October 13, 1954 Dr, J. B. Clark Dept. Plant Sciences University of Oklahoma Borman, Oklahoma Dear Dr. Clark: I am happy to enclose a culture of W-478, which I hope responds to your request of September 30. Diploids are rather troublesome to maintain, and all that had been isolated earlier from the Het crosses published in '49 (PNAS) and '51 (J. Bact. and C.S.H. Sygpos.) are gone. From time to tims, we have made new diploids by the same procedure, as the need for them arose. While W-Z78 seems now to give a rather lower yield of diploid progeny than it did earlier, it is still a feasible source. The most important consideration in maintaining diploid cultures is that they have a strong propeasity to segregate and that, on complete medium, the segregants will rapidly outgrow the parent diploid. Therefore, diploids are isolated which are balanced for nutritional requirements and also, say, for lactose utilization, o.g., M- T+L+B,+ Lact// M+ T~L-B,~ Lac-, ami these are maintained as far as possible on ~lactose medium. This discourages the typically auxotrophic or lactose-negative segregants sufficiently to allow one to obtain suspensions with as much as 95% diploid celle and to maintain the cultures in the diploid condition. It will be necessary to repurify the cultures from time to time, preferably by plating for colonies on EMS lactose agar: unfor tunately, protwtrophic lactose-positive segregants occur by crossing— over from time to time, ani thereBy displace the diploids eveh on migimal. To isolate diploids in the first instance, I would cross W-478 x W-1177 on EMS lactose and proceed as outlined in my '51 CSH paper at p. 421. Lac+ pro- totrophs (especially those that are slightly delayed) are picked and streaked out in parallel on EMS and EMB lactose agar. When streaks are fount that are suspected of variegation (see fig. 2), single colonies on the corresponding EMS plate are saved and tested in the sam manner. I need hardly emphasize that a variety of details on diploid behavior are summarized in the papers mentioned. Since the heterozygous compound VPA," is sensitive to phage Tl, while each parent is (more or less) resistant, another expedient would be to make suspensions of lac+ prototrophs from the cross plates and cross—stroke them against Tl, rechecking any that give a sensitive reaction on EMS (on EMB, resistant segregants and background growth will obseure the result. The ultimate criterion of diploidy is not so mich the variegated appearance as the proof of segregation from a single cell, as shown either by repeated single colony isolations or? perhaps more simply by single cell isolation. You should have no trouble, though there will be a bit of work at the beginning. If you are in any doubts about the characterization of a particular isolate, Z will be happy to check it for you. Alternatively, we will doubtless have some occasion during the next several months to prepare some Het-—cross diploids again ourselves, ami I will be happy to send you some of the material then. But I can't promise how long this will be. I am sopry not to have any appropriate material on hand at the instant, but the loss is not so great. It is hardly less trouble to make and keep one's eyes on a diploid than to isolate it in the first instance, and the latter experience may be worthwhile as such. You asked whether there would be any conflict of prdgram— phobably not. I was interested in the radiobiological behavior of the diploids, as outlined in the '51 CSH paper, but the problem turned out to be uhexpectedly complex. In particular, there was no special difference in the kinetics of UV sterili- zation of diploids vs. haploids, possibly owing to the probably mltiplicity of targets (nuclei?) shared by each. Also, there were other, very complex genetic effects for the full interpretation of which, I felt we needed mich better and more intimate knowledge of B. coli cytogenstigs. Some light 1s begianing to glimmer through in this. You my be interested that it has recently been possible to discover the conjugal padrs in exceptionally fertile crosses of K-12 with other strains. As in Paramecium, the mates disjoin after and hour or two. Both generally survive to give viable clones, but recombinants have been found only in the progeny of the F~ axcanjugant. £2/ti¢/dddddé/ Thus unlike Paragecium, fertilization is unilateral, At the moment, 1 am trying to work out pedigrees to study segregation in the exconjugant clones. I would be obliged if you would keep me current on your work in bacterial genetics through reprints. Yours sincerely, é posta Lederberg — Professor of Genetits P.S., I probably will get back to the UV/diploid work eventually. Larry Nprse Came up here as a student to work on this a few years ago, but he got involved un a more interesting probaem and is writing hés thesis now on a transduction 12.