IMPERIAL CANCER RESEARCH FUND LABORATORIES

Registered Chanty No. 209631

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Tel. : 01-242 0200

Dear Inder:

  Many many thanks for sending the cDNA so prompfly---it arrive(
only 3 days after I spoke with you. Though all the exposures of
the three filters I annealed with it are not yet out, I have comex
to some tendative conclusions that I will share with you.

38 revertants were isolated from the EE*X ASV transformed

rat cells infected with Natalie Teich's stock of MuLV (said to
be Moloney,  obtained from Jan Hartley and p~ apparently cloned
fairly recently).
digests of the single provirus (ASV). Four have lost the entire
ASV provirus and all but seven of the rest have a perfectly normal
provirus and are probably nonconditional XXJXIE: src mutants. This
acconds well with results with revertants obtlitmadd from the same
line without MLV infection---a couple of those MEXE had lost the
provirus by an unknown mechanism (prob. chromosome lsss I would guc
and the rest had src lesions (we could rescue td virusk, the RNA
was normal, p60 generally present but kinase activkty reduced or
absent).

   The seven interesting,ones fall into three groups. The first
was represgnted by a single revertant which has an insertionof
ea. 5.5~10 Wr near the upstream end of the ASV provirus. The inse!
appears to anneal with MLV cDNA, lacks RI or Xho sites, has Bam
and Bgl I1 sites in positions vdhich do not accord with the MoMLV
clone 1 map.  In addtign, the left end of the ASV provirus (upstre;
end) is deleted (ca.10 da,ltons gone). TlGtXX~~ There seem to be
about 2 or 3 MLV proviruses'in ,this clone. The second type was
isolated three times, all presumably the same cell in view Bf the
restriction data. This ales has an insertion of about 5x10 mr, bul
not quite at the upstream end of the ASV provirus (prob about 500-
1000 bases from the end). The insert has sites for Bam, RI, and
Bgl I1 which don't bear any obvious relation to $he Mo-MLV map,
but the new fragments dbtected with ASV appear to be detected with
MLV cDNA. The only difficulty is that there are a lot of new
MLV proviruses (?around 10) and the superimposition of bands could
be a coimcidence. So far, there is no evidence for a deletion in
the ASV at the insertion site, but more mapping is in progress---
a couple of hundred bases could easily be gone without my having
detected the deletion thus far. The three subclones of this type
have very similar if not identical patterns of fragments seen wit?)
ASV and MlLV probes; hence my belief that theyare identicalx specic
isolated three times (they are from the same infectinn and from tli
same plate after BUdR selection against transformed cells).
The thr8d type f is represented by three clones.
unequivocal evidence for a delletion of at least 0.4~10 Mr from the

Thus far I've screened about 33 with restrictior

(7

Therg is

left end of the ASV provirus, but thus far khae all the data can
be accounted for without invokkng ax&& an insertion. Interestingly,
&he three have the same ASV pattern (with three engymes) but very
different (totally different save for endogenous bands) MLV patterns,
suggesting that the event initiating reverskon antedated the infeetior,
I think an unusual deletion of about 1.1~10 Mr (mostly viral DNA,
?some linked cell DNA) occurred in these cells prior to infection,
and that I then isolated the revertant three times, each cibone
bearing MLV proviruses at different (irre1evant)sites. But more
data is pending.

   Needless to say, we will need to clone these oddbal5s to
underatand them properly. In addibmbn, Nancy Quintrelf is currently
looking at the ASV RNA by Northern transfer. Doubtless I will soon
ask for various MLV DNA's to look at the IWBI MLV RNA's. If the
3'5' structure is a promoter at either end of the proviriJs, x;i;fxrk9,
we would expect to find RNA's with the 5' sequence of r7jj"Z aiid
linked ASV swquences, though it is possible such transcripts
are destroyed rapidly if unsuitable for processing (splicing).
We have seen candidatex RNA's of this sort in proviruses inserted
into cellular DNA, since they anneal with cDNA5' but not with
cDNArep. I am about to 610 two additional things: look for
reversbion of tkeseco
consequence of excisio * and to try to retransform type I or
type I11 by superinfecdcm with MLV , hoping to place a 3'5'
unit at a proper position to allow expression of ASV src.

whatever might be done collaboratively when I return in the
first or second week in July.

type to the transformed state as a

We'll talk more about this and how you want to work

Cheers,