Champe here revealed that he had resumed research with the rII mutants of the bacterial viruses T2 (phage), which had been
used since the early 1950s in genetic mapping experiments. These experiments were designed to break up genes into their component
parts, eventually down to the level of individual bases--at a time when biochemical means of sequencing the bases of DNA were
several years in the future.
One goal of researchers had been to map changes in the mutant strain rII to changes in the amino acid sequence of the protein
for which it coded. (Upon infection viral nucleoprotein, an assembly of nucleic acid and protein, enters the bacterial cell,
where it induces the synthesis of more viral nucleoprotein, which is released when the cell ruptures.) However, the protein
for which rII coded had never been found. Champ and his co-workers resumed the search in the fall of 1966.
Number of Image Pages:
2 (177,551 Bytes)
1966-10-09 (October 9, 1966)
Original Repository: Wellcome Library for the History and Understanding of Medicine. Francis Harry Compton Crick Papers
Although I had vowed never again to look for rII, I broke down last winter when a bright and energetic young student by the
name of William McClain started to work for me and could not be dissuaded from having a look. We decided it might be easier
to detect a tryptic peptide of the rII protein rather than the protein itself if a labeled amino acid were used which occurs
rarely in proteins but which pretty surely is in rII. Tryptophan was a good bet because there are rII ambers which are weakly
or not at all responsive to 5FU and these should have arisen from tryptophan.
The initial approach was to chromatograph double labeled tryptic digests of the entire infected cell, comparing a nonsense
mutant far to the left in A (H^3) with a nonsense mutant far to the left in B.(C^14). A trypt requiring, tryptophanaseless,
non-permissive host was used, and at the beginning we used only ochres since this host had an amber suppressor. The cells
from the two cultures were centrifuged after labeling, mixed, and taken up in cold 90% formic acid. After dialysis, the material
was boiled, digested with trypsin and chromatographed on Technicon peptide resin (essentially Dowex 50).
The attached Fig. 1 shows such a chromatogram comparing N55 with UV375.
The H^3 and C^14 track very closely except in the region around fraction 125 (component 30) where there is a striking increase
in the H^3 to C^14 ratio, suggesting that component 30 is a product of the B cistron. Note that the control N55 (H^3) vs N55
(C^14) shows no such peak.
Our attempts to locate the region controlling component 30 are summarized in Fig. 2 where + means that the mutant makes 30
and 0 means that it doesn't. Note that 1589 fails to produce 30 indicating that 1589 overlaps the controlling region.
This is consistent with further results using phase shift pairs FC(1,38) and FC(1,58)
shown in Fig. 3. The fact that the former pair is + while the latter pair is Q implies that the left terminus of the region
is between FC38 and the end of 1589. (For this experiment an A cistron mutation was inserted into the FC doubles to prevent
phage production and possible secondary consequences.)
Fig. 4 is a plot of two separate single label runs showing that double label is not really necessary and that the effect is
not some funny business due to double label counting. That component 30 is in fact a peptide has been shown by recovering
tryptophan fairly quantitatively after pronase digestion.
We have tried to produce a chromatographically altered component 30 by looking at presumed missense mutations within the controlling
region but have not been successful- either it is present or absent. Difficulties arise in re-chromatography of comp 30 for
further analysis since it is often altered by the manipulations (probably drying) required to concentrate enough of it for
Our main aim now is to use component 30 as an assay for the intact protein and thereby try to purify the intact protein. This
is just beginning and there is nothing yet to report. I think this approach offers the best hope of obtaining the left tip
of the B protein.
I would certainly be most interested in having a copy of your manuscript. The authoritative map would be very useful to have.
I have a preliminary one from Drake who said there were probably some inaccuracies. Our assumption that FC 58 is to the right
of 1589 came from this map and I wonder if this is really true. Our crosses of 1589 with FC 58 have not convinced me that
they are separate. In any case it would be useful to have an additional +- pair like (1,58) but where the right mutant is
further out than 58 if you could supply one. Also if you will send X655 we will see if it makes component 30.