EXPERIMENTAL MOLECULAR BIOLOGY OF THE CELL

Background Information about Restriction Enzyme Mapping and Pencil Exercise/Questions

    Plasmids are extrachromosomal, self-replicating double-stranded DNA molecules. Most plasmids exist as supercoiled molecules (CCC = covalently closed circular DNA). Although small, plasmids encode a number of important gene products. Some may confer selectable phenotypes to their recipient cells such as resistance to certain antibiotics or heavy metals, which indicate that the cell has been transfected with plasmid DNA. Other plasmid genes are essential to maintain the copy number (the number of plasmid DNA molecules per cell) or to provide origin sequences which function in the initiation of plasmid DNA replication. Generally, plasmid replication and gene expression depend entirely on the preexisting host factors required to promote these processes. Many plasmids are said to be cryptic if they do not express selectable phenotypes in cells. Such plasmids are, in general, useless as cloning vehicles since plasmid transfection is not readily assayable.

    Although discovered initially in Prokaryotes, several plasmids have been isolated from, or constructed to work in, lower Eukaryotic cells such as yeast and in some plants. In addition, so-called shuttle or bifunctional plasmids have been constructed so that they can replicate in more than one species of bacteria. To be useful as a cloning vehicle for the amplification of inserted foreign genes, plasmids should be low in molecular weight, have selectable phenotypes and a number of unique restriction nuclease cutting sites.

    In part I of this experiment, you will isolate two plasmids from cultures of bacterial cells. The plasmids you will be isolating are called expression plasmids. This means that they have been constructed so that, when introduced into bacterial cells under appropriate conditions, the inserted gene can be transcribed and translated by the cells and generate the protein coded by the inserted gene. The protein you will be generating is T4 lyozyme. The expression plasmid has been constructed by inserting the cDNA (complementary DNA, containing only the DNA sequences complementary to the mRNA of the gene) of T4 lysozyme downstream of a promoter in the plasmid that is inducible by a chemical called IPTG. When IPTG is present in the culture, transciption of the cDNA is initiated from the promoter and the mRNA is made and subsequently translated into T4 lysozyme protein. The two types of plasmids you will use and analyze will be discussed thoroughly with you in preparation for the laboratories.

  You will do Part I of this experiment in Lab 3 . Part 1 consists of breaking open the bacterial cells and extracting and purifying the plasmid DNAs. During Lab 5  you will cleave the plasmid DNAs with restriction enzymes and separate the DNA fragments produced on agarose gels. By examining the pattern and sizes of these DNA fragments, you will be able to determine which plasmid is which.

   Restriction endonuclease digestion of DNA has been extremely useful in the characterization of these molecules since the DNA can be broken down to manageable sizes using them. Because these enzymes recognize a specific nucleotide sequence in DNA, the same enzyme will always produce the same fragments of a certain DNA. Usually, the first step in the analysis of a new DNA is to construct a restriction endonuclease map using one enzyme initially, but eventually using several. In order to construct this map, it is necessary to determine the sizes of all enzyme-generated DNA fragments by agarose gel electrophoresis. A map showing the positions at which the endonuclease cuts the DNA can be created by ordering the fragments on the DNA.

   The pH of the electrophoresis buffer used in running the agarose gel is 7.5, therefore, all of the DNA fragments will have a negative charge and will migrate towards the positive electrode. The smaller the size of the DNA fragment, the faster it will move through the pores of the agarose. You will find that the migration during electrophoresis is proportional to the inverse of the log of the molecular weight (i.e. migration distance = K/log MW where K is a constant for each gel condition). The DNA fragments are easily visualized during electrophoresis by including ethidium bromide (EtBr) in the gel. EtBr interchelates between double-stranded DNA and, as a consequence, fluoresces brightly when illuminated with UV light. Although we will not do this, one way to order the fragments is to digest the DNA only partially so that not all potential cleavage sites are used. The "partial" fragments are then isolated, redigested completely with the same enzyme and the resulting fragments are analyzed again by gel electrophoresis. If the "partials" are overlapping, a map will be produced. Another way to order the fragments is to completely digest the DNA separately with two different enzymes. Each limit fragment is then cleaved with the other endonuclease and the resulting fragments are analyzed again. Alternatively, you can include both enzymes in the same digestion and compare the disappearance or appearance of bands to deduce the cutting patterns (we will use this approach). As a pencil exercise, use the following data to order the fragments generated in a virtual experiment and produce a restriction map.

Pencil Exercise:

    A solution of Virus B DNA (linear double stranded molecule) has been completely digested with EcoRI. An agarose gel of this restriction digest shows the following limit fragments: A limit fragment is what is generated when the DNA is cut at all its EcoRI sites.

 ____  8.6 Kb
 ____  7.9 Kb

 ____  2.3 Kb

Figure 1

Fig 1.  Agarose Gel of EcoRI digest of Virus B DNA. Conveniently, these fragments have had their size determined by the use of Lambda Hind III markers-- data not shown.)

Each of these fragments was eluted from the gel and redigested with Taq I.
The following patterns were produced:

____   7.1 Kb
                                                        ____   5.0 Kb
                                                        ____   2.9 Kb
____  1.5 Kb                                                                                         ____   2.3 Kb

Figure 2                                        Figure 3                                            Figure 4

Fig 2.  Agarose gel of Taq I fragments from the 8.6 Kb EcoRI fragment of Virus B DNA

Fig 3.  Agarose gel of Taq I fragments from the 7.9 Kb EcoRI fragment of Virus B DNA

Fig 4.  Agarose gel of Taq I fragments from the 2.3 Kb EcoRI fragment of Virus B DNA

In addition, a Taq I digest was done on the intact viral DNA, giving rise to the following limit fragments:

____ 10 Kb

____   5 Kb
____ 3.8 Kb

Figure 5

Fig 5.  Agarose gel of Taq I fragments of Virus B DNA


1.  Using the data given above, construct a restriction map showing the locations of all Taq I and EcoRI sites and giving the distances (in Kb) between them.  Remember that the bands shown represent the sizes of the DNA pieces made after cutting the DNA with a restriction endonuclease.

2.  For this experiment, you will be doing a several double digests. Think about how this differs from the technique described above.  Diagram a gel of the restriction fragments that would arise from a double digest  using TaqI/EcoRI  of the Virus B DNA.  What do you perceive to be the advantages or disadvantages of each technique?

3.  How do these approaches (double digest, limit digest/elution/redigestion with a different enzyme) compare to partial digests?

4.  Many viral and bacterial DNA molecules are circular rather than linear.  How would this influence the degree of difficulty experienced in construction of a restriction map?  For purposes of illustration, change our linear Virus B DNA molecule into a circular molecule by ligating the ends.  What happens to the various patterns of fragments generated?  Show using gel patterns and a restriction map.  (Assume ligation does not generate a Taq I or EcoRI site.)