Phage Lambda as a Model Organism

• Why viruses are studied?
• What are viruses?
• Why is phage lambda studied?
• The life cycle of phage lambda
• Lambda's lytic program
• The regulatory protein N
• The Lysogenic Program
• Biomedical Applications

Why viruses are studied?

1) They are harmful to humans and other organisms so scientists what to learn how to control them.

2) Viruses are important experimental model organisms. Virus lambda has been used to address important biological questions. For example: How are the sequence of events during development programmed? All organisms develop as a result of a programmed order of steps. Similar to an automobile assembly line. How is the sequence of events during development programmed. What insures that the parts of the program are carried out in the proper order?

The answers to these questions come from the understanding the behavior of regulatory proteins in phage lambda. What we learn in phage l helps us to understand not only the behavior of other viruses but of the host cells they infect&emdash;in organisms ranging from bacteria to humans.

• Viruses are [obligate, intracellular] parasites.
• They are composed of genetic information (DNA or RNA - never both)
• Surrounding this information is a protein coat (capsid)
• They are the simplest "living organisms" capable of reproduction, but only when their genetic information enters a living host cell&emdash;a process called infection.

One way to characterize viruses is by the type of host cell they infect. A virus that infects a bacteria is called a bacteriophage.

Phage lambda

Viruses are also studied because they are the simplest organisms capable of reproduction.

There is a bigger payoff when one can learn a lot about a simple system rather than just a tiny bit about a hopelessly complex one.

In fact studies of phage taught us that DNA is the genetic material.

1. it is completely safe for humans to work with -- (Why)
2. it is easy to grow - replicating only in the bacterium E. coli, which is also relatively benign, and easy to work with.
3. Lambda's genome is small and has been completely sequenced - the 48,502 bases for all 50 or so genes. The genes have been mapped and most of their proteins' functions discovered. This degree of knowledge allows investigators to ask very precise questions.

Thus a framework is available for asking similar questions about complex systems which are much more difficult to tackle directly.

Individual phage particles are very small - about the millionth the size of a freckle, and can only be seen by an electron microscope (magnification of 20,000 x or more)

The phage life cycle produces about 100 new phages for each infected cell, and takes about 45-60 minutes. Thus given a sufficient population of bacterial cells to infect, a single phage virus can increase in numbers very rapidly - exponentially

Therefore, experimentally the effects of these populations can be readily visualized.

Such huge populations make the study of extremely rare mutant individuals possible. - example lambda-virulent occurs only 1 in 10 billion. Getting 10,000,000,000 mice together for such a study would be impossible.

A) One method of observing the growth of phage.

Given two flasks containing abundant cultures of E. coli the flask inoculated with phage soon becomes clear, while the uninfected flask grows more turbid.

How does lambda infect the E. coli bacteria?

1. The phage particle attaches to the outer surface of the bacterial cell wall.

The specific site where phage attaches to the outer surface of the bacteria is called a phage receptor.

2. DNA of phage is injected into a cell as if from a syringe. (Most animal viruses are brought into a cell by the host cells own phagocytic activity or endocytosis.
3. The ends of the phage DNA join to form a circle, and begins replicating virus DNA using the ATP energy of the host cell. The new phage DNA is used to make head and tail proteins
4. The phage DNA is then packaged inside the polyhedral heads.
5. The various phage subunits spontaneously join to produce about 100 new phage particles.
6. The host cell lyses in approximately 45-60 minutes after infection releasing 100 or so new phages.

B) Another method. Phage growth can also be seen on a petri plate. First an agar gel is pored into a petri dish an allowed to harden. Then a mixture of nutrient agar containing a large number of bacterial cells and a very small number (10-50) phage particles is spread evenly over the agar.

The initial result is a uniform layer of bacteria called a:

Lambda's lytic program.

We know that lambda DNA has the instructions for making more phage particles

What determines when each of the 50 genes of lambda are read? The order makes a big difference.

The 50 lambda genes can be organized into functional groups according to the timing of their expression.

1. see below.
2. Some early genes produce proteins which control DNA replication.
3. A set of later genes are then activated for making the proteins used in the construction of the head and tail subunits.
4. Finally 2 genes are activated to help lyse the host cell.

What insures the proper order for the reading of phage genes?

Two factors are involved: the sequence of genes and the protein products of a regulatory gene which is read as soon as the viral genome enters the host cell.

The first regulatory protein, N, immediately produced by the RNA polymerase of the host cell, insures that the early genes, O, P, and Q are read first. O and P are necessary for viral DNA replication, while Q another regulatory gene activates transcription of the late genes.

This ordered sequence of events called the lytic program occurs inside the bacterial cell after infection. The bacteria makes an enzyme called RNA polymerase. These host enzymes bind to 3 specialized sites on the phage DNA, areas called promoters. They determine where transcription begins. The result is the production of mRNA - instructions for the N protein. (this ends the first stage)

N protein's job is to allow the RNA polymerase to go past the stop signals so the early genes O, P and Q can be transcribed into mRNA.

O and P proteins can initiate the replication of phage DNA. Q is the other regulatory protein. Its job is to turn on the late genes which code for the proteins necessary for viral head and tail segments and finally those proteins necessary for lysis.

Experiment: Can head and tails self-assemble in vitro, outside a living cell (test tubes instead of in a living cell {in vivo})? The experiment found that by adding separate solutions of purified heads to purified tail segments they did indeed self-assemble into active phage particles.

How did the results confirm the presence of active phage particles?

A modified version of this in vitro process is an important step in recombinant DNA technology - can you describe how lambda is used?

Occasionally the host cell survives after infection. In which case the host cell has acquired new properties. These new properties are the result of stable association of the DNA of the virus and its host cell.

Bacterial cells that have been infected by phage but survive are called lysogens. Lysogens contain lambda genetic information in a latent (resting state), and are said to carry a Lambda prophage. Lysogens have 2 important properties.

1. They are immune to further infection - no other lambda phage can infect the lysogen
2. Lysogens can be induced to produce active phage. This is called prophage induction

When plaques are observed more closely they are found to have a turbid center which is caused by the growth and reproduction of surviving bacterial cells (lysogens).

When lambda choose the lysogenic program the formation of Lambda prophages results.

How does this program work?

1. The circularized lambda DNA recombines with the bacterial chromosome. A specific recombinational event that occurs between a special site on the phage DNA called the phage att site and a special site on the bacterial DNA called the bacterial att site.

This results in a lambda prophage (see below).

2. The prophage synthesizes the cI (cee one) protein - a repressor protein coded for by the cI gene. The cI protein is called the lambda repressor because it binds to special sites on the prophage genome called regulator sites which overlap the early promoters (see above)

The operator site overlaps (includes) the early promoter region.

The cI protein prevents the transcription of the early genes of the prophage by physically preventing RNA polymerase from binding to the promoter sites. If another lambda genome enters the cell the cI protein will bind to its early promoter sites as well.

Experiment 2: using lambda virulent a mutant of the wild type (normal) lambda virus which is able to somehow ignore the cI protein.

Environmental damage such as UV radiation can somehow stimulate the formation of a particular lambda protein called RecA. RecA can remove the cI protein cleaving it. Thus the repression is lifted and RNA polymerase begins transcription of the N gene and the lytic phase begins.

This is an example of a genetic switch.

AIDS, caused by HIV may also have a latent phase. A regulatory protein in HIV called Tat may operate like the N protein in lambda.

Herpes simplex virus . Herpes infects nearly 80% of the human population. Herpes simplex primary infection occurs on the skin or oral cavities. Latent infection occurs in a nerve ending where it is carried back to the nerve cell body in the central nervous system through an axon. Here it will last forever.

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