Working with Data:
Lederberg and Tatum’s Experiment
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Introduction
To demonstrate that genetic recombination can
occur in bacteria, Lederberg and Tatum grew
two auxotrophic strains of E. coli. Strain 1
(met– bio– thr+ leu+) required methionine
and biotin for growth 8+on minimal media, while
strain 2 (met+ bio+ thr– leu–) required
threonine and leucine for growth on minimal
media. |
The researchers mixed the two strains together,
grew them in media that contained methionine, biotin,
threonine, and leucine, and then selected for colonies
that could grow on minimal media without supplementation.
Results showed that some of colonies were able to grow
on this media, suggesting that these prototrophic colonies
arose by genetic exchange between the two strains. To
confirm that these prototropic colonies did not arise
from reversion mutations, Lederberg and Tatum also included
an important control. In addition to plating the mixture
of bacteria on minimal media, strain 1 and strain 2
were also plated individually on minimal media. While
plates containing the mixture of strains grew colonies
at a frequency of approximately 1 in 10 million, no
colonies arose on plates containing either strain 1
or strain 2 alone, demonstrating that reversion mutations
cannot restore prototrophy. Together, these results
suggested that some form of recombination of genes had
taken place between the two strains to produce prototrophs.
Importantly, the controls performed by Lederberg and
Tatum were specifically designed to identify reversion
mutations that arose in the absence of any type of mutagen,
a chemical which can induce permanent genetic changes
in cells or organisms. However, substances that are
mutagenic can induce reversion mutations with a high
frequency. In the early 1970s, Bruce Ames and his colleagues
at the University of California, Berkeley developed
the Ames Test, a procedure used in the laboratory as
an initial assay to screen a substance for its mutagenicity.
Similar to Lederberg and Tatum's control experiment,
this procedure often uses as a marker the reversion
of the growth-dependence of bacteria on a particular
amino acid to growth of the bacteria in the absence
of that amino acid. Substances that are mutagenic, and
therefore likely carcinogenic, will induce reversion
mutations in the Ames Test.
Links
National Library of Medicine: Joshua Lederberg Bacterial
Genetics
http://www.nlm.nih.gov/hmd/lederberg/bacterial.html
National Library of Medicine: Profiles in Science:
The Joshua Lederberg Papers: The Development of Bacterial
Genetics http://profiles.nlm.nih.gov/BB/Views/Exhibit/narrative/bacgen1.html
National Center for Biotechnology Information: An
Introduction to Genetic Analysis: Bacterial Conjugation
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=iga.section.1304
Howard Hughes Medical Institute: BioInteractive:
The Animation Console: Bacterial Conjugation
http://www.hhmi.org/biointeractive/animations/conjugation/conj_frames.htm
San Diego State University: The Ames Test
http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/rev-sup/ames.html
Original Papers
Lederberg and Tatum’s first paper on bacterial
recombination was a note without data, just describing
the rare appearance of wild type bacteria (prototrophs)
in the mixed cultures as shown in above Figure.
Lederberg, J. and E.L. Tatum (1946) Gene recombination
in Escherichia coli. Nature. 158: 558.
http://profiles.nlm.nih.gov/BB/G/A/S/Z/_/bbgasz.pdf
A more extensive and formal investigation was reported
shortly thereafter:
Tatum, E.L. and J. Lederberg (1947) Gene recombination
in the bacterium, Escherichia coli. J. Bacteriology.
53: 673–684.
http://jb.asm.org/cgi/reprint/53/6/673
Analyze the Data
From the second paper, here is the definition of the
genetic strains:
When two triply-mutant strains were mixed together
in culture (see parental types, below), the authors
found various phenotypes (and, therefore, genotypes)
arising rarely:
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The total number
of cells on the plates was 100 million. What was
the rate of appearance of prototrophs (all wild
type)? |
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The
spontaneous mutation rate for each of these
six genes, from mutant back to wild type (also
called reversion), is 10–6, meaning that
1 cell in a million spontaneously mutates for
a given gene. What would be the spontaneous
mutation rate for all three genes simultaneously,
resulting in prototrophs without recombination?
Compare the result of this calculation with
the actual data (answer to question 1). What
does this indicate about the likelihood of spontaneous
mutations accounting for the data? |
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See text, pages
292–293, for the process of gene transfer
from one bacterium to another. The genes T and
L map near one another on the bacterial chromosome
and are near the point where transfer begins.
How does this account for data in Table 2? |
