(16) _. Diploid studies: The preceding evidence points to a chromosomal (*) localization of the Lp lysogenicity determinant closely linked to a serics of Gal loci, Evidence for the segregation of a prophage linked to the Gal, Locus ruled out the possibility of a random distribution of cytoplasmic particles in cells carrying A.Qo). These observations have since been extended to Galo and Galy, hybrids (all heterozygous Lp /s), and also Gal) "Lp" /Galy,“Lp™ diploids (table 10). | A study of such diploids segregating out distinguishable types is in preparation, Preliminary evidence also has been obtained elsewhere from crosses with lysogenic parents, one _earrying a mutant d (or one "doubly lysogenic") the other doubly sensitive, which yielded Gal/Lp progeny in parental couplings (1) ° The mitational independence of Gal and Lp was also examined in the doubly homozygous diploid. Comparable experiments with the closely- Lac, and Ve loci have already been reported, Lac* -reversions were selected in Lac“VgF/Lac"Vg% diploids. The resulting doubly heterozygous diploids were of two types: Lac"V. fbac"V8 and Lac“U,¥ /Lac’¥,", and with equal frequency (11). A double homozygote Ga g“Ip®/oal,-1p®, also segregating a few other markers, (end unfortunately also Lp») was prepared by stepwise exposure of (17) the double neterouygote to U-¥ (1) and the isolation of suitable (a4 "reorganized" diploids, The resulting diploid, H-331 was infected with he Several Gal.,“tp*/el.,“tpa isolations, A to G, were then allowed to paplllate on FMS galactose agar. Independently occurring Gal* were selected, and the segregation pattern of Lp and Galy of the resulting double heterozygotes was tested. Tre incidence of mutation to Gai* on the Lp* chromosome (coupling phase, or cis configuration) was com= pared with that on the Ip* chromosome (repulsion phase, or trans- configuration). The analysis included a single Gal” and a single Gal segregant from a large mumber of diploids, (pair analysis) and the examination of many segregants from a single mags diploid culture (random - analysis), From diploid B, 5 cis configurations and 6 trans configurations (table 11) were scored, The conclusion from this evidence/is that the condition of the Lp locus, whether lysogenic er sensitive, has no significant bearing on which one of the 2 Gal~ alleles will mtate to cat*, (These pre liminary data will be expanded, and also extended to a corresponding study of diploids first made heterozygous Gal, “Lp*/Gal,*Lp®, and then infected with },) 23) The above studies provide tio Iinds cf Lp’/lp*; Gal”/Gal” diploids: \ coupled on the one hand with Gal” (cis) and on the other, with Gal,” {trans) if the ectivity of from "trans" bacteria is confined to non Galo recipient caolis, & chromosomal but not melear limitation to \ specificity is indicated. ALL Gah” including Gal” is expected to respond to cis Ac A difference in » from these diplelds which are phenotypically identical, and geretically identical except for the arrangement of component parts established a -” ‘position effect." So far, only \ tron the trans~type diploid has been prepared, Table shows that while Gal,“ (Gal.9"Gady,”) cells are subject to transduction, only rere Gal,” transductions were recovered, The develop~ ment of an adequate diploid culture to satisfy the mitritional prerequisites for U-¥ induction in K-12 (3,5) and an intermediate growth period nec- essarily permits some selection for haploid segregants, The yield of X» | obtained very probably includes a limited portion derived from Gal“Lp” and Gal. Lp* haploids, The latter crossover types may account for these transductions which were found. The data so far allow the tentative con- clusion of a position effect hypothesis and strengthen the concept of an intimate relationship of ana Gal at a specific action site on the chromosome, Transductions of the double homozygote H-331 and lysogenic erivatives has apparently teen obtained, The analysis is complicated by the Pact that dinloid~haploid instability can be confounded with trans~ duction instability. COMPARATIVE GENETICS OF Lp AMD Gall IH OTHER LINES Anong the independently isolated erossable strains of E, coli (12) the wild type of threo lines (28,17, and 51) were sensitive to \earried by line 1, A fourth, line 31, threw off rough variants which were all \ sonsitive. These strains occurred in nature ag F° but could be altered to Fo by gvowsh with K-12 or eultable derivatives, So far, at least ong Gal” metant is subject to transduction. Preliminary intra- Line#-? crosses established an Lp locus like that of K.12, and a Cal-Lp linkage, Very little mapping work has been completed among these strain, and the enphasis so far in these studies has been the genetic behavior of (in outerosses with B-12, Sensitives of each line are readily lysogenized by K~12 Jvut these lysogenics show a reduction of eop on K-12 sensitive indicators, This system is entirely analagous to host modification demonstrated for T2 (19) and \ produced by strain C (2). The terminology established for these systems will be used to describe the properties of our strains, (20) Lt (80 3 lines 28,31, end 47 can be designated as je lysogenic or ht sensitive, (et Lire 1 sensitives are more resistant to he than to type } he can be introduced at low rates into | sonsitive hests, but normal rather than he is recovered, Siailerly, normal Ais converted to fe after a gincle passage in Ke sensitive hosts. The four phenotypes are readily dis- tinguishable in cross~brush tests as follows: Reaction with: ~gens. \tmsens. Cc Example Type bacteria bacteria IN \s line 1 lysogenic A + + R R line 47 sensitive B = “ S s line 1 sensitive C - - s R line 47 lysogenic D ~ + R R +/+ = lysogenic or not; R/S = resistant or sensitive Two najor hypotheses can be tested by intercrossing these types: I Up controls all reactions: the types A-D are determined ata Single locus, II Lp controls lysegenicity/ sensitivity; another locus, Mp, controls resistance or sensitivity to Me (a) Both \and Ae are fixed at'Lp in phenotypes A and D, (bv) )is fixed at Lp in type A; \# 4s fixed at Mp in type D, (21) N @ The cons quences of these hypotheses are shown in table 12, The critical evosses for I and II are Ax BandC xD, The only decisive cross for II a vs. {TbisAxD, II » would be favored by the recovery of sensitive recombinants as well as a novel genotype whose phenotypic effects are unpredictable. Since there is a possibility that Lp and Mp are closely linked a large sample of progeny many be required, One must bear in mind, in reviswing these intercross data that the prototrophs represent recom= bination of as yet uamepped mtritional factors, In addition, chromosome and other irregularities correlated with interstrain hyorids have not been analysed. Effective transductions have been achieved in these strains, Gal- in lines 7 and 31 have been used as recipients, for A produced by Line Ll, 28, 31, and 7, A reduction ir the effectiveness of traneduction to line 1 recipients is parallel with the reduced effectiveness of lyso- genization, In general no important differences with the K-12 mechanism have been demonstrated, Hypothesis II b is doubtful.so far, The dife ferentiation of the \* of different Lines is still to be tested, A single intercross shows no genetic difference so for, In preparing this report, it has been necessary to make numerous references to the unpublished work carried on in this leboratory by Professor J, Lederberg, Mr. M. L. Morse, and others, under other auspices, These are cited by mmber to the bibliography, Table 1 @ Characteristics of F (compatibility factor) and A (virus) Criterion F status ) (effects) (1) Yield of recombinants Decisive None (2) ‘Bype of recombinants Decisive None (3) Transmission to veconbinants 100% Segregated according to linkage with selected mitritional markers; behaves as a genetic locus, (hk) Transmission by infection Rapid and Results in mixed clones (3). fixed (5) Cell-free preparations Not yet Easily filtered. accomplished (6) Effect of antiserum Slight if Blocks adsorption any {7} Role in Gal* transduction None Decisive Table 2 fe Effect of Xd on % Gal” Progeny HGal” parent sensitive x T-L-Th-Gal* parent lysogenic 8.0 6.3 607 irmmane Tod 6.3 10.1 Table 3 Linkage of Gal, Lo, and Hfr _ TUB By Mtr yr carteconchy Malti p® by, Vy Part As Genotypes vecovered+ Total Gai Ip F + + a 1; % = 3 “ 29 % + gs + 5 ~~ + - oO 4 g « h Part B: 2x 2 contingencies Gal” Gat” = Total SP Total Fe "20% 0 20 re 9 3 ho to” 15% 0 15 13s 5 is Lp? VW 2% = ho 6 33% 39 Lac* 28: 5 31 20% 9 31 Lac” iD 26% 30 Ft 3 WF ix 9 320 1x 9 10 V8 28 ale 9 2 20k 43 Eylo- Oe 1 0 ye 62 9 Zylo” 20 30% «50 1600 J 23 % Parental conbination 1 Selected as Gal* and Gal” prototropha, Table Lysogenization in Transduced and Nontransduced Lp® Part At Gait and Gal™ from single papillae Gai* /aal” Pair type Number Gal” Lp® Gal” Lp* Lp*/Lp* 13 Gai* Ly 2 3 Lp*/Lp- 5 Lp? /Lp* 3 Gal* Lp* 17 13 Lp®/Lp® 2 Ly* /Lp§ 2 % Galt sensitive 15,2 % Gal~ sensitive 17,2 A, fawi frien ‘. Part Br Lysogenization of transduced and inserted Gal” 5 Ay, No, Cay* recovered | x Lp” strains Gentrol Treated# Types in mixture No. tested ® lysogenic Gal*Lact.. | 109 92 Gal*Lac*’ (inserta) 16 68.5 ertefar nue > datcfurs Gal“Lac™ 1... LLine 432 Gal"Lae® (original) mn ho 72.5 Me Ce ecane GT Kixtureint 105.5 hig Gal*Lac* (transdttctions) 103 100. * 10°) wt Spontaneous reversions per 108 inoculum su% 108 Gal-Lac- and 109 GalLac*. Table 5 Transductions to Gal” Immune~I: Segregation Patterns Exp. 385: Strain 192k: 27 Gal” Number lysogenic Nunber semilysogenic ‘Number nonlysogenic (1ys) semi) (non) #2 fn A Coleny generation 7 1 4 “~~ ae 1 Gal” non 1 19 Gal” iys 1 Gal“ lys 18 noi 1 Gal” semi 1 Gal” sent 1a”, L (Gai* + and Gal / and Gai™) ee 2 Gal” lys non + 2 Gal™ non II Gal” non 1 Gallys : Gal" ron Gal“lys 2 Gai* 2Gal° 19 Gal* 19 Gal* non lys non non Til Gal“1ys Gal“non 43 1y8 3 non 1 semi Iv = ys” non Vv ys” Noon non we™ Vi lys non Exp. 31: Strain 210: 38 Gal*: 28 non, 1 semi (#23), and 9 iys Segregation patterns all Gal” lys, all Gal™ non: 2 of lys all Gal* lys, all Gal= lye: § all Gsi” lys, Gal” lys and non 2 both Gal* and Galv non: #23 Table 6 Survival and Transduction with Irradiated oY Xeray* (=x 10° r) u-vt 50 100 «1502S 00 16.9 11,667 3,975 377 100 0.013 328 3,13 0.297 0.008. 1 7 OK avony- Bava FN Untreated No phage phage Ay, plaques/ml x 10° ° 127,000 @ survival we 100 Ly® bacteria No. Gal. papillae 20 1,000 eo" " 0.5 100 170 250 85 3030 3 25 176 6 Lp” bacterla No, Gal” papillae 39 60 gon " 65 100 - 135 Ww 2 2205 «(191.7233 20 mirmtes, sterilanp 2 103 r/min. at 250 K.V., courtesy A. Novick, Radiobiology Inst., U. of Chicago. Table 7 x Segregation of Gal, Lp,... diploids A. He32) By H-325 Segregation of Lpo, By, not Segregation of Vé, Mtl, Lpo, By not tabulated, tabulated, Galo Galot — tp Mel Zyl M T,L_ Galy~ Gal,+ a 0 OW? $f ob Fm om a 69 0 1 + + ~ - = 0 0 o 1 bt + ow + 2 0 0 0 + + tw 0 1 1 0 + 4 to + + 0 0 2 0 + + - 4 O 0 25 G B + wm 13 0 9 1 Ss + bm 13 1 3 0 s ~« -~“ ~~ Qo 0 6 0 5 + + - + 7 o 1 Q s ~ + + + oO 0 é 0 8 ° + + + 3 0 0 0 Ss «~ -~ = + 12 0 So 50 Total tested S1 521 Table 8 | Allelic Specificity of the Gal -)fransduction at the Gal 2, Gal 2, and Gal b lock. A- donor bacteria Recipient cells Gal 1 Gal 2 Gal h 1.24) + 1+2-])+ 1+2+h- + am *+ +> + + ° ue + + —_ + + + o ¢ + ~ dipicids: + oa + Lp” No 2 (21)* + (300)% ; ; Tips data (22) (300) . (trans) v ~ 4 Lp” ? : = tps (cis) No data * Gal + papillae per 40" XL Table 9 Sumnary of Current Allelism Tests Total! No. Exp. No. Gal” type F™ parent F* parent progeny Gal” Maxin.,% Galt 535% Llxh W750 Lpt W223) Lp® 5000 17 0.3 563% . 2000 15 0.75 53) exh W-1210 Lp* W..223], Lp® 6000 25 0.) 563% 1600 he 0.68 580: 21,00 8 0.3 535 x3. WeSi8 Lp” We2315 Lp’ 807 6 Oo7h 582 hx33 W518 LpS W.2315 Lp® 5000 0 oO 6700 5 0.06 583 1x? We229L Lp® W.583 Lpt 7603 2 0.026 * AU Ga* recombinants in these experiments are Lp®, xtEstimated total, Table 10 | Behavior of Gal and Lp in Lac +/~ Diploids Type of cross F (TL Th) M Lacy Lac), Galy Gal), Lp Gal Lp - 1. Het diploids (a)(Het) + - a ee the fe or a fo UV S/ + * os oe + + = 8 (b)(Het) + + & © + «© 4& core/o + oe ~ * + ; ; +fo or ~/ | not segregating 2, Lacl» x Lac), ~ (a) | ° ° * * : ; . ; Mostly +/e Mostly +/c 2f * oe ~~ , . Lo 2 te) : + : . + ; : ; Mostly. +/s Mostly s/o 2/ 3. Haploid x auxoe (a) oy «fo +/u t/a aft + +fo +/o Gai* Lp? / Gal-Lps (linked) 3/ trophic diploid + + ~ - + + ~ 8 (b) Same, except M- parent is Ipt Gal* Lp’ / Gal-Lp® (inked) 1/ In Het crosses, Lp does not segregate. Gal 1 and Gal h, two closely linked loci also differ: Gal segregates, ™ but Gal 1 does not, 2/ Diploids resulting from delayed disjunction revealed by heterozygotes of two Lac pseudo alleles show no segregation of Gal or Lp, Reversal of F status reveraes the polarity of the Gal, Lp segregation. 3/ The only successful. demonstration of heterozygosity of Gal and Lp, L/ Asration phenocopy. 5/ +/+ indicates purity for +, whother hemizygous or homozygous, Table 11 Segregation Patterns of Gal* Reyersions in Gal,“Lp®/Gel,"Lp* Diploids — 2 2 D iploid Total Gal” a al” Gai* ‘ G ai” G al* G al” Inferred number spegre- ‘ * + type of gants Lp* Lp? Lp? Lp® Lp Lp,® Ups” py” Mal” Mal“ Mal” Mal” diploid yf Al 161 is) 6 3 96 15 0 39 0 1 653 1736 cis Bi 1 2 3 6 2 s2.C«# 60 i 38 20 6. oo trans BQ ‘73 0 ho hi 0 32 7 31 0 33 7 PP 0 trans B 3 16 61 h i 10 65 0 57 5 65 Q kh 18 cis Cl 48 1 23 om o 3 1 oh oO 9 1 oh (0 trans El 60 30 0 3° OF a hy 2h 6 30 0 1606 ol eis E2 2h 0 12 12 0 12 0 12 0 6 & 12 0 trans E 3 23 2 Oo ¢ch 12 «0 ll oo 120 (OO 3. «8 eis Fi 66 3 ol 8M a0 30 «3 32 ot oo (ue cis PQ ho 0 oO 2 i9 0 0 2 860 2 2820 7 #3 cds F 3 93 2 #0 0 12 06 10 0 1220 3. 8 cis F i 18 mo 1 6 io. 2 0 7 ll 7 30 cis Table 12 Genetic Determination of Host Modification: line i (XY lines 28, 31, h7 Hypothesis I Lp locus with Genotypes Under Hypothesis IIa fixed at Lp, Hypothesis IIb fixed at Lp in line l, alleles modified by Mp at Mp in other lines Phenotypes Symbol Lp Lp Mp Lp Mp lysogenic A + + r + or sensitive B st g 8 8 8 sensitive Cc s 8 r 8 r lysogenic# D ‘Hit + 8 8 + AXB None c, D c, D BXC None None None cxD None A, B A, B AXD None None B and Lp’Mp” EXPTL, RESULTS: Lines crossed Type A B COD Gal. char, Expt. No. Lx 2 A Gal” xB O 6 1 0 > 18 0 0 0 - C Gal? xD oO 0 oO 3h + 2 8 18 3 - his ix3l AGaly>xB 3 3 26 1 No record 20 AGal~xB kh 22 2 12 Gal* only 423 AGal~ xB 8 2 1. 37 + 0 1 0 0 - 23 CxDGal- 2 2 3 £0 (and 28 Lpp*) B or C hhh CGal=xD 5 9 a9 9 mostly Gal” 502 BGal~xC 0 15 £13 0 + o 13 68 0 “ hh3 BL x 31 BxA 0O 26 oO 1 68 1x h7 Ax B Gal~ 52 O 0 6 + 5 0 2 2 3 - A Gal” x 7 1 9 + 2 = 327 hh 0 0 2 - 528 BxCGalm= 0 13 £417 0 + 0 8 2h o o 529 CGal~xD 3 2 2a + 2 2 2 0 - 523 AGal” xD 8 0 0 8652 + 37 0 o 19 - F~ parent underlined, Table 23 Genetic Control of the Semiresistant Phenotypes : Nonlysogenie (W~21h7) and Lysogenic (W-2172) Part I Hypothesis I Hypothesis II A new allele at Lp»: A 3rd locus, Lp3, is involved: Phenotype symbol Lp, Lpo Example Lp Lp9 Lp3 A + 3 Type Lysogenic + s 8 B + r Imaune~2 lysogenic + Yr 8 G + Pp W2172 mutent + 8 Pp D s 3 Type sensitive s 8 8 E s r Immune=2 8 r 8 F Ss p W217 mitant 8 s P BzF Yields: B, F, E, C progeny Yields B, F, E, C, A, D CxE * " Results: Bx F No, of Progeny CxE & B Cc D E P A B C D E F Ma* 55 1 il 1 0 1 22 2 2 26 0 1 Mal” o 58 Oo Oo 1 0 0 0 0 o 659 Part II Linkage of Lp3 to Lp,-~Gal and Lpo--Mal ? No. of Progeny Parents Mal" tpF Ma tpy* Mal Epp? Na” Lp,” F He* x B Mal” h 56 1 58 C Mel* x E Mal~ 27 25 59 0 Mal” Ip = Mal* Ips? Mal~ pp? = Mal” Lpo® F Mal* x B Main 59 L 0 59 C Mai* x E Mal™ 5L 2 0 59 Mal" Lp3? = Mal* EpsP Maal Lp,° Mal” Ip,P ‘F Mal” x B Mal” 57 3 59 C Mal* x E Malm 50 2 50 0 C Gal* x D Gal> Gai* Lpy* Gal* Lp)5 Gal~ Lp,* Gal~ Lp8 60 0 0 28 Gal* Ip, Gai* Lp,P Gal~ Lp,8 Gal~ Lp,P 37 23 37 26 The above data are consistent with the to het a that an Lp3 locus separable from Lp) and Lpo modifies the reaction to \«1 ard \o2. This locus is not linked to Lpy--Gal or Lpo~-Mal. Le 2u 36 90 10. 12, Ly 15 | 30 (mgt By) 5 (ooypret) (1S (page 04 ) maby re a De Soap. 0) MOA, | werk (Me LMR LMR she) N 3 (a9. acy) | Bo | Ar | °/e | 14 ¥ L7 + 4 serge o/¢4 2478 3 tL re YN _ 247 A st Foe 7 FFs po FS OB _ ee ote _f# F Fo See A . : ee Oe 24/ Stee = Bee ogamrh ater / ae 23be FT J Sgt OY ee _ 2th tO oo : eg ee oe oe OF i eee ot a a ee : | ine 4. S/¢ L428 J ee Fu meenies /w_\ | lqa ee a ; off Owed Colrmabecs 7 Hwee fete See yo ° 30 Th we D ; yt ic La oO mh Ge Sgt spa can a 7 : % 2:04 lew ora} at Cot 7 - ee NO ¥ 4 ; t we ff te © — : I~ & bt ‘ we ni ee eee eee o7 a eel : i : , : : # sin: ’ i i - 3 ATG 7t | D4 hor 4 i - FX FX. FRR —\krv. IXY TOR. ~ ORS. OF BOM 240 Pe AVA Rr Mo Feb pt (7%) Ko Fata Up (rt) (t). n~ Gy by‘ (arn) yo" J Me Gag by Ct) OM Gee Bhd m- Fu” pt Qe) Fo ee TE Gao yt (ut) TAR? oh Wy (urd), ch Gs GEC) 3. as os TR? Gar yt TR OAT FE (a) GF cer TE Gch ut) I 360k THI Uy? avy Ce). Lo. Pr 0 Oo o 2 Pa a GR _ xt ca hero (957 | Bt ae Oe Y wo 6ST Ob 1034 ca 4 SY ca Tbs ca, VETTE aoe iyo 877 / oO - 6 0 0.0% ¥_ oO Oo. Vo. / 3 ow 0.)3 VA eos we Oe 0.3F yo ToL G.e _ a LN NNN oN NEO OO eee Eee , 3 (1) a et) hh er / ADO oe ASQ an on oo oe ge BSED Ca. [STO ‘Bah ae (etd TSS , Ot 0.t2 _ J ees, J ve Finbte 0, 06% 60.138 Ae ——— BX) & en lis | (*) 0 tm 5 wd ae vn ST En QO) 4 Oo. 1034 bit GA 0.