June 25, 1955

Dr. J. Lein
Bristol Labs.
Syracuse 1, N.Y.

Dear Joet

Gongratulations to Pat and yourself. Do you really mean to take a vacation,
or just one from the lab? Anjhow, I hope this message won't distract you.

You hardly need to estplain to ms any delay in our correspondence. The wonder
is that you can ever break away from the routine-- perhaps part of my job is to
reaini you to do it. At any rate, as long as I am on a retainer, you may rather
hawe to explain to your bosses than to myself.

Thanks for forwarding the divers materials. It was something of a shock to
read the Branscripte— it always is; how could I ramble so? I hope and trust
they are not that bal to listen to. I hope you will feel free, as Ido, to rely
on any method of communication. The main point 1s that J don't have a secretary
I ean call on for this work. But I will try to orgahize may material somewhat

bettete1s very kind of you to look out after the antibioties. I did not mean for
you to go to any partichlar trouble. Grisein sounds as if it might be wrthwhile.
We have had quite a bit of experienee with strpptothriein already, limited minly
by uncertainty of supply. The crose-resistance with streptomyoin, and thef fact
that a miltistep system also seems to operate 4n K-12 has discouraged us from
doing moh with 1¢ as a marker, but sometime we may need to do more with it.

I am not sure how well I can help you with your request for purine-... mutants,
I am sending what we have along this line, but with no warranty about the cultures
which we have not examined for a long time. Demerec, at Cold Spring Harbor, or
Hob Guthrie (at Roswell Park now) might be able to help you if these don't pan
out, We have never looked into tetracycline resistance in any way. I amsed my-
self by trying to guess what you're after:: don't bother to confirm or deny, but
my hunch is you're going to look inte accuglation and excretion ff purine bases in
relation to tetracycline syfnthesis.

As to the screening program, I woter Lf you have concerned yourself why colonies
should fail to grow on transfer; perhaps there is a group of organisms thet needs
special soil extract factors ? or other nutrients from exosymbiotie organiams?

I was somewhat relieved at your experience with Nobel agar:: my om had not been
as good as you had claimed; perhaps there is a good deal of lot—to-lot variation.
Thanks especially for the flowsheet and. the programming data. I have not
fyhly digested the same,and will defer coment.

One point does occur to me: 1) the large dropout between preliminary charac-
terization of positives, and those kept fam after time study. I was delighted
that you do not discard the saxgatiness low-potent isolates at this point, It seens
to me your competitors are mst likely$ to ignore these altogethey and that they
might therefore represent the most unique material. But what to do about it? The
weakest point in the whole program 1s how to handle these low producers. 1. You've
already mentioned medium study. 2. Are you convinced that the extraction and assay
procedures are beyond improvement, for dunubtatx handling these items? (On my next
visit Inwill try to learn a little more about these from you]. 3. Is not this per
haps the most likely point to apply mitation procedures?

To enlarge on 3), I don't know if 1t would be flopeless to do a full analysis on
overy weakly potet culture. However, these are most likely o include antibiotics
that have been overlooked by other workers. Two approaches are needed: a) means of
soreebing out lilsly repeats even in low-activity broths, b) but regardless, a
fair sample of these should be screened in at least a rough way— say 10 plates
per cultured, from UV'd spores— for posaible improvement in yield. It is uneer-
tain whether there will be any marked correlation between increasing the xkax zone
of inhibition with Kelner's method and improvment of yield in liquid fermentation.

Experience may already have told you that most low-yielders are not qualitatively
unique; if so, 3a will have to be perfected before 3b is mu worth much.

Finally, after time study, I notice how few broths are rejected as being familiar.
Unless your eriterda at this stage are quite reliable, might it not be better to
omit charac terizag$ion tests at this stage, or note them as xonly advisory to Biochem,
and rely on the chromatography? If you do continue the prelim. echaract., wouldn't
your tentative conclusion simplify the later extraction steps enough to make it
worthwhile to do a more rigorous analysis? Aren't you taking an undue chance, in
relation to the coat, of missing a good find? Y am not mach impressed by the deep
agar column, by itself, as a disagnostic tool, since an unknown new substance might
very well. mimic the others. The same for cross-resistance, especially with broad
spectrum antibidtics/

urs, sincerely,

(AA Pm
Soshua Lederbegg

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