time without oxygen. Halobacterium salinarum performs light-mediated ATP synthesis. This works due to a membrane protein called bacteriorhodopsin, which contains retinol. Retinol can bind protons from the cytoplasm. When light hits the retinol, the retinol changes position from the inner flap to the outer flap of the membrane, due to a conformational change from trans to cis conformations of retinol. Retinol does not hold protons particularly well, so it drops this proton outside the plasma membrane. These protons can then run an ATPase. 3. Growth and development - Halobacterium salinarum grows by elongation in preparation for cell division. It doesnt develop. 4. Reproduction reproduces by binary fission 5. Adapted to its environment - Halobacterium salinarum lives in very salty conditions. It exists in places such as salt evaporation ponds, salt lakes, marine salterns, and on salted foods such as sausages, fish, and salted pork. It can withstand high concentrations of various ions. It actually requires these high concentrations in order to live; it must have between a 1.5 and 5.5 M concentration of sodium. Why? Halobacterium salinarum has negatively charged glycoproteins, similar to Thermoplasm volcanium. These glycoproteins bind the sodium ions. But in the absence of sodium ions, the glycoproteins would tear the cell apart. Thus, Halobacterium salinarum is an obligate halophile. But how to deal with sodium ions that enter the cell? Potassium ions help to balance the osmotic pressure difference caused by keeping sodium ions out. But then how can the cell deal with the potassium ions in the cell? Halobacterium salinarum deals with the high potassium concentration in the cytoplasm by having acidic proteins inside the cell that counteract the charge. There are very few hydrophobic amino acids in the proteins, preventing protein clumping. To maintain salt balance, a couple transmembrane transporters are used: The bacteriorhodopsin operates a proton pump of sorts, and these protons are used in a proton/sodium antiporter to remove sodium ions from the
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cell. This is why Halobacterium salinarum cannot live without oxygen for very long because although it can use its proton gradient for ATP synthesis, it needs the gradient for sodium homeostasis. So it can only use the proton gradient for ATP synthesis for short periods of time. The rest of the time, it uses oxygen in oxidative phosphorylation. The other transporter in the plasma membrane is the sodium/potassium symporter, which uses the sodium concentration gradient to bring potassium ions into the cell. Halorhodopsin is another transporter. It also has retinol, and uses light the same way as bacteriorhodopsin does, but instead of transporting protons across the membrane out of the cell, it shuttles chloride ions into the cell, with the retinol going from trans, getting light-activated to cis to bring the ion across the protein, then drops the ion and reverts to its trans form on the starting side of the halorhodopsin. Chloride acts as a counter ion for potassium, and this protein allows the cell to regulate the amount of this chloride counter ion that is in the cell. 6. Responds to environment phototaxis. Halobacterium salinarum moves toward light required for its bacteriorhodopsin (green light) while avoiding harmful light (UV) in order to avoid DNA damage. It has two photoreceptors: a) Htr1 attracts to red light and repels from UV light b) Htr2 repels from blue light. It has a two-component response system, a sensor kinase CheA and a response regulator CheY. The repel causes a tumble flagella response, the attract causes run. 7. Summary - Halobacterium salinarum is an archaea, prokaryotic, with a glycoprotein cell wall. It is lophotrichous and rod-shaped. It is a strict aerobe chemoheterotroph, a strict aerobe that performs glycolysis and oxidative phosphorylation. In low oxygen, it performs light-mediated ATP synthesis with bacteriorhodopsin. It grows by elongation, no development. It reproduces through binary fission, adapted to hypersaline, liquid environment with negatively charged cell wall proteins and multiple ion transport systems. It performs phototaxis. October 13 Description: This was a Jewish holiday, so I was unable to take my own class notes. Luckily for me, my notes list became popular early this year, and I was in contact with enough people that I actually got more than one version of the notes, along with what I remember from class (which is a considerable amount this time). I attempted to slow Brazill down by asking enough questions that he wouldnt be able to teach quite as much. But then he ended class early for the first time all semester! So a win for everyone Notes: New archaea: Methanopyrus kindlari: 1. Organization - Methanopyrus kindlari is a prokaryotic, rod-shaped archaea. It doesnt have a nucleus, nor does it have organelles. It has a cell wall that is gram-negative, but its gram-negativity does not tell us anything about its structure. But we know that its cell wall is made of glycoprotein. It has no flagella and is not motile, living near steam vents in the bottom of the ocean.
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2. Metabolism - Methanopyrus kindlari is a strict anaerobe. Oxygen is deadly to it. Instead of oxygen, it performs a process known as methanogenesis. Methanopyrus is a methanogen, meaning that it can make methane simply from carbon dioxide and hydrogen. Methanogenesis begins with reducing carbon dioxide to a usable level. Two basic types of molecules are involved in this process: a) C1 binders the molecules that bind to the carbon molecule, called thus because they bind to the CO2. b) C1 reducers the molecules that actually reduce the molecule. This process begins with carbon dioxide, O=C=O, and the reducers gradually reduce the various C1-bound CO2molecule through a series of chemical forms, formyl, methylene, and methyl, until it is finally released as methane. The binders act as cofactors because they provide a scaffold for the reducers to produce their function. And it seems that for this day someone actually took pictures of Brazills projector drawings!
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Carbon is reduced to a lower form. Methanofuran (MF) binds to the carbon dioxide molecule. In addition, hydrogen is used to help reduce the carbon dioxide, producing water. This takes carbon dioxide to the formyl form. Next, the formyl group is handed off to another C1 binder called methanopterin (MP). All thats occurred is that C1 binders have exchanged places. The next step is another reduction step in which formyl is converted to a methylene, still bound to methanopterin (MP). This time the reduction is done by coenzyme F420, and hydrogen is again used to help with the reduction. While still bound to the methanopterin, the carbon molecule is again reduced, again using F420, so that it is now at the methyl stage. There is another switch in the C1 binders. Coenzyme M (CM) takes the place of methanopterin. Finally, hydrogen, along with another enzyme, releases the methyl group creating methane along with the coenzyme M and HTP. These are a series of steps with reduction occurring, and a series of steps involving the rotation of C1 binders. In this process carbon is reduced to a usable form, methane, but protons are also pumped out of the cell via the electron transport chain, creating a proton gradient used to synthesize ATP. ATP is produced nearly the same way in almost all organisms. The way these protons are pumped out varies from organism to organism, but generally its about setting up a proton barrier. Methane is used to create the organic molecules that the cell requires. The methane is converted into acetyl CoA, which is used for biosynthesis. Methanopyrus is the primary producer for the food web in which it is found. They are the carbon fixers for this ecosystem. 3. Growth and Development growth in size only, no development. 4. Reproduction incomplete binary fission. This means that the archaea of Methanopyrus kindlari do not actually exist in the environment as lone cells. They exist as filaments of multiple cells with connected cytoplasms. 5. Adapted to its environment - Methanopyrus kindlari is a strict anaerobe, since no oxygen is present. Oxygen on the surface cant diffuse all the way to the bottom of the ocean. Since there are no photosynthesizers, this microorganism is the main carbon fixer for this environment. It must deal with high temperatures and needs to be able to deal with the stresses that high temperatures create. It needs to keep its proteins from denaturing, losing their shape and not being able to function properly. There are a number of different methods that it can do to prevent this: a) It can raise the number of hydrophobic molecules in the core of the protein. Hydrophobic molecules tend to band together because they dont want to interact with the surrounding water. This means that they aggregate in the center of the protein, making it less likely that the protein is going to unfold due to high temperatures. b) In addition to this, this microorganism increases the number of salt bridges that the proteins have. This increases the amount of electrostatic interactions, in which positive and negative interactions help maintain rigidity. In addition to changing the composition of the proteins, Methanopyrus kindlari also has increased amounts of proteins called chaperonins. This is a special class of proteins whose job it is to refold unfolded proteins. All organisms have chaperonins, however Methanopyrus
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kindlari possesses many more chaperonins than most other organisms do. These chaperonins are used to refold those proteins which have become unfolded. There is also the problem of DNA being degraded under high temperatures. An enzyme called reverse gyrase does this. The function of reverse gyrase is to super-coil the DNA. This enzyme twists the DNA even more tightly than it already is coiled, with the idea being that the more tightly the DNA is wound, the harder it is to denature. In addition to supercoiling the DNA, it also has a chemical called K+ cyclic 2, 3diphosphoglycerate. This protects the DNA from being depurinated (bases spontaneously falling off of the DNA). Methanopyrus kindlari also has histone-like proteins which help compact the DNA, making the harder to denature. You also have to keep the membrane intact in high temperatures. Like other archaea, this organism has tetraethers (a lipid monolayer) along with a special unsaturated lipid called geranylgeraniol which helps makes the cell membrane a lot more rigid. Brazills note: (Methanopyrus can deal with very high temperatures, but is there a limit to a temperature at which life must cease? No one can say for sure, but the idea is that life cannot exist above 150oC, because ATP and NAD+ cannot survive at these temperatures. If life were to exist above 150C it would have to use something other than ATP and NAD+, having a different metabolism.) 6. Response to its environment Methanopyrus kindlari does not move. Its only response to the environment is a variety of heat-stress coping mechanisms, and we dont know all of them. 7. Summary - Methanopyrus kindlari is a rod-shaped, prokaryotic, non-motile archaea, with a protein coat for its cell wall. It is a strict anaerobe, a methanogenic chemoautotroph, growing by elongation without development and reproducing through incomplete binary fission. October 20 Description: Thursday was also a Jewish holiday, so although I was quite surprised to find pre-exam that my notes are now being used as the class textbook, I could not take notes on this day. And as of Tuesday, I still havent received notes from anyone else. But thank you to Aleks for supplying the notes for this day. Notes: Organism: Plasmodium falciparum is the organism that causes Malaria. Malaria was officially diagnosed as a disease at around 500 B.C., but it wasnt until 1880 that Plasmodium was discovered to be the cause of the illness. This disease-causing organism creates a major health problem because it is localized to lowland swamps. According to Brazill, about 40% of the worlds population lives in areas near lowland swamps, making malaria a huge problem. There are about 300-500 million cases each year, 1 3 million of which end up being fatal An attempt to eradicate malaria (much like polio) once took place, but was eventually abandoned in 1931(?) because the organism developed drug-resistance. 1. Organization Plasmodium is a Eukaryote, which means that it has a nucleus. Its genome can either be haploid or diploid, depending on what stage of the life cycle it is at. It has 14 chromosomes, approximately 24,000 kb (base pairs) and 5300 genes.
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Plasmodium also has all the regular organelles as well as a specialized organelle called the apicoplast, whose job is to perform fatty acid metabolism. The apicoplast seems to have evolved from a chloroplast. All Eukaryotic microorganisms are grouped in 4 basic groups: a) Protozoan Plasmodium is classified as a protozoan. b) Algae c) Fungi d) Slime molds These groups are based on certain characteristics. Any microorganism that possesses chlorophylls is classified as algae. Fungi have cell walls but no chlorophyll. Slime molds form fruiting bodies and protozoa encompass everything else. If an organism doesnt have chlorophylls, fruiting bodies, or cell walls, it is classified with protozoa. 2. Metabolism Plasmodium is a strict aerobe, meaning that it requires oxygen. It performs glycolysis and oxidative phosphorylation. It is a chemoheterotroph, meaning that it gets its energy from oxidizing organic molecules and its carbon from organic molecules. 3. Growth and development Interestingly, these cells only grow during certain parts of their life cycle and possess a very complex developmental cycle. Plasmodium spends part of its life inside a human being and part of its life inside a mosquito. This microorganism only infects a certain genus of mosquito called Anopheles. Because we dont have this particular genus of mosquito in NYC, we also dont have malaria. Plasmodiums life cycle begins when a mosquito bites a human. During the bite, the mosquito allows sporozoites into the blood stream. The sporozoite is just one of the forms in which Plasmodium can exist. A mosquito needs to inject an anti-coagulant into the bloodstream to prevent clotting during its feeding time, and Plasmodium is injected along with the anti-coagulate. The microorganism then travels into the human liver cells. At this point Plasmodium is haploid. Once in the liver, the haploid cells begin to reproduce and as they do this, they begin to undergo a form of development in which they differentiate: they are no longer sporozoites but become merozoites. After they mature into merozoites, they then leave the liver cells and again enter into the bloodstream. At this point they are now ready to infect red-blood cells, and because of this process, this stage of their life cycle is called the erythrocyte stage. (An erythrocyte is a red-blood cell.) The merozoites find and infect red-blood cells because the latter contain hemoglobin, which merozoites feed on. The way they actually infect the red-blood cell is by binding to the cell using an apical complex, which releases proteases allowing a plasmodial cell to enter the red blood cell. Once it is able to enter into the red blood cell, it is able to fully integrate itself into the cell. All of this is initiated by the apical complex, which is a series of proteins inside the plasmodial cell. Once inside the red-blood cell, the merozoites are able to reproduce. They continuously multiply until they reach at point at which they lyse the red blood cell, releasing huge numbers of merozoites into the bloodstream. The newly released merozoites go on to infect other red-blood cells. Then entire process from infection to release takes about 42 78 hours. This happens to be the period in which victims of
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malaria experience a cycle of chills and fevers associated with the onset of the disease. It is the release of the cell debris and toxins that cause the chills and fevers. Besides the chills and fevers long term damage involves anemia, as well as spleen and liver damage. Some merozoites undergo mitosis to become what are called pregametes (or gamonts). At this point they are now haploids. In the pregamete stage, if they are picked up by a mosquito they can now develop into a full-fledged gamete. This transformation from pregamete to gamete can only occur inside a mosquito. The gametes fuse to create a zygote which moves to the midgut of the mosquito to complete its development. The complete development of the zygote is the production of sporozoites. The zygote enlarges, divides, and differentiates into sporozoites. The sporozoites leave the midgut and travel to the salivary glands from which they can be transferred into a new host, starting the infectious cycle all over again. 4. Reproduction Plasmodium has both sexual and asexual reproduction. They exhibit asexual reproduction when the merozoites reproduce in the liver, merozoite reproduction in the red-blood cell, as well as the sporozoite reproduction in the midgut. Sexual reproduction occurs when the gametes fuse to form the zygote, where two haploids create a diploid. What are the advantages and disadvantages of these two processes? One advantage of asexual reproduction is that its quick. There is no search for a mate. The other advantage is the conservation of your genome. By conserving your genome you preserve those qualities that allow you to survive in the environment in which youre already comfortable. This is most effective in an environment that is not changing (e.g. the merozoites in the liver cells) In contrast, sexual reproduction provides variability. The totally different genome produced is very beneficial if going into an environment that is going to be changing (e.g. the sporozoites in the midgut.) The idea is that you dont know where youll end up and you want to increase the chance of your offspring surviving, so you shuffle up your genome, hopefully producing some cells that are able to survive. 5. Adapted to its environment - Plasmodium has a very complex life cycle to match the fact that it exists in both mosquitoes and humans. In addition, Plasmodium is able to develop drug immunity. For the longest time the drugs used to fight off malaria were Quinone derivatives. Quinine was one of the original drugs used against malaria. Originally tonic water, which has quinine in it, was given as a precaution against malaria. British soldiers in India would drink tonic water with gin because the former was so bitter, and thats where gin and tonic comes from. The quinone derivative enters the endosome of plasmodium and disrupts its heme digestion. The partially digested heme is toxic and kills the plasmodium. The problem is that plasmodium develops immunity by developing pumps that removes quinine from the endosome. It is a constant battle to find new quinine derivatives that can be used against malaria. In addition to being able to develop drug immunity, Plasmodium is also able to avoid the immune system. It does this by changing its coat. When plasmodium infects a red-blood cell, it causes the red blood cell to express a protein on its cell surface. This protein is called PfEMP1. This protein causes the red-blood cell to get stuck in the capillaries. The idea is that if the infection-carrying red blood cells will amass in one area, thereby
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hiding from the immune system. Eventually, of course, the infection is recognized and an immune response is mounted. Therefore every generation of plasmodium has a small percentage of offspring which change their PfEMP1, the idea being that while 99.9% of the plasmodia are attacked by the immune system, .1% isnt, and this percentage ends up repopulating the host. This is a major problem because there are around 60 genes that code for different version for PfEMP1. Gaining immunity to all sixty genes is practically impossible. Humans have also developed some adaptations for dealing with Plasmodium. One of these is sickle cell anemia. Sickle cell anemia is caused by the change on one amino acid in hemoglobin a change from a glutamic acid amino acid (hydrophilic) to a valine (non-polar hydrophobic) amino acid. The end result is the hemoglobin stops carrying around as much oxygen as before. The levels of 02 are therefore lower than normal. These slightly decreased levels of oxygen mean that the plasmodium can no longer grow as well. People who have sickle-cell anemia tend to be more resistant to malaria infections. In general though, this is not a good thing. Heterozygotes do fine at sea-level but begin to have problems at higher altitudes (because oxygen levels at high altitudes are lower). In general however, people who are heterozygotes and carry the gene stay around low altitude. The problems begin with the homozygotes in the population who dont do as well. Without treatment, they dont live past their 20s. Another adaptation that humans have developed is 6-P(phosphate) dehydrogenase deficiency. This enzyme is involved in glucose metabolism. The heterozygotes build up oxidants in the red blood cells, which end up destroying the plasmodial membrane. This ends up killing the plasmodium. 6. Responds to its environment - Plasmodium falciparum can chemotax at various points of its life cycle. It chemotaxes to the midgut of the mosquito, to the salivary glands of the mosquito, to the human liver, and to the human red blood cells. 7. Summary Plasmodium falciparum is a eukaryote, a protist. It is a parasite that lives and reproduces in its host. Part of its life cycle is spent in the human, and part of its life cycle is spent in the mosquito. It has development between sporozoite and merozoite, and goes through both haploid and diploid types. October 25 Description: Today was a bit of a lighter day. Brazill summarized the information on Plasmodium, and then we started on the next microorganism, a yeast. And we all laughed at the terminology of the yeast, at the fact that yeast cells schmoo toward each other. They stretch toward each other. But of course its a weird word, that caused several laughs when the word was used in various ways in various sentences. October 25 Notes: Todays microorganism: Saccharomyces cerevisiae: this is a yeast that was recorded as the first microorganism to ever be cultivated by humans. It has many uses, including brewing and baking, as a source of B vitamins and vitamin D, as well as being a protein supplement. They are also used in the industrial production of recombinant proteins, as well as in biological and biomedical research.
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1. Organization eukaryotic and ovoid, with a nucleus. It has haploid and diploid parts of its life cycle, and has 16 chromosomes. Although it doesnt have the specialized organelles of Plasmodium, Saccharomyces cerevisiae has organelles. It has a cell wall, but no chlorophyll, and is a fungus. Its cell wall is made of polysaccharides and some proteins, and it resists osmotic stress. Saccharomyces cerevisiae has no flagella, and is non-motile. 2. Metabolism - Saccharomyces cerevisiae is a chemoheterotroph. It gets its carbon from organic molecules, and gets its energy from oxidizing organic molecules. It is a facultative anaerobe that performs glycolysis and oxidative phosphorylation, while also being capable of performing fermentation. The carbon dioxide molecules produced in oxidative phosphorylation cause bread to rise. 3. Growth and development - Saccharomyces cerevisiae grows by increased diameter. It develops, too, with development triggered by starvation. It develops into filaments that become invasive, being able to penetrate the interior of the fruit upon which it lives. Filaments are created by incomplete budding. 4. Reproduction - Saccharomyces cerevisiae divides by simple budding. It grows, a bud forms, the bud increases in size, until it is able to fully bud off of the mother, leaving two cells, the mother cell and the daughter cell. It can also replicate through mitosis. This is the haploid part of its life cycle. But Saccharomyces cerevisiae can also reproduce through mating. Haploids can mate to form a diploid. There are two mating types: a and alpha. The a type will mate with the alpha type. Mating type is determined by the genes in the mating type locus. Normally, in the alpha type, the alpha genes are just ahead of the promoter, and produces its alpha genes. And this mutes the a genes farther down the DNA, not producing the a genes and only producing the alpha genes. In the a type, the alpha part just ahead of the promoter is removed by the HO endonuclease enzyme, and the a part gets copied. The copied a part is then put in the place of the alpha part in front of the promoter These parts switch on occasion, so cells can switch their mating type. To return to the alpha type, the alpha part is copied from its place on the DNA, and is put in its place ahead of the promoter in place of the a part. Only a mother cell can switch mating types. After a cell buds off of the mother cell, with the daughter cell keeping the mothers mating type, the mother switches its mating type before it buds again. This mating type switching occurs in order to keep an equal number of a cells and alpha cells. How this mating type switching works: the a part codes for the protein a1, while the alpha part codes for the proteins alpha1 and alpha2. These proteins have very specific jobs. The alpha1 protein is a repressor of a-specific genes. It represses these genes that are only active in a cells, prevents them from being transcribed. The a1 protein has no job in the haploid, nor does the alpha2 protein. But in the diploid, these two proteins come together to form a complex between the a1 and alpha2 proteins that represses alpha-specific genes while activating diploid-specific genes. The alpha and a diploid cells move toward each other, in a schmoo. They schmoo toward each other. The cell walls touch and fuse, and then the nuclei fuse, making the combined cell a diploid. The diploid can do one of two things: it can bud like a haploid, making more diploid cells through simple mitosis. Or starvation can trigger to cell to undergo meiosis, splitting into
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haploid cells after exchanging genes in chiasma. Four spores are formed in an ascus, two a and two alpha cells. These spores are dessication, heat, and UV resistant. In order to mate, the two cells must be able to know that a partner is near, be able to contact the partner, fuse the cell wall, fuse nuclei, and this is done through the use of secreted pheromones. A and alpha cells produce mating pheromones. A cells make afactor, an a-specific gene. Alpha cells make alpha-factor, an alpha-specific gene. Cells know that their partner is near when they can sense the opposite pheromone. So a cells sense the alpha-factor with their alpha-factor receptors, and alpha cells sense the afactor with their a-factor receptors. The a-factor or alpha-factor receptors, Ste3 is the alpha-factor receptor and Ste2 is the a-factor receptor, activates a G protein that amplifies the signal through protein kinases to the protein Ste12. When the Ste12 protein is phosphorylated, activated, it is a transcription factor, causing schmooing, cell wall fusion, and nuclear fusion. 5. Adapted to its environment - Saccharomyces cerevisiae lives on the surface of fruit. So it has fairly easy access to food, and there is no need for motility. The main stress in its life is osmolarity problems due to excess rain or lack of rain. It deals with this osmolarity with its cell wall. The other major stress is starvation, and it deals with that by sexually reproducing into spores or forming filaments to invade the interior of the fruit. 6. Responds to its environment - Saccharomyces cerevisiae can convert into a filament in response to poor nutrition. It can sporylate in response to poor nutrition or in response to dessication. And they can schmoo in response to pheromones. 7. Saccharomyces cerevisiae is a eukaryotic, ovoid fungus. It exists in both the haploid and diploid states, but does not have flagella. It is a chemoheterotroph, a facultative anaerobe that can perform glycolysis, oxidative phosphorylation, and fermentation. It grows by increasing in diameter, and can develop into filaments. It can reproduce both sexually and asexually, with the diploid form allowing for adaptation to life on the surface of the fruit by forming spores. It can respond to its environment by either undergoing development, by mating, or by sporylating. October 27 Description: Today we covered another organism, a slime mold. During the course of the lecture, Brazill twice showed us videos of this slime mold, first expelling water and then phagocytosing yeast. The class laughed at one of the names in the series of phases of the fruiting body formation, the Mexican hat phase. And then he showed us another video, this one of an aggregate being formed around the presence of cAMP. And another video of cells chasing a needle of cAMP. And then a couple more videos. In a laugh-filled light class, we finished by laughing about the regulator called CRAC that stands for Cytosolic Regulator of Adenylyl Cyclase. Way to ruin the fun Brazill. Notes: New microorganism: the slime mold Dictyostelium discoideum: 1. Organization Dictyostelium discoideum is a eukaryotic microorganism, with haploid and polyploidy phases. It has no structure, and is amoeboid, constantly changing its structure. 6 chromosomes of DNA. It has a contractile vacuole in order to cope with
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osmotic stress. Its vacuole fills with water, then fuses with the plasma membrane and releases the water to the environment. Slime molds form fruiting bodies. They can be cellular or acellular. The cellular slime molds can be made up of thousands of cells that come together. The acellular slime molds are made up of a single cell with thousands of nuclei. It has no cell wall for most of its life cycle, and has no flagella. It moves by amoeboid movement, cell crawling by moving part of itself forward and retracting its back end. It extends a pseudopod that reaches forward and creates a connection with the surface. And then it moves itself into the pseudopod, squeezing its cytoplasm into it, causing the cell to move forward. 2. Metabolism - Dictyostelium discoideum is a strict aerobe. It requires oxygen, performs glycolysis and oxidative phosphorylation, but cannot perform fermentation. It is a chemoheterotroph that receives its carbon from organic molecules and its energy from oxidizing organic molecules. It is a predator that feeds upon other microbes. Dictyostelium discoideum is a soil amoeba, and there are plenty of bacteria and fungi that it can eat in the soil, through a process called phagocytosis. In phagocytosis, the cell extends itself on two sides of its prey. It envelops its prey in a vacuole, that fuses with a lysosome and becomes an endosome for digestion. 3. Growth and development - Dictyostelium discoideum grows by enlargement, and can develop into a fruiting body. The process of fruiting body formation is triggered by starvation, in order to make and disperse spores. Steps of fruiting body formation: a) Individual cells eating bacteria, until the supply of bacteria starts to dwindle. Starvation signals the formation of the fruiting body. b) Aggregate the cells come together. They must cross large distances to come together. A chemoattractant is used, cAMP, cyclic AMP. When cells begin to starve, they secrete cAMP, calling the other starving cells toward it. c) Mound the aggregating cells tighten toward each other to become a mound. d) Differentiating mound the cells begin to differentiate into spores in the mound. e) Tipped mound f) If not on surface, the tipped mound falls on its side and becomes a slug, which phototaxes to reach the surface. In this phase, the cells are partially diferentiated, and will have a preference for becoming stalk cells or spore cells, being called prestalk and prespore cells, respectively. They can still become a cell of the opposite type, as they are still differentiating, but they have a preference for one or the other. g) Mexican hat on the surface, the tip extends until it looks rather a bit like a Mexican hat and Brazill is careful to note that he did not invent that term. h) Early culminant the fruiting body shape begins to form. i) Late culminant the cells are terminally differentiated into spore cells and stalk cells. j) Fruiting body two types of cells. Stalk cells, around 20% of the cells, sacrifice themselves for the good of the remaining cells. The other 80%, the spore cells, are lifted off the ground by the stalk cells for better spore dispersal. k) Spores the spore cells decrease their metabolism, and make their spore coat. They become resistant to dessication, heat, and ultraviolet light.
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l) Stalk cells the stalk cells vacuolate, with their contractile vacuole becoming very large. They make a cellulose cell wall, and are very rigid, allowing the stalk to be strong enough to support the spore mass. Cell response to cAMP: a) Sense cAMP cAMP cannot diffuse very well. But it can diffuse a short distance. And another cell closer to the cAMP will receive it, and will start creating its own cAMP. The first cell to make cAMP is called the pacemaker. b) Move toward cAMP c) Release cAMP This entire process of fruiting body formation takes approximately 24 hours from starvation to fruiting body formation. 4. Reproduction two types of reproduction occur. Asexual reproduction is the normal mode of reproduction, taking 4-12 hours in binary fission, haploids producing haploids. Sexual reproduction is triggered by starvation. There are two types, matA and mata. The matA and mata can fuse to become a diploid cytophagic cell that secretes cAMP to attract other cells (endocytes), which it engulfs and eats. But the endocyte nuclei are not destroyed, becoming a large polyploidy cell. The cytophagic cell becomes a macrocyst once all the endocytes have been engulfed. It undergoes meiosis and releases haploids when the macrocyst spore germinates. 5. Adapted to environment - Dictyostelium discoideum is a soil amoeba that hunts other soil microbes. It can deal with a lack of food, with spore formation and macrocyst formation. And it has a contractile vacuole for dealing with osmotic stress. 6. Reponds to environment - Dictyostelium discoideum chemotaxes to cAMP. It can sense cAMP, move to cAMP, and make cAMP. It has a receptor, cAR1, cyclic AMP receptor 1, which is connected to a G-protein. G proteins have 3 subunits, the alpha subunit, the beta subunit, and the gamma subunit. When the cAR1 receptor activates the G protein, it splits into its alpha2 subunit and its beta/gamma combined subunit. The alpha2 subunit activates guanylyl cyclase, which turns GTP into cGMP, which causes cell movement. The beta/gamma subunit needs help activating adenylyl cyclase, help from Cytosilic Regulator of Adenylyl Cyclase, or CRAC (yes, very funny.) Adenylyl cyclase converts ATP into cAMP. Dictyostelium discoideum also performs folate chemotaxis. This is used to hunt bacteria, as bacteria release folate as a byproduct of its metabolism. The folate receptor causes a different G-protein to separate into its alpha4 and beta/gamma subunits, with the alpha4 activating guanylyl cyclase leading to movement (beta/gamma subunit does nothing).
Notes: New organism: Chlamydomonas reinhardtyl: 1. Organization it is an ovoid eukaryote, with a haploid/diploid nucleus. It has 17 chromosomes, and 15,000 genes. It has special organelles, chloroplasts, the sites of
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photosynthesis. It has thylakoid stacks, with an outer membrane and an inner membrane, with the thylakoid space inside that acts as a storage place for protons. Chlamydemonas reinhardtyl has a contractile vacuole, in order to deal with osmotic balance. It also has a pyrenoid, an organelle that is not membrane-bound, in a localized part of the cell that converts bicarbonate to carbon dioxide in order to be used in photosynthesis. Chlamydemonas reinhardtyl also has stigma, eye spots that act as photo-receptors for phototaxis. Chlamydemonas reinhardtyl has a cell wall, made of cellulose and polysaccharides, along with glycoproteins. It has two flagella in the anterior part of the cell, different from bacterial flagella. It is a type of algae, with chloroplasts, and is a chlorophyte. It has two different chlorophytes, A and B, and absorbs different wavelengths of light. Green algae is the phylum Chlorophyta. (Four different phyla of algae, dinoflagellates with chlorophyll A and C and xanthrophytes, brown algae with chlorophyll A and C, brown algae with chlorophyll A and C and different xanthophytes, and red algae with chlorophyll A and D with phytocyanin.) 2. Metabolism Chlamydemonas reinhardtyl uses photosynthesis. It is a facultative anaerobe, able to function with or without oxygen. It is a photoautotroph, getting its energy from light and carbon from carbon dioxide. It performs oxygenic photosynthesis, creating oxygen in the process of photosynthesis. But there are differences between this and Chromatium vinosum: Water is the reducing power instead of hydrogen sulfide, it needs two wavelengths of light instead of just one, and it is oxygenic instead of anoxygenic photosynthesis. There are two parts of photosynthesis: the light reaction and dark reaction. a) Light reaction creates ATP and NADPH. This occurs in reaction centers, located in the thylakoid membrane. It makes ATP, the energy for sugar production, as well as NADPH, the reducing power for sugar production. Two water molecules become four protons and a diatomic oxygen molecule; this is the source of reducing power. Light is harvested by antenna complexes, funneling light to reaction centers. There are two reaction centers for oxygenic photosynthesis, thus requiring two different wavelengths of light. Each reaction center has a chlorophyll molecule, an electron donor, and an electron acceptor. In the light reaction of photosynthesis, light is converted to energy for the cell in the form of ATP. Photosynthesis starts at Photosystem 2. Photosystem 2 has a chlorophyll molecule, p680. There is a pheophytin that does not have a magnesium atom in its heme group. When light hits the reaction center, it sends the electron in chlorophyll to a high level. This electron is donated to pheophytin, and the electron in chlorophyll is replaced by an electron from water. The electron in pheophytin gets passed to quinols, from where it gets passed to Cytochrome bf. Protons are pumped from the stroma to the lumen of the thylakoid, setting up a proton gradient that is used in oxidative phosphorylation. The electron is then passed to plastocyanin, which passes it on to Photosystem 1. The chlorophyll in PS 1 is p700. In PS1, the electron donor is plastocyanin, and the electron acceptor is chlorophyll A0. This passes it to phylloquinone, which passes the electron to a ferrodoxin. Ferrodoxin passes the electron to a ferrodoxin-NAD reductase, which passes the electron to NADP+, turning it into NADPH, for reducing power in the cell. But if there is a low amount of
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3. 4.
5.
6.
7.
ATP in the cell, the electron in ferrodoxin can be passed to Cytochrome bf, resulting in more ATP production without NADPH production, using the same electron to make ATP numerous times. This is cyclic electron flow, or cyclic photophosphorylation. The point of the light reaction is to create ATP and NADPH. b) Dark reaction the purpose of the dark reaction is to make carbohydrates. This occurs through the Calvin cycle. The Calvin cycle has three parts, carboxylation, reduction, and regeneration. Growth and development - Chlamydemonas reinhardtyl grows by increasing diameter, with no development. Reproduction - Chlamydemonas reinhardtyl can reproduce both sexually and asexually. In asexual reproduction, the cell is going from a haploid state to another haploid state, in binary fission. But unlike in previous examples, Chlamydomonas reinhardtyl performs two rounds of binary fission before the cells actually separate. One cell becomes 4 cells. In sexual reproduction, Chlamydemonas reinhardtyl goes from a haploid state to a diploid state. In sexual reproduction, the haploid cells differentiate into gametes. The only difference between a gamete and a normal cell is that the gamete is differentiated. There are two mating types: mt+ and mt-. Mt+ fuses with mt- to form a diploid zygote. The zygote undergoes meiosis to become haploids. The differentiating of gametes and formation of zygotes occurs when there is nitrogen starvation, when the cell cannot make new amino acids or nucleic acids due to nitrogen starvation. When the zygote is again placed in nitrogen, it will undergo meiosis, creating haploids. Adapted to environment - Chlamydemonas reinhardtyl lives in fresh water. It has flagella and is an excellent swimmer, able to phototax to light. It performs photosynthesis, so it doesnt need another source of carbon. Responds to environment - Chlamydemonas reinhardtyl chemotaxes to maltose, sucrose, xylose, manitol, and ammonia, but the mechanism for doing so is not known. It also phototaxes, swimming toward light. When light hits the stigma, which has chlorophyll molecules in it, that light activates the stigma, which causes an increase in the amount of calcium inside the cell. This increase of calcium in the cell causes an increase in calcium in the flagella, which causes the flagella to change direction. Summary - Chlamydemonas reinhardtyl is an ovoid eukaryote, an algae with specialized chloroplasts and a contractile vacuole. It has a pyrenoid, stigma, two flagella, and a cell wall. It is a facultative anaerobe, a photoautotroph that performs oxygenic photosynthesis. It grows by increasing diameter, without development. It reproduces both sexually and asexually. It is adapted to fresh water, and both phototaxes and chemotaxes.
November 8 Description: We started the class with a pile of announcements from various sources encouraging us to look at programs, including one from the pre-health post-bac organization telling us about a CPR training. When we started class, Brazill finished up the last microorganism, Chlamydemonas reinhardtyl, and started a new one, Trypanosoma brucei, which causes sleeping sickness. And of course this introduction wouldnt be complete without Brazill showing us pictures of it and the tsetse fly up close and personal. And when we
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covered the symptoms of Trypanosoma brucei, he mentioned that it causes exhaustion, insensitivity to ain, a need to lie down, drooling, and death if left untreated just like taking organic chemistry! Notes: New microorganism: Trypanosoma brucei, which causes sleeping sickness in humans, and anemia in cattle. It is transmitted by the tsetse fly, a common biting insect in Africa. 1. Organization - Trypanosoma brucei is a eukaryotic amoeboid protozoa, with a haploid nucleus. Its DNA has around 26,000 kilobases, and 10,000 genes on 11 chromosomes. It has a single, very large mitochondrion, with a kinetoplast containing the mitochondrial DNA inside the mitochondria. In addition to the mitochondria, Trypanosoma brucei has a glycosome, which holds the glycolytic enzymes. Unlike most microorganisms, in which glycolysis occurs in the cytoplasm, in Trypanosoma brucei glycolysis occurs in this glycosome. It has a single flagellum, and variable surface glycoproteins that are used as a hedge against the immune system. 2. Metabolism - Trypanosoma brucei is a strict aerobe that requires oxygen. It performs glycolysis and oxidative phosphorylation, a chemoheterotroph that gets its carbon from organic molecules and its energy from oxidizing organic molecules. 3. Growth and development - Trypanosoma brucei grows by enlargement, but has a more complex developmental cycle. Part of its life cycle is spent in the mammal, and part in the tsetse fly. The fly carries it from one mammal to another. It can be transmitted to and from humans and other mammals. Four developmental forms of the Trypanosoma brucei: a) Slender only found in mammals, in the blood. This is the actively dividing form, where Trypanosoma brucei is replicating itself. It causes disease symptoms, parasitaomia. It is found in the bloodstream and in other tissues, but not in the actual blood cells. It can cross the blood/brain barrier, with access to the neurons of the central nervous system; this causes the symptoms of the sleeping sickness that Trypanosoma brucei causes (exhaustion, insensitivity to pain, need to rest, drooling, eventually death if untreated). It uses glucose, solely using glycolysis. All the enzymes for oxidative phosphorylation would have been shut down. It only divides to a certain limit, then it stops multiplying and differentiates into the stumpy form. b) Stumpy only found in mammals, in the blood. The slender form converts to it when it reaches a certain population. The stumpy form does not divide, and is arrested in the G1 phase of the cell cycle. It is not infectious and cannot cause parasitaemia. There are drastic changes in the cell: the flagellum shortens, the kinetoplast moves to the posterior of the cell, the mitochondrion changes, enlarges and increases the number of christae, preparing to perform oxidative phosphorylation. It makes enzymes required for the electron transport chain and the citric acid cycle, It changes the source of carbon from glucose to protein and alphaketoglutarate. This helps it to prepare for life in the fly. If it is taken up by the fly, it will convert to the procyclic form. If not, it can convert back to the slender form. c) Procyclic only found in tsetse flies, in the midgut of the fly. The signal for the conversion from the stumpy to the procyclic form is a drop in temperature and
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change of nutrients. The procyclic form infects the midgut of the fly, and Trypanosoma brucei replicates there. d) Metacyclic only found in tsetse flies, in the salivary glands of the fly. In this phase there is no reproduction, as the protozoa awaits injection into the human to convert back to the slender form. Disease progression: initial infection occurs in the slender form. The slender form causes parasitaemia. The slender form converts to the stumpy form, and stops dividing. The immune system removes some of the stumpy form, some of it goes through apoptosis, and the levels of parasite drops, which triggers the stumpy form to convert back to the actively-multiplying slender form. Thus, there are cyclic levels of trypanosomes, causing the cyclical nature of the symptoms from the sleeping sickness that it causes. 4. Reproduction simple binary fission. 5. Adapted to environment - Trypanosoma brucei only uses glycolysis where glucose is plentiful. It has a developmental cycle to give efficient transmission. It has cycles of remission and resurgence to keep the host alive for a longer period of time so that more of it can be transmitted. And it has antigenic variation, with variable surface glycoproteins that cannot as easily be recognized by the immune system as they change rapidly. The immune system may kill the majority of the cells, but a few will change their variable surface glycoprotein (VSG) and evade the immune system. There are more than a thousand genes that code for VSGs. 6. Responds to environment - Trypanosoma brucei can change form between the mammal and the fly. It can change form when there are too many of the slender form present. It can change form when it moves from the midgut to the salivary gland. And it can change form when it moves from the fly to the mammal. November 10 Description: Today we talked about viruses. Brazill has decided that even though many of us actually bought the textbook, he wants to test out a new version of the textbook. Notes: Viruses have evolved to exploit different environments. Considering the characteristics of life: viruses have organization, but dont have metabolism. They dont grow or develop. They cant reproduce by themselves, and are adapted to their environment. They respond to their environment. And they can be classified as either an animal virus, plant virus, or bacterial virus or bacteriophage. The Baltimore Classification is a method of virus classification. It is based on the genome of the virus. It depends on a few factors: 1. Form of viral genome DNA versus RNA, single-stranded or double-stranded. a) DNA-based double-stranded DNA use host DNA polymerases to replicate its genome. Single-stranded DNA viruses need host DNA polymerases to make the complementary strand before replication. Plant double-stranded DNA viruses have plant reverse transcriptase, which uses an RNA intermediate in replication. All of these use the host polymerases. b) RNA-based these fall into four categories:
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a) Double-stranded RNA these need a viral RNA-dependent RNA polymerase. b) + Stranded single-stranded RNA this is the coding strand, viruses that use mRNA molecules as their genome. The + strand is already mRNA that can be translated directly to make proteins. It needs RNA-dependent RNA polymerases only to make more viral genomes. c) Stranded single-stranded RNA this is the non-coding strand. Both + and single-stranded RNA viruses require RNA-dependent RNA polymerase to replicate it and translate it. But single-stranded RNA viruses need the coding mRNA to be made from it first, before it can be translated. These viruses need RNA-dependent RNA polymerase to both make more genomes and mRNA. d) Retroviruses they use viral reverse transcriptase to make double-stranded DNA. This double-stranded DNA can then be transcribed to make mRNA and the viral genome. A virion is a virus particle. It has two parts: 1. Genome Virions are much smaller than cells. Their genomes are also much smaller. They dont have as much of a need for DNA baggage or proteins for major metabolic processes, as these processes are not existent in viruses. 2. Protein coat which surrounds the genome this protein coat is made of several different subunits that fit together in modules to make a full coat. This is an efficient use of the small genome of the virus. Viral reproduction: The virus must be able to do the following in reproduction: 1. Attachment the virus must be able to identify, attach to, and interact with the target cell that it is attacking, the host. How do viruses recognize and bind to a host cell? It has been observed that two different phages (T2 and PP01) infect two different types of E. coli (K12 and 0157:H7, respectively), but cannot infect the other type (PP01 cannot infect K12, for instance). This observation led to the hypothesis that specific phage proteins dictate host specification. To test this, an experiment was performed, switching attachment proteins on a phage, and attempting to discover whether these phage proteins are indeed the deciding factor in which type of bacteria are infected. This hypothesis was proven to be correct, proving that the attachment protein dictates host specificity. Also, each host cell has specific proteins on its surface with which the virus may interact. This is another reason why certain viruses can only infect certain types of cells. Specific viral proteins only bind specific host proteins. 2. Penetration it must be able to enter the cell, uncoat itself, to be able to begin its replication. 3. Replication it must be able to replicate both its genomic material and its proteins (protein coat and associated proteins). 4. Assembly it must be able to put the protein coat around the genome, have the associated proteins in the coat, and self-assemble without help from the host. 5. Release the virions must be able to leave the cell.
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1. Attachment done by tail fibers 2. Penetration bacteriophages inject their genome, double-stranded DNA, into the bacterial cell. The virus particle never actually enters the cell, not having to break the cell wall or lyse the cell. 3. Once the DNA of the virus enters the cytoplasm, two different things can occur: a) Temperate phages the DNA of the phage genome integrates into the bacterial genome. The phage genome gets replicated along with the bacterial genome, and every time the cell divides, the viral DNA gets replicated along with it. b) Virulent phages once the DNA enters the cell, the phage DNA is replicated multiple times, all the phage proteins are synthesized, self-assembly occurs, and the virus lyses the cell. 4. Assembly the DNA and proteins come together to make new virions. 5. Release the phage proteins disrupt the cell wall, and the cell lyses, releasing viral particles. Some phages can have both temperate and virulent states, inert in the temperate state, becoming activated as it enters the virulent state. Or it can recombine with other viruses, and become virulent this way. November 15 Description: Today was cookie day! So I arrived just on time for the class start, with large amounts of cookies! And I handed them out. People love my cookies! Notes: A found-in-eukaryotes DNA virus is called a papiloma. Stages: 1. Attachment as with most other viruses, the virus recognizes the host cell, some proteins on its coat have specificity for proteins on the hosts cell surface. 2. Penetration the virus sometimes has an envelope, a lipid bilayer around the viral particle. The envelope allows fusion of the viral envelope with the plasma membrane, to release the virus directly into the cell. The virus can then uncoat, leaving its viral DNA genome in the host cell. 3. Replication in order to replicate its DNA genome, a DNA virus must have polymerase proteins. These proteins exist in the host nucleus. So the genome travels to the nucleus. It uses host DNA polymerase to replicate its genome, resulting in new viral genomes. In order to replicate viral proteins, mRNA must be created, Host RNA polymerases are used to make mRNA. The mRNA must be brought to a ribosome, which translates the viral mRNA into new viral proteins. 4. Assembly the genome is in the nucleus, with the viral coat proteins in the cytoplasm. The coat proteins are imported into the nucleus, and the genome and viral coat proteins self-assemble. 5. Release the virus leaves the nucleus, leaves the cell through the plasma membrane, and takes a bit of the plasma membrane with it as a new viral envelope. This is called budding. The cell survives, as opposed to bacteriophages where lysis normally occurs.
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RNA viruses dont need to enter the nucleus. Most eukaryotes dont have RNA-dependent RNA polymerases, so the RNA viruses must bring their own RNA-dependent RNA polymerases. But they never need to reach the host cell nucleus. RNA viruses come in several flavors, one of which is non-coding-strand single-stranded mRNA. Influenza is of this type: 1. Attachment hemagglutanin on the virus binds the receptor on the cell with sialic acid. 2. Penetration the virus gets endocytosed and incorporated into an endocyte vesicle. Normally, endocytic vesicles are used to feed, and they fuse with a lysosome as they pump in protons to make the pH drop. As the pH drops, the viral proteins on its envelope change their conformation, triggering the membrane fusion. The viral envelope fuses with the endocytic vesicle membrane, releasing the viral particle into the cytoplasm, where it can uncoat. 3. Replication this is a negative single-stranded RNA, a non-coding strand. It must be turned into a positive coding single-stranded mRNA intermediate in order to replicate. So, it brings its own RNA-dependent RNA polymerase packaged with the virus, which is used to make a positive coding mRNA. Some of this positive coding RNA is transcribed by RNA-dependent RNA polymerase to make more original negative-stranded noncoding RNA viruses, and some is translated by ribosomes into proteins. 4. Assembly self-assembly of negatively-stranded RNA with protein coat, and including more RNA-dependent RNA polymerases. 5. Release budding. In positive single-stranded RNA viruses, in replication, the positively-stranded virus genome is the mRNA. It doesnt need to bring RNA-dependent RNA polymerase; these can be translated directly from the genome. Once the polymerase is translated, RNA-dependent RNA polymerase makes non-coding strands that can be used as a template to make more positive-stranded mRNA viruses. Retroviruses: HIV is one of them: 1. Attachment the host protein CD4 and the viral protein gp120 bind. 2. Penetration binding triggers gp41 to become active. Gp41 fuses the viral envelope with the plasma membrane. The virus is released into the cell, and uncoats. 3. Replication the RNA retrovirus is packaged along with proteins. Reverse transcriptase makes DNA from the RNA retrovirus, making a provirus. This provirus is integrated into the host genome. This provirus could do nothing and replicate with the host genome. Or it could actively make viral genome and proteins. It uses the host RNA polymerase to make mRNA, which can be translated to make new protein or used as a genome. 4. Assembly self-assembly. 5. Release budding. To conclude the virus section: 1. Attachment host protein binds viral protein 2. Penetration getting the viral genome inside the cell and uncoating the virus; this occurs through several mechanisms.
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3. Replication making more genome and making more viral proteins, and this depends on the genome form of the virus. It doesnt depend on the individual virus; it depends on the genome TYPE of virus. 4. Assembly viruses self-assemble, even in test tubes. 5. Release phages lyse the bacteria. Viruses in eukaryotes bud, for the most part. November 22 Description: Today was the first class after an exam, and we talked about a new phage, bacteriophage lambda. Notes: A new virus: bacteriophage lambda. Bacteriophage lambda can be lysogenic or lytic. Lysogenesis does not kill the host; the virus inserts its genome into that of the host, and is replicated with the host. Whether it performs lysogeny or lysis depends on the Multiplicity of Infection, MOI, the number of phages per bacterial cell. A high MOI favors lysogeny due to low levels of uninfected bacteria, while a low MOI favors lysis due to large numbers of uninfected bacteria. Bacteria detect MOI by detecting the number of copies of viral genomes in the cell. 1. Attachment the host protein is its maltose receptor. The phage attaches with its viral tail fibers. 2. Penetration in penetration, the myelin sheath contracts. This sheath contraction causes the DNA to be injected into the cell. The phage capsid does not enter the cell only the double-stranded DNA genome enters. Once it enters the cell, the viral genome circularizes, with its cos section ends binding. 3. Replication two cycles: a) Lytic pathway as soon as the virus enters the cell, two promoters become active: Pr and Pl. These promoters cause the production of two transcripts of the genome, L1 and R1, leftward and rightward, until they reach the termination sites. Transcription occurs until the termination sites. The proteins at these termination sites are proteins N and cro. N is an anti-transmitter. It allows transcription past the termination sites, allowing C3 protein along with C2, O, P, and a little bit of protein Q, due to the fact that the right-hand terminator is leaky. O and P are used for viral DNA replication. Q is an antiterminator; if it is created, transcription of late proteins are allowed late proteins that include structural proteins for assembly and lytic proteins for release. Cro acts as a repressor, blocking transcription of Pl and Pr. There are three bindings sites for cro: sites 1, 2, and 3. It has preference for sites 3 and 2 over site 2. This binding is cooperative; binding to site 3 must occur before binding to site 2, in turn before binding to site 1. Only when cro is bound to site 1 will transcription be repressed. High levels of cro are needed in order to stop transcription. Thus, transcription can only be stopped late in the infection. Once Pr and Pl are blocked, C3 and C2 can no longer be made; these are the proteins required for lysogeny. Once cro is present in large quantities, the phage is locked into the lytic cycle. b) Lysogenic pathway this stops the production of the late genes, and the viral genome is integrated into the host genome. The C1 protein controls this. The C1
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protein is made from the PE locus. The PE requires C2 to become active. C2 is degraded by FTSH, and C3 helps in stabilizing C2. C2 must be active in order to activate PE to make C1. With high multiplicity of infection, high MOI, each genome is making C2 and C3. There is therefore a large amount of C2 present, overwhelming FTSH. C1, which requires C2 to be made, binds PL and PR, like the cro protein. C1 prefers to bind to site 1, then site 1, then site 1. Binding at site 1 represses PL and PR, repressing the viral protein, so no more N, cro, C3, C2, O, P, Q, etc. It shuts down production of all viral genes, preventing the production of late proteins, preventing lysis from occurring. C1 can activate its own transcription from its own promoter, PRM, so that it no longer needs C2 after it binds sites 1 and 2. In lysogenesis, the viral DNA must be integrated into the host genome. The int protein, the integration protein, does this integration. It is made early in the infection, along with C3, C2, O, and P. Once the other proteins transcription has been prevented, integration can occur. In integration, the phage genome is integrated into the host genome. At this point, the phage is called a prophage. At this point, the bacteria considers it to be part of its genome, and it is replicated along with the rest of the bacterial genome. Thus, the phage is still replicating, albeit not quite as rapidly. In order to integrate, the phage requires a phage protein, called integrase. Integrase catalyzes site-specific recombination between the phage DNA and the host chromosome. Once integration occurs, the phage only makes one protein: the C1 protein. PR and PL have been deactivated, so none of the genes required for lysis can be produced. In addition, PRM is active, which only makes C1. Once integrated, the host is immune to lysis by other infecting bacteriophages. The prophage can be excised from the host chromosome when the host is in danger of death. If there is an increase in the amount of DNA damage in the host genome, then the prophage will excise. DNA damage in the host causes a host SOS response, which repairs the hosts DNA damage. RecA becomes active; this is a protease, needed to repair DNA damage. It also cleaves C1. As C1 levels decrease, PR and PL are no longer suppressed by C1, and become active, activating the transcription of the N and Cro proteins, allowing O, P, and Q to be made, which allow for late gene transcription, leading to phage reproduction and cell lysis in replication, assembly, and release. November 29 Description: Brazill started the class by saying, to loud awwws, that the exams will not be returned until next week. Notes: The last virus type: prions: Prions cause a number of different diseases, including mad cow disease (bovine spongiform encephalopathy), scrapy (when it infects sheep), chronic wasting disease (deer and elk), kuru (humans), and other diseases in other animals. In order to detect the causative agent (whether bacterial or viral), they sort based on size (bacteria are far larger than viruses), and filtered infected fluid through a filter that separated bacteria from viruses. For this disease, prions, the infectious agent went through the filter, meaning that it is a virus. To determine whether it is an RNA or a DNA virus, the virus can be
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treated with DNAses or RNAses to determine whether it is a DNA or an RNA virus but it retained its infectivity under both DNAses and RNAses. They even bombarded it with radiation, which destroys nucleic acids, and it still retained infectivity. So they then treated it with protease, and it lost infectivity, proving that the infectious particle is protein-based. They purified the infectious agent, proving that it is a 30-kilodalton protein, PrPSC, which is very stable and even resistant to large amounts of protease. Prolonged exposure to protease is required in order to destroy it. This infectious protein is found normally in plenty of animals, mammals and birds, in for form PrPC. We dont know the normal function of this protein, except that we know that it binds calcium and may be involved in stem cell regeneration, longterm memory, and neuronal repair. It has the exact same amino acid sequence as PrPSC. It is natural;y found in stem cells and neuronal tissue. The two proteins, PrPC and PrPSC have the same primary protein structure but different secondary and tertiary structures. PrPC is protease-sensitive, while PrPSC is protease-insensitive. An energy hurdle must be overcome in order for the prion to go from one to the other. Progression in the disease is detected as C is converted to SC to cause prion disease. SC is the lower energy state; SC cannot be converted back to C. Conversion is catalyzed by PrPSC. Multiple theories as to how this occurs: 1. Heterodimer theory start with two forms, which bind each other to form a heterodimer. The heterodimer converts to a homodimer of PrPSC, which splits. But monomeric PrPSC is never found. 2. Polymerization model the homodimer grabs another molecule of PrPC and converts that to PrPSC, repeating this again and again many times over. Once this chain of PrPSC gets long enough, it breaks. We dont know why it would break. Infection: There is a long incubation time, months or decades. It is very rare, infecting only around one per million people each year. It can be acquired, contracted when the body comes in contact with infectious PrPSC, through such things as tainted meat and cornea transplants. The other way it occurs is through sporadic, random conversion of PrPC to PrPSC; this is rare as a lot of energy is required to perform this conversion. A third way that this disease can be contracted is familial some families have predisposition to the disease on a genetic basis. Mutations in the PRNP gene makes it easier to convert from PrPC to PrPSC. The disease requires the presence of PrPC; mice without PrPC are immune to prion disease, while mice that overproduce PrPC have rapid onset of the diease when it is infected. Two phases of infection: 1. Colonization of immune system the first signs of the infection is in the immune organs, such as in the spleen, lymph nodes, tonsils, and appendix. Mice without an immune system are immune to prion disease, as the disease is transmitted by B cells. But if PrPSC is injected directly into the brain, they could still get the disease. 2. Transferred to nervous system in the brain, spongiform encephalopathy is caused, as the prions cause neuron cells to die, leaving holes in the brain. Severe neurological symptoms are seen, with convulsions, dementia, ataxia (loss of balance and coordination), personality disorders, and eventually death. We do not know how it gets from the immune system to the central nervous system.
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December 1 Description: Today we continued our discussion on prion disease and its detection and treatment. And Brazill told us that our exams will be given back on Tuesday. Notes: (Detection and treatment of prion disease: Detection is difficult. Normally, disease agents are detected by PCR, which identifies very small amounts of DNA or RNA that belongs to a virus or bacteria, and replicates it extensively. But prions are not DNA- or RNA-based, so they cannot be detected in this way. Another way to discover disease-causing agents is through antibody-based tests, but the protein that causes prion disease is the same as the one in our bodies an antibody would be needed to tell the structural difference between PrPC and PrPSC. The levels of PrPSC in the blood is too low to detect easily even with antibodies. There is no treatment for it; drugs are being developed which block polymerization of PrPSC. It slows down the rate of progression of the disease. And genetically-engineered cows are being made that cannot code for PrPC at all.) Microbial genetics: the interaction of different genes within one microorganism or between different microorganisms. (our basic understanding of genetics is based on work in microbial genetics, uncluding on gene function, inheritance, and gene regulation. Microbial genetics is important for molecular cloning and biotechnology. It allows us to understand the nature of virulence and gene transfer.) Three methods of gene transfer: 1. Transformation DNA can be introduced into a cell. The cell takes up the naked DNA, and may or may not integrate the DNA into its own chromosome. This is a natural process. Competence is a cells ability to be transformed. Cells produce competence factor, CF, a large precursor which gets cleaved and secreted. The concentration of competence factor acts as a quorum factor, a way for the cell to ascertain how many cells are nearby. As the CF concentration increases, it can eventually bind to the receptor, ComD. ComD is part of a 2-component system, just like flagella. When it binds its CF, it becomes phosphorylated. ComD then transfers that phosphate group to ComE, which when activated causes the transcription of the gene sigH. SigH causes the transcription of genes coding for the translocasome that brings DNA into the cell. Thus, a cell can introduce variability and survivability into its genome when cell density is high, and can ensure the survival of the species when the cell density is low. Transformation can be artificially induced during heat shock or electroporation. 2. Transduction introducing DNA using a virus. This occurs naturally, as phages will have extra DNA in its genome. We can artificially introduce DNA into phages (and remove its lysis genes) to artificially inject it into the bacteria. If we use a lysogenic phage, we can get this DNA integrated into the bacterial chromosome. 3. Conjugation introduction of DNA through a sex pilus. This is only seen in bacteria, and involves the transfer of an F plasmid, an episome. The F plasmid is copied in the F+ cell, and one copy is sent through a pilus to the F- cell, leaving two F+ cells. If the F plasmid is inside the chromosome, integrated into the host genome, then the strain is called Hfr. (In his writing, the OriT is the origin of transfer, the OriV is the origin of replication, the Tra are the genes required for transfer, and the IS is the insertion sequence.) We get recombination between the F plasmid and the chromosome, specifically at the host
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insertion sequence. The insertion sequence site on the episome and the chromosome are homologous. Within each episome, there is only one insertion sequence. However, within a single bacterial chromosome, there may be several different insertion sequence sites. Transfer moves away from the transfer genes. (In lab, we did interrupted mating, allowing transfer and then stopping it at different time points.) Transfer can be interrupted by breaking the pilus interaction by shaking. If the Hfr donor has certain genes that the F- recipient doesnt have, these genes can be transferred along with the rest of the F-containing chromosome. Thus, we can use interrupted mating to map genes on a chromosome. We plate matings on selective media, and count the number of colonies (mating/integration event) against the time of interruption. Thus, as it takes longer for arginine to be seen than it takes for leucine to be seen, we can assume that leucine is closer to the insertion site. There are multiple insertion sites, and different Hfrs with episomes at different insertion sites, so there will be different start times depending on which insertion site on the Hfr is used. December 8 Description: On Tuesday, Brazill had sent out an email telling us all that he wasnt feeling well, and that class was to be cancelled. And he didnt get to grade our exams yet. So well get our second exams back this Tuesday. Which means that today is our last lecture!!!!!!!! Notes: Some more microbial genetics: in the last lecture, we discussed transformation (naked DNA), transduction (viral vector), and conjugation (through a pilus). Microbial mating: Mating is the mixing of different genomes. Two haploids fuse to create a diploid. By mating two deficient haploids, due to the fact that every organism receives more than one copy of the genome, the diploid will have a healthy copy of both genes. A mutation is a heritable change in the DNA of an organism. A mutant is a strain that carries a mutation. Wild types are normal, without mutation. Mutations can be detrimental, neutral, or positive in terms of their effects on the organism. The genotype is the description of the genes, while the phenotype is the observable properties of an organism. These two are not necessarily correlated. One type of microbial genetics that is heavily studied is the abilities of microorganisms to synthesize nutrients and resist antibiotics. Prototrophs can synthesize the nutrient in question, such as amino acids or nucleic acids. Auxotrophs require a specific nutrient. These are negatively-selectable mutants, being selected against as they cannot grow where the wild type can, as they cannot synthesize a nutrient that might not be available. Positively-selectable mutants have things such as drug resistance that allow them to grow more at the expense of the wild-type cells. Non-selectable mutants simply have different characteristics, but there is no effect on their ability to grow, so large numbers of cells must be searched to find few mutants. There are several different genes in the pathway to make a given nutrient, and if even one of them is negatively mutated, then the cell will not be able to make the nutrient. But two mutants, each negatively mutated for a different enzyme in the pathway, can mate to complement each other and be again able to synthesize the nutrient. Only mutants that have mutations in different enzymes can make a healthy diploid.
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A gain of function mutation occurs when the gene product can do something different from what the original gene product would have done. This does not complement. (We can use complementation analysis to uncover the number of genes in a pathway. To do this, we randomly mutate wild-type cells, and select for mutants in the pathway. We search for mutants that cannot grow where the wild type cyan, and mutants that can grow where the wild-type cannot. We isolate negatively-selectable mutants by replica plating, using a velvet to move parts from every colony in a culture from a non-selective medium to a selective medium, we compare the plates, and where mutant cells do not exist in the selective medium, that is the position of the mutant cells in the first medium. We then mate the various mutants in pairwise combinations and look for complementation. The number of complementation groups is equal to the number of genes in a pathway. Each complementation group represents mutations in one specific gene.)
And that finishes BIOL200!!!!!!!!!!!!!!!
Page 47

BIOL200Brazill Notes

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BIOL200Brazill Notes

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BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill

By Barukh Rohde. Qs/comments: barukh94-school@yahoo.com

BIOL200 Notes Professor Derrick Brazill