Original Repository: Stanford University Libraries. Department of Special Collections and University Archives. Paul Berg Papers
Reproduced with permission of Theodore R. Breitman.
Medical Subject Headings (MeSH):
Protein Synthesis, Tumor Viruses, and Recombinant DNA, 1959-1975
Letter from Paul Berg to Theodore R. Breitman (May 2, 1961)
Letter from Paul Berg to Theodore R. Breitman (June 6, 1961)
May 19, 1961
As far as I know the only system available at present for separating the 2' and 3' isomers of pseudo-U is ion-exchange
chromatography, using systems similar to the one I used in my paper (Biochem. Biophys. Res. Com. 3, 504 (1960)). The 2'
isomer that I originally sent you was isolated fron commercial yeast RNA by this method. The fractions containing pseudo-U
were pooled and then rechromatographed. The 2' isomer I sent you was isolated from the valley between the 2'-3'
isomers. As you can see from the figure in the paper it is very doubtful that there is contamination with uridine 2',3'
phosphates. There are two better possibilities for contamination. The most likely candidate is ribothymidylic acid. Since
the enolic pK's of pseudouridylic and thymidylic acids are = 9.43 and 10 respectively, pseudouridylic acid should have
a greater electrophoretic mobility at a pH of around 9. In your letter you did not give the conditions for your electrophoresis
or the mobility of the "uridylate?" contaminant relative to the 2' isomer. I would be interested in knowing these
conditions. Also could you give me an estimate as to the amount of this contaminant? Because of its methyl group, ribothymidylate
would be expected to have a greater mobility than pseudouridylic acid in a paper chromatographic system. From personal experience
this would hold for the isopropanol-HC1 and isobutyric-NH3 systems. Again, what systems have you used and what is the relative
mobility of the 2' isomer to its contaminant? I have found that the 3' isomer I sent you is chromatographically pure
Another possible contaminant of pseudouridylic acid is UMP-5'.
According to some workers, UMP-5' has been found in alkaline digests of RNA - the fact that the extent of this contamination
is not reproducible would indicate that it is probably due to incomplete extraction of the acid solubles. If this is the
contaminant, then the UMP-5' will have greater electrophoretic mobility in borate buffer, and a greater Rf in isopropanol-HC1
or isobutyric-NH3 solvents. Periodate consumption or treatment with a 5' phosphatase would be further confirmation if
there is 5' UMP, besides, of course, typical u.v. curves.
In regards to your contribution to Methods in Enzymology - Fred Bergmann had given me the impression that you were writing
an article that dealt with the isolation of a.a. acceptor RNA. I have a copy of your procedure using sodium lauryl sulfate,
norite, and DEAE. I was wondering if you had made any recent modifications in this procedure. What kind of yields of a.a.
acceptor RNA do you get (mg RNA/wet wt. cells)? Have you tried other methods - such as phenol extraction - to prepare acceptor