We have already discussed how to make cDNAs with reverse transcriptase. Today we're going to extend the discussion to discuss how RNA is extracted and purified from cells, and how bacteriophage vectors are used to clone cDNAs.
From the finest sewers of Paris, direct to your benchtop!
The usual picture we carry in our minds of viral infection, is one of death and destruction. We think of viruses that enter a cell, take over and subvert the macromolecular machinery, and produce hundreds of progeny viruses that explode out of the dying cell.
In fact, there are a variety of viral life cycle strategies, and not all of them involve certain death for the cell. We discussed one such example in a previous lecture, when we looked at the filamentous bacteriophages such as M13, fd and f1.
Temperate bacteriophage may enter a cell and produce no progeny virus whatever! They lie silently, allowing the DNA replication machinery of the cell to copy their genomes during the course of the normal cell cycle, and having little discernable effect on the health of the host. At some point, in response to an environmental trigger, the virus leaves its cryptic state (see #7-8 below) and enters a lytic cycle that leads to host cell death and virus release (see #3-6 below).
The most thoroughly studied temperate bacteriophage is lambda, which was pulled out of a Paris sewer 50 years ago by Lwoff, Jacob and Monod. They found that certain strains of E. coli, when exposed to ultraviolet light, generated viral plaques on a plate - that is, small areas of bacterial lysis. The word "lysogeny" was coined to describe this type of cryptic infection. Plaques generated by lambda are typically "turbid" rather than "clear" because after an initial round of lytic growth on rapidly dividing E. coli, there follows an overgrowth of bacteria carrying lambda lysogenically. These lysogenic bacteria are "immune" to lytic lambda infection, because they already harbor the virus!
|Lambda can be engineered to carry DNA into cells.||
Do you recall the definition of bacterial transformation? That's when bacteria take up free DNA from their surroundings. If a virus injects its genome into a cell, that's clearly a different type of "uptake" of DNA (or RNA, as the case may be) - we would call that "infection" of course. What term do we use when the infecting virus is carrying DNA that is not normally its own? We call it "transduction" and with integrating viruses like lambda we distinguish between the transfer of genomic DNA adjacent to the normal integration site (specialized transduction) and tranfer of essentially random fragments from the E. coli genome (generalized transduction).
Let us look at an example of a vector designed to carry foreign DNA - a sort of "engineered transduction". Note the gene marked "434" in the figure, and containing a unique EcoRI site.
The "434" refers to an immunity type of the cI repressor (there are different lambdoid phages in the world, with different classes of immunity, which you may be relieved to know isn't important for this discussion). In any case, the unique EcoRI is a marvelous cloning site in this phage, and an insertion causes disruption of the cI (434) gene. How much foreign DNA can be inserted at the site? The packaging limit is 78% to 105% of the wild-type genome size of lambda, which is 48 kbp. Since lambda gt10 is 43.34 kbp in size, we can fit in an additional 0 to 9 kbp of foreign DNA.
Practical aspects to using a lambda vector for cloning.
Lambda vectors can accept larger pieces of DNA than traditional plasmid vectors, and that is a tremendous advantage. On the other hand, one cannot efficiently introduce lambda DNA into a bacterium by transformation because the 50 kbp size makes it sensitive to shearing or breaking. (It should be noted that infecting a cell by transforming it with DNA, a process called transfection by bacteriologists, is not impossible -- it just isn't feasible on a grand scale)
How then, do we introduce the lambda DNA into cells? We can package the ligated DNA into viral capsids in vitro, then infect the cells with the newly created infectious particles. There are several commercial sources of "packaging extracts" that permit you to package ligated lambda concatamers into viral capsids.
The process is detailed in the figure below:
The packaged lambda phage can then immediately be combined with E. coli, to introduce the recombinant phage vector efficiently into cells.
Consider the vector lambda gt11, shown below, which has a cloning site at a unique EcoRI (at 19.60 kbp) in a lacZ gene:
The size of gt11 is approximately 43 kbp, so the vector can accept insertions of 0 to 9 kbp.
In this phage vector, interruption of the lacZ gene is the hallmark of successful cloning, and we add IPTG and X-gal to the bacteriological plate to develop the blue-white screening method:
The only difference between this assay and what we discussed in the previous lecture is that the color develops in a plaque (area of lysed bacteria) rather than in a colony.
One additional feature of the EcoRI cloning site in lambda gt11, is that DNA sequences containing open reading frames become fused to the lacZ open reading frame.
The result is a chimeric (split) gene, which may encode a fusion protein (provided that the orientation and reading frames of the lacZ and insert are matched):
If the inserted DNA is matched in orientation and reading frame with the lacZ gene, so that it becomes expressed as a fusion protein in E. coli, then we can screen the plaques using antibodies directed against the foreign protein (i.e. encoded by the inserted DNA) as a probe. We'll have a chance to talk about that sort of thing later, but here is a summary of how you can use this method practically:
One important point to consider, when expressing foreign proteins in E. coli, is that many introduced sequences turn out to encode proteins that are toxic to the bacterium. That is, your clone may kill itself if the fusion protein is expressed!