CHAPTER 6 QUESTIONS:
- During cell division in eukaryotic cells (mitosis), the mitotic spindle makes sure each daughter cell gets a copy of each chromosome. How is this accomplished in binary fission?
In binary fission everything in the cell is duplicated and then the cell divides into two, separating all of the doubles of the cell contents.
- Which type of microbe mostly utilizes budding for reproduction?
Yeast generally utilizes budding for reproduction.
- How does the rate of growth of microbes differ in each phase: lag, log, stationary, decline?
The lag phase is the first of the phases and it is the portion where the organisms grow in size, but not in numbers. The Log phase is the second portion where cells grow and divide at an exponential or logarithmic rate. The stationary phase is the third phase where new cells reproduce at the same rate that old cells die (resulting in a flat line because cell numbers remain the same). The death/decline phase is the fourth and final phase of the bacterial growth curve where cells lose the ability to divide and then die.
- What is the difference between synchronous growth and nonsynchronous growth?
Synchronous growth is a hypothetical pattern of growth during log phase where all of the cells in a culture divide at the same time. Nonsynchronous growth is a natural pattern for growth during the log phase where every cell in a culture divides at some point during the generation time, but not simultaneously.
- You want to calculate the concentration of microbes in a sample so you perform a serial dilution to dilute the sample by a factor of 103 and then plate it. It forms 15 colonies. What should you do next?
You need to re-do the serial dilution because you want a plate count of between 30 and 300 bacterial colonies.
- If you dilute the sample above by a factor of 102 you achieve a plate count of 213 colonies. What is the concentration of the original sample?
The concentration of the original sample is 21300 colonies or 2.13x104 CFU.
- Why is it important to shake a suspended culture prior to removing a sample to dilute, plate, or count it?
It is important to shake a suspended culture to ensure an even distribution of the bacteria suspended in the media.
- If you added 20 mµ (microliters) of sample to a hemocytometer and observe the number of cells indicated in each chamber, what is your total cell count per mililiter?
You average each of the corners, so (13+14+10+14)/4= 12.75
- A technician performs the MPN on a water sample and obtains the following results: five turbid samples at 10ml (101 dilution), 4 turbid samples at 102 dilution, and one turbid sample at 103 dilution. What is the most probable concentration of microorganisms being tested for in the water sample? (Hint: use page 154 in your book)
- What are seven physical factors that must be considered when culturing bacteria?
Seven physical factors that must be considered when culturing bacteria are: pH, Temperature, Oxygen, Hydrostatic Pressure, Osmotic pressure, Moisture, and Radiation.
- What are five nutritional factors that must be considered when culturing bacteria?
Five nutritional factors that must be considered when culturing bacteria are: Carbon sources, nitrogen sources, Sulfur and phosphorus, trace elements, and vitamins.
- What is the difference between an obligate organism and a facultative one?
An obligate organism must have the specified environmental condition. A facultative organism means that the organism can adjust to and tolerate the environmental condition, but it can also live in other conditions.
- What are exoenzymes?
Exoenzymes are enzymes that are produced for use outside of the cell. These include extracellular enzymes (act in the medium around the organism) and periplasmic enzymes (act in the periplasmic space). Most exoenzymes are hydrolases that add water as they split large molecules into smaller ones. They are often the first step in digestion for some of the bacteria (before bringing smaller molecules into the cell for use).
- What is the purpose of sporulation?
The purpose of sporulation is to preserve the cell when the environment is not suitable to the bacteria (too hot, not enough food, too acidic, etc.)
15. Describe each step of the sporulation cycle.
1. DNA is replicated and forms a long, compact, axial nucleoid.
2. The core of the spore is made my RNA and protein molecules gathering around the DNA. (The core is the living part of a spore.)
3. An endospore septum grows around the spore, enclosing it in a double-thickness cell membrane (does not have a cell wall).
4. A laminated layer (cortex) is formed, protecting it from changes in osmotic pressure.
5. A spore coat is laid down (protects from many different chemicals) around the cortex by the mother cell.
6. In some spores an exosporium (a lipid-protein membrane) is formed outside the spore coat by the mother cell.
- When is it better to use the pour plate method than the streak plate method?
It is better to use to pour plate method on microaerophiles that cannot tolerate exposure to oxygen in the air at the surface of the medium.
- What is the difference between defined media and complex media? If you add blood serum to a medium what type would it be?
Defined media: is a medium that contains known specific quantities and compositions of substances.
Complex media: medium containing well-defined materials, but whose chemical composition varies slightly.
If blood serum is added to a medium it becomes a blood or chocolate agar.
- Compare and contrast selective media, differential media, and enrichment media.
Selective media: a medium that encourages growth of some organisms and suppresses the growth of others.
Differential media: growth medium with a constituent that causes an obvious change in color in the medium when a particular chemical is released.
Enrichment media: medium that contains special nutrients that allow the growth of a particular organism.
- What are stock cultures, preserved cultures, and reference cultures?
Stock cultures are reserve cultures used to store an isolated organism in pure condition for use in the laboratory. Preserved cultures are cultures where the organisms are maintained in a dormant state. Reference cultures are preserved cultures used to maintain an organism with its characteristics as originally defined.
- What is lyophilization?
Lyophilization is regularly used to preserve vaccines, pharmaceuticals, and other proteins. Freeze-drying is also used to preserve special food products, eliminating the need for refrigeration. Freeze-dried food is eaten by mountain climbers and astronauts. Lyophilization is used by botanists to preserve flower samples indefinitely. Because the process of freeze-drying removes most of the water from the sample, freeze-dried materials become highly absorbent, and merely adding water can restore the sample to something close to its original state.
CHAPTER 7 QUESTIONS:
- What is the difference between a chromosome and a plasmid?
A chromosome is a threadlike molecule of DNA that contains all of the information needed for an organism to survive and reproduce. A plasmid is a circular piece of DNA that only contains nonessential genes.
- What is unique about the chromosomes of Vibrio cholerae and Deinoccocus radiodurans?
Deinoccocus radiodurans is resistant to radiation and has two chromosomes. Vibrio cholera has two circular chromosomes (one large and one small).
- Which microbe was the first to have its genome completely sequenced? When?
The first microbe to have its genome completely sequenced was Haemophilus Influenzae. It’s sequence was published in “Science” in 1995.
- What is special about the genome of retroviruses? Why must they possess the gene for reverse transcriptase?
The genome of retroviruses is unique because it can make DNA from the virus’s RNA by using reverse transcription (use RNA to make DNA). Reverse transcriptase is required because it needed in the process of reverse transcription.
- Why is DNA synthesis said to be “semiconservative”?
DNA synthesis is said to be “semiconservative” because it makes a new strand of DNA that is complementary to the existing one. Therefore each new double-stranded DNA has one old strand (the template) and one newly synthesized complementary strand.
- What role do DNA polymerase, DNA primase (a type of RNA polymerase), helicase, topoisomerase, RNase H, and ligase play in DNA replication?
DNA polymerase: slides along the leading strand in the 3’à5’ direction synthesizing the matching strand in the 5’à 3’ direction.
DNA primase: creates a single RNA primer to start replication
Helicase: breaks the hydrogen bonds
Topoisomerase: unwinds the DNA
RNAse H: degrades the RNA primers
Ligase: enzyme that catalyzes the connection of two Okazaki fragments.
- What is the difference between how the leading strand and lagging strand are copied during DNA replication? Why do they have to be synthesized differently in this fashion?
Leading strand: Topoisomerase unwinds DNA and helicase breaks the h-bonds. DNA primase creates a single RNA primer to start the replication. DNA polymerase slides along the leading strand in the 3’à5’ direction synthesizing the matching strand in the 5’à 3’ direction. The RNA primer is degraded my RNase H and replaced with DNA nucleotides by DNA polymerase, and then DNA ligase connects the fragment at the start of the new strand to the end of the new strand.
Lagging strand: Topoisomerase unwinds DNA and helicase breaks the h-bonds. DNA primase creates RNA primers at spaced intervals. DNA polymerase slides along the leading strand in the 3’à 5’ direction synthesizing matching Okazaki fragments in the 5’à 3’ direction. The RNA primers are degraded by RNase H and replaced with DNA nucleotides by the DNA polymerase. DNA ligase connects the Okazaki fragments to one another (covalent bond phosphate in one nucleotide to deoxyribose of the adjacent nucleotide.
The have to be synthesized in a different fashion because DNA can only be “read” in the 3’à 5’ direction.
- What would happen if insufficient RNase H were produced by a cell? What if insufficient ligase were produced by a cell?
If insufficient RNase H was produced by a cell there would be holes or gaps in the strand of DNA. If insufficient ligase was produced there would be “chips” missing out of the joined bases (especially noticeable in the lagging strand)
- Where do transcription and translation occur in prokaryotes and eukaryotes?
In prokaryotes: transcription and translation both occur in the cytoplasm.
In eukaryotes: transcription occurs in the cell nucleus and translation occurs in the cytoplasm.
- How does transcription in prokaryotes differ from eukaryotes?
In prokaryotes, transcription is done in the cytoplasm. In prokaryotes RNA polymerases are bound to different sigma factors (aka transcription factors) which as specific to different promoters.
In eukaryotes, transcription occurs in the cell nucleus. Eukaryotes must process the mRNA before it can leave the nucleus. Transcription factors assemble at the promoter forming a “transcription initiation complex.”
- What are four key differences between DNA polymerase and RNA polymerase? (“they are difference molecules” doesn’t count as one!)
DNA polymerase: can only add to the 3’ end, polymerase must jump to the end and work backwards, synthesizes new DNA stands complementary to the old strands, proof-reads the new strand.
RNA polymerase: binds to strand of DNA, catalyzes the synthesis of RNA, directs proteins synthesis
- Compare and contrast codons and anti-codons?
Codons are a sequence of 3-bases in mRNA that specifies a particular amino acid in protein synthesis.
Anti-codons are 3-base sequences in tRNA (complementary to one of 3 mRNA codons) forming a link between each codon and its amino acid.
- What is alternative splicing? Why is it necessary in eukaryotes?
Alternative splicing is a process by which the exons of the RNA produced by transcription of a gene (a primary gene transcript or pre-mRNA) are reconnected in multiple ways during RNA splicing. The resulting different mRNAs may be translated into different protein forms; thus, a single gene may code for multiple proteins
- What is an operon? In what ways is it similar to alternative splicing?
An operon is a sequence of closely associated genes that includes regulatory sites and structural genes that control transcription. This is similar to alternative splicing because they both control what genes are and are not needed.
- During translation, what amino acid sequence would the following mRNA segment be converted into: AUGGACAUUGAACCG?
The amino acid sequence would be converted into: met-asp-ile-glu-pro
- How come there are only 20 amino acids when there are 64 different codons?
There are 64 DNA codons (possible sequences of the 3-letter nucleotide bases A, U, C , and G) but only 20 possible amino acids because of the possibility of mutations that would replace one nucleotide base with another.
- How come prokaryotes can both transcribe and translate a gene at the same time, but eukaryotes cannot?
In prokaryotes, transcription and translation can occur at the same time because they both occur in the cytoplasm. This does not work for eukaryotes because transcription occurs in the nucleus, while translation occurs in the cytoplasm.
- Which regulatory mechanisms occur at the DNA-level, which occur at the protein-level?
DNA-level: transcribing only the genes that are needed by the cell, genetic regulation (turning on and off genes to be transcribed), regulatory sites (DNA sequences involved in enabling and disabling structural genes)
Protein-Level: Feedback inhibition,
- What is feedback inhibition?
Feedback inhibition is the regulation of a metabolic pathway by the concentration of one of its intermediates or its end-product. The end-product of a chemical pathway inhibits the first enzyme in that chemical pathway.
- What are the two types of DNA modifications that block transcription of a gene?
DNA modified to block transcription is either too tightly condensed for the enzymes to get close to the bases OR the DNA is shielded by chemical groups (i.e. methylation).
- What is the difference between a repressor and an activator and how can each be affected by an inducer?
Repressors may also be used by binding to the regulatory sites blocking the attachment of other transcription factors or RNA polymerase. Repressors are controlled by inducers.
Activators turn on genes by binding to regulatory sites near and/or far from the promoter. Activators are also controlled by inducers (i.e. inducer binds to repressor, inactivating it, therefore allowing the activator to bind and turn on the gene).
Inducers are substances that bind to and inactivate a repressor enzyme.
- What is an enhancer and how does it help control how much of a particular protein is made?
“Enhancers” are specific DNA sequences associated with a gene that will only respond to the presence of specific signals to cause activation of that gene’s promoter.
- How does the presence of lactose control the production of lactase?
In the LAC operon the presence of lactose acts as the inducer to remove the repressor to turn on the genes to break-down lactose in order to use it as energy.
- What is the difference between spontaneous mutations and induced mutations? What type are caused by ultraviolet radiation?
Spontaneous mutations are innate mutations incurred during DNA replication.
Induced mutations are caused by mutagens (i.e. chemicals or radiation, so UV radiation is an induced mutation).
- How accurate is DNA replication? (That is, how many errors typically result per nucleotide? Per gene?)
DNA replication is very accurate. Errors per nucleotide are 1:100,000. After standard repairs error per nucleotide is approximately 1:10 billion. Errors per gene are between 1:10-3 and 1:10-9.
- What type of mutation is shown here? AGTGCCGTCAC
TCACGGCCAGTG
This is a frame-shift mutation.
- Why are addition and deletion mutations typically more harmful than substitution mutations?
Addition and deletion mutations are typically more harmful than substitution mutations because a deletion or insertion of one of more bases in DNA changes entire sequences of codons and greatly alters that amino acid sequence. It can introduce a terminator codon and produce useless polypeptides instead of normal proteins (needed to make certain processes in the cell run).
- Compare and contrast the four types of chromosomal mutations.
Chromosomal mutations can fall under two categories: breaks (which includes deletion, duplication, inversion, and translocation) and nondisjunction. Deletion causes a loss of genes. Duplication causes multiple copies of genes per chromosome (upregulation of proteins). Inversion can cause upregulation or downregulation of genes by placing them next to new promoter regions (switch or change direction of a gene). Translocation can cause upregulation or downregulation of genes by placing them next to new promoter regions (mix and match portions of a gene).
- Compare and contrast the four types of chemical mutagens listed in table 7.4.
Base analogs substitute “look-alike” molecules for the normal nitrogenous base during DNA replication (resulting in a point mutation). These can be caused by caffeine or 5-bromouracil.
Alkylating agents add an alkyl group (i.e. methyl group) to nitrogenous base, resulting in incorrect pairing (point mutation). These can be caused by nitrosoguanidine.
Deaminating agents remove an amino group from a nitrogenous base, resulting in a point mutation. These can be cause by nitrous acid, nitrates and nitrites.
Acridine derivatives insert into the DNA ladder between backbones to form a new rung, therefore distorting the helix and causing a frame shift mutation. These can be caused by acridine dyes and quinacrine.
- What is the difference between a mutagen and a carcinogen?
A mutagen is an agent that increases the rate of mutations, while a carcinogen is an agent that can cause cancer.
- Compare and contrast the ways high-energy radiation and lower-energy radiation affect DNA.
Lower-energy radiation (i.e. UV radiation) and link adjacent pyrimidines to each other and can impair replication. High-energy radiation (i.e. x-ray and gamma ray) can ionize and break molecules in cells to form free radicals, which in turn break up DNA.
- How would the results of a fluctuation test differ if the mutation you are looking at is induced rather than spontaneous?
The flucturation test is used to determine that resistance to chemical substances occurs spontaneously rather than being induced.
- What is the purpose of the Ames test? How is it performed?
The Ames test is a test used to determine whether a particular substance is mutagenic based on its ability to induce mutations in auxotrophic bacteria.
The bacteria being studied are spread onto an agar plate with a small amount of histidine. This small amount of histidine in the medium allows the bacteria to grow for an initial time and then have the opportunity to mutate. When the histidine is depleted only bacteria that have mutated to gain the ability to produce its own histidine will survive. The plate is incubated for 48 hours. The mutagenicity of a substance is proportional to the number of colonies observed.
- What is the purpose of PCR? How is it performed?
The purpose of PCR is to synthesize many copies of a segment of DNA from very small quantities of the source DNA.
First the unknown DNA is heated, causing the paired strands to separate leaving single strands now accessible to primers.
Next you add large excess of primers relative to the amount of DNA being amplified, and cool the reaction mixture to allow double-strands to form again and due to the fact that there is a large excess of primers, the two strands will always bind to the primers, instead of with each other.Then you add to a mixture of all 4 individual letters (aka nucleotides), add an enzyme which can "read" the opposing strand's "sentence" and extend the primer's "sentence" by "hooking" letters together in the order in which they pair across from one another - A:T and C:G. This particular enzyme is called a DNA polymerase (because makes DNA polymers). One such enzyme used in PCR is called Taq polymerase (originally isolated from a bacterium that can live in hot springs - therefore, can withstand the high temperature necessary for DNA-strand separation, and can be left in the reaction). Now, we have the enzyme synthesizing new DNA in opposite directions - BUT ONLY THIS PARTICULAR REGION OF DNA.
After one cycle, add more primers, add 4-letter mixture, and repeat the cycle. The primers will bind to the "old" sequences as well as to the newly-synthesized sequences. The enzyme will again extend primer sentences ... Finally, there will be PLENTY of DNA - and ALL OF IT will be copies of just this particular region. Therefore, by using different primers which represent flanking regions of different genes of various organisms in SEPARATE experiments, one can determine if in fact, any DNA has been amplified. If it has not, then the primers did not bind to the DNA of the sample, and it is therefore highly unlikely that the DNA of an organism which a given set of primers represents, is present. On the other hand, appearance of DNA by PCR will allow precise identification of the source of the amplified material. (http://people.ku.edu/~jbrown/pcr.html)
