JUN 9 1965

Children’s Cancer Ksearch Soundation, Sne.

35 Binney Street, Boston, Massachusetts O2115

REgent 4-6000
. Sidney Farber, M. D.

Director of Research

Boston June 2(staying until June 23)
1965

Dear Joshua:

Thank you for your letter of April 16 with the questions
about the cytochemistry of human chromosomes. I share
your views about the importance of that field and also its
potentialities for automated large scale surveys in the future.

During the last 2 1/2 years I have had a projects ,
running, very close ro gue ideas. Af this momen m,just
writing up the results. sar Lonel wot Mpg iL ant fo

The basic problem was to find out which accuracy one cdéuld get
at the determination in ordinary metazooan metaphase chromosomes
of 1f DNA 2 total mass and eventually 3/ RNA. Regarding 1/
the obvious difficulty is in the fACT Phat the chromosome
dimensions are so close to the wavelength of the light to be
used and thus the measurements afe on the very limit of the
theorethically possible. Physical theory is of no real
use here as the theory of the microscope image is not completex
in that range of dimensions. We have thus made an empiric approach
using Chinese hamster, rye ,nd human chromosomes. The spectrophoto
metry has been made with somewhat modified models of our high
resolution instruments. Quite a large amount of work has gone into
that - a numbe r of technical problems arose - but I believe
that we are as close to the physical limit in the measurement
as 1s possible. ,

fT include as an example Feulgen and UV measurements
on (RNA-free) hamster chromosomes presented graphically.
Feulgen determinations can thus be done with an accurady of
about (or possibly somewhat less than) 5 %. UV gives somewhat
lower accuracy due to adhering extrachromosomal substances.
However, while Feulgen, because of its inherent irreproducibility,
gives only information of which #-age of the DNA of the total
metaphase plate which is present in one chromosome, the UV
gives fahrly good absolute values. Our material on Chinese hamster
chromosomes is fairly comprehensive. Total mass determiniTIONs
can be made interferometrically, ho,.ever with an as yet only
estimated error of more than 10 % We hope to apply the by us
for the purpose several years ago developed electrommicrosctpic
chromoseme-~mass-determination procedure in the near future but
h,ve not started yet. For RNA we have at long last now a good
fluorimetric technique which permits the direct determination of
RNA in a mixture with DNA - as you know no digestion procedures
work well in such systems. JI look forward to apply that on different
chromosome problems next year.

Rye was used specifically in order to investigate the
possibility to xdemkkfyxxx follow the DNA-distribution along
the chromosome for the purpose of identification aN@, in the
first line, work just now being started on chromosome changes in
virus infected materials. It might amuse you to see in the
enclosed she®t giving the DNA-distribution along four different

rye chromosomes how the chromosomes distinctly differ (reflecting aha
details &tsq not visible to the eye) fromeach other and how
chromosomes with the same number give reasonably similar patterns.

Regarding the human chromosomes we have also some material.
That object is more difficult than the Cyinese hamster and the
accuracy which can be reached’is not enough to t#mmxx characterize more
than a limited number of individual chromosomes. It would take too long
to go into details here, but I am convinced one could get further here
with some technical investments, which I am sorry to say we are not
able to make just now. Anyhow we have during the last weeks in
fast considerably increased the possibility to get good measurements on
the smakl human chromosomes bya a new arrangemet for quantitative fluo-
rimetry. This arrangement might possibly come closesé to your idea
of A automated measurements in human materials.

The above is cited as an indication that one can, I believe,
already now fairly well define what is technically feasible on the field
you outlined. We are now applying the experiences won as yet
on virus problems and on differentiation and agbing problems - 4 long
and tedious work no doubt, but the technical possibilities for
work on metaphase chromosomes have in the work cited above
proved to be considerably better than I believed 3 years ago.

Thank you also for your reprint which contains many
refreshing thoughts. ; .

With best personal regards [I am

Ss urs “

Tord ) on —

*) ow the DVA~ Letorm t nat ton

Excl Oo |

P.S. I believe several people are interwsted in this type of
work and as we are now writing it up after a quite long
period of quite hard work I would appreciate your keeping

the data confidential. ,