Original Repository: Stanford University Libraries. Department of Special Collections and University Archives. Paul Berg Papers
Reproduced with permission of Robert L. Sinsheimer.
Medical Subject Headings (MeSH):
Protein Synthesis, Tumor Viruses, and Recombinant DNA, 1959-1975
Letter from Paul Berg to Robert L. Sinsheimer (May 1, 1963)
May 22, 1963
With regard to publication we will not be presenting any data, at CSH, but this should not inhibit you at all. As you point
out we can still submit two papers together to JMB.
With respect to the Tm - our figure is 77.5 - 78 degrees in 0.01M sodium citrate pH3 7.0 p1us 0.02M NaCl. This is 0.05 M
Na[plus] which I gather from you graph you would say gave a Tm of 75 degrees. The general shape of our melting curve is similar
to yours with perhaps a little shaper bend on the low temperature side.
Our experiment on the replication of a hybrid was done by incubating H3-hybrid plus enzyme plus C14-tri-PO4 and examining
the product (approximately 1.7 x as much RNA was made as was in the original hybrid) in a sucrose gradient. The hybrid which
has an S about 18 now contained both Cl4 and H3 (molar ratio c14/H3[about]1.6). Other counts were in free RNA (5[about]5-10)
and C14/H3 molar ratio[about]3. This seemed like a mixture of conservative and non-conservative replication (with conservative
favored) although as Mike pointed out we could partially explain this by assuming that the enzyme, present in limiting amount,
selects substrate at random so that after a few C14 hybrids have been made, they could be used - i.e. we could not talk about
the replication of individual molecules. Your experiment with 5 BUTP and C14 CTP is a better one, but would require careful
We have tried to approach this by using saturating amounts of enzyme so that one might assume all hybrid primer molecules
were engaged. However this got us into another difficulty - our enzyme preparations do have a small activity in the absence
of added primer. With high enzyme concentration and low primer concentration this seriously confuses the result.
At present we are trying to determine the result kinetically with special emphasis on low levels of incorporation. I will
let you know how this comes out.
We have recently been studying the incorporation of deoxyATP into what appears to be a [phi]X-DNA-RNA hybrid. You state in
your paper with Mike that deoxynucleotide-triphosphates do not work, but at least with [phi]X DNA, and one deoxytriphosphate
this seem to go fairly well. The product has a reasonable density. My only concern is that the C14-dATP we are using might
have some rATP in it - however reconstruction experiments suggest it would have to be as much as 5% which seems unlikely.
We are doing the appropriate degradation experiments on the product which should provide a definite answer. Such products
should be of considerable value in sequence studies.
With best regards,
R. L. Sinsheimer
Professor of Biophysics
P.S. I hesitate to ask but I wonder if in return for the [phi]X DNA we could obtain either some E. Coli cells or some polymerase
from you. Our problem is that our [. . .] has been out of order for three months now [. . .] 'fired' twice and each
time has not survived a full run--and I begin to [. . .] when it will be functional. In the meantime we are now virtually
out of enzyme.