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Second report on lac- study
N. Morton
Jan. 10, 1957
New stocks. Five new lace stocks have been prepared by UV-induced mutation
in W3236 and called W3267,8; N39-1,2,3. Vs stocks were derived from each of
the Hfr lac- stocks by selection in broth, In future, a ve stock will be
used as source of new lace mutants to assure identity of the ve marker,

Lac~ stable derivatives of the two allelic mutable stocks W3120 and
W1950 were obtained after UV treatment (N36). Of 10 non-papillate colonies
of each type tested, 2 of each stock failed to recombine with other lac-1
mutants. These colonies were labeled W3269-72, %.

Mapping of V6, lac, P, A cross was made between W1366 % lacy (VESTER, )"Fe
and W3236 Hfr-1 P- M- on D-O+proline. The scoring of 218 recombinants
gives as the most likely order i

(Her) V6___—_idac P vt) (TLB)
35" 15 Be “31 38

In several other experinents. was | some selection for P+, but the same order
and approximately the same distances are indicated,
ONPG tests

Single colony isolates from B-O were grown overnight on a rotator in
YZ broth + 0,5% lactose +0.5% glycerol, These cultures were spun down, the
pellet resuspended in 1 ml. water, and the cells autolysed by shafling with
1-2 drops of benzene. To 0.1 ml. of this mixture were added 2 ml. of an
ONPG solution (30mg./l00m1,) and the color read by eye after 10 mimtes at
37° C, ALL of the single-step lac-1 mutants were ONPG+, Of the two-step
lae-1 mutants, W3159 and Y70 were also +, but W3229 and W3269-72 were ONPG-.
All of the remaining lac mutants were ONPG- except W3267, which contained
reversions, W3268, which is a weak fermenter of several sugars, and W3239, a

lace stock received from Borek, Lac-3, lac-5, and N39-1,2,3 were not tested.
Aliquots of all stocks were tested for lac+ reversions before autolysis;
only W1950 and W3267 contained a detectible number of reversions,
Fermentation tests

W3268, N39-1, and lac-3 ferment galactose, maltose, glucose, and
arabinose weakly or not at all, N39-3 does not ferment maltose, but does
ferment the other sugars, Lac-5 does not ferment galactose or maltose but
does ferment glucose and arabinose, The remaining lac- mutants will ferment

all the other sugars tested,
Crossover suppression in two-step mutants

Of the 2 ONPG + two-step lac-1 mutants, Y70 shows the “recombination
pattern as ¥53, and W3159 covers all of the recognized lac-l region except
W196, W3229 and W3272 cover all of the lac-l region and presumably extend
beyond it, since they are ONPG-. However, they have not been shown to be
allelic with any ONPG- single-step mutant, W3269-71 do not recombine with
lace2 and lac-l; or with the majority of lac- mutants of unknown location,
Either the region of crossover suppression is large » or most of the lace
mutants are in the neighborhood of lacel.
fransduction tests

If an M lac plate is spread with two drops of an F- lac- protetroph and
half of the plate respread with 1 drop of a Pl lysate obtained from a non-
allelic lace Hfr ve by the Lennox modification of the Adams layer plate
technique, then a yield of from 20 te 300 colonies will be obtained with Pl
at a time when the control half of the plate is blank or has at most a few
colonies, Allelic lysates give zero yields, In all combinations so far
tried, the proportion of v, among lac+ colonies is less than 1%. No per-
sistant heterogenotes have been obtained. |
Recombination tests

More than 20 crosses have been made of Hfr Ve M- lece X Fe lace prototroph
300

on M lac, the colonies being crossestreaked, without purification, against
T6 on M lac, The Hfr parents have been W3229, W196, W3146, and W3120,
In every case, the proportion of vs among the recombinants is less than 50%,
with no evident reversal of ratios in reciprocal matings,
Discussion

So far attempts to map the lac-1 region have been unsuccessful, This
may be due to multiple adjacent crossovers in pairing regions, to proximity
of the Hfr-] elimination region to Ves or to heterogeneity in the location
of V, mutants, It is not clear what the most expeditious way to investigate
these possibilities will be. Tentatively it 1s proposed to use W3133 as a
universal receptor for all Pl lysates and as a universal F- for all Hfr-1
stocks, excluding lac-1 lysates and stocks, This procedure is suggested by
the fact that W3229 ve gave no V; recombinants with W3215, W3151, W315h, and
W3112, indicating perhaps that there is a V6 locus closely adjacent to the
crossover -suppression region of W3229 and W3133. If at least one cross gives
an excess of Yes this will give some hope that the region can be mapped with
existing stocks, If there is no such cross, the most hopeful possibility
would seem to be that the Hfr-1 locus is responsible for the mapping difficulty,
and either transduction with F- donors and receptors, another fertility
system, or the use of P as an unselected marker may be tried, To introduce

P or another Hfr into existing P+ stocks would be difficult,
nes

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