Froblea: If the unite (autone) of an allele each produce a
gael] part of an engyme ani they ere put together in a particular
sequence, it is urlerstandable why the necianian is stopped if one
muton in the saxue chromosome ie defect. The rroblem ia whether, in
ease the defect muton ie supplied fro= a virus ( N » Bore synthesis
of scuplete engyme can teke place, 1,€., without recombination to
a cigform., In other words, ig it poesible to devise more sensitive
methods than cell division and coloniformetion for the study of
ensyme synthesia? Accordins te vacc!, at least 102° molecules af
glucose are reeded to sake ore “acterial coll Uvide, wherees less
then 2 x 10” molecules sre needed to produce SC phages, The follow-
ing mthod is proposed,

Kurahasht has found thet Lederberg's nunber 83091 and W309 of
Kyo) are MGel uridyl trenaferase~less. CrosaA 3091 with Kyo 309k,
wash and starve; then induce with U.Y. light, teke @m alicuot con-

taining about 10° celle and plate on galactoee agar which hae in
advarce been inoculeted with a very large amount of Gal + £. cold

to which \'is virulent. In a population of about 1000 K, yA there
should be between © and 5 big plaques due to reeembinants. If the
transduced but nonerecombined Kyo also can produce enzyme, but auch
less effectively, a mmber of severel hundred small plaques shoujd
appear after an induction period of 1 to 3 hours, for instence. “ince
many enayuee have rete eccnstente (number of soles substrate reacted
per nole ensyne protein) amounting to between 20° to 10” substrate
molecules per hour, only between 10 and 1000 protein molecules of
g@lactokinase need be produced duting 1 hour to make enough vorcal
to start a mall plaque. Thia is a true trigger reaction in the
genge thet one induced Kyo ) being lysed will lysocenise about 5 to
SO surrounding sensitive §, coli end so on,

Controls: ‘Transduced w3091/W3091 (>) or W309k/ W309k, 1.0.
teronses! of sane auton should not «tre any plaqes,. An experisent
af the sane type, am well ag on controls, could actuslly aso be run
on the sutents of gelecto kinase. Te outlired technicuws mi-ht also

be used on mixtures of protoplasts inside s eellophane container,

ueing heavy ineculation beth of induced Kyoh » Gale and of sensitive
K

E, gol, gal? jis

This principle should also be considered for use in trying te
pick up phenotypic segregation of f-glucurenidase in spernatcscos
from hybrid sice.  “permetosoos are incubated with ¢lucoseeglucuronide
at pil bhe3. About 200 spermatesoos are spread on agar without eny
earboen source and pleted with starved 5,). induced Eo hae indicator
strain (heavy bectorial inoeulete). The spermatozoo is the feeder
of the Kaphe In a hybrid population one should observe two types
of pleques: lerge en! small (difference in siset 1 to 3 or more).
In homomyg., OG (i.e. high f-glucuronidese titer) only lerge pleques
should be present, in homesyg., eg (low glucuronidase titer) only
small plaques sheuld appear, If the spermatezee population of a
single individual show only small fluctuations, such a technique
should be able toreveal phenctypic segregation in heterosygetes (Og)
ie@s, SOE large plaques end 50% emall. One prerequiatte wich is
important ig that the ebeteates used, whether it ia galactose or

eellobiuronie acid, must be wery pure. Any trace of glucose wuld
disturd greatly.

* Another variation of this experiment would be to use Kyp gal + (3 ))
aa the indicator strain on the g@ actos agar wd intues with 0.9,
several tims after plating. This would in effect més the Gal+
sensitive after lysogenisation, ¥ Meeli strain which is sensitive
te xwithout any induction, 4% would obviously be prefershle),