ROYAL EDWARD LAURENTIAN HOSPITAL

FOR THE STUDY. PREVENTION AND CURE OF TUBERCULOSIS

PATRON
HER MAJESTY THE QUEEN .
HONORARY PRESIDENT
HIS EXCELLENCY THE RIGHT HONORABLE VINCENT MASSEY, C.H. MONTREAL DIVISION

PRESIDENT: HOWARD MURRAY, 0.B.E.
VICE-PRESIDENTS: NORMAN J.DAWES

THE HON.HARTLAND DEM MOLSON,O.8.E. TEL.VI.9-520)

W.TAYLOR-BAILEY

L.N.BUZZELL 3674 St. URBAIN STREET
HON. TREASURER: R.E.HEARTZ, B.Sc¢.,M.E.1.G.,LLD, MONTREAL 18

ADMINISTRATOR AND SECRETARY: H.D.JACK,
MEDICAL DIRECTORS: HUGH €.AURKE.M.D. (MONTREAL DIVISION)

F. LEARN PHELPS,M.D. (LAURENTIAN Division) April 22, 1959

Dr. Joshua Lederberg
Professor of Genetics
Department of Genetics
Standord University
Stanford, California, U.S.A.

Dear Doctor Lederberg:

Enclosed herewith I send you a preliminary note regafding my
recent research, Since this is my first excursion into the domain of
classical bacterial genetics, you must not be surprised to find signs
indicating my lack of experience, I, therefore, would greatly appreciate
your valuable advice and severe criticism. I realize some of the short~
canings of this work: as for instance, I am not quite happy about the
insufficient number of selected markers, However, I have not been able
to establish new ones, especially with BCG. Modification of the known
techniques to some extent became necessary in view of the particular
pattern of growth of mycobacteria,

What appears to be of interest to me is that I recently suc-
ceeded in conferring the streptomycin resistance to M. phlei via a cell-
free filtrate of the resistant BCG strain (both in the presence and in
the absence of streptomycin), First es tigations of the frequency of
occurrence show that about 1-10 out of 10° M. phlei cells are affected
by this change. I should like to mention that the new strains thus pro-
duced yield about twice as many colonies on agar media in the presence
of streptomycin as in the absence of this antibiotic, This, however,
happens only if platings are made immediately after the appearance of
growth on a selective medium, Approximately 8 hours later (i.e. eight
to twelve generations) the reversal of this ratio can be observed,
-2-

Serial transfer to a non-selective medium will also result in decrease
of the number of organisms maintaining the transferred streptomycin-
resistance, I wonder if this might account for what you call the
“heterogenote" stage, or do other mechanisms play @ role in it? The

_ Similarity of behaviour of resulting streptomycin-resistant clones sug=
gests a possible similarity in the mechanism of transfer of this single
trait when either intact cells or cell-free filtrates are carrying the
genetic information, even though the frequency of transmission is
different, Another interesting point is that while the streptomycin-
resistance of the BCG strain was acquired in four or five steps, the
highly increased streptomycin-resistance of the new organism immediately
emerged after isolation in both cell-to-cell contact or cultivation in a
filtrate, Some of the facts presented here bear a resemblance to that
of the interspecies induction of streptomycin-resistance in the genus
Hemophilus as shown by Leidy, Hahn and Alexander, Although at present

I do not think that the mechanism by which the streptomycin-resis tance
is transmitted among mycobacterial species is a DNA-transduction, I plan
to examine in the near future the effect of DNAse on the whole phenomenon,
It is worth mentioning that so far no transfar of characters could be
achieved by a cell-free filtrate of M. phlei. Uni-directional transfer
seems @iso to be operating in this case,

Also enclosed are three plates, as follows:

Plate I: Typical multiplication of BCG, 72-hour process, Generation
time is minimally 18 hours.

Plate IT: Usual elongation of "cocci" of M. phlei and their subsequent
division when transferred to a fresh medium, 6-hour
process, Generation time: 5 ~ 8 hours,

Plate III: aims to give a hint at the possible mode of conjugation of
individuals belonging to M, phlei and BCG strain respectively,
18-day process,

In addition to the distinct cellular morphology of M, phlei
("cocci" at the beginning which later elongate to form rods

and divide like rods) and M, tuberculosis (longer rods), growth
at roon temperature was chosen as marker character, After
conjugation has taken place (fig. 6), temperature was set at
37°C, Shortly after this, a small coccoid form resembling

the morphology of the M. phlei organism appeared at the opposite
end of the conjugating BCG rod, A new cytogenetic approach
employing additional marker characters became necessary in

order to elucidate the genetic significance of the emergence
~3-

of such forms. Do they actually result fran the conguga-
tion process or, as is seen in other instances, are they
merely representing the re-organization into rods of
amorphous mycobacterial masses? Such questions and other
controversial points raised by the above continuous obser-
vation of the conjugation process have to be cleared,

I am anxious to know your opinion about this work, which

is still in a very early stage,

SJsMB

Very sincerely yours,