Name three types of mutations that can occur spontaneously.

-A change in nucleotide or nucleotide sequence that may not lead to any observable change is called a silent mutation.

-The change in a cell's genotype and phenotype to its orignial state through a change in the mutated gene is termed reversion.

- Mutations would be observed much more frequently than they are if it were not for several different repair mechanisms that all cells possess.

Define the event base substitution.

Base substitution - A mistake during DNA synthesis when an incorrect base is incorporated into the DNA.

Name three types of mutations that can result from base substitutions.

Point mutation - Mutation in which only a single base pair is involved.

Missense mutation - A point mutation which results in the substitution of a different amino acid being incorporated into the protein coded by the gene.

Nonsense mutation - A mutation which generates a stop codon, resulting in a shortened protein.

How would the growth requirements change in a cell that has a silent mutation in a gene for histidine synthesis?

It wouldn't change the growth requirements because the mutation is silent meaning the mutation can be seen in the genotype but is not expressed in the phenotype.

How would it change if the mutation were a knockout?

It would cause a frame shift mutation, which would be deadly to the cell.

Would the addition of three bases to DNA always lead to a frameshift mutation? Explain your answer.

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Name four mechanisms by which mutagens act on DNA.

Chemical Mutagen:

Base analogs - Incorporates in place of normal nucleotide in DNA.

Intercalating Agent - Inserts between base pairs in either the template or new strand.

Chemical modification of bases - Converts amino group to keto group in adenine and cytosine.

Transposons - Random insertion into any gene.

Describe the effect of UV light on DNA.

Ultraviolet Irradiation - Intrastrand thymine dimer formation. This distorts the shape of the DNA and prevents replication past the dimer.

How does UV light affect cells?

The major mutagenic action of UV light, results from the cell's attempt to repair the damage by a mechanism termed SOS repair.

If you wished to isolate a point mutant, what mutagen would you use? To isolate a knockout mutant?

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Do you think that mutations caused by reactive oxygen should be considered spontaneous or induced mutations? Justify your answer.

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Explain how DNA polymerase can correct base substitutions and prevent misincorporation of nucleotides.

DNA polymerase has a proofreading function.

In mismatch repair, an endonuclease cuts out the damaged single-stranded fragment and a new DNA strand is synthesized.

Explain the three mechanisms by which UV light damage can be repaired.

In light repair, a light-requiring enzyme breaks the two covalent bonds (photoreactivation)

In dark, or excision, repair the single strand of DNA containing the thymine dimer is removed and destroyed. The newly synthesized strand is joined to the end of the original strand by the enzyme DNA ligase. Light is not required, the newly synthesized strand is not methylated immediately after synthesis.

Explain how a mutation caused by reactive oxygen is repaired.

DNA synthesis by a new DNA polymerase bypasses damaged DNA. The cell survives but numerous mutations are generated.

Distinguish between light and dark repair of thymine dimers.

Light repair (Photoreactivation) - breaking of covalent bond forming thymine molecules.

Dark repair (Excision repair) - Excision of a short stretch of single-stranded DNA containing thymine dimer and synthesis of a new strand by DNA polymerase.

Give an example of a mutagen that causes a major distortion in DNA; a minor distortion.

Major distortions:X-rays causing single and double stranded breaks in DNA, chemical modification of purines and pyrimidines such as nitrous acid.

Minor distortions:

If you wish to maximize the number of mutations following UV irradiation, should you incubate the irradiated cells in the light or in the dark, or does it make any difference? Explain your answer.

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Explain how mutations relate to natural selection.

Mutations provide a mechanism by which organisms, with their altered characteristics, can respond to a changing environment.

If the rate of mutation to streptomycin resistance is 10^-6 and to penicillin resistance is 10^-4, what is the rate of mutation to simultaneous resistance to both antibiotics?

The rate of resistance is independent of one another:

So the probability of simultaneous resistance to both antibiotics is 10^-4 x 10 ^-6 , or 10 ^-10 .

Is it as effective to take two antibiotics sequentially as it is to take them simultaneously, as long as the total length of time that they are both taken is the same? Explain.

Combination therapy - is when two or more drugs are administered simultaneously in the treatment of disease. Any mutant cell or virus resistant to one antimicrobial medication is likely to be sensitive to the other and therefore will be killed by the combination of the two antimicrobials.

Distinguish between direct and indirect selection and how mutant of each type are selected.

Direct selection - involves inoculating cells onto a medium on which the mutant but not the parent can grow; these are the easiest kinds of mutants to isolate.

Indirect selection - is required when the desired mutant does not grow on a medium on which the parent grows.

Distinguish between the kinds of mutants that can be isolated by direct and indirect selection.

Mutants resistant to antimicrobials are usually very easy to isolate by direct selection.

In replica testing a technique is used to indirectly select auxotrophic mutants.

When does penicillin enrichment not work?

Penicillin only kills "growing cells" - most of the cells that have no growth factor requirements will grow and so will be killed, while the non-multiplying auxotrophs will survive.

The Ames test measures the reversion of a mutant gene. Would it be just as good to test for the generation of a mutant gene rather than its reversion? Explain.

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How could you demonstrate by replica plating that the environment selects but does not mutate genes in bacteria?

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Describe the process of DNA-mediated transformation.

The donor DNA comes from a cell that is streptomycin resistant (Str ^R). The recipient cell is streptomycin sensitive (Str^s ).

What effect would adding deoxyribonuclease to the culture have on transformation?

The nuclease will cleave the cell's single-stranded DNA on either side of the donor DNA. This fragment of DNA is released into the cytoplasm where it is degraded by nucleases.

If cells do not become naturally competent, can they still be made to take up DNA? Explain.

Through Electroporation the electric current makes holes in both the cell wall and cytoplasmic membrane through wihch the double-stranded DNA can pass. These holes are then repaired by the cell, and the DNA becomes incorporated into the chromosome of the cell. pg 207

Can you devise a test using DNA-mediated transformation that could test chemicals for their mutagenic activity?

Purified DNA containing a selective marker is treated with the suspected mutagen. The DNA is used then to transform cells along with untreated DNA. The frequency of transformation is compared in the two cases. If the chemical damages the DNA and by inference is a mutagen, the transformation frequency will be lower when the treated DNA is used as the donor.

Explain how dead cells confer virulence on avirulent living cells.

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Describe the process of bacterial gene transfer by transduction.

Any piece of the chromosomal DNA of the donor cell can be transferred in this process. All of the DNA molecules of the bacterial virus and the bacteria are double-stranded. pg.208 fig.8-21

Distinguish between generalized and specialized transduction.

In generalized transduction any gene of the donor cell can be transferred.

In specialized transduction - only a few specific genes can be transferred.

What effect would the addition of deoxyribonuclease to the donor cells have on this process?

Deoxyribonuclease cleaves the replicated phage DNA into the proper size fragments to be enclosed in the coat protein of the phage.

However, it also cleaves the bacterial chromosome into fragments, which also can become surrounded by the phage coat, taking the place of phage DNA.

Two genes are transduced simultaneously. What does this suggest about the location of the two genes relative to each other? Explain.

They must be close together on the genome because the virus can only package a small piece of the genome.

Describe the properties of resistance plasmids which make them so much of a problem in a hospital environment.

Plasmids confer resistance to many anti-microbial medications

and heavy metals found in the hospital environment.

Explain the ways in which plasmids differ from bacterial chromosomes.

Bacterial Chromosome

1. It is linear in shape
2. They have thousands of genes
3. They have a centromere and two sister chromatids
4. It is covered with protein
5. Size: It is very large in size usually expressed in mega units.
6. They cannot be used as gene carriers
7. Function: They carry genetic information

Plasmid

1. It is circular in shape

2.Gene number is relatively smaller

3.They do not have a centromere and chromatids

4.It is not covered with protein

5.1.0 kb to 200 kb or greater (kb-kilobase pairs)

6.They can be used as gene carries (vectors) to carry foreign DNA into host cell. Used in genetic engineering

7.They are involved in bacterial conjugation and production of antibiotics

What single function must all plasmids encode?

Code for resistance to various antimicrobials, plasmid replication, and plasmid transfer.

What functions must a plasmid code for in order to be self-transmissible?

The Resistance Transfer Factor portion contains genes that code for pilus synthesis, the origin of transfer, and other mobilization genes, all of which are required for plasmid transfer.

What does the phrase, "reservoir for R plasmids" mean when referring to plasmids carried by non-disease-causing bacteria?

"Healthy people" act as the incubator of non-disease causing bacteria with R-plasmids, which then can be transferred to disease-causing organisms.

If a cell lost all its plasmids, would it be able to survive? Explain.

In the short-term yes, but over time the lack of genetic adaptation to the environment will lead to its death.

Compare an F⁺ to an F^- cell in terms of their morphology and gene content

An F⁺ cell is the donor cell, which contains the F plasmid (fertility capability). The F plasmid codes for the sex pilus (protein appendage). Donor cells can transfer their F plasmid, but not their chromosome into recipient cells.

The F^- cell does not have any of this.

Compare the state of the F plasmid in a cell that transfers only plasmids with a cell that can transfer chromosomal DNA, and with a cell that transfers both plasmid and chromosomal DNA.

Plasmid transfer - Cell-to-cell contact required, entire plasmid transfered.

Chromosome transfer - Cell-to-cell contact required; only certain cells can be donors (Hfr). Variable fraction of chromosome is transferred, and no sensitivity to DNAase.

The F' plasmid has the transfer properties of the F plasmid but carries chromosomal DNA. This process is reversible.

What two transfer functions does the F plasmid encode?

The F plasmid codes for synthesizing a sex pilus

Explain why DNA is transferred in only one direction in conjugation.

The "donor" cell contains the F plasmid, where as the "recipient" cell does not. So the flow of transfer is in one direction, in a polar fashion.

Would you expect conjugation to be more efficient if cells were plated together on solid medium (agar) or mixed together in a liquid shaking flask? Explain.

Probably the solid medium, because it will be easier for the pilus to attach to the recipient cell.

Why must chromosomal (but not plasmid) DNA that is transferred be integrated into the chromosome of the recipient cell? Explain.

Unlike plasmids, the piece of DNA that is transferred is not a replicon and therefore, in order to replicate, the fragment must integrate into the chromosome of the recipient cell through homologous recombination.

Explain how transposable elements contribute to the spread of antibiotic resistance.

Transposable elements often transfer antimicrobial-resistance genes from a chromosome to a plasmid. Plasmids can be readily transferred by conjugation to unrelated bacteria.

Describe the feature that is common to all transposable elements.

The sequence in one strand of DNA is the same as the other strand, only going in the opposite direction. Such a sequence is termed a palindrome or inverted repeat.

What are two features common to all insertion sequences?

An insertion sequence contains a gene that codes for the enzyme transposase.

A composite transposon consists of one or more genes flanked by insertion sequences.

How does transposition promote gene transfer between bacteria?

The composite transposon, can move from one replicon to another in the cell and become integrated through non homologous recombination, and can be readily transferred to other cells.

What would you think is the substrate for the transposase enzyme?

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