Genetics Lecture notes (9-Mar-1998)

Biology 210

GENETICS

9 March, 1998

Chapter 8a (pages 307-328) Bacterial Genetics

Outline:

1. Introduction

2. Bacterial Transformation

3. Bacterial Conjugation -or-

SEX in a Blender
4. Transduction

1. Introduction

Bacteria have four important advantages for "traditional types of genetic experiments": (page 308)

1. They are haploid (no masking).

2. New generation is produced every 20 minutes.

3. Easy to grow in ENORMOUS NUMBERS.

4. Individual members of these large populations are GENETICALLY IDENTICAL (or very nearly so).

A few notes about working with bacteria:

Bacteria are easy to grow:

There are THREE MAJOR TYPES of genetic transfer found in bacteria:

1. Transformation - this is what Avery characterised - remember? Somehow or another, the bacteria can take up externally provided DNA.

2. Conjugation - DNA is transferred from one bacteria cell to another, via "sex pilli".

3. Transduction - this is where DNA is introduced into bacteria by injection from a bacteriophage. (We'll talk more about this on Wednesday.)

2. Bacterial Transformation

"Transformation" is simply the process where bacteria manage to "uptake" or bring in a piece of external DNA (somehow or another). Usually, this process is used in the laboratory to introduce a small piece of PLASMID DNA into a bacterial cell.

figure 14_2a from Griffiths et al., 1996

DNA is the gentic material -
The First demonstration of bacterial transformation.
Experiments done by Frederick Griffith (in London) in 1928 found there were two different types of the bacterium Streptococcus pneumoniae:
An "S" or SMOOTH coat strain, which is lethal to mice. figure 11_1a from Griffiths et al., 1996

figure 11_1a from Griffiths et al., 1996

An "R" or rough strain, which will not hurt the mouse. figure 11_1b from Griffiths et al., 1996

Griffith found that he could heat inactivate the smooth strain. figure 11_1c from Griffiths et al., 1996

However, if he were to take a mixture of the heat-inactivated S strain,
mixed with the R strain, the bacteria would die. Thus there was some
Material in the heat-killed S strain that was responsible for "transforming" the R strain into a lethal form. figure 11_1d from Griffiths et al., 1996

figure 11_1d from Griffiths et al., 1996

Fred Griffith (and a lab co-worker) was killed in their laboratory in 1940 from a German bomb. However, their work continued on in the U.S., and in 1944, Oswald Avery, C.M. MacLeod, and M. McCarty carefully demonstrated that the ONLY material that was responsible for the transformation was DNA - thus, DNA was the "Genetic material" - however, many scientists were still not sure that it was REALLY DNA (and not proteins) that was the genetic material.

figure 10-16 from Griffiths et al., 1996

The genetic transfer of streptomycin resistance (str^r) to the streptomycin sensitive (str^s) cells of E.coli. The recovery of str^scells depends on the concentration of the str^r DNA.

McCarty,M., The Transforming Principle - Discovering that Genes are made of DNA, (New York: W.W.Norton & company, 1985) - Although this book was written about 40 years after the experiments took place (1985), it is an excellent history of the research that was going on in the early 1940's. I would strongly recommend the reading of this text.

Click here for a link to a Biology 101 lecture on the "Central Dogma", where this was taken from.

Co-transformation is simply the simultaneous transformation of two different DNA fragments.

First, you must obtain you DNA - you can do this by isolating DNA from a nice bacteria that has some DNA you want to use....
Hartl & Jones, figure 8-5

figure 8-5b
Now you do the transformation and have a look at the products - if you're transforming into a strain that lacks the gene of interest (which you usually are), then the process is quite easy - just look for colonies that carries the trait of interest:

3. Bacterial Conjugation

"Bacterial conjugation is the pocess in which DNA is transferred from a bacterial donar cell to a recipient cell by cell-to-cell contact. It has been observed in many bacterial species and is best understood in E.coli, in which it was discovered by Joshua Lederberg in 1951." (from page 314 in Hartl & Jones).

The ability to transfer DNA by conjugation is dependenton the presence of a cytoplasmic entity termed the fertility factor, or F. Cells carrying F are termed F⁺; cells without F are F^-. F is a small, circular DNA element that acts like a minichromosome. It is an example of a class of elements termed plasmids, which are self-replicating extrachromosomal DNA molecules. F contains approximately 100 genes; these give F several important properties:

1. F can replicate its DNA, which allows F to be maintained in a cellular population that is dividing.

2. Cells carrying F produce pili (singular, pilus) - minute proteinaceous tubules that allow the F⁺ cells to attach to other cells and maintain contact with them.

3. F⁺cels can transfer the newly synthesized copy of the circular F genome to a recipeint (F^-) cell that lacks such a genome; note that a copy of F always remains behind in the donating cell. When a donor cell transfers a copy of its cytoplasmic F to an F^- cell, the recipeient cell also becomes an F⁺ cell, because it now contains a circular F genome.

4. F+ cells are usually inhibited from making contact with other F+ cells and do not usually transfer the F genome to F⁺ cells.

5. Occasionally, F leaves the cytoplasm and integrates itself into the host bacterial chromosome. When this occurs, F can also transfer the host chromosomal markers to the recipient cell along with its own DNA.

(the above section is from Griffiths et al., 1996, page 277)

compare this with figure 8.7 in your text - it is essentially the same.

"SEX in a Blender"

By some clever use of timing experiments, it is possible to generate a genetic map of E.coli!

figure 10-7 from Griffiths et al., 1996

Chromosome Mapping

4. Transduction
We'll talk more about this on Wednesday, but the general idea is outlined here.

Link to Lecture on GENOMICS (Biology 101, 16-Feb-98) Link to Escherichia coli complete genome EcoCyc: Encyclopedia of E. coli Genes and Metabolism Other Genomes that have been completely sequenced.

Link to DNA Atlases for complete genomes.
(New link - added in Feb. 2000).

Back to the Genetics Syllabus

Last modified on: 3 February, 2000 by Dave Ussery