UNIVERSITY OF CALIFORNIA, SAN FRANCISCO

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BERKELEY * DAVIS - IHVINE * LOS ANGELES - RIVERSIDE - SAS DIEGO - SAS FRANCISCO

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SANTA BARBARA . SASTA CRUZ

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SCHOOL OF MEDICINE
DEPARTMENT OF BIOCHEMISTRY AND BIOPHYSICS

SAN FRANCISCO. CALIFORNIA 94143
(41 5) 666-4324

May 26, 1977

Dr. Daphne Kamely
Office of Recombinant DNA Activities
Bldg 31, room 4A52
NIGSM, NIH
Bethesda, Maryland 20014

Dear Dr. Kamely:

We would like to undertake a project involving recombinant DNA. Under the present
NIH guidelines, this project would appear to require P4 containment. We request
that the Recombinant DNA Advisory Committee review the following considerations which
we feel would warrant P3 containment conditions.

We propose to use polyoma virus as a vector to clone the thymidine kinase (TK) gene of
pseudorabies virus (PRV) in mouse cells. DNA of a polyoma mutant temperature sensitive
in an early function (ts-a) will be purified. Restriction endonuclease(s) will be
used to delete a portion of the genome that codes for late function. A PRV restriction
fragment that codes for TK will be identified and purified using transfection of TK-
mouse L cells to HAT resistance as an assay of activity. Recombinants of the polyoma
and TK fragments will be formed using conventional procedures. TK- L cells will be
infected at the restrictive temperature and cells selected in HAT medium to obtain
clones that express TK activity. These cells will be shifted to the permissive tem-
perature and closed circular DNA isolated from the Hirt supernatant.

The following considerations apply in evaluating the biohazards of the proposed experiment:

1. PRV, a herpes virus, is not known to be pathogenic for man, but is patho-

2. Viral strains with highly attenuated virulence for livestock and labora-

3. Polyoma with a deletion of late function will be used.

4. No virions will be generated.
5. A partially defined fragment of PRV DNA will be used.
6. Mouse cells are permissive for both polyoma and PRV, hence both genomes

genic for pigs and cattle.

tory animals have been described and will be used if available.

have had the opportunity to coexist in a single host before the introduc-
tion of recombinant DNA technoloqy.

In view of these considerations, we request permission to conduct this series of exper-
iments under P3 containment conditions. This project will be conducted by the undersigned.

Sincerely yours,

ERBERT W. BOYER

Dept. of Biochemistry

PHILIP COFFIN0
Dept. of Microbiology

HAROLD VARMUS
Dept. of Microbiology