UNIVERSITY OF WASHINGTON

SEATTLE, WASHINGTON

Department of Medicine
Division of Oncology

December 27, 1974

Harold Varmus, M.D.
Department of Microbiology
University of Cal i fornia
School of Medicine
San Francisco , California

Dear Harold:

We have repeated some df the competition experiments related to the
question of the repregentation in normal  cells of the "X piece" sequences
of RSV.
interest.  The fight7hand panel depicts competition of unlabeled RNA from
td  r-C RNA and paTental Pr-C RNA in the hybridization reaction between

of .5 mg of fragmented norma17chick cell DNA, 2,000 cpm of     labeled
RNA (specific activity 4 x 10  cpm/ug), and increasing quantities of
unlabeled competitor RNA in 5 x SSC, 50% formamide.  Re ctions were in-

of radioactive RNA acquired RNase  resistance (after subtraction of a
"zero time" background) in uncompeted control  reactions.  RNA from both the
transformation defective and parental viruses competed virtually completely
with the labeled parental 70s RNA.
of RNA at the competition plateau there was about an 8 cprn competitive
advantage for the parental type RNA (out of a total RNase resistant hybrid
of 600 cprn).
as significant, that would suggest that a little more than 1% of the entire
RSV genome is represented in the DNA of normal cells, but not in the RNA of
the transformation defective mutant.

/

I encloze abcopy of the results of these experiments for your

labeled Pr-C RNA and normal chick DNA. Reaction mixtur consisted

cubated at 49OC to an uncorrected C& value of 1.5 x 10 4 . About 600 cpm

           Averaging the 3 points for both types


If one chose to accept this tiny increment in radioactivity

The right-hand panel depicts, for comparison, the same competition experiment
run with RSV induced wing web tumor DNA of the norm  DNA. We have repeated
the control homologous competition curve with the IpJ5 labeled probe, but
otherwise the data is the same as was described in the paper we published in
3. VIROLOGY.

hybrids between I   labeled Pr-C RNA on wing web tumor DNA was not competed
out by a large excess of unlabeled RNA from the transformation defective
mutant.  These results are distinctly different than those obtained with

The ordinate is normalized in this gr ph and represents a 60%

uncompeted hybridj55tion at a Qvalue of 1.5 x 10 8 . About 15% of the

Mailing Address: Division of Oncology, Providence Hospital, 500 17th Avenue, Seattle, Washington 98122
                  Telephone (206) 322-3140

Harold Varmus, M.D.
December 27, 1974
Page Two

normal chick DNA. On the basis of this data I have to conclude that at
least 90% of the "X piece" sequences which we are measuring in these
reactions are not detectable in normal chick embryo DNA.  This does not
exclude the possibility that 10% or less of the transformation-specific
sequences in RSV are still endogenous to the normal chick genome.

I suppose the possibilities that I have suggested before for rationalizing
the differences between your data and mine remain viable, and I look forward
to hearing more about your results as they progress.

Sincerely yours,

Paul Neiman, M.D.

PN/lm
Enclosure