Department of Genatics University of Wisconsin ENCLOSURE: CULTURES OF ESCHERICHIA COLI Madison 6, Wisconsin (a harmless bacterium) for scientific investigation To: Dr. L. L. Cavalli Istituto sieroteranigo Milanese Via Darwin 20 ‘March 28, 1953 wilano, Italia ear Luca: I enclose a number of cultures as mentioned in previous correspondence. I apologize for taking so long, but I made a number of attempts to reisolate H-313, which have failed. It would be easier to repeat the cross, and I will do this at an early opportunity. I assume, however, that you are more interested in the variety of segregant types than kn this diploid itself. I am therefore sending you a culture which represents the unpurified mixture of segregants from the H-313 stock culture. I was note able to recover the original diploid itself, which is rexognized as protobzophic, Mal+ on EMS maltose; but Mal v on EMB maltose agar. Attempts to reisolate 11313 from this mess now probably will lead only to new crossings. In addition, as noted, there arm is a group of isolations previously made from this culture. Their designation as Hfr is ten- tative, but my records sfiow them to be very active F+ phenotypically, but non- infective. You should have no s@kffeime in securing prototrophic Hfr recombinants also from the mess. To avoid confusion, I repeat the correct pedigree: W-1895 (your Hfr) X W-1177 gave H-310, a Lae v S™ noticed in a cross on EMB Lactose + sm. H-310 appears to be segregating only for lac, and is pure for the other markers of ‘-1177 (whether homo- or hemi-zygous I do not yet know}, All its segregants so far tested have been F—, but H-310 itself behaves as an Hfr. It is relatively stable, and can be purified easily by picking hazy—mottled colonies on FMB lactose. These rarely throw off typical Lac+ and Lac—. H-310 x W+1895 on EMS Lac. *f@ or Mal. 1/12 was Mal v H313. H313 is pure Lac+ (not surprising as it comes from Lac +/— x tas"), but segregating for M, TL, S, Mal, Méh,amixf{ioommisxemmsxx Xyl and V,. [Note, inter alia, that it has a full genotypic contribution from each parenti, Only five segregants have been tested, each behaving like Hfr as mentioned above: Vi, Mal.Xyl.Mtl S LB, WM 2057 r ~ s - + These are not a random sample e058 . _ r _ + of segregants as I was looking for | special types. 2059.3, 1895, not included 2060 r + s - + W-2061 r + § - ~ The remaining cultures are the partially analysed issue of passages through 2 tubes each of motility agar (formula in Zinder and Lederberg '52). W~ From 2206 58-161 This shows very high rate of recombination (not quite as high as 42895, although one does find Lac+S™ recomb. x “=1177) but is still infective F+. It may possibly have a snecial F+ agent; this needs to be checked, as does its purity (possibility of its being a mixture of 4fr and F+, but doubtful). 2207, 2208 " seem to be typical F-. 2209 ‘im 1678 (Proline-, glycine(or serine)-). This one is curious. It is very infertile, but does give some prototrophs X W+1L77F+. after being grown with J-l177F+, it becomes moderately fertile with W-1177, less with W-1817. This could be explained if ¥preceding pages of that symp. independently of becoming F-, this stock also:picked up some modifier that reduced its overall productivity. The original W-~167& is extremely fertile (not quite Hfr) x 1177, much less so x W1177F+. Jim Watson sent me his ‘Watson—Hayes opus. I have not wanted to polemicize with him, but cannot accept the underlying theory. F+ x F- crosses have given diploids which are deficient for a Mal-S segment from the F-—_ parent, as well as a few which are vial-S crossovers. This seems to necessitate a post-zygotic elimination, and certainly one which is not absolutelg dependent oh F-~polarity. As to the nugber of linkage groups, an ‘[.A. student (Phyklis Fried—— now working for Ryan) completed an sxckmmxk extension of Rothfels' work last June in which S, M, P (proline—), and TL were variously used as elected and unselected markers. We could not confirm the M-Lac linkage, which is based entirely on the segregation ratio of Lac into prototrophs, so the markers seem to fall into the following groups: Vv S--H---B, and pPac-———-V]-——-TL [Mal-XyL-Mt1! The detailed ordering is not-entirely worked out. To explain these data, and the unselected Hfr x W-+1177, one has to postulate a tkeamtsst polarized segrega~ tipn, controlled by F, and directed at two points: one near S, the other near TL. To counter the possible argument that the diploids mentioned on the 6th line of this page somehow resulted from a ceoss of inverted polarity, following Ftransduction, 1 am trying to obtain F~ Het stocks (by the semisolid passage technique) so that we can secure diploids from the non-infective Hfr x F—Het cross. But I have aimost given up trying to explain this reasoning to Hayes, etc. I would almost rather leave him to make some definitive enough assertion that it will be possible to test it. Goncerning the cultures included, I have of course no objection to your discussing or demonstrating them with anyone, but feel that the same considerations apply to their distribution as to Hfr. I have word thirdhand that you have recovered a Bmi- 58-161. Is this so? I propose we raname our current Bt culture now \i-6 and regard it as a (genetivally unahalyzed) reversion from the proper 58-161 type. I have not forgotten our is. Thank you for your reprint and microfilms which arrived about the 24th. By the way, I think Umbreit#is all wrong (and not en- tirely forthright) about the metabolism of ST’ mutants: at least as concerns their non-aerobic growth responses. They have had such a culture, but this behavior had nothing to do with Sf: subsequent isolates seem perfectly normal, and they claim to have lost the original S°. I was once interested (at Stanier's suggestion) to test indirectly selected Sf to determine whether streptomycin had any direct effect on the aerobic metabolism ( a 14 Ephrussi), but could not confirm the premiss. Oginsky sent her strain, with same negative results. But I would not want to bother with this in print. Sincereky, P.S.: I have a Pyrex filter on order. when it comeg shall I send it direct, which would be much safer, or have shua Lederberg S my own glassblower make the U-tube, which will be more hazardous to ship?