Department of HEALTH, EDUCATION, AND WELFARE o Public Health Service
      National Institutes of Health o Bethesda 14, Md.

NATIONAL INSTITUTE Of ARTHRITIS AND MEIXBOLIC
NATK)NALd$ANCER INSTI1IITE
NATIONAL INSTITUE OF DENTAL RESEARCH
NATIONAL HEART INSTITUTE
NATIONAL INSTITUTE OF MENTAL HEALTH
NATIONAL MICROBIOLOGICAL lNSTl"E
NATIONAL INSTITUTE OF NEUROLOGICAL DISEASES AMD BwIDII?sF
THE CLINICAL CENTER
DIVISION OF RESEARCH GRANTS

December 13, 1955

Mrs. Maxine F. Singer
Ilepartment of Biochemistry
Yale University
333 Cedar Street
mew Haven, Connecticut

Dear Krs. Singer:

Here are the "Project Suggestions" that I promised you:

Project:
in several bacterial systems, with nucleoside 51-diphoephates 8s precursor
compounds (1).
extracts of animal tissues although the results of isotope incorporation 3

Net synthesis of polynucleotide material has been demonstrated

No comparable success has been achieved with soluble

experiments are encouraging.

9 i,&A '

I propose to stEdy nucleic acid synthesis in animal tissues by

searching for a soluble systeln catalyzing the phosphorolysis or pyro-
phosphorolysis of polynucleotides. A number of possible substrates in-
clude virus, yeast or animal ribonucleic acid, the various polymers
described by Ochoa and Manago (1) or the small polynucleotides terminated
with a 51-phosphomonoester group which have recently been obtained by
Heypel, Ortiz and Ochoa (2).  There is reason to believe that poly-
nucleotides terminated with a 5t-phosphomonosster end group are more
apt to be involved in nucleic acid biosynthesis than the n31-endedtt
polynucleotides which result from the action of pancreatic ribonuclease
on rnA.

The problem outlined above is difficult because the nucleic acid

synthesis system is pobably weak compared with the activity of interfer-
ing nucleases, which act on polynucleotides, and the many enzymes which
would remove nucleoside 5' diphosphates. However, studying the reaction
in the direction of polynucleotide phosphorolysis should offer a good
chaxzce of success.  Yith purification the various interfering activities
should be removed and the reaction mechanism can then be examined in
detail.

(1) M. Grunberg-hianago and S. Ochoa.

J. h. Chem. SOC. n, 3165 (1955).

o (2) L. Heppel, P. J. Ortiz, and S. Ochoa, Science, In Press.

Mrs. Maxine F. Singer

Page 2.

An Alternative Suggestion: It has been suggested that ribonucleic acid
synthesis may take place  in the nucleus and that the newly synthesized
polymer is then transferred to the cytoplasm. Accordingly it becomes
of interest to study the enzymes of nuclei preparations which act on
ribonucleic acid arid ribopolynucleotides.

The most active diesterase activity of nuclei is the hydrolysis

of cyclic mononucleotides and cyclic polynucleotidee to give the non-
cyclic derivatives (1).  This activity is fax greater than the rate of
hydrolysis of various internucleotidic phosphodie ster linkages.
hydpolysis is very curious in that it yields exclusively nucleoside-2'
phosphates, which were once considered artefacts of alkaline hydrolysis.
It would be of considerable interest to purify this activity and to study
it fn detail.

The

Another strange property of relatively crude liver nuclei preparations

is to hydrolyze yeast BTA to give exclusively nucleoside-3' phosphates,
whereas the n51-endedt1 polymers of Ochoa are qlit to give exclusively
nucleoside-5' phosphates and small polynucleotides terminated with a
phosphornonoester groxp at C-5'.  Study of tnis phenomenon should be very
prof itahls indeed.

  Suggestion (1) could be expanded. Point out that enzymatic and
other studies of large polymers are complicated by the uncertain nature
of the material isolated. With small, chemically defined golpucleotides
of different types (i.e., 5'4nded and Jt-eoded) one might have a better
chance of working out the reaction mechanism for polynucleotide synthesis
and degradation.

I hope these rather tentative suggestions me useful. I sent on

the sponsor's form to Dr. Scmtlebury.

We all enjoyed your visit very mch and hope that your fellowship

applications will be favorably acted upon.
everything goes.

Please let BEZ how how

Sincerely yours 8

Leon A. Heppel

M:lh