October 4, 1977

Dr. Aaron KIUgp F.R.S.
MRC Laboratory oL Molecular Bidlogy
University Postgraduate Medical Sohool
Cambridge CB2 PQH England

Dear Aaron,

Thlss is the letter I pa=ombs&d you &bout Bak and Zauthen. Z ncaed not
disauss their personalities as you have already met them.  Bak has a
research student (medically qualified) also called Bak, but relation,
He is the one who actually makes all masurmntrs -- b said hs sees
unit: fibre in hie dreams!

Talking with them and looking at their slides soon revealed a number of
things which my prcevious sxtenfaive eorrsspondance ha8 not uncovered.
In the first p2.aeel although the yield of chromoisonw?~ QPJ high, they are
a mixture of very different atages of condensation.  This 5.8 because
the coloemfd irJ applied for a very long time (19 hours?), 80 eiaat some
of the mitotic chromosomes have seen it only tor a short time and others
for a long tine, depending on when that cell entered mitosis.  W*P+
the ahromsomes tend to stay together in groups, presumably because
parts of the spindle are still there. However the really bad feature
is the extremely low yield of unit fibres (perhaps I%)$ thar rest of
the material being 9n irretgukaw clumps,
fixative (methanobaaetio acid). Zt? lookfsr kc3 me at3 a@8e is B race
between unfolding (due to pH?) and Pixation,

These are produced by the

3 told them that it was essential that their yblds should be h%gher:
at lleaest SO% and hopefully 90% greater. I told them to try acid
buffers of different pH.  I alea suggested that they attempt to dirinperea
the ofucamoaomes somewhat, before fixation, by a cold treatment in the
hapa it might depolymerize the microtubules. Alas Z suggest that they
try (there are trfeks to do this) to get the mitotic chromosomes
more uniform before they start and to v&y conditiom to see if the
less condensed chromosomes unfolded mrb, or less, easily than the Illore,
aondrsnmd ones,

Xf they found hhht a pH OS, say# 5-0 WOUM unfold the ohrorrPosamsalhJ to
give unit fibres then they might try other fixative@.
try the effecte of various salts, etc.

Also they could

October 4, 1977

Dr. Aaron Klug, F.R.S. -2-

I told them it was useless to pursue elm work (except to try out
methods) until they had a pure preparation of fibre? For this they
should certainly get the yields up and even then might have to try
fractionation (to remove unfolded or clumped chromosomes) perhaps
'using a sucrose gradient.

In short, my message was to stop measuring and instead to try lots 6f
things, Also to work with smaller samples so that they could do many
quick experiments,
do careful rneaauremnts when the coadition8looked promising.
younger Bak liked this idea because I think h& was sick of measuring!
Zauthen got the various points and I think will act on them, though my
impression was that the elder Bak didn't me what all the fuss was
about ,

Also to work briefly to explore conditions and only

The

Anyway, it wagl a lesson to me not to Write papers with people you've

The pofnt ier 80 elementary.

c cc L*L.n e' never met 1

ft never-to me to ask what their yd&lBs were.

Incidentally the "doubleness" in the microscope means vsxy little if
anything. As you go out of focus the image broadens as it appears
double. X suspect almost anything aylindrica3. will do this, Xn spite
of! all the above, I think it's worth their while to try to clear it
all up. A major question is whether a unit fibre comes from one
chromatid or a pair of chromatids (a metaphase chromosome consists of a
pair of chromatid8 joinsd only at the centromere),
this up is to meamre the mass/lenglh or DNA/length,  but these measure-
ments are very difficult to do accurately.
Zeuthen's preliminary measurements. A better way is to watch the
formation of the amit fibre under the microscope and, eventually, make
a video tape of the process,

--l---

One way to clear

I was not impressed by

I am trying to arrange fox Dan Linderlsy, of UCSD, to send them a
Drosophila strain with a translocation to make the three major
chromobiomes of very different lengths, This should produce unit fibres
of three clearly distinct, size classes,  Of course if they really can
produce unit fibres in good yield,  there is no reason why they shouldn't
try a drot of other interesting spcim, to say nothing of aoing good
e/m work,

I've just spoken to Micqael Zevitt,
My only other new8 is w.  I've been on a diet -- I've lost 12 lbs!
Apparently I need only loss another 4-5 lbs, Then T can buy some new
trousers.

He seems cheerful but time-shifted.

F, E. C. Crick
Rieckhefer Distinguished

Research Professor