I am writing to ask whether it would be possible for you to supply me with a hopefully modest amount of some hybridization
reagent useful for detection of endogenous rat leukemia virus DNA in Southern DNA transfer experiments.
The problem is as follows. Last year, while on sabbatical leave at the ICRF, I carried out an experiment (suitable only for
someone on such leave) designed to ask whether retroviruses can serve as insertional mutagens. First John Wyke and I characterized
an ASV-transformed rat-1 cell (called B31) which was found to carry a single ASV provirus and to revert to normal phenotype
at low frequency. (Revertants constituted about 10^-5 of the cell population and had either lost the entire provirus or undergone
mutation in src. These mutants are interesting, but that is another story.) The B31 cells were then infected with a high
titre stock of Moloney MuLV which I obtained from Natalie Teich (she had gotten it from Janet Hartley and passaged it only
a couple of times since). The cells were grown for 1-2 weeks, to insure infection of every cell, and then put through selection
procedures for the isolation of revertants.
As expected, most of the 35 or so revertants characterized appear to fall into the two classes observed spontaneously (four
have no ASV provirus, over twenty have probable point mutations in src), but a few have curious lesions in the ASV provirus.
In particular, at least two show clear evidence by restriction mapping of insertions of ca. 5-6x10^6 Mr. In both of these
cases, the insertion is present near or at the left end (3'5'-gag region) of the provirus, causing reversion by interference
with the synthesis or processing of src mRNA (this has now been documented by Nancy Quintrell). In one case, the insertion
is accompanied by deletion of ca. 10^6 Mr from the left end of the provirus, including the 3'5' repeat and possibly
some flanking cellular DNA; in the other case, the insertion can be mapped within two restriction sites (ca. 250 bases apart
near the gag start point, but I can't say whether any deletion has occurred.
I am obviously interested in determining the nature of these inserts, but I have been confounded by several problems. First,
it was apparent from the mapping data that the inserts must have restriction sites which differ from those reported for Mo-MuLV
clone 1. Secondly, Inder Verma kindly supplied me with some MO-MuLV cDNA which showed that most if not all of my revertants
have 1 or more copies of MuLV DNA, but again it was apparent that the maps of these proviruses do not conform with published
In the "interesting" revertant with an insert but no evident deletion, there are so many new MuLV proviruses that
it is difficult to say whether the insert is MuLV DNA; ASV and MuLV cDNA's detect superimposible fragments produced by
several enzymes, but there are enough MuLV bands to make the data less than totally convincing. I am in the process of isolating
Eco RI fragments from this line for secondary digestions (and ultimately cloning in bacteria) to try to resolve the issue
in this case.
The other "interesting" revertant, with an insert and a deletion, clearly has three new proviruses detectable with
Inder's MuLV cDNA, but none of these seem to be the insert. Since the insert appeared after infection and is the correct
size for a provirus, I certainly favor the idea that it encodes a retrovirus, but which one? An obvious possibility -- at
least, with your help, a testable one -- is that the MuLV used for superinfection rescued an endogenous RaLV from the ASV-transformed
rat-1 cells and that the pseudotypes formed then spread through the culture. I would very much like to test this possibility
using labeled RaLV cDNA to anneal to already existing filters bearing "signature" fragments for the insertion (the
insert is not cut by Xho I or Eco RI), to the isolated and secondarily digested Eco RI fragment bearing the insert (this is
about to be run on the gene machine); and to the cloned insert (if and when). If you are willing to give me some help with
this, it would probably be easiest if we could chat on the phone about the details (amounts, specific activities, dates, etc.).
I am at 415-666-2824 at least between 9 and 6 PST almost any weekday.
I gather that Ed is now off on an English sabbatical. How is he enjoying it? Incidentally, some of your recent work was
the topic of a journal club here this week--it is obviously of great interest to us.