Many many thanks for your letter dated September 14. I have informed Koteswar about the staining procedure. Poly A solutions
though slightly viscous are working alright.
We have already commercial samples of poly I, poly C and alternate poly AU. So we are ready to search for RNase III type of
activity. In case you have already started please let us have your advice. I am also keeping track of Schlessinger's reports.
Unfortunately, however, our ultracentrifuge which arrived about a month ago is not yet put into operation, As soon as it is
working we hope to look for similar system in S.typhimurium. Yes we know that S.typhimurium RNase II is
extremely thermolabile. So we can easily get rid of it.
Perhaps you have forgotten that we use phage P22-infected spheroplasts as our starting material for our RNase II preparation
(B.B.E.A. , 212, 102, 1970) for two reasons. On conversion of cells to spheroplasts there is loss of considerable amount of
RNase I, as expected. Further, on infection of these spheroplasts with phage there is still some more lowering. Even the intact
cells on phage infection loose some (not much, we did not make quantitative measurements) RNase I. We feel that the enzyme
is released due to some type of physical shock as a result of phage infection. There is similar report in literature. In case
you are further interested I shall send you the reference.
The enzyme RNase I that gives only 2 bands is not 100 fold purified, it is about 500-1000 fold purified. You have missed
the point in the report that the enzyme purified though our standard procedure (J. Bio. Chem., 243, 1133, 1969) was further
purified through phosphocellulose column and this column eluate gave only two protein bands. Unfortunately RNase assay on
gels is not working in our hands.
Have you any experience with it? Please let us know.
Our progress with RNase is slow. With a new colleague (Aloke Dutta) we tried to repeat our earlier results of having multiple
peaks of RNase I from DEAE cellulose column. We get 2 to 3 distinct peaks all behaving like RNase I activity but it occurred
to us that they may represent free RNase and RNase bound with the ribosomes. Actually DEAE cellulose column has been used
for purification of the ribosomes. Since we know that Mg++ is required for the latency of the enzyme we prepared extracts
in presence and absence of Mg++ . Elution pattern varies but multiple peaks are there in both the cases. It is unfortunate
that the ultracentrifuge is not working. We could have then easily solved the problem. Anyway we then incubated the extracts
in presence of EDTA to destroy the ribosomes (we have indirect evidence that RNase I destroys the ribosomes
under these conditions) but still multiple peaks of RNase I are obtained. This may be an artifact. So we are planning to concentrate
each peak and put it back on the column to find out whether the multiple peaks really represent multiple forms of RNase I.
You mentioned about some report on similar line. Will you kindly send the reference?
Another new colleague of ours (Khandekar) is searching for phage P22-specific DNase phage-infected S.typhimurium extracts.
He is using P32-DNA as substrate.
Large amount of endonuclease (though inhibited by tRNA) and another E.coli exonuclease type of activity are creating problems.
We have, however, indication of increase of a DNase activity specific for single stranded DNA. The work is however, in very
It is now high time for me to plan my trip. So please let me have approximate idea about the timing of Gordon Research Conference
and Cold Spring Harbor meeting. Please let me know the name and address of the person to whom to write regarding G.R.C.
Maharani is excited to learn about the robe. I am always excited to receive your letter as it contains lot of food for thinking.
So boss! please be regular in correspondence.
Maharani joins with me in sending our best regards to Dan and love to the children.