June 9, 1966

Dr. Jaraes Caatles
The Presbyterian Hospital
622 West 168th Street
New York, N. Y. 10032

Dear Jim:

I'm sorry that it has taken so long for me to get down to writing this
letter.
you and then you can spend some time both now and after you arrive
thinking about them before you settle on one.  Aside from inherent
interest, all of them appear to have aeveral advantages.
acquire diversified experience in techniques and you have same assurance
of results, although of course the results themselves may be more or
less exciting than we might anticipate.
to read through the following t-m books as a good introduction to the
whole area:
Ingrm, The Biosynthesis of Macromolecules. Both are published Ln paper
binding by W. A. Benjamin, Inc., Mew York. Copies of both are on the
shelf in my office should you want to borrow than.

There are several projects which I would like to outline for

You would

In any case it would be useful

3. I). Wat~on, Molecular Biology of the Gene, and V. M.

Here ere the projects.

(1) RNA degradative enrrymes in Lactobacillus arabinosus. A wide variety

of microorganitma have been tasted for presence of the enzyme poly-
nucleotide phosphorylase.
enzyme is &. arabinosus. Furthermore, it has been reported that
extracts of &. arabinosus from which ribosomes have been removed also
lack the common postasslum activated RNase XI.
then, this bug locks both enzymes now believed to be responoible
for the breakdown of messenger RNA, at least in E. coli. What, then,
is the rnecbnhn of mRNA breakdown in &. arabinosus.  Recently we
made sme &. arabinosus extracts for another purpose,  ad I looked at
the ribosomal rather than the soluble fraction. I believe that there
may indeed be $cane phosphorylase end X am certain that there is an
enzyme canparable to RNase II.
Certainly membrane fractions should be teetad.  Whatever enzyme or
enzymes are found should be charactarized-with a view toward the
mechanisn of messenker breakdown.  This could dsvelop into a very
interesting problem--on the other hand it might turn out dull-
just sho-wing the same enzymes a8 in coli but with a different
distribution.
could then easily anbark on an alternative problem.

The only organism reported to lack the

On the face of it

You might then look at this closely.

In any case you should know that fairly quickly and

An important recent tool is eo-called hybridization of DNA to RNA.
The theory is that a single strand of DNA and a siqle strand of
RNA having exactly complementary sequences will form a hydrogen-
boded double helix, much like DNA itself. Operationally, whan
looking for such identity, the extra, nonspecific RNA ie degraded
away with pancreatic ribonuclease, after hybrids are formed. The
theory is that RNA in a double-strand is not susceptible to
pancreatic RNase. In fact, this is only relatively true. The
-- E. coli RNase I1 which we hava in highly purified state seema con-
pleely inactive on double stranded RNA (eee papers by Toltert and
Singer).
inactive on DNA-RNA double helices, and if so,  it should be a better
reagent for hybridization studies than is pancreatic RNase.
project would involve you in the preparation of synthetic polymer8
in order to have suitable model substrates, and also in preparation
of hybridizable DNA6 and RNAs,  There is an excellent review of
hybridizatlon in Volume 1 of Progress in Nucleic Acid Research,
edited by Davidson and Cohn.

It ie of great interest to see whether it is equally

This

The article is by Marmur et al,

(3)

Several years ago ~JB looked at the phosphorolysis of transfer RNA
(sRNA) by polynucleotide phosphorylase.
presumably because it is hydrogen bonded and polynucleotide
phosphorylase prefers randan coil substratess. Nevertheless it was
phosphorolyzsd some 20-30 percent. The same conclusion was arrived
at concernin6 sRNA from a variety of 8ources-by a variety of
workers, The following question aroee: are 20-30% of the chains
degraded canpletely--or are all the chains degraded 20030%.
results on amino acid incorporation given in the paper with Cantoni
(no reprint available, Biochim. Biophys. Acta 3& 568, 1960) suggest
that at least some of the chains are intact.
that in a11 probability the sRNA was badly contaminated.
thing should now be looked st carefully with highly purified sRNA
which can now be prepared.
hydrolysis of clean tRNA with the 9. coli RNase 11.
review of sRNA chemistry in Volume 2 of Progress in Nucleic Acid
Research-an article by Brown.  And in one of the 1966 issues of
Biochemistry there are two relevant papers by Muench and Berg.

sRNA is a poor substrate-

The

Hindsight now indicates

The whole

At the same time, we should look at

There is a good

(4)

Dave Logan in our lab has been looking at the following problem.
It has been hypothesized that messenger RNA is protected €ram
degradation in vivo by its association with riboeomes.  He ha8 then
looked at the degradation of synthetic messenger--namaly polyuridylic
acid, by purified RNase 11, and looked for protection by riboaanass.
He has some very exciting results indicating that one does get
protection, but it is dependent on the presence of tRNA and protection
increase8 with protein synthesis.
write about; a1'11 have to give you the full significance some other
the.
be looked at and appear very promising.

All this is too complicated to

In any case, there are many ramifications of this which should

The really bad bugs are

worked out and a anticipate that rapid progress could be made.
For exmple, the same thing should be looked at with polyaucleo-
tide phorphorylaee. Also, it now seems possible, by synthesis of
very specific polynucleotides, to be able to prepare complexes of
synthetic mRNA and ribosomes,  such that the ribosome and tRNA sit
at very specific posittone on the messenger.
to study the dfrcction of messenger reading,  the rata and PO on.
It may or may not be helpful, but I'm including a preprint of our
review on meeeenger RNA.

Thio should allow us

This should give you plenty to look at.
be making a preparation of polynucleotide phaphorylaee and Mr. Tolbert
making a prep of RNase XI.
intereBted in, you may want to work along with one or the other of them.
It would be good experfence and help break up the tedium of reading.
Either one of them would be happy to have your help.

Early In July, Dr. Klee dll

Depending on the problem you think you are

1'11 see you on July 25.

Best regards to your wife,

Sincerely yours,

Maxine Singer

MS: peg