Table & Summary of the analytis of segrezants by transduction test, lysate test and G) by creseing test Recip, Trned. 4 Classification of segregant by Culture Lysate frned. Lysate Cross “ Idiotypic Allotypic (+) fot. Prot. (+) fot. Prot. Galo- Lp* wild (1)Gal,- Gal,- 0 7805 - - (2) " " 0 4992 7 - (3) * " 0 106 - - (4) _* 7 0 4652 - = Galo~ Lp* wild (1)@al,- Galp~ 0 4070 - - (2) " 7 0 5384 - - (3) # * 0 2072 - - (4) 3 £ Q 6988 _ = - Galj- Ip® wild (1)Galj- Galj- 896 - - (2) 8 " 0 918 - - (3) ® " 0 1134 - - (4) # a 0 863 - - Galy- Ip* Galj- (1)Gal,- Gal,- 0 2786 3 3183 (2) * " 0 2675 2 3471 (3) * " 0 3485 23 5342 (4) * x 0 5952 1 2665 (5) _# & 0 5000 1 891 (2) _" " 10 4364 0 1187 Galy~ Lp Galo (1)Galj- Galj- 0 16104 3 1389 (2) * 4 0 5730 1 164 (3) * i 0 3358 0 202 (4) 8 ft oO _12848 1 171 (1)Galo- Galo- 1 11200 0 827 (2) " " 6 10608 0 718 (3) * " 3 5000 0 409 4, Gest ef Ye Segrgent 2. Tesk sf lysake of Mee , wits jew cullnres 3. Test crossing aust fe Ly sakes of a Kuoun culbures Seq reg aut ogawust fer own curlheves Galy~ HB 3\ 63) 20 20 Gal,- - Galg- (3)% 6 1 7 (4) 2 0 1 Gal,= 1 0 i Gal.- (5) 2° 36 6 42 (6) -18 3 21 Galo~ 2 Gal,- 20 0 20 aad Gal,= 21 1 1 23 + (7)¢° Galy- 19 2 0 21 14 3 2 19 (9) © Galy- 22 1 0 23 9 7 0 16 Gal,- Galo (11)'17 19 . (12), 37 ig Ay 21 (13),-16 19 (14)°15 18 (i),(8) (10), caltures of W2175. (2),(7).(9), cultures of W1210 (3) .(6),.(11), lysates of ¥1210. (4),(5),(12),(13),(14) lysates of W902. W902 is the Lpo* parent of W2175, Table 12 Seerezants in table 11 whose classificatior was confirm the action of their lysates on Ne ° 33 eas known ,caltures Recipient Lp Trnad, Segregants cells 20 idio ¢ allotypic tot Gal,- + wild 5 0 5 Gal,- + (1) . 0 5 (2) 4 0 4 Gal,- 8 4 0 4 + 4 0 4 Gal; + Gal, (3) ty 5 9 (4) 0 3 3 Gal,- s G@al,,- 0 | 1 1 + (5) Gal- 0 2 2° (6) Gal,~ 4 0 4 (7) 0 1 1 Galy- 8 Gal,- (8) 16 3 19 (9) 0 z 1 + (10) 15. 4. 18 60 19 79 (1),(5),(6), cultures of W2175, (2),(7), cultures of W1210 (3),(8),.(10), lysates of W902. (4),(9), lysates of W1210 Table 18. Gaisotese peentive cultures giving BFF lysates ol NN HF? Recipient Trused, ature o uFT Sature of Galt re__cell ysate Gp evergions segrersar aversion HFT peg. Gak,~ Gal,- Gal,- unstable Gul,~ stable Gal,~ Gal “s Gal. ~” unstable Galj-, Gal e stable ery Gal, - Cal Gal 2" - Calg Gala- Gal,- unstable Saig- table - Gal,- Gal.- unstadle Gal, -Gal,- none observed Caly~ Galp~ unstable Gal,-Gal,- none observed Gal;- Ga.o- unatable Gal,- shoo Gal,~ Gal,- unstable Gal,- ( sholeky Qal,- RRR" * unstable Gal - Gal), on unstable Gal.— stable “Cady . Fa1,-- Gal, - Salim - Baas ——_ —_— —_. Gal.- Gal,= wat dane Coty - she * Transduction made with a mixture of HFT Gal,- and al,- lysates, ** These lysates were from ao mixture of cultures, . Table iy Sorrelation of lysomenicity with tramadnotion using dysntes giving a bich frequency of transduction ¢ d2_—he_transductions ells Poat tumber of colonies observed Exposed Expogure Gai(-) _Gal(+) Gal (-)partiably lysed Broth ld x 10? 3280 0 0 HFMysate* 3.51207 280 32 Sh ‘ Exam 4 ns 5 t neo Lan s ar Ae Feate expo str Colony Sumber of Munber of colonies Mts Lx* Lyte dart) 31 31 0 0 Gal (+) | 26 0 23 3 * Lambda plaque titer waa 1.2 x 1074 One ml of 0811 suspension was added to one mi of lysate and the mixture incubated at 570 for 10 minutes, The cella were then centrifuged down, the superhatant discarded . and the celle resuspented in one mi broth. The suspension was then diluted and plated on EMB galactose medium, ) Fe cs Table 16 Te WERE interaction between Gal and Jah, torre te Trned, omber of colonies in* colle _ HPP Jyante Gal(+) Gal (-) ~) papi 0 broth al,- Sakge 0 b65 0 Gal,~ broth 4) KO 0 ait a ~ “trae, — Mi oaluchoas nemmtlee of searegaate oO patirlent mr iemie Sas = Gale Gai3-0a),- Gal (~) papilleting Galy~ Gad)— 10 2 0 1 COLLEGE Typing COMPANY S827 STAT MADISON - € STREET WISCONSIN . _ ALpine 5-7497 Planographing Lettez Service Multigraphing Addvessocrazn Assembling Addressine Folding Tyving TO: Melvin L. Morse Date: Dec. 27, 1954 Your Order No. Our Invoice No. L074 TERMS: Cash Job No, Day Description of Work Amount Typing of PhD Thesis in Elite Type: 31 Pages Double Spaced @ $0.30 per page $9.30 3 Pages Single Spaced @ §0.40 per page 1.20 16 Pages Tables: 6 hours © $1.50 per hour 9.00 50 Carbons on Bond Paper @ 30.06 3.00 100 Garbons @ $0.05 5.00 $27.50 Pp. lyv--v9- “cy | _—_ - [ Here) ee Tv Ge, ~ Gere . = oat . , 5 _ Ye sao (8) = fhe © se a aie (®) — = =} ats j3¥- be id tho 2 Gays (7) % © i yor 0T 07 4t @ 3(7) _ ote S&S Tar Oe 30 - _ t+ - —= GR) ee - 4 _ . eS “ete @. pt _ _ ; : ON : 4 - ~ : ; Pobuc Eft . ee + Tovah 2 aa ” ue Corday - Goqeenk fe a Nomad Oe Bo Ste ers ene. 20 I i 1 i | t i | } { | a t ' } } I | ' . ; . | a Quads bbw 6G en _ 3°Th 2 BT fe ta ane, Mp me ID, FAD apt SOD BAD 3% (Gs) 27 pat pest ee dtr — 343 6K IT a S44 AR KO 1 IB 5s ¢ 0 7 Y Hl (2 WA 4 —xe Tv ~ ee (bye ET 2 CJ) 68 as + WERE x 4 sn 3 (8) tg) AQ) 6 8 e+) a Ww . i . tore TI OI _ a —— nen | c Cc q Oden m ( C ‘t z- ft q 2 a * 0 : (7 yt L ¢ 2. j got, (&) SOL (2) 100% (r4-) 32 (v2-)@ 9%G--) go GC) SE) Se). a2 be 92 (re) 5% (4) __ _eY (v4) 5, (+) Sh (t) ee PBC) Hye“) %H“). ar )0 : , le — qwhes) @ toloew) Se ge) 3o% (¥) Klee) Mr) () 9® bs G@:) 4 E--) 38% (==) SM (re) ~ZE-)@ wh G) 2% (4) @ 2 ) / : discovery of a compatibility mechanism (15), a lysogenic system subject to genetic control (10), and a systen of Limited transduction by temperate phage (22) comparable to that of Selmonella (28), These three phenomena involve transfer of heritable factors by infection in contrast to bacterial mating which involves the entire genotype. The clarification, differentiation, amd interrelationships of these mechanisms were emphasized in this in- vestigation. I The LYSOGENIC SYSTEM IN E. COLI K-12 The re‘ationship of a temperate phage, ds to a specific locus, Ip, (latent phage) has already been reported (10). In summary, the prin- elpal reaction types of bacterial strains are: sensitive (ip*) ’ lysogenic {Lp*), aml the non-lysegenic resistant type, Immne-I (Lp"). In crosses they behave as a system of multiple alleles , ‘linked most closely with Galj,, This Linkage has been confirmed in a Gal* Lp” x Gal” Lp® cross | in another laboratory (27). In addition, the two factors segregated out of heterozygous diploids in the parental coupling. This evidence points, therefore, to a genic determinant regulating the maintenance of ) provirus. (2) Fron a mimber of direct and imlirect experiments it is known that ali these types adsorb X. A second locus; Epo» controls resistance or sen- sitivity to \~2, a virulent Amutant, and is situated in the Mal,--5 region of the chromosome. As tps? strains caymot adsorb ds they are therefore not suoject to any consequences whose initial reaction requires adsorption; Ip, does not interfere with the maintenance of A previously established in Lp* strains, The genotype Lp®Lpo” is consequently indistinguishable from Lp*Lpy* types with respect to lytic effect of he Cross-reactions of \wwith )-2 antiserum have been observed, New Data on Inmune-1: The status of the various isolates of inmune~l1 strains has been reported, and the interpretation of their constitation with respect to prophage had been reserved pending evidence of a “eryptolysogenic® phage that normally faiis to mature to give rise to lytic virus, The segregation pattern of Gal"Lp"/Gal,, “Lp” diploids ; also heterozygous for Mtl and Mal, (table 7 ) is identical with similar lp’ /ip® results, ‘The hypothesis that Lp* types may carry a non-reproducing prophage is supported by experiments in which a low titer of i was recovered by U-V induction of ab least one (22). Lp” types are also subject to transduction, and the results of these studies will be deferred to that section. (3) Incidental Veriant Tynes: No wey evidence bearing on the problem on the "semilyscgenic’ strain (10) can be presented, Tests to determine whether host-modified was carried (section IIT) were negative, An intermediate host reaction, semiresistant to both Xana 2, comparable to the one in Shigella paradysenteriae (26) and the v,” allele of K-22 (12) has been clarified, Standard X suspensions have a reduced efficiency of plating {eop) on this mtant such that the plaques produced are reduced in size and number, and also show a reduced efficiency of transduction, | The mtants have been successfully lysogenized, but are still semiresistant to \-2, The protocols for crosses which establish 2 mutation at a new Lp3 locus not linked to Lppelial or Lp, « Gai, and conferriag partial resistance to As are presented in table 13, Mechanism of Infection; Mutation and Selection vs. Induction: Breeding experinents ani diploid segregations reveal. only the chromosomal determi- nant of lysogeniclty. The facility of the change Lp® to Lp” encourages the possibility that \ directly induces (rather than selects) Lp’ among the numerous survivors of exposure to phage. The following types of evidence would be useful in elucidating the primary infection process: (1) identification of a "prelysogenic" genotype in the absence of phage y (hi) would encourage the mutation hypothesis. It wonld te chsracterized as an apparent iumune-~1 that would be converted to a stable lysogenic after treatment with). (2) a careful study of the dynamics of infection, in- cluding the isolation of clonal pedigrees of single cells exposed to r which engender lysogenics, A pure lysogenic pedigree would favor the induction hypothesis, Attempts to identify the prelysogenic genotype in Kel2, and hybrids of K-12 and other crossable lines have been unsuccessful, Preliminary experinents of the infecticn process (10) have disclosed lysogenic colonies contaminated with sensitive cells and free phage long after initial con- tact with}, These mixed clones have since been confirmed in K-12 (18) ard Salmonella (1h,21,238). The possibility that spontaneous alteration of the bacteria predisposing to a lysogenic dscision plays some role in the recovery of lysogenics is thus not yes excluded, However, the simplest conception remains that the genetic elexents of the phage are directly incorporated in, or attached to the bacterial chromosome as we have been able to find no indication of an extrasmelear Inheritance of lysogenicity. The Effect of hk and P on Crossing Behavior: . The presence of d in one, both, or neither of the parents of a cross does not influence the yield of recombinants, As noted earlier (8) sensitives were not eliminated as lethal vhenetypes, mab tho progomy of lyscgenic x sensitive included both parental types, and no others, in ratios depsndent on the selected avuxotraph markers, On the other hand, the compatibility fector (F) determines not only the yleld but also the segregation pattern of many overtly unselected markers, Prototrophs are recovered only when at least one parent is F; F also seems to direct the elimination of certain chro- mocomal segments after the formation of the hyorid zygote (15,23). The important distinctions of F and are summarized in table 1 e These are emphasized to mitigate any confusion that might arise from the suggestions that have been recorded elsewhere that A may Play a direct role in sexual recombination as vell as to en the distinction be~ | @zeen the ) controlled transduction of restricted genetic factors and the Fecontrolled sexual recombination. The independent transmission of these factors was demonstrated by the recovery of (1) F*Lp® cells on the one hand, and F"lp on the other, from mixtures of genetically Labelled F"Lp® and F'ip*, and similarly, (2) Lp*F” (but no Lp®r* or Lp*F*) as sur- vivors from F"Lp® exposed te \wcontaining filtrates from F*Lp* cultures. (6) II TRANSDUCTION Cell-free filtrates derived from suitable Salmonella strains were espable of transferring unit eenetic factors to a competent recipient (23). A wide range of imlependent markers has been equally subject to transduction. Additional analysis has shown that the temperate phage of the donor strain ig the vector of the genetic naterial (16,25). Attempts to detect trans- duction in &.12 among the survivors in the turbid centers of J plaques were negative (10); but by using high-titer lysates obtained by U-V induction (20), a succeesful transduction was achieved (22), Two striking contraste with the Salmonella system were demongtrated: (1) the restriction to a single genetic character, galactose fermentation, and (2) a striking | > instability manifested by mosaic Gai*/Gal” colonies after transduction GesplLte repeated single colony purification on EMB galactose agar. The incidence of persistent inotability, rarely if ever encountered in Salmonella (uh), varies with the recipient strain, Confourling of Transduction with Recombination ?: The conditions required for transduction are generally precluded in crossing experinsnts, Moreover, the unstable mosaic Gai* /Gal™ colony characteristic of trans- Quction has rot been so far recovered among recombinant progeny. A (7) Fae more careful inquiry inte the effect of rw and Gal segregation was necessary, oo however, in view of the transduction phenomenon, since it may provide an alternative interpretation of the Gal-Lp cosegregation ratios currently satisfied by a linkage explanation, Crosses of genetically related parsnts differing only in the presence or absence of Awere therefore studied, Table 2 demonstrates no significant deviation in the yleld of Gal” re~ combinants where parents vary only for the Lp marker, is Transduction a Selection Artefact?: Interaction of genetic factors on reverse mutation of entirely independent leci have been re- ported before ( 15). An analysis of the Gal- segregation from the un- stable transduction, the allelic transduction, reported below, as well as many other types of evidence (22) rule out the interpretation that the transduction is a selection artefact. The most convincing evidence, hevsver, has been the development of specific Gal” transductions in Gai” recipient strains by means of A with extraordinary bigh frequency of transduction (22), when the d donor was Gal”, Transduction and F~transfer: Just as lysogenization is independent of the conversion of F” into F* strains, the trensduction mediated by dX is unrelated to the F status of either the recipient or the donor cells, Crosses of PF" x FY by standard techniques are completely sterile, How. ever, recombination of two nonallelic Gal” mutants can be indirectly deme onstrated by transduction. Lysates from Up'GalF” were completely functional. in introducing the Gal* factor to GalF* cells. Similarly, nonalleliva of wo Gal F” strains can be established by the formation of Gal* in transduction experiments whereas the sexual sterility of the cross would block. cel recombination in tote. . Crosses of a strain characterized by its enbeneed fertility, Her, (15) displayed a Linkage of the Hfr trait to Gal. (12), These data were verified (sabe 3) for Galo, Despite this Linkage, efforts to trans port the Hfr ‘and Gal’ factors simulteneously into Gal" F"Lp® recipient cells via \ prepared from Hfr bacteria were unsuccessful, The conversion of F° to F* by \ filtrates fron F strains yas examined by crossing the Gal* transduction with F* tester strains and was Likewise unsuccessful. The competence of an transduction therefore continues to be confined a to the Gal cluster. the Concurrence of Transduction and Lysogenizationt Observations ‘on the Ey colt system, as in Salmonella, are consistent with the hypothesis that the vector of transduction consists of temperate phage. As a rule, (8) es (9) sna teenccuetions isolated from GalLp® nactorie exposed to A are con- (WN sistently pure, stable lysogenics, aepive the persistent instability of the Ga* trait; the ensuing Gal~ segregants are algo aysogenic. Lyso- genization occurs very much more frequently than transcuction, but the correlation of the two remained to be explored as evidence bearing cn the hypothesis. In the first experiment (table h , part A) transductions were picked as cal* paptlice and streaked out on ee galactose agar. A single Gal” (representing non-transindueed celts) and a single Ga* lihe successful transduction) were cach tested for lysogenicity on an appropriate ip® indicator. In experiment B, marked Gal Lp® celis in he approximate proportions expected tron trenstuctton were introduced — with the Gal” and the mined eulture on EMB galactose plates. With the assumption that both Lp® strains would adsorb and be equally affected bys & disparity in lyscgenizations of the two ensuing Gai* classes was leoked for, Whereas ell of the transduction Gal* were lysogenized, only up to 70% of the artifically inserted Gal* or of the original Gal” had been infected, Both parts of the experiment show a distinct corre~ lation of lysogenization with transduction; the incidence of lysogenization is almost higher in these than in the control bacteria on the same plates. (20) s not so far been observed (up to (ay 500 tests) from these simultaneously transduced and lysogenized recipients, CA QO d c py ct 3 Ss .o ry ¢ e a [s) Cy E G 4 : 8 pte fa < we x 5 0) This evidence argues that Aas the passive vector of genetic material from its source strain, This material is injected to the bacterium by the phage, In Saimonella the transduced genetic factors seen to undergo an immediate substitution for the homologues in the recipient bacterium, if they are successful at all, In E, coli K-12, however, an intermediate stage is perceived where one can detect simultaneously the presence of the original recipient and the new transduced genetic factors in the same cells by virtue of their subsequent segregation, The relationship between this replacement of genetic material and the conversion of virulent Aanto its prophage stage { "reduction" 6) has not yes been completely worked a As will be described below, however, these processes have been separated and are therefore not mitnally dependent, Lysogenization of fmemne-1 in Transduction Exveriments: When immune-1 strains such as W+1027 and W-192) are exposed to A, no evidence of their lysogenization is ordinarily perceived, However, under conditions where transductions can be selectively isolated about 5% of these altered bacteria ave alco found to have been lysogenized, Repeated serial segregation of the resulting transductions showed that in some cases, lysogenicity failed to segregate. In others, lysogenicity and Gal segrepate together, while in a single instance a lysogenic Gal” segregant was found which con~ timed to segregate Lp™ colonies, Sometimes a very weak lysogenicity is observed (None-plaque types" in cross~brush tests)» which is completely lost after a few transfers. Some of these atypical cases are presented in table 5, and suggest the following alternative interpretations: (1) Ip” cols _ genetically lysogenic but carry a modified prophage, Theee cells ere generally resistant to infection with ), However, rR may be exceptionally introduced simultaneously with the Ga* fragment and there may displace the avirulent form of the prophage, or when Lp segregation is observed, both prophages persist together for the time being, (2) The Lp is a "mil" allele. In transduction, Lp* and Cal” factors are introduced, but the lysogenicAmmmne segregation eccurs when Cal segregates. This hypothesis can not account easily for the Gel“Lp’/" types except by devising a complicated scheme involving crossingover, (3) Iumunes may or may not be genetically lysogenic. The production of Lp” signifies the occurrence of a double transduction at tue loci, Gal and Lp, (a) ordinarily these linked factors would tend (11) ay (12) to be Lost as a bleck in the ensuing segregation, or (b) a linked transe (x ducticn dces not operate, By a two-step process, two effective perticles have penstrated; one fragment carries Gal®, the other Lp*, Independent segregation is permitted and a mechanism requiring the breakage of a.2= factor linked fregaent as in (2) is not called for. In any event, special assumptions must be made on the avidity of the ip” locus for pro») te account for the failure of transductions to Ly® to segregate Lp* /up® along with Gal.*/Gal”. However, the Lp” may ‘only block the propagation of hor its reduction to pre-\, Hypothesis (1) accounts for the occurrence of immunes which can be induced by U-V (22). The recovery of unstable Lp* traneductions in non~transinduced Gal» would tend to support hypothesis 3, The most decisive elucidation of whether transduction displaces a mutant phage particle with a wild type Aor whether a normal Lp* allele is substituted for a mutant or nuli host Lp’ gene would be provided by experiments with genetically distinguishable )\ preparations, Lp /tp® transductions were prominent with irradiated ) , tending to support hypothesis 2, irradiation effects: Quantitative assays of transducing potentiality of phage preparation are necessarily based on plaque counts, The survivel (13) after various treatments of plaque-~producing particles and transducing ‘43 varticles are not identical either in Salmonella (28) or K-12 (22). In fact, 1% is known from both studies that transducing power may be. increased at some intermediate dosages. A comparison of the effects of U-~T and X-radiation is given in table6. A U-V dese reducing plaque 10 5 assay from 1/2 x 10” to 16.9 x 10° per mil yielded 170 transductions fron an initial titer of 10° / wi. A comparable X-ray dose was found to be between 150 3000 and 200,000 r, No recognizable transductions were recovered at the latter exposure. Two viewpoints are indicated: (1) the lytic and transducing principles in Aave separable by their independent survival, and (2) avirulent A particles are produced but they are damaged only to the extent of virulence for the host cell, Conclusive evidence favoring one or the other viers of Lp, however, is not yet at hand, A decisive chemical and genetic separation of the transducing material from the virus particle has not yet been experi-~ ° : mentally achieved, whether or not it is at all theoretically possible, GENETIC DEFINITION OF THE GAL LOCI } . Recombination: Attention was focused on galactose nonfermenting mutants because of the colncidence of the first recognised A-sensitive (i) mutant in Gal”), (#4518) ; and the subsequent observation of linked @ segregation of lp and Gal), (10). Gal mutants have been isolated directly by inspection of surviving colontes after U-V treatment on EMB galactose agar and also as non»papillating variants of Lac” mutabile recovered on EMB lactose agar plates, Interaction of Gal™ and Gai* on the phenotypic expression and reverse mutation of Lac, and Lacy alleles have been deseribed (9}, Recombination analysis provided the evidence for a cluster of four linked Gal loci (7), Galy and Gal), show a very low order of crossovers. Preliminary data could only differentiate then on the basis of behavior in Het crosseaj Lp and Gal, are both hemizygous, while Gal," /Gaty,~ heterozygous diploids are readily obtained (iabie 7 ). Yransduction: Transduction tests reinfores standard allelism tests (table 8), ani in fact have tentatively identified several new loci, now awaiting cont tems thon by recombination analysis. Whether the relative yield of Gal” transductions is proportional to the map dis- tance between Lp and the Gal locus is in question, The results of large-scale alleliam tests made available to date by new techniques to facilitate crossing are summarized in table 9, Ths ins tability characteristic o% the Gal” transduction results in the mosaic colony already noted and deserves further comment. Despite passage through a large number of serial single colonies, Cal- segregants are almost always thrown off. In transductions from Gal”, Ls@o Gal” —x Gai", these Gal” segregants have been identified as alleles of the locus of the original recipient strain, both by crossing and further transduction tests. Wo other kinds of Gal” have been recovered, On the other hand, if the donor is a non-allelic Gal”, both donor and recipient cal appear among the sogreganta from the Gal* transduction (22). For example, Galp --x Galj,” gives galactose~fermenting intermediates , presumably of the constitution Galy Gal) “/Galy Galy,”. The segregants in all these tests are identified by (1) crossing experiments with Galp~ and Gal,” testers, (2) deriving and subjecting the testers to its action, and (3) applying A tron Gal”, Gal”, Gel)“, ete. The Gel,” Gal)”; a crossover type, has not been conclusively and consistently established, This double mutant would be identified as one which is subject to transduction by ) trom Gal* and from any Gal” other than Galg” or Galy”, and would yield no Gal* recombinants in crosses with Gal,” and Gal, ~ testers,