1. Gene Expression and Replication in Medium DNA Viruses
Phage Lambda
Adenovirus

2. What can the medium sized viruses do that the small ones can not
What can the medium sized viruses do that the small ones can not? Adenovirus: more sophisticated gene expression strategies Lambda: two pathways for infection

3. Phage Lambda Bacteriophage isolated from human gut-Andre Lvov?
dsDNA, 48.5 kbp
Linear, sticky ends “cos”
Complex capsid
Unusual biological properties noticed
Model system from 1950’s
Cloning vector

4. Evergreen State-Olympia Mansfield State (Ohio)
The Evergreen State College Phage Lab has been a center for undergraduate research at Evergreen since Betty Kutter came here in 1972, one year after the college opened. Today, there are generally students involved in work in the lab under the direction of Kutter and faculty colleague Andrew Brabban. We are focusing particularly on phage ecology, with the aims of understanding the infection process under anaerobic and other conditions better reflecting those in the natural environment, as well as on interactions between phages of different families during simultaneous infection and the building of cocktails for potential therapeutic applications.
Dr. Elizabeth Kutter: Dr. Andrew Brabban:
Evergreen State-Olympia
Mansfield State (Ohio)
Bacteriophage Therapy Inst-Tbilisi
Bacteriophage therapy was used broadly in the Soviet Union, particularly the Republic of Georgia, which has been the global center of phage expertise for over 80 years.

5. Lambda Entry and Uncoating
Non-contractile
Lambda J protein uses Lam B as receptor
DNA enters through mannose permease complex

6. Lambda genetic map Shortly after entry the linear DNA forms a circle
Cos sites are complementary with one another
Assembles functional regions

7. Lambda’s two developmental pathways
In some cases lambda follows the “usual” pathway in which the host cell is killed straightaway-the “lytic” pathway.
In other cases lambda DNA inserts into the bacterial chromosome to form a “prophage”.
The prophage remains in the chromosome as a passenger until the host cell is stressed or “induced”.
Then it lyses the host cell to release new phage.
The latter pathway is called the “lysogenic” or “prophage” pathway. (temperate, vegetative)

8. Lysis and Lysogeny for Phage Lambda
What are the advantages of lysogeny for lambda?
How does lambda make its decision for lysis or lysogeny?

9. Phage lambda regulatory region
Key region regulates developmental decision
P = promoter
O = operator
T = terminator

10. Operon Model Shown for lactose operon
Repressor protein binds to operator
sequence:
No mRNA so gene is off!
Inducer causes shape change
in repressor
Repressor drops off operator
mRNA produced so gene is on

11. Gene Expression
Immediately after infection, N and cro are transcribed by host RdRp (“immediate early genes”)
N is an antiterminator- it allows transcription and expression of cIII, cII, O, Q (“early genes”).
Cro is a repressor. It can block transcription from Pr and Pl.
N says “go”, cro says “stop”

12. Lytic pathway
If cro builds up quickly enough it prevents further synthesis of N and of cro itself.
Q allows expression of “late” genes that encode for lytic functions.

13. Establishment of Lysogeny
If cII builds up fast enough it activates the transcription of cI and also some integration functions.
cI is called the lambda repressor. It shuts off all lambda genes except itself and allows lambda DNA to integrate into the host chromosome.

14. Lysis and Lysogeny Compete
If cro “wins” then late genes are expressed and lysis follows.
If N, cII, cIII win then the lambda repressor is expressed from cI and lysogeny follows.
This medium-sized virus is complex enough for two developmental options.

15. Post Script:Lambda repressor binding
Binds to operator as a homodimer
Helix-turn-helix protein
Up to 3 dimers can bind
Binding is cooperative

16. Cooperative repressor binding produces
steep response curve-”all or nothing” response
R E P R E S S I O N
REPRESSOR CONCENTRATION

17. When enough repressor is around is turns off its own synthesis
When repressor levels drop-more is made
Induction involves destruction of repressor
Superinfection immunity

18. Lambda DNA Replication
Lambda uses a combination of host and viral proteins.
Theta strategy for first few rounds of replication.
Then shifts to rolling circle.
Long tail converted to ds concatemer.
Cleavage occurs at cos sites during packaging.