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Bact. 303 Study Questions IV

University of Wisconsin - Madison

Bacterial Genetics


109. What is mutation? Distinguish between spontaneous and induced mutations. How frequently do spontaneous mutations occur? What natural environmental factors might cause mutations?

110. In a discussion of mutagenesis explain the molecular basis of mutation and identify several classes of mutagens and the types of mutations that they cause.

111. Distinguish between point mutations (i.e., substitutions) and frame-shift mutations.

112. A mutation in a gene which controls the production of a given enzyme may result in either the complete absence of this enzyme or the production of a modified form of the enzyme. Explain.

113. If a mutant or recombinant bacterium had no selective advantage over the parent strain in a medium in which the two are growing, is it likely that this mutant or recombinant will ever replace the parent strain? Explain.

114. What are auxotrophic mutants? How can the techniques of penicillin enrichment and replicate plating be used to isolate auxotrophs? How would you isolate an auxotroph of E. coli which requires arginine and phenylalanine for growth?

115. Outline a procedure to detect and isolate a strain of Bacillus subtilis which is tetracycline resistant (TCR) and auxotrophic for the amino acid methionine (met). Start with the wild type which is tetracycline sensitive and does not require methionine for growth.

116. What are insertion sequences (IS) and transposons which occur in bacterial DNA? How can the insertion of a IS or transposon into bacterial DNA lead to mutation?

117. Explain how it is possible for a frame-shift mutation early in a gene to be corrected by another frame-shift mutation farther along the gene.

118. Indicate by a + or - in the columns below whether growth would or would not occur if Escherichia coli cultures with the indicated characteristics were inoculated onto each medium.

E. coli						Medium contains					
Characteristics	B,L,T		M,L,T		L,T,SM 	B,M,L		B,M,L,T,SM
B- M- T+ L+ SMR
B+ M+ T- L- SMR
B+ M+ T-L-SMS
B+ M- T+ L- SMR

B- means that the organism cannot make biotin and must be supplied with it to grow.

B+ means that the organisms can make biotin and can grow if biotin is not in the medium. L=leucine, T=threonine, M=methionine, SM=streptomycin, SMR=streptomycin resistant, SMS=streptomycin sensitive.

119. Normal wild-type E. coli does not require the amino acid tryptophan for growth. A microbiologist isolates a mutant that requires tryptophan. Explain how a change in a DNA base sequence of the wild type might result in the occurrence of this tryptophan requirement.

120. Explain the occurrence of antibiotic-resistant strains of bacteria in our gastrointestinal tracts, and discuss the significance of this finding from the point of view of evolution.

121. What is the purpose of the Ames Test and how is it performed?

122. Identify each of the following terms:

  • transduction
  • transformation
  • conjugation
  • lysogeny
  • lysogenic conversion

    123. Outline an experiment which might show that genetic recombination could occur between cells of E. coli and cells of Shigella. If you determined that conjugation did occur, would there be any important medical implications in your findings?

    124. How is it possible that a "wild type" or prototrophic organism could be obtained from mixing these two strains of E. coli? Hfr met-, arg-, his-, X F- phe- trp- cys-

    125. Describe the steps in conjugation in bacteria.

    126. Identify each of the following mating types of Escherichia coli: Hfr, F+, F', F-.

    127. Illustrate or describe the possible outcomes of mating between the following strains of E. coli:

  • Hfr X F- F+
  • X F-
  • F' X F-

    128. What are R-factors? What is their relationship to F-factors and to plasmids in general? What is RTF (resistance transfer factor)? How do you think R-factors and RTFs become constructed in nature? What is the medical significance of the existence of R-factors and RTFs?

    129. In conjugation between an Hfr and an F- E. coli cell, what determines which genetic markers are the first to enter the recipient?

    130. The following experiment was done: 108 cells of strain 1 of a bacterium requiring the amino acid proline (P) and the vitamin thiamine (T) were mixed with 108 cells of strain 2 which requires the amino acids methionine (M) and leucine (L). After 30 minutes incubation the mixture was spread at various dilutions onto plates of minimal medium (containing glucose as the only organic substance). After overnight incubation 310 colonies were obtained on the plate containing a 10-3 dilution, and 34 colonies on the plate containing a 10-4 dilution. Each of the following statement refers to the above experiment; indicate whether each is true or false:

    ___If each requirement is considered due to a single gene, the genotype of strain 1 for the four characteristics is P- T- M+ L+.

    ___This experiment provides evidence that gene transfer in bacteria is unidirectional.

    ___In this experiment the results could have been due to transformation, transduction, or conjugation.

    ___It is impossible to calculate the frequency of recombination from the date given.

    ___Strain 1 is an Hfr strain.

    ___This experiment has shown that genetic recombination has occurred by gene transfer after cell-to cell contact.

    131. Two cultures are mixed, one which is B- M- T+ and SMR, and the other which is B+ M+ T- and SMS. After being mixed they are plated on the media indicted. Show by + or - in the columns below whether any growth will be observed if genetic recombination between the two cultures does or does not occur.

    											
    				Growth if no				Growth if
    Medium contains		recombination			recombination
    				occurs				occurs
    											
    B, T, SM
    SM
    B, M
    T
    T, SM
    No additions

    B- means that the organism cannot make biotin and must be supplied with it to grow. B+ means that the organism can make biotin and can grow if biotin is not in the medium. T=threonine, M=methionine, SM=streptomycin; SMR=streptomycin resistant.

    132. Explain how it is possible to use the interrupted mating procedure to determine the relative order of genes on a bacterial chromosome.

    133. Explain why this statement is false: Genetic change in bacteria occurs only through processes of mutation.

    134. Describe the phenomenon of transformation as it was first observed by Griffith in 1928 in Streptococcus pneumoniae. Give a current explanation of Griffith's observations. In what other bacteria has transformation been observed?

    135. Do you think that the process of transformation is strictly a laboratory phenomenon, or where do you think that it might occur in nature. Why?

    136. From what you know about cell wall and membrane structure in bacteria explain the problem a DNA molecule would encounter during uptake if the transformation process were to occur.

    137. What is competence in bacteria, and how is it measured?

    138. What is meant by the term natural transformation, as opposed to artificial transformation? In what bacteria is natural transformation known to exist? Explain the overall molecular events, emphasizing the differences, in natural transformation in Haemophilus influenzae and Streptococcus pneumoniae.

    139. Compare the lytic and lysogenic life cycles of bacteriophages.

    140. Transduction is a widespread phenomenon in bacteria. Describe this process, relate it to lysogeny, and indicate its importance in the bacterial world.

    141. Distinguish between restricted (specialized) transduction and generalized transduction. What types of bacteriophages are involved in these processes?

    142. For each of the following characteristics, indicate whether plasmids are similar to or different from temperate viruses: a. ability to replicated independently of the host genome: b. transmission by cell-to-cell contact; c. ability to integrate into the host genome; d. ability to acquire host genes.

    143. What are restriction enzymes? What is the prime function of a restriction enzyme in the cell which produces it? How does a restriction enzyme differ from a DNase? From an exonuclease? How is it that the restriction enzyme in a cell does not cause degradation of that cell's DNA?

    144. Describe (with suitable diagrams if useful) a general laboratory procedure for construction a genetic recombinant of E. coli B containing a plasmid bearing genes for streptomycin resistance (SMR) and tetracycline resistance (TCR) using these three bacteria: E. coli B (SMS, TCS), Staphylococcus aureus TCR, and E. coli SMR. In Staphylococcus, the TCR genes are born on the chromosome which has several restriction sites for EcoR1 but none within the genetic region coding for tetracycline resistance. In E. coli SMR, the genetic information for streptomycin resistance is contained on the SMR plasmid which has only one restriction site for EcoRl, and this is not within the region coding for streptomycin resistance. E. coli B (SMS, TCS) is receptive to conjugation or transformation. E. coli SMR is nonconjugative and cannot be transformed. In your discussion consider also the media you would use to selectively isolate the recombinant.

    145. Describe in words the basic procedure involved in cloning and expression of a synthetic gene in a bacterium such as E. coli or B. subtilis.



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    Edited with Frontier Applications on a Macintosh on Fri, Mar 14, 1997 at 9:13:51 AM by Kenneth Todar University of Wisconsin-Madison Department of Bacteriology.