UNIVERSITY OF CAMBRIDGE PROFESSOR R. A. FISHER, Sc.D., F.R.S. DEPARTMENT OF GENETICS WHITTINGEHAME LODGE MISS M.F,.1.SPEYER, B.Sc. 44 STOREY’S WAY Research Assistant and Secretary CAMBRIDGE Tel. $5822 27th ay 1950 My dear Lederberg, : Thank you for your letter and for your strains,which arrived in egcellent conditions. Since when I wrote you,the small capacity of py laboratory has been entirely absorbed by the new strains, th that I have nothing to add con— cerning mapping work. However, I am giving in an apvendix data concernigea WV 836 crosses. I forgot to tell you in my last letter, concerning mapping work, that Tf mapved sore time ago an azide-resistant mutant wpich was localized between Viand TL. A chloromycetin-resistant mtant showed uxigenx roughly in the same resion( but in the latter case ,selection was by successive subeulturings and more than one locus or step may be involved). While chloromycetin resistance work is being con- tinued ( selection by successive transfers shows a nearly perfectly continuous increase of resistance!) I have discontinued azide-resistance, because it seemed to me that there is too little a gap between sensitiveness and resistance . Chlo- ronycetin resistance was so far weeless for selection of recombinants according te your streptomycin-azide metiod. Re W 1113 strain, I had little experience with it, since cros- Sings to K12 always yielded very few or no prototrophs, I have never tested then with sugars, so that I covld not tell you about theh mich more than that. I drop- ped work with W 1113 because T found so little antigenic difference between it and A K-12, If you are interested in a confirmation,I shall repeat these crosses,which te appeared to me to give some, although scanty results. tes | Ww The new strains have been rather deceptive. Finding nom narked Vu antigenic difference between fart interfertile strains kmown at that time ,we set up a patient search of fertile strains among coli-strains Wnown to be antigeni- cally different. Eventually,two were found ( marked by Yauffmann O-antigens 3 and 5) that seemed to Shue consistently miriads of prototrophs,ywhen crossed at high titres, On dilution, a smaller number of"protétrophs" anneared,but these colonies, which I should call S#seudoprototrophs", were always small ,not gret@@@ than 1 ma in diameter, and grew adbrly and badly,or not at all, on transplantation to fresh mininal mediun. true Marking with sugars has confirmed suspects, that no/recombina tion is proba- bly taking plece among them. At present. two Gas fees biochemical mutants are dvaae “lable for each of the two strains 3 and 5 3 pseudoprototrophs are formed in the cross within coli 3gnot neinsenxtwithin coli 5 jand in three out of the four pos- Sible crosses between 3 and 5 with these strains. What these pseudoprototrophs are, if recombination will be entirely exc!uded yi. could not say 3 I heave been thinking eu! of unstable heterokarions,althovgh. the mmax mode of division of coli seems to prevent the possibility of formation of heterokarions having a minimun.of sta- bility2. Association with perhaps partial. back mutation seems then’ the only el‘er- native, IT hope to be able to decide soon between extracelinlar or intracellular syntrophisp, _ Contrels of the strains are. satisfactory,of course. Although deceptive from ene recombinational point of view,at least so far, these "crosses" have been found exciting from the antigenic point of view. For in stance five Ay six psevdoprotutrophs better then the others were found to have —- and keep after six successive platingsgon co:nlete - the antigenic reactivity of both parental strains. Decision between reconbination, cyto olasvic inheritance,or extracellular transformation partly depends on the decision about the nature of the- se "prototrophs". I hope you will not mind receiving information of a research which is still at such an early stage . It will help me to know if you have any "experience of svch pseudoprototrophs. I have an inpression that some of the smaller truz vrototrovhs in FK12 crosses may be of the kame type. I found an early nibtrogen mustard resistant mutant {which is ineapeble of crossing, to be non-motile.tmfortunately,decisions on motility are not the easiest, in coli, and flagelle staining not very satisfactory. Yours sincerely SUMMARY OF OUTCROSSES TO W 836 a) Cross W 705 x W 836 Ata yf Lac V_ Gal Mal Xyl No.prototrophs Exp. - @.0.(additional to 1. c.0. between M and MlyT) - rr - + + 313 314.0 none. + 8 + + 69 .. 68.4 IT + 8 - + + 3 7e9F II- ~ 8 - + + 15 12.8 Tif - r + + + 3 1.7 I,ItI * r * + + 4 2.8 I, III + P - + + h 3 II, III - 8 + + + 0 01 I, II, III 08 of 408 prototrophsmt, 162 from plates supplemented with tryptohane; none was Ty=. Expectations calc.on basis of order:M-MlyT-G al=Lac-Vy 5 b) Cross W 705 x W 677 es Of 108 prototrophs, all Gal=- ; 24 Mal+,14 Xyl+. c) Cross W 677 x W 836 ve 4 No.of prototrophs we Lac Vy Gal no addition with B, c.0. + 8S + 25 8 I + s - 52 25 II - Ss - 70 69 ITI - r - 31 79 IV ~ s + 6 1 I, II, III - r + 1 O I, II, IV + r + 9 0 I, III, IV Mh My 2 186 182 of which 18 are B+ ailing cae B+ in cross with B), 11 are Lac+v}. C.oeregions given assuming order :Miyf B,~ MLy Dee Gal - Lac - V, - LE II III “IV Other possible order : By, GalMlyTLacV,LT, then stronf/negative interferen- ce between Gal-MlyTr and MlyTr-Lac. Data available for Mal and Mtl show linkage,not complete,with Gal. sed . and W 836 : M-MlyTr-Gal-Lac-V onsere prope WH W GT? : B, Gal(Mal ete. VMLyTr-M-Lac=V., -LT % The major difficulty encountered in assuming the same order,i,e, M-Gal-Lac-V_-LT for all the three strains is in the comparison of frequencies éf c.o. for the same regions in different crosses. For instance, M-Gal is greatly exagger&ted in one Ststance and depressed in the other. Also,there always is negative interference between B-M and M-Lac in any cross where such regions are marked. It could be explained by double c.o. within ‘the imversion loop. Double c.o.in the inversion. loop could also explain partial linkage of Mal, Gal,Mtl,Xyl etc. -