Robert Morton, an Australian biochemist, had recently found that baker's yeast contained a cytochrome (a protein involved
in electron transfer in cell metabolism) with a DNA component. That DNA proved to have different base ratios than the yeast's
nuclear DNA. He had suggested that this DNA could be used in understanding nucleotide sequences, and Kornberg had been working
with some samples Morton sent, looking at their ability to prime DNA synthesis with his polymerase. In this letter, he reported
some of his findings to Morton.
Number of Image Pages:
3 (226,803 Bytes)
1961-11-16 (November 16, 1961)
Morton, Robert K.
Original Repository: Stanford University Libraries. Department of Special Collections and University Archives. Arthur Kornberg Papers
Reproduced with permission of Arthur Kornberg.
Medical Subject Headings (MeSH):
"Creating Life in the Test Tube," 1959-1970
Letter from Robert K. Morton to Arthur Kornberg (November 29, 1961)
November 16, 1961
I feel a report on our progress with the cytochrome b2 DNA is overdue even though we know less than I thought we would by
this time. First let me acknowledge with thanks your last two shipments of the yeast and cytochrome b2 DNA's. Each arrived
frozen and we're impressed by the ingenuity of your packaging.
We have used bacterial alkaline phosphotase (BAP) to determine how much Pi can be released. We wasted time and material learning
how to reduce the Pi content of the enzyme, DNA and reagents so that estimates of 1 m[Mu]mole or less could be trusted.
Pi released by BAP: 10.5 m[Mu]moles/ml. (see enclosed curve)
We are reasonably confident that the BAP has no diesterase since we get theoretical values for pTpT despite large excesses
of enzyme and incubation time. We are also encouraged that excess BAP and time in the DNA digestions release no additional
Pi. If we assume that all DNA chains terminated in a monester P and were split, then the average chain contains about 100
residues (0.0105 [Mu]moles Pi/1.0 [Mu]moles nucleotide). We would now like to subject the DNA to the action of splenic diesterase,
an exonuclease (Razzell and Khorana, J. Biol. Chem. 236, 1144 (1961); Hilmoe, J. Biol. Chem. 235, 2117 (1960)) which acts
only on chains with a free primary hydroxyl group on the terminal nucleoside (i.e., XpYp----). The experiment will be done
with and without prior exposure to BAP. Unfortunately our current supply of the splenic enzyme is inadequate for the job
and we are working up a fresh batch.
The nearest neighbor analyses were not very revealing. We can see no striking discrepancies in any of the sequences from
what is predicted on the basis of random distributions in a DNA of that base composition. What is remarkable is the spread
of values for the base compositions of your bulk yeast DNA, the cytochrome b2 DNA and a sample we prepared from bakers'
Yeast DNA (Morton) 1.65
Yeast DNA (Fleischmann) 2.48
Cytochrome b2 2.14
As you will recognize there is also considerable variation here with your published values. I am also enclosing a copy of
a preprint which Mahler sent me which documents these variations in base composition still farther. I'm almost convinced
that we must analyze yeast DNA carefully by density gradient centrifugation to determine whether more than one major component
is present. What are your ideas?
The Mahler paper is also of interest in the experimental data and conclusion that the DNA is single stranded. Offhand I don't
think his data are inconsistent with a double-helical DNA of unusually small size.
So far we've used up only about 2 ml of the 15 ml you sent us. Our contemplated die6Lmase studies will require very little
material. We won't need a lot until we're encouraged to think the DNA is homogeneous and identification of a deoxynucleoside
end group is possible. We'll let you know.
We are slightly troubled by a variation between the two samples you sent us in their priming ability for polymerase, the recent
one being only 1/3 as active as the first. Here again we'll gain more experience with these samples in due time.
Many thanks again for your enjoyable and stimulating visit.