Unit 2
What is DNA a polymer of? List the three components of this molecule. Where is it synthesized?
DNA and RNA are composed of polymers of nucleotides. A single nucleotide contains a nitrogenous base, sugar unit, and phosphate group. DNAis a nucleic acid made up of nucleotides joined into long strands or chains by covalent bonds. There are two ways nucleotides are synthesized in our cells. The first way is called de novo synthesis, which is the synthesis of nucleotides from simple molecules, including carbon dioxide, glucose, ammonia, bicarbonate, and amino acids. The second way nucleotides are synthesized is through salvage pathways. Salvage pathways recycle nucleotides obtained from our diets or from degraded DNA and RNA to form new nucleotides that are needed by our cells. These synthesis reactions occur in the cytoplasm and are catalyzed by enzymes. As you might guess, the nucleotide synthesis pathways are tightly regulated. If there is an insufficient level of nucleotides, then our cells will be unable to synthesize RNA and DNA. At the same time, our bodies do not want to waste energy to produce nucleotides in excess when they are not needed. Nucleotide synthesis is regulated by a process called feedback inhibition. Products of the nucleotide synthesis pathways can inhibit specific enzymes that catalyze earlier reaction steps to control the level of nucleotides produced. In this way, our cells can produce just the right amount of nucleotides.
What is a free radical? The superoxide radical or anion? How does the supperoxide anion form? How does the supperoxide anion damage the cell? What is SOD? Do all organismscontain SOD? Draw an equation illustrating how SOD neutralizes the superoxide anion; what is the end product? What is the function of catalase? Draw an equation illustrating how catalase neutralizes the H2O2; what is the end product?
- Oxygen has the capacity to form free radicals, which can be damaging to obligate aerobes and humans alike. Free radicals may also contribute to cancer and the aging process. - Superooxides: the elctron transport chain, which is found in the inner mitochondrial membrane, utilizes oxygen to generate energy in the form of adenosine triphosphate. Oxygen acts as the terminal electron acceptor within the ETC. Anywhere from 2 to 5 % of the total oxygen intake during both rest and exercise have the ability to form the highly damaging superoxide radical via electons escape. - Superoxide radicals attempt to "steal" the electrons of susceptible bilologic targets, including lipids, proteins, and nucleic acids. - Because superoxide is toxic; nearly all organisms living contain superoxide dismutase ( SOD) an enzyme that catalyzes the production of hydrogen peroxide and molecular oxygen, as illustrated via the following reaction: O2˙ ¯ + O2˙ ¯ + 2H----> + H2O2 + O2 Catalyase H2O2--------> H2O + O2 (OH ˙ (the hydroxyl radical); also can kill obligate anaerobes).
Describe the following and include the pH ranges anbd general locations as to where each is located.
1. Acidophiles: 0.1 -5.4 ( H.pylori). 2. Neutrophyles: 5.4 - 8.0 ( E. cloi, S. aureus) 3. Alkaliphiles: 7.0 - 11.5 Most human pathogens are neutrophiles.
List and describe the function of four different nutritional factors that affect bacterial growth.
1. Carbon: It is the backbone of all living matter. Photoautotrophs utilize carbon dioxide to produce glucose; both authotrophic and heterotrophic organismscan obtain energy from glucose via glycolysis, fermentation, the Krebs cycle, etc. 2. Nitrogen: All organisms and microorganisms require nitrogen for the synthesis of enzymes, other proteins, RNA, DNA, and ATP. 3. Sulfur: necessary to synthesize sulfur-containing amino acids. 4. Phosphorus: necessary to synthesize ATP, phospholipids, RNA, and DNA
Describe the following, and include the temperature ranges and general locations as to where each is located.
1. Psychrophiles: 15-20C ( listeria monocytogenes, 5C). 2. Mesophiles: 25-40 C 3. Thermophiles: 50-60 C 4. Extreme thermophiles: 100 - ? Most human pathogens are mesophiles.
Orthomyxoviridae
: Includes influenza virus; causes the "flu" each winter; attachment sites mutate.
What is a mutation? Are mutations good, bad, or both?
A mutation is an alteration that occurs to the base sequence of DNA. Mutations are the result of many environmental agents such as chemicals and radiation; they can also be inherited. b) Mutations always change the genotype of an organism, but may not affect the phenotype. This depends often on the type of mutation and it's affect on protein synthesis. c) Mutations occur regularly in DNA and in general, DNA repair enzymes identify and correct mutated DNA. If the mutation is not repaired, it may have a silent, deleterious, or beneficial effect.
Describe the structure and function of plasmids
A plasmid is a small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. Plasmids naturally exist in bacterial cells, and they also occur in some eukaryotes. Often, the genes carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance.
Describe the structure and function of both capsids and capsomeres. How do capsomeres affect viral shape?
A protein coat called a capsid surrounds the viral genome; each capsid is composed of protein subunits called capsomeres. Capsomeres often determine viral shape; most are either polyhedral (many sided) or helical (spiral staircase). Capsomeres are commonly utilized for viral identification and classification.
What is a vector? Give some examples of vectors. Can they self replicate?
A vector is a vehicle utilized for transferring genetic material into a cell. Vector examples include both plasmids and viruses. Vectors are self replicating. Mosquitoes are vectors for malaria, as they transmit a microbe (protozoa) to the host.
When do tRNA molecules stop bringing amino acids to the ribosome?
After stop codon
What are chromosomes? Genes? Alleles? Is all genetic information composed of DNA? Why or why not?
Alleles: one of two or more alternative forms of a gene that arise by mutation and are found at the same place on a chromosome. Chromosomes are typically circular (prokaryotes) or linear structures (eukaryotes) that carry hereditary information via genes. Genes are the basic units of heredity; all of the information for the structure and function of an organism is coded in its genes. Genes are segments of DNA or RNA that code for specific functional products, such as proteins and enzymes, etc. All information necessary for life is stored in an organism's genetic material, which is primarily DNA or in the case of some viruses, RNA.
Describe the 3 steps involved in creating a clone. Are competent bacteria used in genetic engineering? Why?
An overview of Recombinant DNA Procedures. 1. The gene of interest is inserted into a DNA vector, such as a plasmid or virus, via restriction enzyme use. 2. The vector is then introduced into competent bacteria. Bacteria (E.Coli) can often be manipulated to become competent via various techniques. In the lab, E. coli is commonly bacteria used due to the fact that it grows easily and that its genetics are fairly well understood. E. coli can be rendered competent via simultaneous mild heat shock and calcium chloride exposure; electroporation. 3. Competent cells that contain the vector of choice are identified, grown and harvested. Large quantities of the gene or proteins of interest can then be retrieved.
List some applications of genetic engineering and describe their function.
Applications of Genetic Engineering 1. Human insulin: Used in treatment of diabetes and is derived from E.Coli cultures. 2. Tissue plasminogen activator (tPA): Used to dissolve blood clots in post-heart attack therapy and is derived from tissue cell culture. 3. Interleukin 2 (IL-2): A potent stimulator of the immune system used in treatment of cancer; derived from E. coli. 4. Interferon (IFN): A potent stimulator of the immune system used to treat infections and diseases such as Hepatitis C, multiple sclerosis; derived from E. coli and yeast. 5. Monoclonal antibodies (Mab): Used in diagnostic testing and potentially in cancer therapy and transplant rejection; derived from tissue cell culture. 6. Hepatitis B vaccine: protects from long term side effects of HBV; derived from yeast cultures. 7. Understanding how a microbe replicates: Led to development of protease inhibitors for HIV/AIDS. 8. Used in agriculture to produce larger, "better" fruits/vegetables by enabling plants to become resistant to pests, weeds, fungi; also used to increase nutrient value of foods. 9. Treatment of certain diseases (cystic fibrosis) via gene therapy. 10. Many, many other uses in science (Cloning humans?).
Compare and contrast the differences between DNA and RNA.
At the chemical level, the DNA of prokaryotic cells, which have no nuclear membranes, and that of eukaryotic cells, which have separate, membrane-enclosed nuclei, is identical. DNA is a long, double-stranded molecule containing a backbone of repeating, alternating sugar and phosphate units. One of four different nucleotide bases, cyclic molecules containing nitrogen, hangs off each sugar unit. Differences between the DNA of the two organism types include its packaging, quantity, replication and information content. Packaging Prokaryotes and eukaryotes package their DNA molecules with protein in structures called chromosomes. A prokaryotic chromosome is circular and resides in a cell region called the nucleoid. The types of proteins found in prokaryotic chromosomes, known as the nucleoid-associated proteins, differ from the histone proteins that appear in eukaryotic chromosomes and cause the prokaryotic chromosomes to form looped structures. Unique eukaryotic chromosome packaging features include tight coiling, dense packing, enclosure within a nuclear membrane and linear rather than circular structures. Quantity Prokaryotes typically have one main chromosome, through it might have a few copies of it. These cells can also have smaller DNA structures called plasmids that contain additional information. The number of nucleotide base pairs in the prokaryotic chromosome ranges from 160,000 to 12.2 million, depending on the species. Eukaryotes frequently have multiple types of chromosomes with many more base pairs. For example, humans have 23 pairs of chromosomes, the maternal and paternal sets, containing about 2.9 billion base pairs in total. Replication Cells must replicate their DNA before dividing. Prokaryotic replication is relatively simple. The copying of the DNA strands begins at a single point of origin on the prokaryotic chromosome, and only one replication fork and bubble is formed during replication. Prokaryotic DNA replication is speedy, about 2,000 base pairs per second. Eukaryotic DNA replication relies on multiple replication origins, forks and bubbles to compensate for a slow pace, about 100 base pairs per second. In prokaryotes, only two proteins are required to initiate replication, whereas eukaryotes use complexes composed of multiple protein subunits. Information Content Prokaryotic DNA gene sequences are organized as operons, each of which can code for multiple proteins. The size, sophistication and number of encoded proteins is relatively small, but a high percentage of the prokaryotic DNA strand encodes proteins. Eukaryotic DNA is organized in genes that each code for a single protein, although in some cases multiple genes might be transcribed at the same time. About 5 percent of eukaryotic DNA codes for proteins or RNA, but much of the remaining DNA helps identify gene and control gene expression. Prokaryotic cells usually have a single version of each gene, whereas eukaryotes normally have two versions, one from each parent.
How many chromosomes are in eukaryotes? Prokaryotes?
At the chemical level, the DNA of prokaryotic cells, which have no nuclear membranes, and that of eukaryotic cells, which have separate, membrane-enclosed nuclei, is identical. DNA is a long, double-stranded molecule containing a backbone of repeating, alternating sugar and phosphate units. One of four different nucleotide bases, cyclic molecules containing nitrogen, hangs off each sugar unit. Differences between the DNA of the two organism types include its packaging, quantity, replication and information content. Packaging Prokaryotes and eukaryotes package their DNA molecules with protein in structures called chromosomes. A prokaryotic chromosome is circular and resides in a cell region called the nucleoid. The types of proteins found in prokaryotic chromosomes, known as the nucleoid-associated proteins, differ from the histone proteins that appear in eukaryotic chromosomes and cause the prokaryotic chromosomes to form looped structures. Unique eukaryotic chromosome packaging features include tight coiling, dense packing, enclosure within a nuclear membrane and linear rather than circular structures. Quantity Prokaryotes typically have one main chromosome, through it might have a few copies of it. These cells can also have smaller DNA structures called plasmids that contain additional information. The number of nucleotide base pairs in the prokaryotic chromosome ranges from 160,000 to 12.2 million, depending on the species. Eukaryotes frequently have multiple types of chromosomes with many more base pairs. For example, humans have 23 pairs of chromosomes, the maternal and paternal sets, containing about 2.9 billion base pairs in total. Replication Cells must replicate their DNA before dividing. Prokaryotic replication is relatively simple. The copying of the DNA strands begins at a single point of origin on the prokaryotic chromosome, and only one replication fork and bubble is formed during replication. Prokaryotic DNA replication is speedy, about 2,000 base pairs per second. Eukaryotic DNA replication relies on multiple replication origins, forks and bubbles to compensate for a slow pace, about 100 base pairs per second. In prokaryotes, only two proteins are required to initiate replication, whereas eukaryotes use complexes composed of multiple protein subunits. Information Content Prokaryotic DNA gene sequences are organized as operons, each of which can code for multiple proteins. The size, sophistication and number of encoded proteins is relatively small, but a high percentage of the prokaryotic DNA strand encodes proteins. Eukaryotic DNA is organized in genes that each code for a single protein, although in some cases multiple genes might be transcribed at the same time. About 5 percent of eukaryotic DNA codes for proteins or RNA, but much of the remaining DNA helps identify gene and control gene expression. Prokaryotic cells usually have a single version of each gene, whereas eukaryotes normally have two versions, one from each parent.
How does replication start? What proteins prevent the unwound DNA from twisting back?
At the origin of replication, hydrogen bonds between complementary base pairs of DNA stands are broken via enzymes called DNA helicases. 2. The separation of the two strands is stabilized by the assorted enzymes (helix destabilizing proteins).
During translation, how do tRNA molecules attach to the mRNA strand? Is it random?
At the start codon (AUG-methionine), tRNA molecules align their anticodon accordingly. (tRNA molecules carry amino acids and have a sequence of three nitrogenous bases called an anticodon; Anticodons are complimentary to codons of an mRNA strand. What is the c
What is complimentary base pairing? Give an example of complimentary base pairing in DNA as well as RNA.
Attached to each sugar ring is a nucleotide base, one of the four bases Adenine (A), Guanine (G), Cytosine (C), and Thymine (T). The first two (A, G) are examples of a purine which contains a six atom ring and five atom ring sharing two atoms. The second two (C, T) are examples of a pyrimidine which is composed of a single six atom ring. A base pair is one of the pairs A-T or C-G. Notice that each base pair consists of a purine and a pyrimidine. The nucleotides in a base pair are complementary which means their shape allows them to bond together with hydrogen bonds. The A-T pair forms two hydrogen bonds. The C-G pair forms three. The hydrogen bonding between complementary bases holds the two strands of DNA together. Hydrogen bonds are not chemical bonds. They can be easily disrupted. This permits the DNA strands to separate for transcription (copying DNA to RNA) and replication (copying DNA to DNA). In our simple model, the entire base pair structure is represented by the single blue rod. Various more elaborate models can be constructed to represent base pairs, including the one above which shows individual atoms and bonds.
Why is protein synthesis regulated at the gene level in bacteria?
Bacteria have developed mechanisms to turn reactions on and off in accordance with their needs. Energy and materials are too valuable to squander; thus metabolic activities are regulated to avoid wasteful excesses. Regulating protein synthesis at the gene level is efficient because proteins are only manufactured when necessary. Many of these proteins are enzymes that are necessary for metabolism.
Describe the five basic steps of viral replication in animal cells; how does it vary from viral replication in prokaryotic cells?
Basic steps of viral replication 1. Adsorption: Viral attachment structures (spikes) and attach to specific receptor sites on host cells. Receptor sites are genetic and thus vary from individual to individual. 2. Penetration: Entry is facilitated by endocytosis (naked virus) or fusion of the viral envelope and the plasma membrane. The viral genome is separated from the capsid via uncoating (eclipse). Host hydrolytic enzymes facilitate this process. 3. Synthesis: DNA or RNA replication occurs; capsid synthesis occurs. In general: DNA viruses: DNA synthesis occurs in the host cell nucleus via viral enzymes; viral protein production occurs in the cytoplasm. RNA viruses: It's complicated. 4. Maturation: Once viral nucleic acid and capsid construction is complete, assembly of virions occurs. 5. Release: If the virus contains an envelope, the virion will not be complete until it buds through a host membrane. With non enveloped viruses, the host cell ruptures (and dies) releasing the viruses.
What is budding? Binary fission? Describe the events that occur during each phase of binary fission.
Cellular division in bacteria occurs primarily by binary fission, where a cell duplicates its components and divides into two cells by essentially pinching itself in half. Each cell is genetically identical. Some bacteria reproduce by budding, fragmentation, or spore formation. b) There are four phases of binary fission: 1. Cell elongates, DNA replicates and forms two nucleoids; each attaches to a different part of the cell membrane. 2. Cell wall and plasma membrane begin to divide. 3. A cross-wall (septum) forms and separates nucleoids. 4. Cells separate.
What is the biological significance of conjugation?
Conjugation is the process by which one bacterium transfers genetic material to another through direct contact. During conjugation, one bacterium serves as the donor of the genetic material, and the other serves as the recipient. The donor bacterium carries a DNA sequence called the fertility factor, or F-factor.
What is conjugation? Who demonstrated this phenomenon and won a Nobel Prize at 33! Describe the structure and function of conjugation pili. Are they always present on a bacterium?
Conjugation: The transfer of genetic material from one bacterium to another via the assistance of conjugation pili and plasmids. Requires physical contact between bacteria. Lederberg experiments with E.coli demonstrated the phenomenon of conjugation. (Lederberg won the Nobel prize in 1958 at age 33 for this huge contribution to science). Conjugation pili are tiny, hollow projections that physically connect bacteria to one another; facilitate the transfer of plasmids. Plasmids are circular rings of extrachromosomal DNA that are not essential for survival. (F= fertility. F + cells contain F plasmids; F¯ cells do not.) Plasmids often contain bacterial resistance genes, and multiple resistance genes can be found on a single plasmid. Resistance genes provide bacteria with the ability to resist deleterious actions of antibiotics etc.
Are viruses larger than prokaryotes? Organelles? What is their size range?
Contain no nucleus, organelles, or cytoplasm. Size ranges from 18-200 nm; Orthopoxviruses tip the scale at 2400 x 300 nm.
What is a culture medium? What is agar? What is a selective media? An enrichment media?
Culture media: is any material that is used to grow bacteria. It is designed to have the proper nutrients, pH, moisture, and oxygen to allow for optimal microorganism growth. The medium must be sterile so that the culture will only contain the microbes that are added to the medium. Culture medium may be liquid, semisolid or solid. The most common media for culturing bacteria is called agar, a complex polysaccharide extracted from marine algae. Agar located in a Petri dish is called an agar plate. Agar is a jelly-like substance that can be used in test tubes and Petri dishes. Test tubes (deeps) are better for testing the microbe in aerobic and anaerobic conditions, or for fermentation end products. Petri dishes have a large surface area, making it easier to differentiate between colonies of microorganisms. The addition of various materials such as a carbon source (glucose), peptides (peptone), vitamins, minerals, etc to agar give rise to specific media. Selective media encourages the growth of some organisms but suppresses the growth of others via the addition of various substances such as salts, dyes, or antibiotics to the agar to inhibit the growth of particular organisms. An example is using a Thayer-Martin plate, which is selective for N. Gonorrhea. Enrichment media contains special nutrients to promote growth of certain organisms that may not be indicated or missed unless encouraged to thrive. Anaerobes require special handling so that the oxygen is eliminated. Their growth media is in an airtight vial and contains chemicals to reduce the oxygen. Viruses, parasites, and some bacteria will not grow on agar plates. They require live tissue cells to grow or other types of media. When doing a bacterial culture, it is important to get a pure culture. This is done by performing the streak plate method. This method "dilutes" out the organisms so that a single bacterium can be identified (you will do this in lab). Without isolating bacteria, identification may be impossible.
What is the function of DNA replication?
DNA makes new, identical DNA
What is the function of DNA polymerase III? DNA polymerase I? Which of the two polymerases functions to remove RNA primers? Which one produces the majority of the DNA on the leading and lagging strand?
DNA polymerase III: endonuclear activity; puts in DNA nucleotides; operates first; leading stands 5-> 3 prime direction. DNA Polymerase I In charge of synthesizing nucleotides onto primers on the lagging strand, forming Okazaki fragments. However, this enzyme cannot completely synthesize all of the nucleotides. - proof reading capability - can fix mestakes - ladding strands 5->3 DNA Polymerase I then removes RNA primers and fills the gaps between Okazaki fragments.
What must first occur prior to transcription?
DNA unwinds, separates, and is stabilized via assorted proteins.
Describe some basic viral characteristics...Do they contain a nucleus? Organelles? Cytoplasm? Are they living or non-living? How do they affect cell's metabolic machinery once inside? Can they be latent?
Derived from the Latin word meaning poison. Contain no nucleus, organelles, or cytoplasm. Obligate intracellular parasites, meaning that they can only replicate inside a living host as they lack enzymes necessary for metabolism. (Nucleic acid, protein, and ATP synthesis). Living or non-living? Size ranges from 18-200 nm; Orthopoxviruses tip the scale at 2400 x 300 nm. Viruses possess the ability to transfer their nucleic acids to other cells, where they multiply in living cells by hijacking the cell's metabolic machinery. May remain latent (inactive) for years. Have the ability to mutate at a rapid rate. (How many HIV strains exist? 40)
Describe the structure and function of viral envelopes; do all viruses have envelopes? Describe the structure and function of spikes and canyons.
Envelopes Viruses may be enveloped or nonenveloped (naked). Composed of a typical bilayer of phospholipids, proteins, and carbohydrates. Glycoprotein "spikes" can project from the envelope; serve as attachment points for viruses to host cell surfaces. Envelopes facilitate viral entry and "invisibility" once inside host cells; however are vulnerable to environmental conditions that destroy membranes. (Temperature, pH, disinfectants).
What is a frameshift mutation? Is this considered a "good" or "bad" mutation? Why?
Frameshift mutations: The deletion or insertion of base pairs.
What are halophiles? List some examples as to where they live.
Halophiles: bacteria that require an environment rich in salt. Typically found in the ocean, (staphylococci)
List the enzymes/proteins involved in DNA replication and describe their function.
Helicase Uses the hydrolysis of ATP to "unzip" or unwind the DNA helix at the replication fork to allow the resulting single strands to be copied. Primase Polymerises nucleotide triphosphates in a 5' to 3' direction. The enzyme synthesises RNA primers to act as a template for future Okazaki fragments to build on to. DNA Polymerase III In charge of synthesizing nucleotides onto the leading end in the classic 5' to 3' direction. DNA Polymerase I In charge of synthesizing nucleotides onto primers on the lagging strand, forming Okazaki fragments. However, this enzyme cannot completely synthesize all of the nucleotides. Ligase This enzyme is in charge of "gluing" together Okazaki fragments, an area that DNA Pol I is unable to synthesize. Telomerase Catalyzes the lengthening of telomeres; the enzyme includes a molecule of RNA that serves as a template for new telomere segments. Nuclease This enzyme is in charge of excising, or cutting out, unwanted or defective segments of nucleotides in a DNA sequence. Topoisomerase This enzyme introduces a single-strand nick in the DNA, enabling it to swivel and thereby relieve the accumulated winding strain generated during unwinding of the double helix. Single Strand Binding Proteins Responsible for holding the replication fork of DNA open while polymerases read the templates and prepare for synthesis.
How viruses enter host cells? What must host cells contain in order for the virus to replicate?
Hosts Viruses are capable of infecting many hosts, or life forms. Bacteria, plants, algae, fungi, and vertebrates etc are all fair game. Viruses that infect bacteria are called bacteriophages (phages). In order for a virus to infect a cell, the following must occur: 1. Host cells must contain specific receptor sites on their surface that are complimentary to viral attachment structures. 2. Host cells must contain the appropriate metabolic machinery necessary for viral replication.
Describe what occurs to a prokaryotic cell when placed in each of the following media.
Hyperosmotic medium: plasmolysis ( shrinkage). Hypoosmotic medium: turgor ( distended). Isosmotic medium: no visible change.
Filoviridae
Includes Ebola virus and Marburg virus; both cause hemorrhagic fever with mortality rates as high as 90%.
Retroviridae
Includes HIV (AIDS) and HTLV (T-cell leukemia); called retrovirus because it replicates from RNA to DNA, while other RNA viruses make copies of their own RNA. Viral DNA is then inserted into host DNA (provirus); viral DNA stays in host DNA.
Herpesviridae
Includes Herpes (HSV-I and HSV-II), Epstein-Barr (EBV), Varicella zoster (VZ-chicken pox) and Cytomegalovirus (CMV). "Herpetic" = spreading; latent (hides in sensory neurons).
Hepadnaviridae
Includes hepatitis B virus (HBV); does NOT include hepatitis A or hepatitis C viruses (RNA viruses).
Papovaviridae
Includes mainly papillomavirus (HPV); also known as genital warts and is associated with cervical dysplasia.
Poxviridae
Includes smallpox and cowpox; smallpox is the first virus to be eradicated worldwide due to vaccination. Pus-filled legions are common.
Picornaviridae
Includes the poliovirus (polio), enteroviruses (meningitis and GI disease), hepatitis A, C (HAV, HCV), and rhinoviruses (common cold).
Eosin Methylene Blue Agar
Lactose and the dyes eosin and methylene blue permit differentation between enteric lactose ferments and nonferemters as well identification of the colon bacillus, E. coli.
Describe the function of mRNA ; what is it composed of? (Triplets?) What does mRNA attach to?
Messenger RNA Carries Information from DNA in a Three-Letter Genetic Code RNA contains ribonucleotides of adenine, cytidine, guanine, and uracil; DNA contains deoxyribonucleotides of adenine, cytidine, guanine, and thymine. Because 4 nucleotides, taken individually, could represent only 4 of the 20 possible amino acids in coding the linear arrangement in proteins, a group of nucleotides is required to represent each amino acid. The code employed must be capable of specifying at least 20 words (i.e., amino acids). If two nucleotides were used to code for one amino acid, then only 16 (or 42) different code words could be formed, which would be an insufficient number. However, if a group of three nucleotides is used for each code word, then 64 (or 43) code words can be formed. Any code using groups of three or more nucleotides will have more than enough units to encode 20 amino acids. Many such coding systems are mathematically possible. However, the actual genetic code used by cells is a triplet code, with every three nucleotides being "read" from a specified starting point in the mRNA. Each triplet is called a codon. Of the 64 possible codons in the genetic code, 61 specify individual amino acids and three are stop codons. Table 4-2 shows that most amino acids are encoded by more than one codon. Only two — methionine and tryptophan — have a single codon; at the other extreme, leucine, serine, and arginine are each specified by six different codons. The different codons for a given amino acid are said to be synonymous. The code itself is termed degenerate, which means that it contains redundancies. Table 4-2. The Genetic Code (RNA to Amino Acids)*. Table 4-2 The Genetic Code (RNA to Amino Acids)*. Synthesis of all protein chains in prokaryotic and eukaryotic cells begins with the amino acid methionine. In most mRNAs, the start (initiator) codon specifying this aminoterminal methionine is AUG. In a few bacterial mRNAs, GUG is used as the initiator codon, and CUG occasionally is used as an initiator codon for methionine in eukaryotes. The three codons UAA, UGA, and UAG do not specify amino acids but constitute stop (terminator) signals that mark the carboxyl terminus of protein chains in almost all cells. The sequence of codons that runs from a specific start site to a terminating codon is called a reading frame. This precise linear array of ribonucleotides in groups of three in mRNA specifies the precise linear sequence of amino acids in a protein and also signals where synthesis of the protein chain starts and stops. Because the genetic code is a commaless, overlapping triplet code, a particular mRNA theoretically could be translated in three different reading frames. Indeed some mRNAs have been shown to contain overlapping information that can be translated in different reading frames, yielding different polypeptides (Figure 4-21). The vast majority of mRNAs, however, can be read in only one frame because stop codons encountered in the other two possible reading frames terminate translation before a functional protein is produced. Another unusual coding arrangement occurs be- cause of frameshifting. In this case the protein-synthesizing machinery may read four nucleotides as one amino acid and then continue reading triplets, or it may back up one base and read all succeeding triplets in the new frame until termination of the chain occurs. These frameshifts are not common events, but a few dozen such instances are known. Figure 4-21. Example of how the genetic code — an overlapping, commaless triplet code — can be read in two different frames. Figure 4-21 Example of how the genetic code — an overlapping, commaless triplet code — can be read in two different frames. If translation of the mRNA sequence shown begins at two different upstream start sites (not (more...) The meaning of each codon is the same in most known organisms — a strong argument that life on earth evolved only once. Recently the genetic code has been found to differ for a few codons in many mitochondria, in ciliated protozoans, and in Acetabularia, a single-celled plant. As shown in Table 4-3, most of these changes involve reading of normal stop codons as amino acids, not an exchange of one amino acid for another. It is now thought that these exceptions to the general code are later evolutionary developments; that is, at no single time was the code immutably fixed, although massive changes were not tolerated once a general code began to function early in evolution.
Discuss the different methods utilized for viral detection
Methods for viral detection 1. Tissue culture: A wide variety of tissue cells can be utilized for tissue culture. Viral specimens are inoculated into cells and placed in the incubator, and cells are then monitored for morphological changes over a period of days/weeks. The visible morphological effect viruses have on cells is called the cytopathic effect (CPE); the CPE is often characteristic for a particular viral group. CPE's are clinically significant for diagnoses. 2. PCR: Polymerase chain reaction amplifies small quantities of DNA that can be used for diagnoses. Expensive. 3. Serology: Serum is evaluated for the presence of viral antibodies. Many blood panels exist to screen for viruses based on symptoms (respiratory viruses, gastrointestinal viruses etc). 4. Direct fluorescent microscopy: A fluorescent stain is used directly on infected tissue; a positive reaction confirms viral presence. 5. Electron microscopy: Used to examine tissue directly, electron microscopes reveal great detail of minute biological structures. EM exam tissue directly and look for virus directly. Expensive.
What is PCR? How is it functionally useful when looking for gene sequences?
Methods of Performing Basic Research with DNA Southern Blot: used in DNA "fingerprinting" to compare DNA from a crime scene with that of a suspect. Polymerase chain reaction (PCR): Takes a small fragment of DNA and replicates it into billions of copies in a matter of hours. Good when only a small amount of DNA is present initially or when dealing with viruses, white blood cells, etc.
What is an obligate aerobe? What are some examples of obligate aerobes?
Must have oxygen to grow. M. tuberculosis
What is a point mutation? Give an example of a point mutation that was discussed during lecture. Is this considered a "good" or "bad" mutation? Why?
Mutations vary and can be classified by the following: 1. Point mutations: A single base substitution. Missense: Results in an amino acid substitution. Nonsense: Results in stop codon formation. 2. Frameshift mutations: The deletion or insertion of base pairs. 3. Spontaneous mutations: Mutations that occur in the absence of any agent; may be the result of mutagens.
Know the following DNA and RNA viral names and examples of viral types found on page 81 of the lecture packet: Adenoviridae
Named after the adenoids; a common cause of the common cold and some GI distress.
What are intracellular obligate parasites?
Obligate intracellular parasites, meaning that they can only replicate inside a living host as they lack enzymes necessary for metabolism. (Nucleic acid, protein, and ATP synthesis).
Okazaki fragments are short sequences of DNA nucleotides which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA replication.
Okazaki fragments are short sequences of DNA nucleotides (approximately 150 to 200 base pairs long in eukaryotes) which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA replication.
What are Okazaki fragments?
Okazaki fragments are short sequences of DNA nucleotides which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA replication.
What is facultative anaerobe? Do they contain simple or complex enzymatic systems? Why? What are some examples of facultative anaerobes?
Ordinarily carry on aerobic metabolism when oxygen is present, but shift to anaerobic metabolism when oxygen is absent. Contain complex enzyme systems.
Why do obligaete anaerobes die when introduced to O2?
Oxygen has the capacity to form free radicals, which can be damaging to obligate aerobes and humans alike.
How does chromosomal shape differ in eukaryotes vs. prokaryotes?
Packaging Prokaryotes and eukaryotes package their DNA molecules with protein in structures called chromosomes. A prokaryotic chromosome is circular and resides in a cell region called the nucleoid. The types of proteins found in prokaryotic chromosomes, known as the nucleoid-associated proteins, differ from the histone proteins that appear in eukaryotic chromosomes and cause the prokaryotic chromosomes to form looped structures. Unique eukaryotic chromosome packaging features include tight coiling, dense packing, enclosure within a nuclear membrane and linear rather than circular structures.
What do the terms F + and F ¯ refer to?
Plasmids are circular rings of extrachromosomal DNA that are not essential for survival. (F= fertility. F + cells contain F plasmids; F¯ cells do not.)
Which three enzymes are utilized in constructing the leading and laggings strands? What is their function?
Primase Polymerises nucleotide triphosphates in a 5' to 3' direction. The enzyme synthesises RNA primers to act as a template for future Okazaki fragments to build on to. DNA Polymerase III In charge of synthesizing nucleotides onto the leading end in the classic 5' to 3' direction. DNA Polymerase I In charge of synthesizing nucleotides onto primers on the lagging strand, forming Okazaki fragments. However, this enzyme cannot completely synthesize all of the nucleotides.
What are prions? What disease are they responsible for in humans? Cows?
Prion is short for: proteinaceous infectious particle. Composed solely of proteins; does not contain RNA, DNA, or a plasma membrane. Resistant to heat, radiation, and enzymes; sensitive to protein denaturing agents. Prions were first discovered in sheep with "Scrapie," progressive neurological degeneration occurs; scrapie infected sheep were fed to cattle....and "mad cow" disease resulted. Consumption of infected cattle results in Crutzfeldt-Jakob disease.
What enzyme binds to the start site on DNA? What is the function of this enzyme?
RNA polymerase binds to the promoter (start site) site on DNA. (Occurs on the anti sense only strand due to the 5′-3′ direction of mRNA production). RNA polymerase assembles nucleotides complementary to the triplets of DNA into an mRNA strand; composed of codons. RNA polymerase continues to assemble the mRNA strand until the stop site (found in DNA) is reached.
What is genetic engineering? Recombination? Recombinant DNA?
Recombination refers to the combining of genes (DNA) from two different cells, which ultimately increases genetic diversity. Insertion of DNA from one species to another produces recombinant DNA. The three main mechanisms by which bacteria acquire new DNA are transformation, conjugation, and transduction.
Do all virions kill host cells when they exit? Why or why not? Can some be latent? How long can latency last?
Replication characteristics of animal viruses The infection pattern of viruses varies, however the following characterizes the majority of animal viruses. 1. Virions are assembled and released from the host cell in a manner that results in host cell death. 2. Virions are assembled and released from the host cell in a controlled manner that results in host cell survival. 3. Viral genomes insert into host DNA, and are latent. Latency can last from months to a lifetime. (Varicella-Zoster)
What are restriction enzymes? How do they function? Are they important molecules in the biotechnology industry?
Restriction enzymes are enzymes that are able to cut DNA at specific sites by recognizing nucleotide sequences. Restriction enzymes are essential; they are the tools upon which the advances of molecular biology and the biotechnology industry depend
What is the function of the ribosome? What two subunits comprise prokaryotic ribosomes?
Ribosomes consist of two major components: the small ribosomal subunits, which read the RNA, and the large subunits, which join amino acids to form a polypeptide chain.
What are spontaneous mutations?
Spontaneous mutations: Mutations that occur in the absence of any agent; may be the result of mutagens.
Describe the events that occur during the lag, log, stationary, and decline phase of growth.
Stages of growth: Once bacteria have adjusted to a medium, they begin to grow in predictable stages: 1. The lag phase: Microbes increase in size and are metabolically active as the synthesis of DNA and enzymes occurs; little or no division. 2. The log phase: Microbes multiply at a logarithmic (exponential) rate; generation time is determined in this phase. Example: You have 10 bacterial cells, how many bacteria can there be in 4 hours if bacteria have a doubling time of 20 min? 12 doublings = 10 x 212 = 40, 960 cells 3. The stationary phase: Microbe population remains constant as the rate of cell death equals rate of growth; nutrients are limited and waste products increase. 4. The decline (death) phase: Microbe population decreases at a log rate as conditions in the medium decline; some organisms can remain viable for months or even years.
List and briefly describe three mechanisms for measuring cell growth.
Standard plate count: Measures Colony Forming Units (CFU's) or colonies of clones derived from a single bacterium. Plate out cells, give time for cell division, and then count number of clones. b) Turbidity check: Measures an increase in the "cloudiness" of broth which correlates with increased growth (but cannot distinguish live from dead bacteria). Best for measuring growth via a spectrophotometer without disturbing the culture. c) Indirect measures: Measure the metabolic activity of a cell population by quantitating end products of specific metabolic pathways. (Detecting gas production via an inverted tube, measuring acid production via pH indicators, etc). Bacteria in the body are rarely fully viable - many have ceased growing due to immune response, lack of nutrients, etc. Bacteria that are the most efficient in growth and living are selected for by the environment.
Describe the effects of a temperate phage
Temperate phage: After infection, phage synthesis does not occur; prophages exist without replicating and destroying the bacterial cell. (Lysogenic cycle) May enter the lytic cycle eventually.
What occurs during general transduction? Specialized transduction?
Temperate phages can replicate themselves either as a prophage in a bacterial chromosome or independently by assembling into new phages. Transduction occurs when some bacterial DNA is packed into the heads of bacteriophages. Bacterial or host DNA can be transferred via: General transduction: Any host genes can be transferred. Specialized transduction: Select host genes are transferred.
Describe the function of tRNA; what shape does it resemble? What is the anticodon? How does the anticodon interact with an mRNA strand? Is it random?
The anticodon of a given tRNA can bind to one or a few specific mRNA codons. The tRNA molecule also carries an amino acid: specifically, the one encoded by the codons that the tRNA binds. The two ends of a strand of DNA or RNA are different from each other. That is, a DNA or RNA molecule has directionality.
What functions to proofread growing DNA strands, DNA ligase, DNA polymerase, or RNA primase?
The function of proofreading is correction of errors such as mismarched bases. Primase Polymerises nucleotide triphosphates in a 5' to 3' direction. The enzyme synthesises RNA primers to act as a template for future Okazaki fragments to build on to. DNA Polymerase III In charge of synthesizing nucleotides onto the leading end in the classic 5' to 3' direction. DNA Polymerase I In charge of synthesizing nucleotides onto primers on the lagging strand, forming Okazaki fragments. However, this enzyme cannot completely synthesize all of the nucleotides. Ligase This enzyme is in charge of "gluing" together Okazaki fragments, an area that DNA Pol I is unable to synthesize.
MacConcey agar
The inhibitory action of crystal violet on the frowth of gram-negaitve organisms allows the isolation of gram-negative bacteria. Incorporation of the carbohydrate lactose, bile salt, and the pH indicator neutral red permits differentiation of enteric bacteria on the basis of trheir ability to fermetn lactose. On this basis, enteric bacteria are separated into two groups: a. Coliform bacilli: produce acid as a result of lactose fermentation b. Desentery, typhoidm abd paratyphoid bacilli: are not lactose fermenters and therefore do not produce acid.
How is a strand of mRNA built from the following DNA strand? When does RNA polymerase stop DNA transcription? DNA strand / mRNA strand?
The mRNA strand attaches to a ribosome. 2. At the start codon (AUG-methionine), tRNA molecules align their anticodon accordingly. (tRNA molecules carry amino acids and have a sequence of three nitrogenous bases called an anticodon; Anticodons are complimentary to codons of an mRNA strand. What is the complementary anticodon to the AUG codon? 3. The ribosome moves down, and a second tRNA molecule attaches to the mRNA strand via complementary codon/anticodon interactions. AA1 and AA 2 form a peptide bond; the first tRNA molecule is free to pick up more amino acids and return to the mRNA strand. 4. The process continues until the stop codon is reached and protein synthesis is complete.
What is an operon? What structural genes and regulatory sites do they include? Describe how an increase of substrate affects the function of the repressor protein relative to the lac operon.
The operon model of gene expression illustrates both enzyme induction and repression in prokaryotes. An operon is a sequence of closely associated genes that regulate enzyme production. Operons include the following: 1. Structural genes which carry information for the synthesis of specific proteins such as enzymes. 2. Regulatory sites (promoter and operator). Promoter: Transcription start site. Operator: The region of DNA that repressor proteins bind to; found in between the promoter and structural genes.
How do amino acids join to one another to make a protein? In other words, what bonds are involved?
The ribosome moves down, and a second tRNA molecule attaches to the mRNA strand via complementary codon/anticodon interactions. AA1 and AA 2 form a peptide bond; the first tRNA molecule is free to pick up more amino acids and return to the mRNA strand. 4. The process continues until the stop codon is reached and protein synthesis is complete.
What is the biological significance of genetic engineering?
The term 'genetic engineering' stands for human alteration of the genetic code of an organism, so that its biosynthetic properties are changed. The major applications are for the industrial production of desired peptides or proteins, or to alter the biological capabilites of the organism.
Are viruses made of DNA? RNA? Can they be single stranded? Double stranded? Circular? Linear?
They can be made of DNA and RNA; single stranded or double stranded; linear or circular.
Mannitol salts agar
This medium contains a high salt concentration, 7.5 % NaCl, which is inhibitory to the growth of most, but not all bacteria other than the sytaphylococci. The medium also performs a differential function: it contains the carbohydrate mannitol, which some staphylococci are capable of fermenting, and phenol red, a pH indicator for detecting acid produced by mannitol-fermenting staphylococci.
Blood agar
This medium is enricheed by blood for the cultivation of fastidious orfanisms such as the Streptococcus spp. The blood also permits demonstration of the hemolytic properties of some microorganisms, particularlythe strptococci, whose hemolytic activities are calssified as follows: 1. Gamma hemolysis: no lysis of red blood dells resultws in no significant change in the appearance of the medium sorrounding the colonies. 2. Alpha hemolysis: incomplete lysis of red blood cells, with reduction of hemoglobin to nethenooglobin, resulrs in a greenish halo around the bacterial growth. 3. Beta hemolysis: lysis of red blood cells.Result cles zone around colonies.
Phenyletyl alcohol agar
This medium is used for the isolation of most gram-positive organisms. The phenylethyl alcohol is partially inhibitory to gram-negative organisms, which may form visible colonies whose size and number are much smaller. It reduces the growth of E. coli and selects for S. aureus.
What are the two steps of protein synthesis? Where do these steps occur in prokaryotes? Eukaryotes?
Transcription is the process of forming a strand of RNA from a DNA template, and is the first step of protein synthesis. b) Steps that occur in transcription are the following: 1. DNA unwinds, separates, and is stabilized via assorted proteins. 2. RNA polymerase binds to the promoter (start site) site on DNA. (Occurs on the anti sense only strand due to the 5′-3′ direction of mRNA production). 3. RNA polymerase assembles nucleotides complementary to the triplets of DNA into an mRNA strand; composed of codons. 4. RNA polymerase continues to assemble the mRNA strand until the stop site (found in DNA) is reached. 5. The mRNA strand detaches from the DNA. c) Steps that occur in translation are the following: 1. The mRNA strand attaches to a ribosome. 2. At the start codon (AUG-methionine), tRNA molecules align their anticodon accordingly. (tRNA molecules carry amino acids and have a sequence of three nitrogenous bases called an anticodon; Anticodons are complimentary to codons of an mRNA strand. What is the complementary anticodon to the AUG codon? 3. The ribosome moves down, and a second tRNA molecule attaches to the mRNA strand via complementary codon/anticodon interactions. AA1 and AA 2 form a peptide bond; the first tRNA molecule is free to pick up more amino acids and return to the mRNA strand. 4. The process continues until the stop codon is reached and protein synthesis is complete. In prokaryotes transcription and translation occur in the cytoplasm. In a eukaryotic cell, transcription occurs in the nucleus, and translation occurs in the cytoplasm.
What is the biological significance of transduction?
Transduction. Transduction, a process of genetic recombination in bacteria in which genes from a host cell (a bacterium) are incorporated into the genome of a bacterial virus (bacteriophage) and then carried to another host cell when the bacteriophage initiates another cycle of infection.
What is transduction? Who demonstrated this phenomenon? What are bacteriophages? Prophages?
Transduction: The transfer of genetic information by bacteriophages. Lederberg and Zinder's experiments with Salmonella demonstrated the phenomenon of transduction. Bacteriophages (phages) are viruses that infect bacteria. Once inside of a bacterium, phage DNA is incorporated into the genome and is referred to as a prophage. Phages may be either virulent or temperate in nature.
What is the biological significance of transformation?
Transformation. Transformation, in biology, one of several processes by which genetic material in the form of "naked" deoxyribonucleic acid (DNA) is transferred between microbial cells. Its discovery and elucidation constitutes one of the significant cornerstones of molecular genetics.
What is transformation? Who demonstrated this phenomenon? What is naked DNA?
Transformation: The uptake of naked DNA via competent bacteria. Griffith's classic experiment with pneumococci and mice illustrated the phenomenon of transformation. (Smooth vs. Rough capsule) Naked DNA refers to DNA that has been released via cell lysis and is cleaved into pieces. Competent refers to the fact that a bacterium is capable of DNA fragment uptake. In nature this probably occurs following the breakdown of dead organisms in an environment where live ones of the same or closely related species are present. Not all bacteria are competent and thus capable of transformation; factors such as cell wall modification and the formation of specific receptor sites on the plasma membrane dictate whether a bacterium is competent.
TSA media
Tryptic Soy Agar (TSA) is recommended for use as a general growth medium for the isolation and cultivation of microorganisms. Tryptic Soy Agar supports the growth of nonfastidious as well as moderately fastidious microorganisms. This medium is recommended for use in the cultivation, storage, maintenance and transportation of pure cultures of microorganisms.
What are the components of a virion?
Viral components a) Virions The combination of the nucleic acid core, capsid, and envelope (if one is present) is referred to as a virion. b) Viral genomes and capsids Viruses contain a nucleic acid core of either DNA or RNA. Viral genomes can be single or double stranded; circular or linear. A protein coat called a capsid surrounds the viral genome; each capsid is composed of protein subunits called capsomeres. Capsomeres often determine viral shape; most are either polyhedral (many sided) or helical (spiral staircase). Capsomeres are commonly utilized for viral identification and classification. c) Envelopes Viruses may be enveloped or nonenveloped (naked). Composed of a typical bilayer of phospholipids, proteins, and carbohydrates. Glycoprotein "spikes" can project from the envelope; serve as attachment points for viruses to host cell surfaces. Envelopes facilitate viral entry and "invisibility" once inside host cells; however are vulnerable to environmental conditions that destroy membranes. (Temperature, pH, disinfectants).
Describe the effects of a virulent phage
Virulent phage: After infection, phage synthesis occurs and results in the death of the cell as progeny are released. (Lytic phase)
How do viruses cause cancer? What genes are disrupted? List some cancers and the viral agents responsible for their manifestation.
Viruses and cancer a) Genetic mutations are the basis of all cancers. Viral insertion into the host genome disrupts proto-oncogenes and tumor suppressor genes. Proto-oncogenes can be activated into oncogenes; oncogenes stimulate cellular division. Tumor suppressor genes can be inactivated via viral insertion. Tumor suppressor genes inhibit cellular division; when mutated, tumor suppressor genes are not functional= stimulate cellular division. Note, genetic mutations can be induced via chemicals, radiation, or be inherited. b) Certain viruses have been implicated in particular cancers. Human papilloma virus: Cervical cancer Hepatitis B virus: Liver cancer Epstein-Barr virus: Burkitt's lymphoma; Hodgkin's disease; MS?
What type of bond holds together the double helix? What is the triplet code?
Weak hydrogen bonds. Triplet code: the standard version of the genetic code, in which a sequence of three nucleotides on a DNA or RNA molecule codes for a specific amino acid in protein synthesis.
What is an obligate anaerobe? What are some examples of obligate anaerobes?
Will die if oxygen is present, ussually killed via superoxide free radicals. C botulinum, tetanus.
Aren't objective questions cool?
Yes, they are.
Define the terms genotype and phenotype. Does a change in genotype always result in a change of phenotype?
a) The term genotype refers to the genetic information contained in the DNA of an organism; the genetic composition. b) The term phenotype refers to the physical characteristics of an organism determined via gene expression. In humans, variations of hair and eye color represent different phenotypes; in microbes, it manifests as proteins found in the cell wall.
What is the generation time? Is it the same for all bacteria? What factors influence the generation time? What is a typical generation time for most bacteria?
c) The time it takes for bacteria to divide is the genetically predetermined generation time, which varies with relation to environmental conditions such as temperature, pH, and availability of nutrients etc. d) The generation time for most bacteria is between 20 minutes and 20 hours, and is typically less than one hour! Growth is in a logarithmic (exponential) progression, and the population of microbes doubles in each generation time. With such rapid division, a microbe can infect a host very quickly.
Know all of the " Diseases of the Cardiovascular, Lymphatic, & Respiratory Systems" causative agents and their basic characteristics, the diseases that they cause, symptoms and most common mode of transmission relative to the chart that you filled out
in the table
What are the three types of RNA? What is the function of rRNA?
mRNA or Messenger RNA mRNA transcribes the genetic code from DNA into a form that can be read and used to make proteins. mRNA carries genetic information from the nucleus to the cytoplasm of a cell. rRNA or Ribosomal RNA rRNA is located in the cytoplasm of a cell, where ribosomes are found. rRNA directs the translation of mRNA into proteins. tRNA or Transfer RNA Like rRNA, tRNA is located in the cellular cytoplasm and is involved in protein synthesis. Transfer RNA brings or transfers amino acids to the ribosome that corresponds to each three-nucleotide codon of rRNA. The amino acids then can be joined together and processed to make polypeptides and proteins.
