Your stream of episodes are a constant source of entertainment to us all. You appear to be having a vigorous and eventful
Martha and I (and Fredi) drove to Seattle a few weeks ago, and got most of the problems for our June
arrival settled. We've found a large furnished house for the summer (to accomodate any Fair
visitors); Martha has signed a teaching contract, and I recarried my grant. It was cut somewhat,
especially the first year, to a total of $50,000 for the 3 years. NSF approved it, but has no funds
now to take up the NIH slack. Perhaps they will be able in the new fiscal year. Actually, I'm
pretty well off, and will have access to any equipment I will need. Only Fredi was disappointed
with the trip. He got to stay in a kennel near Seattle, which didn't suit his idea of a vacation.
The research is building up to a lengthly program in Enzymology, I believe. I do not have the
repeat experiment of the labeling by serine-3- C14 yet (it will be run tomorrow), but there seems
to be no doubt that B. subtilis has an active tryp. Synthetase. I can routinely demonstrate high
activity in whole cell suspensions (higher than wild type E. coli), but the activity is lost when
the cells are broken up. The secret to showing acticity is to use a very low level of tryptophan
for growth, since the enzyme is totally repressed, by 5 mg/ml, and 2 mg/ml give cells with only low
activity. The 3 carbon moiety is specifically serine, and the product formed is tryptophan.
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I've also prepared C14Oalenine[?] labeled cells protein, and should have an analysis on this
before you get back.
The most suprising aspect of the research concerns the his sensitive (his s) locus. All 3 of our
his s strains (all recombinable) are also sensitive to Oalenine. The mapping of this locus is not
complete yet, but it appears to be to the right of tyrosine. It definitely is not at the 5MTR
locus, which appears to be probably to the left of the aro 2 (shk 1) locus. I've started to analyze
the growth characteristics of the his 5 mutants in terms of colony size of high dilutions of cells
on the appropriate media. In the first experiment of this kind, the ratio of the colony sizes on
the different media (after 24 hr.) are approximately:
Oal + tyr - 10
Oal - 0
Oal + his - 0
tyr - 6
tyr + his - 8
D.O. - 8
The Oal + tyr colonies are very clearly the largest with all 3 strains. Suprisingly, colonies are
larger on D.O. than on D.O. + tyrosine, which does not fit in well with the growth studies in
liquid. The age of the plates are not comparable however (tyrosine were the oldest) and this may
account for it. I'm repeating it with freshly poured plates, and will also check the growth of SB
32 on his + Oal and his + tyre. SB 19 (in liquid cultures) was not inhibited appreciably by any
amino acid (except slightly by DL serine, and threonine), and Oalomine or tyr. did not stimulate
In this connection, you may recall that we have either histidine or oalamine (and are leaky on
D.O.). When the following cross is performed the results are as follows:
32 SU ---> 168
Selecting on Oal + his of 99 colonies picked --
74/100 -- growth on his; Oal, but not D.O. (Donor chose his - Su +)
21/100 -- his + Su + -- grow on D.O.
4/100 -- his - Su - -- growh on his, but not Oal (32 ---> 168 gives no colonies able to grown on
Obviously, the present concept is that a suppressor, closely linked, but separable from the his-
locus is responsible for the new genotype in SB32. Whether there is any relationship between this
phenomenon and the his 5 locus is obscure at the present time.
I think that further work on these problems must revolve around a study of the enzymatic reactions
of histidine and aromatic amino acid biosynthesis. I'm particularly interested in the effects of
histidine on prephenic dehydrogenase and the transinase. It will also be interesting to determine
whether we can detect enzymatic activity to determine whether we can detect enzymatic activity of
other type enzymes in extracts.
We're also actively working on the kinetics of competency, penicillin killing any transformants.
Nothing of interest to report on these studies.