August 12, 1949

Dr. Joshua Lederberg
Department of Genetics
University of Wisconsin
Madison, Wisconsin

Dear Dr. Lederberg:

I have started the crosses, and I felt that you would
be interested to hear the results thus far obtained. As you
shall see things have not turned out to be as simple as was
expected. he crosses were all done in EMS lactose with Bl, and
no streptomycin. The genotype of the parents was BM lace plus
Fis by TLB1 lac negative f1lR with streptomycin resistance and
dependence gn either or both stocks, depending on the specific
cross involved. ‘When resistant was crossed with resistant all
the prototrophs were streptomycin resistant, indicating thatthe
genes involved were alleks. The yields were surprisingly low ( wf runt
and the scoring for the lactose character somewhat difficult
because of the mucoid character in the resistant stocks which ™ 4
was segregatéad(Qin the progeny. fol )

The data of lac and the Tl segregation are below.

BN” lnes YO “S" X ThE ge. US “s*

lee + Y¥* le - U* lo. + lo. ¥*

9-1 X 5-3 sl 36 § 9
G-2K 53 G ¥ ° }
D-AOX SAG 1G 1S +o G
$-qJoX das (4 ls y lo
S-19K S25 py ly / x
S-197K SAK AS Is a =
S-apx ses 45 /o t >
S-aiKSR2& AF 13 / y

TER 36S ey 244% 47% (ES 7
robe

2

Going under the hypothesis that these were alleles seg~
regating and having no effect upon the lactose Ti segregation
I took the liberty of summating these figures and calculated
the recombination percentages from these. As is obvious the
segregation of lac and tl has in some way been altered. The
percent recombination,both for the singles and the triples,
is in accord with your data. The gross excess of the one parer
and the deficiency of the other, plus the consistency of the &

atait appears as if some semi-lethe] was linked to Tl or that
the population dynamics tesepplying acsptrong selective
pressure against the lac- Tlr class.

The next set of crosses were between resistants and
dependants. Here again all of the prototrophs were streptomych
resistant, once again indicating that a single "locus " was
involved.

(614) BM bee US *S" X THR mr) 4 sa” Gey)

on a UY (+ U,® a. -~ UR

Aor Sf 7 3

(sd) BOM” beet YP "sa" X TAB fe -U*% “s"

4 lo 0 ¥

In order to equate everything the procedure outlined
below was put into effect.
( sd we AU

cdg 2 SF sdiy aS ho

gate =

Jo ds
Ke. quiere

4t this point I tended to believethat the so called
streptomycin resistant was partially dependant (qualitative
observations on the growth of resistants in the presence and
absence of streptomycin) and that the segregation above
was only the extreme of that previously mentioned.

de , FN ut
it . x
" of Dptherem |)
$14 = Sas s Saat sal]

sd as

  
3

The crosses off Sd by Sd yielded no prototrophdana at
this point Dr. Bemerec left for his vacation happy and secure
in the knowledge that he was dealig with a single complex
gene,the queer lac Tl segregation not interesting him.

The prototrophs from the outcross were to my gbeat
amazement all streptomycin resistant. “dequate controls
on the medium used and the parental reactions were run.
The lace Tl segregation was consistant again.

5¥-/6/ x TAB bk -U% Ws 0

Ae uP wo yt yuk Ree UF
— 4G Vy Ra S oe
Lerenssty KoSd Ae pike May

ce

WwSISX RN “toe V,> “s
/@e (~,A)

o
’o

5 69 10

o Gs
6 */o as °/, . s hy

The mucoid character again interfered with the lac scoring.

Several hypothses came into mind:
l- There may be many genes involved or just two, one f
at each end, but Rhkese are supposedly single steps -
2-prototrophs were heterozygotes but this is invalidated
by even the extremely small % lac- ob 7
Sewe are dealing with an extra genic factore¥® one coup]
with a gene

xauxx
Though somewhat wild I8ve been thinking strongly along

this latter line as it might alse explain some queer data
Bertani has been obgaining with reversions from dependance.
The protocol I've decided upon for the remainder of my stay is
as follows:
i- Repeat the outcrosses( not another Ravin)
2-Cross the wild types as a control of their genetic
constitution especially in regard to the lao Tl
5-Cross resistant by dependant on streptomycin medium
to determine if I can recover both types or once

again only resistants
$-,futagogsthe dependant sand proceed with an analysis
4

of the type mentioned above (these latter two
showing nothing much if they give both types but
indicatory if only one occurs)

Se If the original data is reproduceable pick
@ sample of the prototrophs and transfer them daily
testing for resistenceas thers might ( cytoplasmic
interpretation)be two types if the factor"c8"is
gene reproduced and hence could be diluted out by
serial transferfrom those only phenotypically res-
istant.
6 Any suggestions will be appreciated
Of cougse the most efficient approach would be to put
@ resistant through a heterozygoteFfrankly 1 don't think that
they will be able to follow through here and 1 must wait for %
Bemerec's return before going into suchand of cousse your okay
on my follow through at Wisconsin.
I ran that experiment with Adems . SW- 87 was streaked #
free of phage and grown up in nutrient broth. It was sub-
cultured into broth contajng the usual sugars, making a faintly
turbid suspension, and 10° phage particles added. Clearing
occured in the lactose and the galactose tube after 35 minutes
(single burst?) and an increase in turbidity in the nutrient
broth and dextrose.This behavior was typical of the parent
(SW-13) and indicates that it is a direct action of the sugars
with no necessity of polysaccharide formation. Absorbtion
experiments are in order as soon as I can obtain a reliable
assay on the phage stock ( produces clearer plaques in Hershey
agar. “inding some difficulty in the @isposal of contaminated
material and noit wanting to further impose on Adams I've let
the matter lie.

My best regards to you and “rs. Lederberg.
Sincerely,

Norton Zinder