February 22, 1972

Ilt. R. J. Huebner
Chief, Viral Qsrcinogenesie Branch
National CSlncer Institute
Bldg 37, Room 2D28
Bethesda, Maryland 20014

Dear Dr . Muebner :

Thank you for sending us a copy of the appraisal of OUT application for renewal
of contract 171-2147.  Needless to say, we were pleased hy the kind words about
our past performence and future promise, and appreciative of your support.  We
feel constrained, however, since you have solicited our cmments,  to make aaee
rejoinders to the remarks ahour our use of reassociation kinetics to detect
viral sequences in cells.

Pirst, the design of these e,periraents is to observe the effect of unlabeled
cell DMA upon the renaturation kinetics of labeled viral polymerase product,
not the reverse as was stated by the reviewer.
copies of WA representing at least 25% of BVIW tumr virus 70s genome@  in
normal chick cells, without 8 detectable increase In transformed cells. As
you know, Gelb et al. have very similer results with a murine system. It is
impossible to compere these numbers, as was attempted, with the results of
Balfuda and Hayak for several reasons: (1) by their own admission, "the actual
number of copes remains undetemined" in their experiments; and (7) their
claim that four additional viral genomes are present in leukemic chick cells is
subject to considerable skepticism since (a) the number is cocsputod from the
weight of I(NA annealed, which may not represent a randam sampling of 706 sequences;
(b) a very ma11 fraction of the incubated arJ& is annealed, again suggesting that
only a mall selected population of sequences is anneeled; (c) the available D?$A
sites are not saturated, taplying an underestimate of the amount of ir;lA capable
of annealing; and, importantly, (d) the hybridization reactions are performed at
relatively low eQt values (not precisely calculable, of course, with filter
hybridization techniques) at whfch only htghly reiterated sequences in the cell
genome would be expected to anneal.
the published DNA-WA hybridization work in this field; we believe that the only
satfsfactory approach to the hybridization of 70s RNA Bind cell DNA is with the
technique of RNA-WA hybridization in solution cat high t&t values as recently
described by Helli and Bishop.
to apply this technique to the problem of detecting Wh tuaror virus sequencea,  3s
outlined in our renews1 application.

We have found about 10-15

- -I

Most of these criticisms apply to a11 of

Bur laboratory and others are currently attempting

Dr. R. J. Huebner
February 22, 1972
Page 2

Our estimates of copy number are jeopardized principally by some uncertatnty
regarding the precise complexity of the DNA probe, and, of course,  the results
are likely samewhat limited because the double-stranded probes may not be re-
presentative of the entire ?OS genome.
dearcribed by hiesberg and Csnnani with unfractimated enzymatic produce are
now being performed in our laboratory to test directly the representation of
viral sequences In the rapidly and slowly reannealing fractions of double
stranded product.  However, the general technique of measuring copies oE viral
sequences in cells ha8 been validated in eeverel ways: with the reconstruction
experiments using SV-40 IMA, performed by Galb c- et al; with single-strand specific
nuclease as a test for duplex formtion; with melting curves of cell-product
hybrids; with expertments employing BUdR labeled cell INA to damnstrate cell-
product density hybrids; and with experiments altering celljprobe ratios to
alter the degree of acceleration of reannealing.
product in the presence of several heterologous cell DNA's does not augment the
renaturation, provgding excellent controls against which to measure CCPY ambers.
The ability to follow a reaction to its camPpletion assures us that ell  the 5NA
Ps participating in the annealing reaction, particularly when essayed with single-
strand specific nuclease, as wsl% as by elution from hydroxyapatite.  Confornation
of the data tQ thearetical expectations of second-order kiaettc6 further suBstantiatee
clsims for the va'lidity of this techniym.

Experhents of the type originally

Zn addition, reanneeling of

The reviewer raises the passltbility that transcription ob viral R??A of cellular
oriRirt, patticufariy free tW$ or 4s RNA in the 70s complex, may by a  $source oE
error'' in our experiments. There are several abjections to this point: (1) the
DMA product anneals to poriffed 70s RNA;  (2) tt does not hybridize to BNA extracted
frm normal chick cells; (3) Its pattern of re%issociatlon is too rrrpid for It to
be copied from a heterogeneous ppulatfon of! cellirlar RNA (or 5N.4) molecules
(although the Cotl/;l alone kwuld not exclude tsanscrbptfon from several tWNA
specbas); and (49 ft reassociates with cell mit at high cell Gt valuss consistent
with ten-fold representation in ehixk cells, fat below the re.teretbon frequancy
demonstrated for tmA genes.

The reviewer suaests that we shauld aynthaalne our probes with purified poly-
merase and purified 80s or 355 BNP template.
mney, and materials Lncurxed by this approach, there is 30 good evidence it:
would offer any advantages. In face, we have recently demonstrated that the
product of. the "purified" reaction ha8 sbmilar reassociation kinetics and is
hmmlogous to the product of ths'+cxude"  reaction.  Mareaver, ws are unable at
present to obtain with the purltffad reaction that smal1 fraction of slowly re-
associating doplhle stranded product,  found in the crude reaction, which presumably
allows us to detect a fivs-fold Iargaz ftectim of the genome.
primer, as the reviewer s~ilggests, with native or melted 70s RNr; as tearplate,
purified polymerase synthesizes product agaPn hmlogous to and now 2s complex

hide from the expense of time,

Usin$ dT as a

tatan She principal product o the emde reactfan.

We want to repeat (PUP thankis for your ftrtereat in our research program and hope
you vi11  eel free to direct to us any suggesttons or questions you may have.

Yours ,

Harold E. Vanaus, M.D.
Department of MicrobLology

HEV :hb

3, nicheel Riahop, M.D.

Department of Mierobfology

Associate Professor