IMPERIAL CANCER RESEARCH FUND LABORATORIES

Registered Charity No. 209631

P.O. BOX NO. 123
LINCOLN'S INN FIELDS.
LONDON, WCZA 3PX

Cables : Cancerch

Tel. : 01-242 a200

Dear Mike, Art, Herman, Nancy---

    Enclosed are copies of the information Chris Marshall
original13 provided me about his hybrid lines. I would still
suggest loeking at one parent (1&47 with wt provirus, B77) and
two hybrids derived from it (1577 and 1578). At this point, the
isolation of "retransformantz" from the hybrid population is the
only evidmce for retention of the provirus which miginally
traq$sformed the cell---in vlew of the evidence that some of
my rat-1 revertants are DNA-minds, it might be necessary to map
provimses in parental and hybrid
find viral RNA or protein. (The
cells could have multiple proviruses, losing the active one duri.
selection ofhybrids and retaining silent ones that might be
reactivated later. Although unlikely, this possibility is testal
and,if indicated,I will do so when I return.)

I have discussed our recent results with the revertants of

ASV-rat-1 cells (clone 31) with J&n Wyke.(l;rChe transformed clon
was originally picked as an agar colony and then subjedted to
single cell cloning.  (Eohn does this by picking single cells
with a micropipette under the microscope, using a dilute soluti,,
of trypsinized cells) the single sells are then grown in a mien.
%itre dish.)  This means that it is virtually impossible for t-
original clone to kve been contaminated with uninfected cells,
implying that the three DNA-minus revertants have truly lcst the
gstm viral genome.  We will attempt to get karyotyping done on
them here; it the karyotgpe is unrevealing, 1 will  consider clo.
ing the Clone 31 provirus when I return,  in order to make a pro'r;
fm the adjacent sequences to see whether one copy of' them is
disordered in the DNA- minus cells as a consequence of excision.

(2) Since no examples of revertants I and N have been tested qQa
biochemically for gene expression,  it would be nice to test thenA
for kinase and labelgd proteins, RNA testing would seem unneces:
ary at this stage.
the DNA-minus cells further (Gg, H, and 0). (3) Three r

(3, I, and J) have been tested for rescue of polymerase'
particles in the absence of transforming activity? they are all
positive, but interestingly the titres of' stock sz from J are

      *higher than stocks from B and I. This seems $ot ti
correlate with Herman's observations !zkkh. about pr76 levels, but
'kkz'x such comparisons are probably premature.

Obviously there is llttle point in testing

(4) John will

a=-

IMPERIAL CANCER RESEARCH FUND LABORATORIES

Registered Charity No. 209631

P.O. BOX NO. 123
LINCOLN'S INN FIELDS.
LONDON, WC2A 3PX

Cables : Cancrrch
Tel. : 01-242 0200

recover T+ virus from revertant E and will prepare clones of
retransgormed cells from E also. It would be of' obvious interes
to test these matarials for kinase activity and levels of pp60.
For comparison, he will rescue nontransforming virus from a
few other clones: J (good pp60 band,?no kinase)+ and B,F, or M
(low pp60, ? no kinase). In additioi;, he WAS% has a stock of
virus rescued from L; this stock is transformink but makes
fusiform foci (as you recall, L is kinase +). When these things
are ready, we will send viruses in chick celgs.

   John was, of course, surprised by the observation that
his double revertant had only a portion of a single provirus,
whereas the putative parent (GE 11, also picked as a single cell)
appeared to have three proviruses, I will try to do a little
more mapping with these) to see if the partial provirus in the
double revertant*his in the parbnt and to get some idea of the
organization of this peculiarly deleted provirus (Steve is about
to reannezl the first filter with cDNA ,&-; please share this
letter with him).  Although John is ce2tain that the double rever
ant was isolated from GE11, some of his other lineages are clearl:
confused (based upon mapping data with revertants of one of two
active proviruses), and he is now redoing the experiflent from t5
beginning, making double transformants (avoiding t'ae helper virus
and defectives used In the previous protgpol) and then select&tdg
both sbngle revertants (pf he & provirusd and double revertmts
(at 35 ) . If any of the&ikf!&?ar from the mapping data and from
retransformtion experiments to be candidates for cellular mutant:
you will be hearing from xs.

that you have put into the clone 31 revertants. When same of the
items discussed above are completed, I will put bogether a b&&ef
description of these revertants for publication.

John and (needless to say) I appreciate the time and effort

It is nice to be back here, decelerating to the & ICRF rkyti-i

Cheers ,